KR20230003688A - Apparatus for promoting apoptosis and method for promoting apoptosis using the same - Google Patents

Abstract

The present invention provides a device for promoting apoptosis, which sequentially generates an irreversible electroporation pulse and an electrolysis pulse, wherein the maximum temperature after the electrolysis pulse and a change in temperature before the irreversible electroporation pulse can be within 25℃, and a method for promoting apoptosis using the same.

Description

Apparatus for promoting cell death and method for promoting cell death using the same {APPARATUS FOR PROMOTING APOPTOSIS AND METHOD FOR PROMOTING APOPTOSIS USING THE SAME}

The present invention relates to a device for promoting apoptosis using irreversible electroporation and electrolysis and minimizing temperature change, and a method for promoting cell death using the same.

To date, tissue ablation has been performed using thermal energy or changes in pH concentration, such as Radio-Frequency Ablation (RFA), High-Intensity Focus Ultrasonic, Interstitial Laser Coagulation, and Electrolysis. An ablation method that causes cell necrosis is used. Electrolysis pulse is an ablation technique that causes cell necrosis through a chemical reaction that occurs while current is passed between two electrodes in millisecond pulses using low voltage. Chemical reaction refers to a phenomenon in which water molecules are divided into hydrogen and oxygen, and the pH concentration is changed by the chemical reaction, which induces cell necrosis.

Cell necrosis is accidental cell death, and refers to the case where cells die unexpectedly due to factors such as rapid temperature change, pH, and toxins. The above-mentioned ablation techniques have excellent local relief effects, but because they depend on thermal energy, normal cells located near the tumor to be resected have side effects such as heat-induced burns and inflammation due to cell necrosis. Due to these side effects, there is a limitation in that it is difficult for new cells to regenerate at the site where cell necrosis occurred.

As an alternative to these limitations, over the past few decades, irreversible electroporation (IRE), which is minimally invasive to the human body and can maximize the areas to be resected and minimize related side effects, has emerged as an alternative.

Irreversible electroporation is an ablation technique that induces apoptosis, not cell necrosis. Apoptosis refers to the natural death of a cell through a pre-determined step. In the case of irreversible electroporation, since it does not generate heat and induces apoptosis, there are no side effects such as burns, inflammation, and bleeding, and cells can be regenerated at the site where apoptosis has occurred.

The limit of irreversible electroporation shows a 90% success rate when a 2000 V/cm voltage is applied to a tumor of 3 cm or less with multiple pulses of microseconds. However, in the case of tumors larger than 3 cm, there is a disadvantage that the success rate is significantly lowered.

In order to overcome this, it is possible to increase the voltage of irreversible electroporation to expand the area, but indiscriminately increasing the voltage of the pulse, there is a side effect of lowering the survival rate, so there is a limit to increasing the voltage.

The present invention generates an irreversible electroporation pulse and an electrolysis pulse sequentially, but the maximum temperature after the electrolysis pulse is generated and the change in temperature before the irreversible electroporation pulse is within 25 ° C. Characterized in that, it is intended to provide a device for promoting apoptosis.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The present invention generates an irreversible electroporation pulse and an electrolysis pulse sequentially, but the maximum temperature after the electrolysis pulse is generated and the change in temperature before the irreversible electroporation pulse is within 25 ° C. Characterized in that, it provides a device for promoting apoptosis.

The irreversible electroporation pulse may be adjusted by one or more parameters selected from the group consisting of voltage level, pulse width, pulse interval, and pulse number.

The voltage level may be 100 V to 2,000 V, the pulse width may be 1 μs to 100 μs, the pulse interval may be 1,000 μs to 5,000 μs, and the number of pulses may be 1 to 10 times.

The electrolysis pulse has at least one parameter selected from the group consisting of a pulse low time, a pulse high time, a pulse tail time, and a relay capacitor. can be regulated.

The pulse low time may be 1,000 μs to 5,000 μs, the pulse high time and pulse tail time may be 10 μs to 5,000 μs in total, and the relay capacitor may be 0.1 μF to 5,000 μF.

The sequential generation of the irreversible electroporation pulse and electrolysis pulse may be repeated one or more times.

The device includes a power supply unit for supplying voltage; A user interface is provided, and one or more parameters selected from the group consisting of the voltage level, pulse width, pulse interval, and number of pulses, and one or more parameters selected from the group consisting of the pulse low time, pulse high time, pulse tail time, and relay capacitor. Pulse control unit for setting; And including at least one capacitor and at least one switching element, by controlling the voltage supplied from the power supply unit based on the value set by the pulse setting unit to control the switching element, the irreversible electroporation pulse and the electrolysis pulse It may include a pulse output unit that generates.

The device may be for treating cancer or tumors.

One embodiment of the present invention provides a method for promoting cell death in animals other than humans by applying a pulse generated from the device.

The promotion of cell death may be for treating cancer or tumor.

The device for promoting apoptosis according to the present invention sequentially generates an irreversible electroporation pulse and an electrolysis pulse, but the maximum temperature after the electrolysis pulse is generated and the temperature before the irreversible electroporation pulse is generated. It is characterized in that the change of is minimized, compared to the case of generating a conventional electroporation pulse alone, significantly high cell death can be obtained without side effects such as burns and inflammation. Therefore, when the present invention is applied to cancer or tumor treatment, it is possible to exert an effect of reducing the burden on the patient with a low voltage.

1A is a graph showing IREEL pulses according to an embodiment of the present invention.
1B schematically illustrates an IREEL block diagram according to an embodiment of the present invention.
1C shows a firmware flow chart applied to an embodiment of the present invention.
Figure 1d shows a pulse output unit circuit applied to one embodiment of the present invention.
Figure 1e shows a test board, PADS design and PCB applied to one embodiment of the present invention.
Figure f shows a device prototype for promoting apoptosis according to one embodiment of the present invention.
Figure 2a shows the irreversible electroporation area of the IREEL pulse in the case of Comparative Example 1 and Examples 1 to 4 at 500 V.
Figure 2b shows the irreversible electroporation region of the IREEL pulse in the case of Comparative Example 2 and Examples 5 to 8 at 1,000 V.
Figure 2c shows the irreversible electroporation region of the IREEL pulse in the case of Comparative Example 3 and Examples 9 to 12 at 1,500 V.
Figure 2d is a graph comparing the irreversible electroporation area of the IREEL pulse in the case of Comparative Examples 1 to 3 and Examples 1 to 12.
Figure 3a shows the irreversible electroporation area of the IREEL pulse in the case of Comparative Example 4.
Figure 3b shows the irreversible electroporation area of the IREEL pulse in the case of Example 13.
Figure 3c shows the irreversible electroporation area of the IREEL pulse in the case of Example 14.
Figure 3d shows the irreversible electroporation region of the IREEL pulse in the case of Example 15.
Figure 3e shows the irreversible electroporation area of the IREEL pulse in the case of Comparative Example 5.
Figure 3f is a graph comparing the irreversible electroporation area of IREEL pulses in Comparative Example 4, Examples 13 to 15, and Comparative Example 5.
Figure 4 shows the impedance change based on 10Hz of the IREEL pulse in the case of Comparative Examples 1 to 3 and Examples 1 to 12.
5 shows the impedance change amount based on 10 Hz of the IREEL pulse in the case of Comparative Example 4, Examples 13 to 15, and Comparative Example 5.
Figure 6 shows the temperature change before and after the IREEL pulse in the case of Example 12.
7 shows the temperature change before and after the IREEL pulse in Example 14.

In order to overcome the above-mentioned limitations, the present inventors combined an irreversible electroporation pulse and an electrolysis pulse to maximize the irreversible electroporation area while lowering the voltage of the pulse, and also found that the temperature change could also be minimized. confirmed, and completed the present invention.

In the present specification, irreversible electroporation and electrolysis are integrated and also expressed as IREEL.

Hereinafter, the present invention will be described in detail.

A device for promoting apoptosis

The present invention generates an irreversible electroporation pulse and an electrolysis pulse sequentially, but the maximum temperature after the electrolysis pulse is generated and the change in temperature before the irreversible electroporation pulse is within 25 ° C. Characterized in that, it provides a device for promoting apoptosis.

"Irreversible electroporation" in the present specification refers to a phenomenon in which an electric field is applied to a cell to permanently permeate a cell membrane, which induces apoptosis. Since irreversible electroporation does not use thermal energy, side effects such as burns, inflammation, and bleeding can be avoided, and since apoptosis is induced and resected, cell regeneration can be expected in the treated area. However, when irreversible electroporation is used alone, the area that can be ablated is small. To overcome this, the voltage can be increased, but there is a limit in that the survival rate is low, so it is difficult to increase the voltage indiscriminately.

The irreversible electroporation pulse is one or more parameters selected from the group consisting of voltage level (VL), pulse width (PW), pulse interval (PI) and pulse number (PN) It is preferably adjusted to, but is not limited thereto.

At this time, the voltage level may be 100 V to 2,000 V, preferably 1,000 V to 1,500 V, and more preferably 1,000 V to 1,200 V, but is not limited thereto. At this time, if the voltage size is too small, there is a problem that the irreversible electroporation area is not properly generated, and if the voltage size is too large, there is a limit point in which the survival rate is lowered.

In addition, the pulse width refers to the time during which the irreversible electroporation pulse is applied, and may be 1 μs to 100 μs, preferably 1 μs to 50 μs, but is not limited thereto. In addition, the pulse interval refers to the interval between the times the irreversible electroporation pulse is applied, and may be 1,000 μs to 5,000 μs, preferably 1,000 μs to 3,000 μs, but is not limited thereto. The number of pulses may be 1 to 10 times, preferably 2 to 10 times, and more preferably 4 to 10 times, but is not limited thereto.

In order to overcome the limitations when using irreversible electroporation alone, the present invention is characterized in that the irreversible electroporation is combined with electrolysis.

"Electrolysis" in the present specification is an ablation method of inducing apoptosis by utilizing a change in pH concentration through a reaction generated while current flows between two electrodes with a pulse of millisecond or longer using a relatively low voltage. Changes in pH concentration can change the pH concentrations of acidity from the anode and alkalinity from the cathode to 1, 13, and extreme pH changes can lead to protein denaturation, collapse of cell structure and cell death. .

The electrolysis pulse has at least one parameter selected from the group consisting of a pulse low time, a pulse high time, a pulse tail time, and a relay capacitor. It is preferable that it is controlled, but is not limited thereto.

At this time, the pulse low time means the time before the electrolysis pulse is applied, and may be 1,000 μs to 5,000 μs, preferably 1,000 μs to 3,000 μs, but is not limited thereto. In addition, the pulse high time means a time when the voltage level is maintained as it is while the electrolysis pulse is applied, and the pulse tail time means a time when the voltage level is lowered while the electrolysis pulse is applied. The total time and pulse tail time may be from 10 μs to 5,000 μs, preferably from 400 μs to 2,000 μs, and more preferably from 800 μs to 2,000 μs, but are not limited thereto. At this time, when the pulse high time and the pulse tail time are too long, there is a problem in that the temperature change before and after the pulse becomes too large. Meanwhile, the pulse high time may be 1,000 μs or less. If the pulse high time is longer, the efficiency is improved, but there is a problem in that the maximum temperature after the pulse is too high. The relay capacitor may be 0.1 μF to 5,000 μF, preferably 0.1 μF to 2,000 μF, but is not limited thereto.

The sequential generation of the irreversible electroporation pulse and the electrolysis pulse may be repeated one or more times, and preferably repeated less than 10 times, but is not limited thereto.

Thus, the device for promoting apoptosis according to the present invention is characterized in that, by the specific heat characteristics of the pulse, the maximum temperature after the electrolysis pulse is generated and the change in temperature before the irreversible electroporation pulse is generated within 25 ° C. It is preferably within 10 ° C, and more preferably within 5 ° C, but is not limited thereto. After the electrolysis pulse is generated, the temperature is increased to a maximum temperature and then the temperature is decreased to room temperature again. That is, the apparatus for promoting apoptosis according to the present invention may satisfy NTIRE (Non-Thermal Irreversible Electroporation) since the maximum temperature after generating the electrolysis pulse does not exceed 54 °C.

On the other hand, the apparatus for promoting apoptosis according to the present invention includes a power supply unit for supplying voltage; A user interface is provided, and one or more parameters selected from the group consisting of the voltage level, pulse width, pulse interval, and number of pulses, and one or more parameters selected from the group consisting of the pulse low time, pulse high time, pulse tail time, and relay capacitor. Pulse control unit for setting; And including at least one capacitor and at least one switching element, by controlling the voltage supplied from the power supply unit based on the value set by the pulse setting unit to control the switching element, the irreversible electroporation pulse and the electrolysis pulse It may include a pulse output unit that generates.

Specifically, the power supply unit is for supplying voltage, and may include a power supply for supplying a high voltage to a boost unit described later, a power supply for supplying a low voltage to a boost unit to be described later, and a power supply for a processor and an IC device of a control unit. . For example, the power voltage of the primary side is +24V, +12V, +5V, and the power voltage of the secondary side is +12V, +5V.

Meanwhile, the step-up unit may generate a high voltage by using a flyback-converter from a rectified primary-side voltage in order to implement a high-voltage pulse generator. The generated high voltage can cause a capacitor bank to be charged in series and parallel connection of capacitors. A capacitor can use a photo-flash to emit an instantaneous current. The reason why the structure of the flyback converter is adopted is that it can have a simple circuit configuration with a minimum of parts, and it is possible to electrically isolate the primary side, which is a low voltage side, and the secondary side, which is a high voltage side, for safety. The reason why isolation is possible is that the transformer used in the Flyback converter acts as a kind of inductor and transfers the energy of the primary side to the winding of the secondary side. For example, the transformer for Flyback used in the production used a PQ3535 bobbin and core, and the primary inductance was 22 μH, the secondary inductance was 2.98 mH, and the air gap was composed of about 0.25 mm with both side gaps. The air gap was controlled using insulating paper.

In addition, the pulse control unit provides a user interface, and includes one or more parameters selected from the group consisting of the voltage size, pulse width, pulse interval, and number of pulses, the pulse low time, pulse high time, pulse tail time, and a relay capacitor. It is for setting one or more parameters selected from the group. For example, these parameters can be controlled by an encoder using a user interface directly produced by IAR Embedded WorkBench (Sweden), a firmware program. The micro-controller located in the control unit used an AVR-type ATMEGA128A (Atmel, USA), and the firmware flow chart is shown in FIG. 1c.

In addition, the pulse output unit includes at least one capacitor and at least one switching element, and by controlling the switching element based on the value set by the pulse setting unit, the voltage supplied from the power supply unit, the irreversible electroporation for generating pulses and electrolysis pulses. The pulse output unit may configure an insulated gate bipolar transistor (IGBT), which is a power component, and a relay, which is an electromagnetic switch, in series. For example, the pulse output unit circuit is shown in FIG. 1D.

The device may be for treating cancer or tumors. Specifically, the pulse generated from the device creates a temporary hole in the cell membrane, and by continuously inducing it, cell death can be promoted, and cancer or tumor can be ultimately treated. More specifically, the cancer or tumor is skin cancer, carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma (mesothelioma), schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoidmalignancy, squamous cell cancer, squamous cell carcinoma ( epithelial squamous cell cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, lung squamous cancer ( squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, glioblastoma ), cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer ), colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney and renal cancer, prostate cancer, etc. Vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract cancer ( biliary tract cancer) and head and neck cancer (head and neck cancer) may be at least one selected from the group consisting of.

How to Promote Apoptosis

The present invention provides a method for promoting cell death in animals other than humans by applying a pulse generated from the device.

"Non-human animal" in the present specification may be one or more mammals selected from the group consisting of mice, rats, pigs, rabbits, guinea pigs, hamsters, dogs, cats, cows, and goats, but is not limited thereto.

Since the device has been described above, redundant description will be omitted.

In addition, the promotion of cell death may be for treating cancer or tumor. Specifically, the pulse generated from the device creates a temporary hole in the cell membrane, and by continuously inducing it, cell death can be promoted, and cancer or tumor can be ultimately treated. More specifically, the cancer or tumor is skin cancer, carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma (mesothelioma), schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoidmalignancy, squamous cell cancer, squamous cell carcinoma ( epithelial squamous cell cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, lung squamous cancer ( squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, glioblastoma ), cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer ), colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney and renal cancer, prostate cancer, etc. Vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract cancer ( biliary tract cancer) and head and neck cancer (head and neck cancer) may be at least one selected from the group consisting of.

As discussed above, the device for promoting apoptosis according to the present invention sequentially generates an irreversible electroporation pulse and an electrolysis pulse, but the maximum temperature and the irreversible It is characterized by minimizing the change in temperature before generating the electroporation pulse, and compared to the case of generating a conventional electroporation pulse alone, significantly high cell death can be obtained without side effects such as burns, inflammation, and hemorrhage. Therefore, when the present invention is applied to cancer or tumor treatment, it is possible to exert an effect of reducing the burden on the patient with a low voltage.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1: Optimization of irreversible electroporation pulse and electrolysis pulse generation

The present invention was experimented with potato, a vegetable tissue, to apply the 3R principles of animal testing: Replacement, Reduction, and Refinement. The reason why potatoes were used is because they are phantoms commonly used in electroporation experiments. Although potato tissue does not match the characteristics of in vivo tissue, it can be a reasonable model for understanding the trend of electroporation ablation range. However, potatoes can have different impedance values due to various factors such as seed, harvest time, region, and storage method. In order to reduce the error of this potato experiment, the experiment was conducted by selecting potatoes with a size of less than 10 cm in width and length among potatoes harvested in the same area stored between 0 and 10 ° C. It is important to maintain the same temperature of the potato so that the experiment can be conducted under the same conditions because the impedance value in the potato changes according to the temperature. If the temperature of the potato is different, even if a pulse of the same voltage is applied, the current value flowing through the potato may be different. This means that the energy delivered is not the same according to the temperature of the potato even though the pulses have the same voltage, and it can be seen that the irreversible electroporation area may vary. If the experiment is performed immediately on frozen potatoes, the temperature of the first potato sample and the last potato sample may be different. The experiment was carried out the next day at room temperature without direct sunlight the day before. Finally, to verify the temperature under the same conditions, the temperature of the potatoes was measured with an Infrared Thermometer (DT8280, OLBO, China) before the experiment, and it was confirmed that the experiment under the same conditions was conducted.

The electrode used in the experiment was made of 19-gauge needle-shaped steel. The total length of the electrode cable is 1.7 m, the diameter of the electrode is 2 mm, and the electrode exposed length is 20 mm. The experiment was conducted by inserting two electrodes into the phantom in parallel at 10 mm intervals.

The pulse experiment conducted in the present invention is divided into two types: an electrolysis pulse time change comparison experiment and an IREEL pulse rate comparison experiment.

First, the pulse specifications used in the electrolysis pulse time change comparison experiment are shown in Table 1 below. At this time, the pulse high time was not separately set.

voltage size pulse width pulse
interval number of pulses pulse
low time pulse
tail time relay
capacitor Comparative Example 1 500V 10 μs 2,000 μs 8EA 2,000 μs 0 μs 100 µF Example 1 500V 10 μs 2,000 μs 8EA 2,000 μs 400 µs 100 µF Example 2 500V 10 μs 2,000 μs 8EA 2,000 μs 800 µs 100 µF Example 3 500V 10 μs 2,000 μs 8EA 2,000 μs 1600 µs 100 µF Example 4 500V 10 μs 2,000 μs 8EA 2,000 μs 3200 μs 100 µF Comparative Example 2 1,000V 10 μs 2,000 μs 8EA 2,000 μs 0 μs 100 µF Example 5 1,000V 10 μs 2,000 μs 8EA 2,000 μs 400 µs 100 µF Example 6 1,000V 10 μs 2,000 μs 8EA 2,000 μs 800 µs 100 µF Example 7 1,000V 10 μs 2,000 μs 8EA 2,000 μs 1600 µs 100 µF Example 8 1,000V 10 μs 2,000 μs 8EA 2,000 μs 3200 μs 100 µF Comparative Example 3 1,500V 10 μs 2,000 μs 8EA 2,000 μs 0 μs 100 µF Example 9 1,500V 10 μs 2,000 μs 8EA 2,000 μs 400 µs 100 µF Example 10 1,500V 10 μs 2,000 μs 8EA 2,000 μs 800 µs 100 µF Example 11 1,500V 10 μs 2,000 μs 8EA 2,000 μs 1600 µs 100 µF Example 12 1,500V 10 μs 2,000 μs 8EA 2,000 μs 3200 μs 100 µF

Second, in the IREEL pulse rate comparison experiment, IRE, the front part of the IREEL pulse, used 1,500 V voltage, PW: 10us PI, 10us PN: 8 pulses, and Electrolysis, the back part of the pulse, used the capacitor energy connected to the relay as a time-constant It was released in an amount of time. The time constant pulse comes out in the form of a time constant because the capacitance value of the capacitor connected to the relay is small. The specific gravity of IRE and Electrolysis of the IREEL pulse used in the experiment was configured as 100:0, 75:25, 50:50, 25:75, and 0:100. The capacitance values of the capacitors used at this time are 0uF, 0.25uF, 0.5uF, 0.75uF, and 1uF. Its specifications are shown in Table 2 below. At this time, the pulse high time was not separately set.

Voltage
size pulse width pulse
interval number of pulses pulse
low time pulse
tail time relay
capacitor energy
(3 sets) Comparative Example 4 1,500V 10 μs 2,000 μs 8EA 2,000 μs 0 μs 0 µF 4.14J Example 13 1,500V 10 μs 2,000 μs 6EA 2,000 μs 32.5 μs 0.25 µF 4.16J Example 14 1,500V 10 μs 2,000 μs 4EA 2,000 μs 65 μs 0.5 µF 4.19J Example 15 1,500V 10 μs 2,000 μs 2EA 2,000 μs 97.5 μs 0.75 µF 4.22J Comparative Example 5 1,500V 10 μs 2,000 μs 0EA 2,000 μs 130 μs 1.00 µF 4.25J

In order to adjust the ratio of IRE and electrolysis of IREEL, the number of pulses can be changed in the case of the IRE pulse, which is the front part of the pulse, but the capacitor value must be changed by calculating the time constant value for the electrolysis pulse, which is the latter part. To set the capacitor value, the voltage versus current through the actual potato was averaged. When the voltage is 1,500 V, the average current flowing through the potato is about 11.5 A. V = IR Using Ohm's Law, it was found that the impedance of the potato was 130 Ω on average. Calculating the energies of the 8 IRE pulses using the formula below gives 1.38J. When 8 pulses are applied in 3 sets, the total energy is 4.14J. The reason why the 8 pulses were divided into 3 sets was that 24 pulses could be applied at once, but the stored voltage dropped as the charged capacitor bank energy was released, so the 8 pulses were divided into 3 sets and applied. The interval between pulse sets is 10 seconds.

First, in order to match the energy of 8 IRE pulses of 1,500 V, a 1uF capacitor with a breakdown voltage of 3300 V was fabricated by serializing ten 10uF photo flash capacitors with a breakdown voltage of 330V. The capacitor used in the fabrication uses a photo-flash capacitor for instantaneous current emission. At 1,500 V, assuming an average load of 130 ohms and a 1uF capacitor, the time constant is τ = R x C = 130 x 1uF = 130us. If the energy value is obtained as in Equation (1), 1.23J is obtained. For 3 sets, it is 3.69J.

(1)

(One)

In order to find out whether the energy of the 5 types of IREEL pulses mentioned above is the same, the energy value was calculated using the formula (energy calculation formula) and the formula (time constant integration formula). The energy value may be slightly different because it was used by replacing it with a commercially used capacitor value.

Example 2: Tetrazolium test

Tetrazolium test is a method of assaying seed vitality, and is an assay method that distinguishes between life and death through tissue respiration by using dehydratase activity in a watered state of seeds. The dehydratase generated in the seed that absorbed water reacts with the substrate to create hydrogen ions and reacts with colorless tetrazolium to produce red formazan, which then colors the seed. Red-stained areas indicate that cells are alive, and unstained areas indicate that cells are dead.

To prepare the tetrazolium staining reagent, put 5 g of TTC measured using an electronic balance (HR-200, A&D Weighing, USA) into a 1,000 ml transparent lab bottle, and then add 1,000 ml distilled water to 5% w/v (weigh per volume) reagent was prepared.

First, after applying a voltage of 500 V to potato, which is a vegetable tissue, in the order of Comparative Example 1 and Examples 1 to 4, a photograph of tetrazolium dyeing for 6 hours is shown in FIG. 2a. In addition, referring to Table 3 and FIG. 2d below, it was found that as the electrolysis pulse time increased, the irreversible electroporation area appeared wide in the potato.

On the other hand, after applying a voltage of 1,000 V to potatoes, which are vegetable tissues, in the order of Comparative Example 2 and Examples 5 to 8, a photograph of tetrazolium dyeing for 6 hours is shown in FIG. 2B. In addition, referring to Table 3 and FIG. 2d below, it was found that as the electrolysis pulse time increased, the irreversible electroporation area appeared wide in the potato.

On the other hand, after applying a voltage of 1,500 V to potatoes, which are vegetable tissues, in the order of Comparative Example 3 and Examples 9 to 12, a photograph of tetrazolium staining for 6 hours is shown in FIG. 2c. In addition, referring to Table 3 and FIG. 2d below, as the electrolysis pulse time increases, it is confirmed that the irreversible electroporation area appears wide in potatoes.

)area
(unit:

) Comparative Example 1 13.413 Example 1 59.468 Example 2 96.265 Example 3 146.713 Example 4 148.283 Comparative Example 2 22.836 Example 5 89.189 Example 6 180.241 Example 7 235.053 Example 8 303.123 Comparative Example 3 162.427 Example 9 169.147 Example 10 237.454 Example 11 258.829 Example 12 339.837

Next, the irreversible electroporation area for the irreversible electroporation pulse in potato, which is a vegetable tissue, in order of Comparative Example 4, Examples 13 to 15, and Comparative Example 5 corresponding to the same energy conditions was analyzed using FIJI, TTC The staining experiment was conducted a total of 4 times, and the resulting photos are shown in FIGS. 3a to 3e, and the resulting regions are shown in Table 4 below. Under the same energy conditions, it is confirmed that the irreversible electroporation area appears wider in the potato when the irreversible electroporation pulse or the electrolysis pulse is used in combination than when used alone.

)average area
(unit:

) Comparative Example 4 313.777 Example 13 372.977 Example 14 377.684 Example 15 360.987 Comparative Example 5 331.611

Example 3: Impedance change measurement

When a high-voltage electric field pulse is applied to the potato, cell membrane rupture is induced, which drastically lowers the impedance within the potato. Since the rate of change in potato impedance can be seen as an indicator of irreversible electroporation, the impedance values before and after the experiment were measured using an Impedance Analyzer (4192A, HEWLETT PACKARD, USA).

First, Comparative Example 1 and Examples 1 to 4 with a voltage of 500 V, Comparative Example 2 and Examples 5 to 8 with a voltage of 1,000 V, Comparative Example 3 and Examples 9 to 8 with a voltage of 1,500 V In the order of 12, the impedance change for the irreversible electroporation pulse in potato, which is a vegetable tissue, was measured, and the results are shown in FIG. 4 and Table 5 below. As the electrolysis time increases, it is confirmed that the change in impedance is large based on 10 Hz.

before experiment
impedance
[kΩ] after experiment
maximum impedance
[kΩ] impedance
amount of change
[kΩ] Comparative Example 1 5.18 5.18 0 Example 1 5.28 4.74 0.54 Example 2 5.28 4.55 0.73 Example 3 5.74 4.92 0.82 Example 4 5.39 4.09 1.30 Comparative Example 2 5.18 5.18 0 Example 5 5.73 4.22 1.51 Example 6 5.43 3.19 2.24 Example 7 6.49 4.13 2.36 Example 8 6.58 3.05 3.53 Comparative Example 3 6.00 4.79 1.21 Example 9 5.94 4.00 1.94 Example 10 5.75 3.18 2.57 Example 11 6.21 2.91 3.3 Example 12 6.80 2.19 4.61

Next, in order of Comparative Example 4, Examples 13 to 15, and Comparative Example 5 corresponding to the same energy conditions, the impedance change for the irreversible electroporation pulse in potato, which is a vegetable tissue, was measured, and the results are shown in FIG. 5 and below. Table 6 shows. Under the same energy condition, when the irreversible electroporation pulse or the electrolysis pulse is used in combination than when used alone, it is confirmed that the impedance change is larger on the basis of 10 Hz.

before experiment
impedance
[kΩ] after experiment
maximum
impedance
[kΩ] impedance
amount of change
[kΩ] Comparative Example 4 4.25 1.70 2.55 Example 13 4.39 1.20 3.19 Example 14 4.86 1.59 3.27 Example 15 3.68 1.50 2.18 Comparative Example 5 3.68 2.20 1.48

Example 4: Temperature change measurement

In order to prove that the IREEL pulse used in the present invention has specific heat characteristics, the temperature of potatoes before and after the experiment was measured. In order to measure the amount of temperature change, it was observed with a thermal imaging camera (ONE PRO LT, FLIR, USA). After the IREEL pulse, it is confirmed that the temperature rises to the maximum and then decreases to room temperature again.

First, the heat change for the IREEL pulse was measured using a FLIR camera for potatoes sliced to a thickness of 10 mm in the order of Comparative Example 3 and Examples 9 to 12, and the results are shown in Table 7. On the other hand, the photograph of measuring the temperature change in Example 12 is shown in FIG. 6.

Since the maximum temperature did not exceed 54 ° C. after the experiment in Comparative Example 3 and Examples 9 to 12 with the maximum voltage of 1,500 V, Comparative Examples 1 to 3 and Examples 1 to 12 were all NTIRE (Non -Thermal Irreversible Electroporation) is confirmed to be satisfied. As in Examples 9 to 12, it is confirmed that the electrolysis pulse time of 1600 μs or less is preferable in terms of minimizing the amount of temperature change.

before experiment
Temperature after experiment
maximum temperature Temperature
amount of change Comparative Example 3 22.9℃ 23.3℃ 0.4℃ Example 9 23.6℃ 24.6℃ 1.0℃ Example 10 23.0℃ 26.1℃ 3.1℃ Example 11 22.7℃ 27.5℃ 4.8℃ Example 12 24.0℃ 48.7℃ 24.7℃

On the other hand, the heat change for the IREEL pulse in potatoes sliced at 10 mm thickness in the order of Comparative Example 4, Examples 13 to 15, and Comparative Example 5 corresponding to the same energy conditions was measured in Table 8 using a FLIR camera. On the other hand, a photograph of measuring the temperature change in Example 14 is shown in FIG. 7 . In Comparative Example 4, Examples 13 to 15, and Comparative Example 5, the temperature change amount was all the same at 0.4 ° C, and it was confirmed that NTIRE (Non-Thermal Irreversible Electroporation) was satisfied.

before experiment
Temperature after experiment
maximum
Temperature Temperature
amount of change Comparative Example 4 20.3℃ 20.7℃ 0.4℃ Example 13 20.7℃ 21.1℃ 0.4℃ Example 14 20.1℃ 20.5℃ 0.4℃ Example 15 20.0℃ 20.4℃ 0.4℃ Comparative Example 5 20.7℃ 21.1℃ 0.4℃

First, according to the results of the electrolysis pulse time change comparison experiment, compared to Comparative Example 3 using an irreversible electroporation pulse using only a high voltage of 1,500 V, IREEL 800 and 1600 at a voltage of 1,000 V with a reduced voltage , In the case of Examples 6 to 8 using 3200 pulses, the area was increased by 10%, 44%, and 86%, and equivalent or better effects were confirmed.

Next, according to the results of the IREEL pulse ratio comparison experiment, compared to Comparative Examples 4 or 5 using an irreversible electroporation pulse or electrolysis pulse alone, IREEL 2EA + 97.5 μs, IREEL 6EA + 32.5 μs, IREEL 4EA + 65 μs In the case of Examples 13 to 15 using , it is confirmed that the area is increased by 14%, 18%, and 20%.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (10)

An irreversible electroporation pulse and an electrolysis pulse are sequentially generated,
The maximum temperature after the electrolysis pulse is generated and the change in temperature before the irreversible electroporation pulse is generated, characterized in that within 25 ℃, apoptosis promoting device.
According to claim 1,
Characterized in that the irreversible electroporation pulse is controlled by one or more parameters selected from the group consisting of voltage level, pulse width, pulse interval and pulse number, A device for promoting apoptosis.
According to claim 2,
The voltage magnitude is 100 V to 2,000 V,
The pulse width is 1 μs to 100 μs,
The pulse interval is 1,000 μs to 5,000 μs,
The number of pulses is characterized in that 1 to 10 times, the device for promoting apoptosis.
According to claim 1,
The electrolysis pulse has at least one parameter selected from the group consisting of a pulse low time, a pulse high time, a pulse tail time, and a relay capacitor. Characterized in that the regulation, a device for promoting apoptosis.
According to claim 4,
the pulse low time is between 1,000 μs and 5,000 μs;
the pulse high time and pulse tail time are between 10 μs and 5,000 μs in total;
The relay capacitor is characterized in that 0.1 μF to 5,000 μF, the device for promoting apoptosis.
According to claim 1,
The sequential generation of the irreversible electroporation pulse and the electrolysis pulse is characterized in that repeated one or more times, apoptosis promoting device.
According to claim 1,
The device
a power supply unit for supplying voltage;
A user interface is provided, and one or more parameters selected from the group consisting of the voltage level, pulse width, pulse interval, and number of pulses, and one or more parameters selected from the group consisting of the pulse low time, pulse high time, pulse tail time, and relay capacitor. Pulse control unit for setting; and
It includes at least one capacitor and at least one switching element, and controls the switching element based on a value set by the pulse setting unit for a voltage supplied from the power supply unit, thereby producing the irreversible electroporation pulse and the electrolysis pulse. Characterized in that it comprises a pulse output unit for generating, apoptosis promotion device.
According to claim 1,
The device is a device for promoting apoptosis, characterized in that for treating cancer or tumor.
A method for promoting cell death in animals other than humans by applying a pulse generated from the device according to claim 1.
10. The method of claim 9, wherein the promotion of cell death is for treating cancer or tumor.

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