Variable-frequency electric field generating system and its application in the treatment of pathological angiogenesis

Abstract

The present invention relates to the technical field of medical devices, and particularly to a variable-frequency electric field generating system and its application in the treatment of pathological angiogenesis, including: an alternating current power generator for generating a sine wave signal with a frequency of 500 - 1500 kHz and an amplitude of 50 - 100 Vpp; a wire, the first end of which is connected to the output end of the alternating current power generator. The present invention suppresses the generation of pro-angiogenic factors at the lesion site by generating a medium-high frequency alternating electric field, and has a better effect on suppressing pathological angiogenesis. Compared with the prior art (such as bevacizumab, injection sclerosing agent, endovascular interventional embolization, surgical resection, radiotherapy, hormone therapy, etc.), the device of the present invention suppresses pathological angiogenesis through an alternating electric field, and has the advantages of non-invasive (used on the body surface) or minimally invasive (implanted in the body), safe, locally applied, no systemic adverse reactions, pure physical and pollution-free, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and particularly to a variable-frequency electric field generating system and its application in the treatment of pathological angiogenesis. Background Art

[0002] Under physiological conditions, blood vessels undertake the functions of transporting blood, nutrients, and oxygen to various organs and tissues throughout the body, and at the same time carry away carbon dioxide and metabolic wastes in the tissues. Otherwise, the organs and tissues will become ischemic, hypoxic, and necrotic. Therefore, angiogenesis is essential for all organs in the body. During embryonic development and childhood growth, the angiogenesis process in all organs is very active. After adulthood, the blood vessels in most organs are in a static state, and vascular endothelial cells generally divide only once every about seven years. In the adult human body, only when an organ is damaged and needs to be repaired, the angiogenesis process will be reactivated to participate in tissue repair.

[0003] Angiogenesis refers to the generation of new microvessels from the existing vascular network. The process of angiogenesis includes the degradation of the vascular basement membrane; the activation, proliferation, and migration of vascular endothelial cells; and the reconstruction to form new blood vessels. The angiogenesis process is regulated by a variety of pro-angiogenic factors and angiogenesis inhibitors. Currently, a variety of pro-angiogenic factors such as VEGF, bFGF, IGF-1, TNF-α, TNF-β, and PDGF have been discovered.

[0004] Pathological angiogenesis refers to abnormally active angiogenesis (vascular hyperplasia) that is not required physiologically, which is a common pathological mechanism of many diseases. Examples are as follows:

[0005] (1) Cutaneous hemangioma: It is a benign tumor with capillary hyperplasia and dilation. It mostly occurs soon after birth, grows rapidly in infancy, and then continues to grow with age, stopping developing in adulthood. Most of them invade the skin of the head and neck, seriously affecting appearance. Clinically, it is divided into four types: nevus flammeus, simple hemangioma, cavernous hemangioma, and mixed hemangioma. Current treatment methods include surgical resection, external radiotherapy, isotope therapy, cryotherapy, laser therapy, sclerosing agent injection, hormone therapy, etc., and often require a combination of multiple methods. Large maxillofacial hemangiomas can also erode bone, and transcatheter arterial embolization or surgical resection is required, with a high risk.

[0006] (2) Retinal hemangioma: In the early stage, small hemangiomas or tangled capillaries appear in the peripheral part of the fundus. The affected retinal arteries and veins are dilated and tortuous. The capillaries at the junction of the arteries and veins are highly dilated to form spherical hemangiomas, which then gradually increase in size. There is localized retinal edema and exudation near the hemangioma, which may be accompanied by small hemorrhages. Due to leakage from the hemangioma wall, the surface of the tumor and the surrounding retina appear grayish white and turbid. As the disease progresses, the water in the exudate is absorbed, lipids are deposited, and large ring-shaped or arc-shaped yellow-white hard exudates appear around the hemangioma. When the exudate affects the macula, vision is significantly impaired. As the hemangioma continues to grow, the exudate also gradually increases, and the retina undergoes exudative detachment. In the late stage, it can cause blindness. Treatment methods include photocoagulation, cryotherapy, and diathermy electrocoagulation to shrink the hemangioma and turn it into a scar, but the effect is not good for large hemangiomas and often causes blindness.

[0007] (3) Cerebral proliferative angiopathy (CPA): It is manifested by diffuse, highly dense vascular network tissue on imaging, with normal brain tissue intermixed between abnormally proliferating cerebral blood vessels. It often affects the entire cerebral hemisphere or multiple lobes. Clinical manifestations include epilepsy, headache, progressive neurological deficits, including limb movement disorders, language disorders, mental disorders, etc. Some patients present with cerebral hemorrhage, with a rebleeding rate as high as 67%. Existing treatments include symptomatic treatment (such as anti-epileptic, analgesic), interventional intravascular embolization, indirect angioplasty and radiosurgery, but it is difficult to cure clinically.

[0008] (4) Cerebral arteriovenous malformation (AVM): It manifests as a malformed vascular mass with mixed arteries and veins, with thick supply arteries and drainage veins, and extremely large blood flow, which often leads to cerebral hemorrhage and epilepsy. Clinically, if the supply arteries and drainage veins are cut off, but the vascular mass is not completely removed, the remaining vascular mass will grow rapidly and re-establish the supply arteries and drainage veins, leading to recurrence. This process reflects vigorous pathological angiogenesis. Existing treatment options include intravascular interventional embolization therapy, surgical resection, and radiosurgery. The treatment effect of large or deep AVM is poor, often leading to cerebral hemorrhage and disability or death of patients; and the above-mentioned treatment methods themselves are also very risky, and massive bleeding, cerebral infarction or perfusion pressure breakthrough syndrome are prone to occur during the operation, which is life-threatening.

[0009] (5) Pathological angiogenesis in malignant tumors: Since the metabolism of malignant tumor tissues is extremely active, they require more oxygen and nutrients than normal tissues, and also need to excrete metabolic products. The growth and survival of malignant tumors highly depend on pathological angiogenesis. When the diameter of the tumor > 2 mm, obtaining the required nutrients and oxygen through tissue interstitial diffusion alone is no longer sufficient. At this time, the local angiogenesis regulatory balance is disrupted, generating many pro-angiogenic factors, especially VEGF, which promotes tumor angiogenesis and provides material support for tumor growth, progression, and metastasis. These newly formed blood vessels supply nutrients to the continuously infiltrating and growing tumor. In turn, tumor cells secrete various substances during growth to accelerate tumor pathological angiogenesis. Due to the abnormal structure and function of the newly formed blood vessels in tumor tissues and the imperfect vascular stroma, tumor cells are easily able to directly penetrate into the blood vessels and enter the bloodstream, forming metastases at distant sites.

[0010] It should be noted that among the existing technologies, the treatment methods for tumors also include electrotherapy. Although electrotherapy also applies an alternating electric field with a certain frequency to the lesion site, as shown in the technical solution of patent document 1 for example, its principle is to act on the tubulin of proliferating cancer cells through a low-intensity, medium-frequency (200 kHz) alternating electric field, interfering with the mitosis of tumor cells, causing the affected cancer cells to apoptosis and inhibiting tumor growth, and it does not have the therapeutic effect of anti-angiogenesis. On the one hand, the electric field frequency used in the present invention is different from that in patent document 1, so the biological effects are different (anti-cancer cells vs anti-angiogenesis); on the other hand, more importantly, the target of action of the present invention is vascular endothelial cells, rather than tumor cells, and it can not only be used for malignant tumor angiogenesis, but also can be applied to benign vascular proliferative lesions.

[0011] Therefore, angiogenesis plays an important role in the development and metastasis of tumors. Inhibiting this process is beneficial to preventing tumor growth, diffusion, and metastasis. Currently, the treatment for this process generally uses anti-angiogenic drugs, including anti-vascular endothelial growth factor (VEGF) monoclonal antibodies, recombinant human endostatin, etc. In theory, anti-angiogenic drugs can inhibit the process of tumor angiogenesis, causing the existing tumor blood vessels to degenerate, thereby slowing down tumor growth. However, in actual applications, the efficacy of existing anti-angiogenic treatments for malignant tumors is still not satisfactory, the survival benefit of patients is very low, and there are systemic adverse reactions such as gastrointestinal bleeding and perforation.

[0012] In summary, pathological angiogenesis is a common pathogenic mechanism for many diseases (including both tumor and non-tumor diseases, benign and malignant, superficial and deep); correspondingly, there are already many therapeutic methods targeting pathological angiogenesis clinically. For superficial, benign, and small pathological angiogenesis, the treatment effect is relatively good. However, the existing technologies have poor efficacy for deep, widely dispersed, and malignant pathological angiogenesis, and some drugs and surgeries themselves also carry high risks; therefore, there is an urgent need to develop new anti-angiogenic treatment devices.

[0013] Prior art documents:

[0014] Patent document 1: CN112553075A, a method and device for inhibiting the rapid growth of tumor cells using a sensitive frequency electric field Summary of the invention

[0015] In view of the technical problems existing in anti-angiogenic treatment in the prior art, the first aspect of the present invention provides a variable-frequency electric field generating system, including:

[0016] An alternating current power generator for generating a sine wave signal with a frequency of 500 - 1500 kHz and an amplitude of 50 - 100 Vpp.

[0017] A wire, with its first end connected to the output terminal of the alternating current power generator.

[0018] At least one pair of insulated electrode plates connected to the second end of the wire.

[0019] Wherein, the insulated electrode plate includes a flexible conductive layer and flexible insulating layers coated on both sides of the flexible conductive layer, and the insulated electrode plate is affixed to the surface of the body part with the lesion or, as an implant, affixed to the surface of the diseased internal organ.

[0020] When the alternating current power generator outputs a sine wave signal to the insulated electrode plate, its rated output power is less than 3 W, and an alternating electric field generated between a pair of the insulated electrode plates is used to inhibit the generation of angiogenesis-promoting factors in the alternating electric field region.

[0021] Preferably, the distance between a pair of the insulated electrode plates and the amplitude of the alternating current power generator are configured to satisfy that the intensity of the alternating electric field generated between a pair of the insulated electrode plates is greater than 0.6 V / cm.

[0022] Preferably, the flexible conductive layer includes a metal layer, and the metal layer includes a silver foil layer.

[0023] Preferably, the flexible insulating layer includes an insulating film, and the insulating film includes a polyimide film.

[0024] Preferably, the thicknesses of the flexible conductive layer and the flexible insulating layer are less than 0.1 mm.

[0025] Preferably, the frequency of the sine wave signal is 800 - 1200 kHz.

[0026] Preferably, the frequency of the sine wave signal is 1000 kHz.

[0027] Preferably, the alternating current power generator includes a sine wave generator, a differential amplifier, a step-up transformer, a communication controller, and a DC / DC isolated regulated power supply. The sine wave generator, the differential amplifier, and the step-up transformer are connected in sequence. The communication controller is connected to the sine wave generator, and the DC / DC isolated regulated power supply provides power for each component.

[0028] Preferably, the sine wave generator uses the high-frequency waveform generator of integrated chip MAX038.

[0029] Preferably, the wire includes a coaxial cable.

[0030] In the second aspect of the present invention, a technical solution is proposed, which is an application of a variable-frequency electric field generating system as described above in the treatment of pathological angiogenesis.

[0031] Compared with the prior art, the advantages of the present invention are as follows:

[0032] 1. Based on the research result that the alternating electric field inhibits pathological angiogenesis first discovered by the inventor team, the present invention inhibits the generation of angiogenic growth factors at the lesion site by generating a medium-high frequency alternating electric field, and has a better effect on inhibiting pathological angiogenesis. Compared with the prior art (such as bevacizumab, injection sclerosing agent, intravascular interventional embolization, surgical resection, radiotherapy, hormone therapy, etc.), the device of the present invention inhibits pathological angiogenesis through an alternating electric field, and has the advantages of non-invasive (used on the body surface) or minimally invasive (implanted in the body), safe, local application, no systemic adverse reactions, pure physical and pollution-free, etc.

[0033] 2. The present invention designs the material of the insulating electrode plate, uses a material with soft texture and high conductivity as the metal layer, and uses a material with soft texture, good insulation performance, relatively large relative dielectric constant and good biocompatibility as the insulating layer, so that the overall texture of the insulating electrode plate is soft and the adhesion is good, and it can be conveniently and firmly attached to the body surface of the lesion site or attached to the surface of the lesion organ as an implant in the body.

[0034] 3. The present invention controls the alternating current power generator to generate a sine wave signal with a frequency of 500 - 1500 kHz, and at the same time, the rated output power is less than 3 W. On the one hand, it reduces the technical difficulty and implementation cost of the equipment, and on the other hand, it ensures the safety of equipment use. When the present invention is in normal use, it can form a high voltage and a high electric field strength. In case the transmission wire or the insulating layer of the electrode is damaged, once the resistance decreases, the voltage will instantly drop to an extremely low value, thus avoiding the possibility of electric shock and injury.

[0035] 4. The transmission wire of the present invention uses a coaxial cable, which reduces the voltage loss.

[0036] 5. Due to the design principle of the extremely low output power of the alternating current power generator, it is more energy-saving. Therefore, the volume of the storage battery used for power supply can be reduced, the weight can be lightened, and the portability of the equipment of the present invention can be increased.

[0037] 6. Pathological angiogenesis is a common pathogenic mechanism of many diseases (including tumor and non-tumor diseases, benign and malignant, superficial and deep), so the present invention has a broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings are not intended to be drawn to scale. In the drawings, each identical or approximately identical component shown in each figure may be denoted by the same reference numeral. For clarity, not every component is labeled in each figure. Now, embodiments of various aspects of the present invention will be described by way of example and with reference to the drawings, wherein:

[0039] Figure 1 is a schematic structural diagram of a variable-frequency electric field generating system proposed by the present invention;

[0040] Figure 2 is a schematic structural diagram of an alternating current power generator proposed by the present invention;

[0041] Figure 3 is a schematic cross-sectional view of an insulating electrode plate of the present invention;

[0042] Figure 4 is a schematic diagram of the use state of the insulating electrode plate of the present invention on the body surface;

[0043] Figure 5 is a schematic diagram of the use state of the insulating electrode plate of the present invention in the body;

[0044] Figure 6 is a schematic diagram of the experimental result shown in Embodiment 1 of the present invention;

[0045] Figure 7 is a schematic diagram of the experimental result shown in Embodiment 2 of the present invention;

[0046] Figure 8It is a schematic diagram of the experimental results shown in Embodiment 3 of the present invention;

[0047] Figure 9 It is a schematic diagram of the experimental results shown in Embodiment 4 of the present invention;

[0048] Figure 10 It is a schematic diagram of the experimental results shown in Embodiment 5 of the present invention;

[0049] Figure 11 It is a schematic diagram of the experimental results shown in Embodiment 6 of the present invention;

[0050] Figure 12 It is a schematic diagram of the experimental results shown in Embodiment 7 of the present invention;

[0051] Figure 13 It is a schematic diagram of the experimental results shown in Embodiment 7 of the present invention;

[0052] Figure 14 It is a schematic diagram of the experimental results shown in Embodiment 8 of the present invention;

[0053] Figure 15 It is a schematic diagram of the experimental results shown in Embodiment 9 of the present invention;

[0054] Figure 16 It is a schematic diagram of the experimental results shown in Embodiment 9 of the present invention;

[0055] Figure 17 It is a physical schematic diagram of the TTFields unidirectional electric field generator shown in the embodiments of the present invention;

[0056] Figure 18 It is a diagram of the chicken embryo chorioallantoic membrane test and other animal test scenarios shown in Embodiment 1 of the present invention. Detailed implementation manners

[0057] To better understand the technical content of the present invention, specific embodiments are given below in conjunction with the accompanying drawings for illustration.

[0058] Combined with Figure 1 and Figure 17 As shown, the first aspect of the present invention provides a frequency-variable electric field generation system, including an alternating power supply generator 100, a wire 300, and at least a pair of insulating electrode plates 200. Among them, the alternating power supply generator 100 is used to generate a sine wave signal, and the frequency of the sine wave signal is a sine wave signal of 500 - 1500 kHz and the amplitude is 50 - 100 Vpp; the first end of the wire 300 is connected to the output end of the alternating power supply generator 100; at least a pair of insulating electrode plates 200 are connected to the second end of the wire 300.

[0059] Among them, the insulating electrode plate 200 includes a flexible conductive layer and flexible insulating layers coated on both sides of the flexible conductive layer. The insulating electrode plate 200 is affixed to the body surface of the lesion site or, as an implant, to the surface of the diseased internal organ.

[0060] When the alternating current power generator 100 outputs a sine wave signal to the insulating electrode plate 200, its rated output power is less than 3 W, and an alternating electric field generated between a pair of insulating electrode plates 200 is used to inhibit the generation of angiogenesis-promoting factors in the alternating electric field region.

[0061] Among them, the alternating current power generator 100 is used to generate a sine wave signal with a frequency of 500 - 1500 kHz and an amplitude of 50 - 100 Vpp to meet the individualized treatment requirements of various diseased tissues, different parts, and different patients.

[0062] Preferably, the distance between a pair of insulating electrode plates 200 and the amplitude of the alternating current power generator 100 are configured to satisfy that the intensity of the alternating electric field generated between a pair of insulating electrode plates 200 is greater than 0.6 V / cm. At this electric field intensity, the inhibition of pathological angiogenesis is better, and it is safe and reliable to use.

[0063] Alternating current power generator

[0064] As Figure 2 shown, the alternating current power generator 100 of this embodiment includes a sine wave generator 1, a differential amplifier 2, a step-up transformer 3, a communication controller 4, and a DC / DC isolated regulated power supply 5. The sine wave generator 1, the differential amplifier 2, and the step-up transformer 3 are connected in sequence. The communication controller 4 is connected to the sine wave generator 1, and the DC / DC isolated regulated power supply 5 provides power for each component.

[0065] The sine wave generator is designed and constituted by using a high-performance high-frequency waveform generator integrated chip MAX038, which has the ability to generate sine waves with a frequency of 0.1 Hz - 20 MHz, can generate a 500 - 1500 kHz sine wave signal required by this device, and its amplitude is 2 Vpp.

[0066] The differential amplifier 2 is responsible for amplifying the 500 - 1500 kHz sine wave signal output by the sine wave generator. Since the signal frequency band is relatively wide and a relatively large output amplitude is required, it cannot be simply realized by using an ordinary power amplifier circuit. For this reason, this embodiment uses two wide-band amplifiers to respectively amplify and output two signals with a phase difference of 180 degrees between positive and negative phases. That is, a differential amplifier is constituted by using 2 amplifiers, and each amplifier outputs a signal of 25 - 50 Vpp, and the 2 differentials generate a signal of 50 - 100 Vpp, thereby greatly reducing the requirements for the performance of a single amplifier.

[0067] To further reduce the requirements for the performance of the amplifier, a solution of adding an output step-up transformer at the output end of the differential amplifier is adopted. Through the step-up of the transformer, an integrated operational amplifier with a lower supply voltage can be used to design a differential amplification circuit, thereby ensuring stable step-up at higher frequencies and wider frequency bands.

[0068] The communication controller realizes the adjustment and control of the output frequency and output voltage, and controls the sine wave generator to output the required waveform.

[0069] The DC / DC isolated regulated power supply is responsible for converting the input DC voltage of 19 - 24V into various voltages required by the machine, such as ±5V required by the sine wave generator, ±50V required by the wide-band amplifier, etc.

[0070] The above alternating power generator is connected to a pair of insulated electrode plates to generate an electric field; since the positive and negative poles are insulated, with extremely high resistance and extremely small current (only a leakage current of <1 μA), the rated output power of the alternating power generator is designed to be <3W. The extremely low output power and high voltage of the alternating power are one of the characteristics of the present invention. On the one hand, it reduces the technical difficulty and implementation cost of the equipment, and on the other hand, it ensures the safety of equipment use: under normal use conditions, high voltage and high electric field strength can be formed. In case of damage to the transmission wire or the insulation layer of the electrode, once the resistance decreases, the voltage will instantly drop to an extremely low value, thus avoiding the possibility of electric shock and injury.

[0071] In this embodiment, the wire 300 is a coaxial cable, which can reduce voltage loss.

[0072] Insulated electrode plate

[0073] As Figure 3 shown, the insulated electrode plate 200 includes flexible insulating layers on both sides in a sealed package and a flexible conductive layer in the middle, that is, there is no gas or other medium residue between the flexible insulating layer and the flexible conductive layer, and the flexible conductive layer is electrically connected to the wire 300. The outer surface of the flexible insulating layer is the surface where the insulated electrode plate contacts the object to be acted on, and is used to form an alternating electric field in the area between a pair of insulated electrode plates.

[0074] In today's market, insulated electrode plates used for other electric field treatments generally use piezoelectric ceramics as the insulating layer because piezoelectric ceramics have extremely high relative dielectric constant and dielectric strength, which can reduce voltage loss and avoid high-voltage breakdown. However, piezoelectric ceramics are thick and hard, have a very poor fit with the skin, are prone to air gaps, or cause skin damage, and are very inconvenient to use; moreover, piezoelectric ceramics are difficult to implant into the body and can only be used on the body surface. Due to the safety of the extremely low-power alternating power supply of the present invention, the insulating layer of the insulated electrode plate does not need to be too thick, and an ultra-thin insulating layer can be used. In this embodiment, the thicknesses of both the metal layer and the insulating layer are less than or equal to 0.1 mm.

[0075] In an alternative embodiment, the flexible conductive layer is made of a material with a soft texture and high conductivity, and the insulating layer is made of a material with a soft texture, good insulation performance, a relatively large relative dielectric constant, and good biocompatibility. The flexible conductive layer includes a metal layer 202, and the flexible insulating layer includes an insulating film 201. The metal layer can be selected as silver foil, etc., and the insulating layer can be selected as a polyimide film, etc.

[0076] The dielectric strength of polyimide is 100 - 300 KV / mm (Reference: https: / / wenku.baidu.com / view / ed2ca4906037ee06eff9aef8941ea76e58fa4a28.html). When the thickness of the polyimide insulating layer is 0.05 mm, it can prevent voltage breakdown of 5000 V, which is much higher than the peak voltage of 100 V used in the present invention, and is safe enough.

[0077] Since the insulating layer is very thin, it does not require an extremely high relative dielectric constant. The relative dielectric constant of the polyimide insulating layer is 3.4. According to the calculation of the experimental electric field distribution, with a total voltage of 106 V, the voltage distributed in the biological tissue reaches 84 V (79.2%), while the voltage lost in the insulating layer is only 22 V (20.8%). Since both the insulating layer and the metal layer are very thin, the insulating electrode plate of the present invention is overall soft and has good adhesion, which is convenient for being attached to the surface of the body part with lesions or as an implant to be attached to the surface of the diseased internal organ. This is not possessed by the existing electric field treatment devices.

[0078] As Figure 4 shown is a schematic diagram of the use state of the insulating electrode plate 200 on the body surface for cerebral arteriovenous malformation. The insulating electrode plate 200 is attached to the surface of the skull, facing each other front and back, and is connected to the alternating current power generator 100 through the transmission wire 300. The diseased part is in the alternating electric field between this pair of insulating electrode plates.

[0079] As Figure 5 shown is a schematic diagram of the use state of the insulating electrode plate 200 in the body for liver cancer. Through the surgical incision 400, the insulating electrode plate 200 is placed into the abdominal cavity as an implant, attached to the surface of the liver, and the transmission wire 300 is led out and connected to the alternating current power generator 100.

[0080] A technical solution is proposed in the second aspect of the present invention, which is an application of a frequency - variable electric field generating system as described above in the treatment of pathological angiogenesis.

[0081] The specific application implementation method is as follows: Taking the lesion site as the center, the insulating electrode plates of the above-mentioned electric field treatment device are attached to the surface of the body surface or internal organs on both sides; through the transmission wire, the insulating electrode plates are connected to the alternating current power generator, so as to form an alternating electric field in the area between the two electrode plates. The electric field strength (E1) can be calculated according to the relative permittivity (∈2) of the insulating layer of the electrode plate, the thickness (d2) of the insulating layer, the relative permittivity (∈1) of human tissues, the thickness (d1) of human tissues, and the voltage (U). The formula is as follows:

[0082]

[0083] According to the experimental results, when the electric field strength > 0.6 V / cm and the electric field frequency is greater than 100 kHz, the alternating electric field begins to exhibit the effect of inhibiting pathological angiogenesis. When the electric field frequency is 1000 kHz, the effect of the alternating electric field on inhibiting pathological angiogenesis is the strongest.

[0084] Preferably, the frequency of the sine wave signal is 1000 kHz.

[0085] In an alternative embodiment, the frequency of the sine wave signal is 800 - 1200 kHz.

[0086] Next, the inhibitory effect of the above-mentioned electric field treatment device on vascular endothelial growth factor will be verified in specific embodiments.

[0087] Example 1

[0088] In this example, the effect of medium and high frequency alternating electric fields on pathological angiogenesis was detected by chicken embryo experiments.

[0089] Specifically, when primary blood vessels appear in the chicken embryo, MCF-7 breast cancer cells are injected into the chicken embryo with a fine needle, and pathological angiogenesis is induced in the chicken embryo by tumor cells. Then, a medium and high frequency alternating electric field is applied to the chicken embryo. The electric field strength of each electric field treatment group is 0.6 V / cm, and the electric field frequencies are 30, 50, 100, 200, 300, 500, 800, 1000, 1500 kHz respectively. The control group is not treated with an electric field; after 48 hours, the generation of secondary blood vessels in the chicken embryo is observed (the test scenario is as Figure 18 shown, the lower part is the chicken embryo chorioallantoic membrane test).

[0090] As Figure 6 shown in the upper two columns above, according to the experimental results of Example 1, it can be found that when the alternating electric field frequency is 1000 kHz, the electric field has an obvious inhibitory effect on angiogenesis.

[0091] Then, U87 glioma cells were used for verification. According to Figure 6As shown in the lower two columns below, by comparing 200, 500, 1000, and 1500 kHz, it was also found that the inhibitory effect of the 1000 kHz alternating electric field on angiogenesis was the strongest.

[0092] Example 2

[0093] The purpose of this example is to detect the inhibitory effect of medium and high frequency alternating electric fields on the proliferation of vascular endothelial cells.

[0094] As mentioned above, the process of angiogenesis includes the degradation of the vascular basement membrane; the activation, proliferation, and migration of vascular endothelial cells; the reconstruction to form new blood vessels and vascular networks, and vascular endothelial cells play the most important role in this process; various angiogenic growth factors such as VEGF also mainly play their roles by promoting the proliferation of vascular endothelial cells.

[0095] To study the mechanism of medium and high frequency alternating electric fields inhibiting angiogenesis, we detected the inhibitory effect of the electric field on the proliferation of vascular endothelial cells through the CCK-8 (Cell Counting Kit-8 cell counting) assay.

[0096] The control group was not treated with an electric field. The electric field intensity of the four electric field treatment groups was 0.6 V / cm, and the electric field frequencies were 200, 500, 1000 kHz, and 1500 kHz respectively. The electric field was continuously applied for 48 h, and then the cell viability was detected by the CCK-8 assay.

[0097] As Figure 7 shown on the left, the 200, 500, and 1500 kHz electric fields had a slight inhibitory effect on the proliferation of vascular endothelial cells, while the 1000 kHz electric field had a significant inhibitory effect; indicating that the alternating electric field achieved the effect of inhibiting angiogenesis by inhibiting the proliferation of vascular endothelial cells.

[0098] As Figure 7 shown on the right, under the condition of selecting 1000 kHz, the higher the electric field intensity, the stronger the inhibitory effect on the proliferation of vascular endothelial cells.

[0099] Combining the above Example 1 and Example 2, it can be concluded that compared with other frequencies, the inhibitory effect on the proliferation of vascular endothelial cells is better under the condition of 1000 kHz.

[0100] Example 3

[0101] Example 3 further conducted experiments on vascular endothelial cells under the condition of a 1000 kHz electric field. Through immunofluorescence chemical staining, it was found that the microtubules of vascular endothelial cells showed a "woolly ball" - like change under the 1000 kHz alternating electric field: Since the classical mechanism of TTFields involves mitotic inhibition, we attempted to determine whether AEF (Alternating Electric Fields) induced HUVEC cell death by affecting the formation of the spindle.

[0102] Combined with Figure 8 the results shown, treatment with AEF for 24 or 48 hours induced an obvious "ball of yarn" - like spindle structure, in sharp contrast to the shuttle - like spindles observed in the control group.

[0103] Notably, the spindle structure induced by AEF was very similar to the spindle formed by vincristine, suggesting a possible common mode of action. It is known that vincristine can bind to tubulin and hinder the polymerization of tubulin dimers, thereby preventing chromosome separation in metaphase. These findings indicate that AEF exerts an inhibitory effect on HUVEC growth by interfering with the formation of the general spindle. Notably, in the experimental group, we did not observe abnormal behavior of histone H3 - labeled chromosomes.

[0104] Example 4

[0105] Example 4 further studied the growth - inhibitory effect on vascular endothelial cells under the condition of a 1000 kHz electric field and found that two - photon imaging showed that the 1000 kHz alternating electric field inhibited and remodeled animal tumor angiogenesis and had a synergistic effect with bevacizumab.

[0106] Although tumor electric field therapy has achieved remarkable success in the treatment of glioblastoma, its in - vivo anti - angiogenic effect has not been widely studied. Based on the highly efficient anti - angiogenic effect of AEF in chick embryo assays and lumen formation assays, we established a glioblastoma mouse model (U87) to evaluate the in - vivo anti - angiogenic effect of AEF (the experimental scenario is as Figure 18 shown, the upper part is the mouse experimental scenario).

[0107] Mice bearing tumors were injected with PBS or bevacizumab intratumorally (i.t.), and AEF treatment was performed by attaching copper plate electrodes to the tumor surface. Bevacizumab is a first - line clinical drug for anti - tumor angiogenesis, which provides an important reference for the anti - tumor angiogenesis effect of AEF.

[0108] One week after treatment of the mouse tumors, two - photon imaging using Texas Red - labeled Dextran was performed to observe the morphological characteristics of tumor blood vessels after bevacizumab or AEF treatment.

[0109] Combined Figure 9 As shown, representative images of tumor blood vessels in our control group (PBS), AEF, bevacizumab, and bevacizumab & AEF groups were analyzed. The blood vessels in the control group showed characteristics of high leakage, disorganization, dilation, and sacculation. In contrast, the blood vessels in the AEF, bevacizumab, and bevacizumab & AEF groups were significantly reduced.

[0110] Among them, bevacizumab treatment mainly cleared large blood vessels and capillaries in the tumor microenvironment but did not change the disordered morphology of tumor blood vessels. In contrast, AEF treatment effectively transformed the disordered blood vessels into parallel capillaries similar to the normal blood vessels found in skeletal muscle.

[0111] Upon observation, the parallel capillaries were strictly perpendicular to the direction of the electric field transmitted by AEF through two pairs of sensor arrays generating perpendicular fields. Notably, most capillaries decomposed into dozens of segments. In addition, AEF and bevacizumab showed a synergistic inhibitory effect on glioblastoma angiogenesis, resulting in fewer blood vessels in the AEF & bevacizumab group.

[0112] Therefore, we conclude that AEF reduces and remodels tumor blood vessels into parallel capillaries and enhances the anti - angiogenic effect of bevacizumab.

[0113] Example 5

[0114] Example 5 continued to analyze the principle of the inhibitory growth of 1000 kHz alternating electric field on blood vessels using other means. Microvascular quantitative analysis showed that 1000 kHz alternating electric field significantly inhibited animal tumor angiogenesis.

[0115] Two - photon imaging enabled us to evaluate the number and diameter of blood vessels in each tumor sample section. According to their diameters, blood vessels were classified into three categories: "capillaries" (≤9 μM), "medium - sized blood vessels" (≥14 μM), and "large - sized blood vessels" (>14 μM).

[0116] Based on this, to comprehensively study the effect of AEF on angiogenesis, we quantitatively analyzed tumor blood vessels through two - photon imaging and 3D simulation charts.

[0117] In Figure 10 In the left - hand column of pictures, representative images of the surface, middle, and inner layers of tumors untreated (Control) or treated with different treatments (AEF, bevacizumab, and AEF & bevacizumab) are shown.

[0118] Each two - photon imaging or simulation chart contains data from three independent mice (R1, R2, and R3), and the number of each type of blood vessel is listed above the chart points.

[0119] We observed that for all treatment groups, the number of blood vessels in the simulated graphs was generally consistent with the two-photon imaging results, except for the control group. The leaky nature of tumor blood vessels in the control group led to the release of the imaging agent into the surrounding microenvironment, generating a strong background that made it difficult for the analysis software to distinguish blood vessels from the background, especially for capillaries. To further compare the average number of the three types of blood vessels among the control group, AEF, and bevacizumab groups, we analyzed the data from two-photon imaging. The results showed significant differences among the groups.

[0120] AEF treatment led to a significant increase in the number of capillaries (37.33 ± 2.52), which was significantly higher than that in the control group (23.33 ± 7.23) and the bevacizumab group (17.0 ± 7.21). On the other hand, the number of medium-sized blood vessels in the AEF group was significantly less (2.3 ± 1.5), compared with higher numbers in the control group (22.0 ± 8.66) and the bevacizumab group (17.0 ± 5.29). Importantly, there were almost no large-sized blood vessels in the AEF group. In addition, the total number of blood vessels in the control group was significantly higher than that in the AEF and bevacizumab groups (two-photon imaging). There were no significant differences among the three types of blood vessels in the AEF & bevacizumab group.

[0121] Therefore, the AEF group could significantly inhibit the growth of capillaries into medium and large blood vessels.

[0122] Example 6

[0123] 1000 kHz alternating electric fields inhibit angiogenesis and tumor growth in animals.

[0124] Combined Figure 11 As shown, to further evaluate the effectiveness of AEF in inhibiting tumor growth, we extended the treatment period and used intravenously injected bevacizumab as a positive control, considering the standard drug administration in clinical cancer treatment. After 14 days of treatment, both AEF (0.42 ± 0.1 g) and bevacizumab (0.88 ± 0.39 g) significantly inhibited tumor growth.

[0125] Notably, compared with the PBS group (1.31 ± 0.33 g), the inhibitory effect of AEF on tumor growth was even higher than that of bevacizumab. In addition, AEF demonstrated the ability to enhance the therapeutic effect of intravenously injected bevacizumab (0.045 ± 0.026 g).

[0126] We believe that, in addition to the individual anti-angiogenic effects of AEF and bevacizumab, their combined application results in a synergistic effect. This effect is at least partially attributed to the tumor vessel normalization induced by AEF. This normalization facilitates the efficient delivery of oxygen and bevacizumab to the tumor, thereby enhancing the overall therapeutic effect. These findings suggest that AEF effectively inhibits the growth of U87 tumors and produces a potent synergistic effect in combination with bevacizumab. This highlights the potential of AEF as an effective strategy for cancer treatment and provides a new approach for improving anti-cancer therapy.

[0127] It can be concluded that AEF inhibits neovascularization and subsequently promotes tumor atrophy.

[0128] Example 7

[0129] Based on Example 5, two-photon microscopy showed that all the blood vessels within the tumor were transformed into a unique pattern, referred to as "parallel capillaries". To further investigate the distribution and organization of these blood vessels, we conducted immunohistochemical studies on tumor samples.

[0130] Considering that the direction of the electric field is known to be strictly perpendicular to the coronal plane of the tumor, we obtained paraffin sections of the coronal plane ( Figure 12 ) and cross-section ( Figure 13 ) for immunohistochemical studies, as Figure 12 shown. This enabled us to closely study the arrangement of blood vessels on different planes and comprehensively understand the impact of AEF on the tumor vascular network.

[0131] In the PBS group, the blood vessels in the middle and surface layers of the tumor appeared large, abundant, and randomly dispersed, without a unified direction. Similarly, there were no significant differences in the morphology and distribution of tumor blood vessels in the bevacizumab group compared to the PBS group, although they were fewer and smaller, indicating the effective anti-angiogenic effect of the anti-cancer drug. In contrast, the AEF treatment group showed a completely different pattern. The blood vessels of the tumor were in a consistent direction, with discontinuous punctate structures, which was in complete agreement with the results of two-photon imaging ( Figure 12 ).

[0132] Furthermore, compared to the PBS group, the number of blood vessels in the surface and middle layers of the AEF group was significantly reduced. Importantly, the tumor cells stained with DAPI and α-tubulin did not show significant changes among the three groups, indicating that AEF mainly affected vascular endothelial cells rather than tumor cells. This observation was further confirmed by the TUNEL assay, which showed that bevacizumab induced apoptosis in both tumor cells and vascular endothelial cells, while in the AEF group, apoptosis mainly occurred in vascular endothelial cells.

[0133] Overall, two-photon microscopy and immunohistochemistry results provided valuable insights into the effects of AEFs on tumor blood vessels. The blood vessels were arranged in a consistent direction, and a clear background appeared after intravenous injection of fluorescently labeled Dextran, indicating the potential of AEFs in restructuring tumor blood vessels in addition to their anti-angiogenic effect.

[0134] Example 8

[0135] Based on Example 5, we further confirmed by TUNEL assay, which showed that bevacizumab induced apoptosis in tumor cells and vascular endothelial cells, while in the AEFs group, most apoptotic cells (green) overlapped with CD31-labeled vascular endothelial cells (red) ( Figure 14 A), meaning that AEFs at 1000 kHz were more inclined to induce apoptosis in vascular endothelial cells rather than tumor cells. By applying trend lines to connect the discontinuous blood vessels, we generated a simulation graph, similar to two-photon imaging, where the direction of capillaries within the tumor was perpendicular to the direction of the electric field ( Figure 14 B). In summary, two-photon imaging (Figure), immunohistochemistry results ( Figure 12 and 13 ) and TUNEL assay again demonstrated that the present invention was capable of remodeling the vascular structure within tumors and reducing angiogenesis.

[0136] Example 9

[0137] Based on Example 5, we performed a comprehensive bioinformatics analysis on the transcriptome data of tumors in the control group and the AEFs treatment group ( Figure 15 A). The volcano plot showed the differential gene expression between the two groups, revealing that compared with the control group, AEFs treatment upregulated 98 genes and downregulated 101 genes. Hierarchical clustering heatmap analysis further highlighted the major differential genes ( Figure 15 B). Among the genes upregulated in the AEFs group, including myosin heavy chain (MYH) 1, MYH2, MYH4, MYH13, myocin-associated protein that binds to actin (NRAP), long non-coding RNA 2603 (LINC02603), tetraspanin 2 (TSPAN2) and actin-binding protein α2 (ACTN2), these genes were mainly related to muscle function and cell motility. The re-expression or upregulation of these genes was related to muscle regeneration or injury, similar to the effect of chronic electrical stimulation. This may partly explain the phenomenon of parallel capillary formation in AEFs-treated tumors, which may involve cell movement in a specific direction. In addition, AEFs treatment led to significant downregulation of chemokines (CXCL1, CXCL2, CXCL3, CXCL5 and CXCL8) and matrix metalloproteinases (MMP1, MMP3 and MMP12) ( Figure 15C). Notably, members of the CXC chemokine family, including CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8, act by activating CXCR1 and CXCR2 and play an important role in promoting tumor angiogenesis. Similarly, matrix metalloproteinases MMP1, MMP3, and MMP12 are key regulators of tumor angiogenesis, vascular structure, permeability, and integrity. Downregulation of these chemokines and matrix metalloproteinases contributes to the anti-angiogenic effect of AEFs. In addition, AEFs treatment reduced the level of interleukin 6 (IL-6), a characteristic of the tumor microenvironment that can promote tumorigenesis by regulating various cancer hallmarks and multiple signaling pathways, including proliferation, apoptosis, angiogenesis, and metastasis. The decrease in IL-6 level indicates the downregulation of cancer-related hallmarks and the inhibition of tumor growth. However, other important angiogenesis-related factors, such as VEGF and HIF-1α, showed no significant difference between the AEFs group and the control group. This indicates that the anti-angiogenic effect of 1000 kHz AEFs is VEGF-independent, making it a complementary therapy to bevacizumab. In addition, KEGG pathway analysis revealed significant changes in pathways with known anti-tumor and anti-angiogenic capabilities, including MAPK, cytokine-cytokine receptor interaction, NOD-like receptor, chemokine signaling, and TNF signaling pathways ( Figure 15 D). Gene set enrichment analysis (GSEA) of tumor gene data showed that the MAPK signaling pathway, cytokine-cytokine receptor interaction, chemokine signaling pathway, and JAK STAT signaling pathway were downregulated in the AEFs group compared with the control group. Figure 15 E). The downregulation of these signaling pathways supports the inhibitory effect of AEFs on tumor angiogenesis.

[0138] The main gene expression changes in tumors after AEF treatment were further confirmed by real-time fluorescence quantitative PCR and immunohistochemistry. The results showed that the expression of many key angiogenesis-related genes was downregulated in the AEFs group compared with the control group. Figure 16 A). In particular, in the control group, the mRNA levels of CXCL1 (P = 0.0112), CXCL5 (P = 0.0039), and MMP12 (P = 0.041) were more than ten times higher than those in the AEFs group, while the expression of CXCL2 (P = 0.0091), CXCL3 (P = 0.0267), CXCL8 (P = 0.0463), and MMP1 (P = 0.0002) was more than twenty times higher in the control group. In addition, the mRNA levels of IL-6 (P = 0.0266) and MMP3 (P = 0.0336) in the AEFs group were significantly lower than those in the control group, with 61-fold and 71-fold decreases, respectively. Immunohistochemistry further verified the downregulation of these gene proteins. Figure 16B). After AEFs treatment, CXCL1, CXCL2, and CXCL8 proteins almost completely disappeared from the tumor. Although some residual proteins of CXCL3, CXCL5, MMP1, MMP3, and MMP12 were still detected, their levels were significantly reduced due to AEFs treatment. Overall, these findings demonstrated that AEFs reduced the expression of angiogenesis-related chemokines and matrix metalloproteinases in glioblastoma tumors, thereby downregulating the signaling pathways and inhibiting tumor angiogenesis.

[0139] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Those of ordinary skill in the art to which the present invention pertains can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope defined in the claims.

Claims

1. A variable frequency electric field generating system, characterized in that: include: An alternating power generator (100), the alternating power generator (100) being used to generate a sine wave signal; A wire (300), a first end of which is connected to an output end of the alternating power generator (100); At least one pair of insulated electrode plates (200) connected to the second end of the wire (300); The insulating electrode plate (200) comprises a flexible conductive layer and a flexible insulating layer covering both sides of the flexible conductive layer, and the insulating electrode plate (200) is applied to the surface of a diseased part of the body, or applied as an implant to the surface of a diseased internal organ; The sine wave signal has a frequency of 800 to 1200 kHz and an amplitude of 50 to 100 Vpp; When the alternating power generator (100) outputs a sine wave signal to the insulating electrode plate (200), its rated output power is less than 3W, and the alternating electric field generated between a pair of the insulating electrode plates (200) is used to inhibit the generation of angiogenic growth factors in the alternating electric field region.

2. The variable frequency electric field generating system according to claim 1, characterized in that: The spacing between the pair of insulating electrode plates (200) and the amplitude of the alternating power generator (100) are configured to ensure that the intensity of the alternating electric field generated between the pair of insulating electrode plates (200) is greater than 0.6 V / cm.

3. The variable frequency electric field generating system according to claim 1, characterized in that: The flexible conductive layer comprises a metal layer (202), and the metal layer (202) comprises a silver foil layer.

4. The variable frequency electric field generating system according to claim 1, characterized in that: The flexible insulating layer comprises an insulating film (201), and the insulating film (201) comprises a polyimide film.

5. The variable frequency electric field generating system according to claim 3 or 4, characterized in that: The thickness of the flexible conductive layer and the flexible insulating layer is less than 0.1 mm.

6. The variable frequency electric field generating system according to claim 1, characterized in that: The frequency of the sine wave signal is 1000 kHz.

7. The variable frequency electric field generating system according to claim 1, characterized in that: The alternating current power generator (100) comprises a sine wave generator (1), a differential amplifier (2), a step-up transformer (3), a communication controller (4) and a DC / DC isolated voltage-stabilized power supply (5); the sine wave generator (1), the differential amplifier (2) and the step-up transformer (3) are connected in sequence; the communication controller (4) is connected to the sine wave generator (1); and the DC / DC isolated voltage-stabilized power supply (5) provides power for various components.

8. The variable frequency electric field generating system according to claim 7, characterized in that: The sine wave generator adopts a high-frequency waveform generator of an integrated chip MAX038.

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