KR20220093918A - Device of accelerating apoptosis by using combination of electroporation and electrolysis - Google Patents

Abstract

The present invention relates to a device for promoting apoptosis, wherein a pulse of electroporation and a pulse of electrolysis occur sequentially. The pulse of the electroporation is controlled by parameters of pulse width and a pulse interval, and the pulse of the electrolysis is controlled by parameters of pulse width, a pulse interval, a pulse tail time, and a relay capacitor (TEV). The device for promoting apoptosis according to the present invention comprises a power unit, a pulse setting unit, and a pulse generation unit. Compared to conventional single application of the pulse of the electroporation, sequential application of the pulse of the electroporation and the pulse of the electrolysis can produce significantly high apoptosis. Accordingly, when the device is applied to cancer treatment, a voltage is lowered to reduce a load to a patient.

Description

Device of accelerating apoptosis by using combination of electroporation and electrolysis

The present invention is a device for promoting apoptosis, in which an electroporation pulse and an electrolysis pulse are sequentially generated, and the electroporation pulse is controlled by parameters of a pulse width and a pulse interval and the electrolysis pulse is controlled by parameters of a pulse width, a pulse interval, a pulse tail time and a relay capacitor (TEV).

Existing interventional medical devices for gastrointestinal cancer treatment have excellent local relieving effects, but are ineffective in areas where stimulation is not transmitted, and there is a risk of damaging the bile duct, blood vessels, gallbladder, and intestines around the treatment probe, leading to cancer metastasis It can also cause many side effects such as burns, bleeding, ulcers, perforations, portal vein thrombosis and pulmonary embolism due to excessive stimulation.

To solve this problem, as non-thermal ablation (non-thermal ablation) electrostimulation treatment or electroporation (electroporation) is in the spotlight as a new technology, basic and clinical research is being conducted worldwide. When a cell is suspended in a DNA solution and a pulse of DC high voltage is passed through it, a temporary hole is created in the cell membrane. A gene introduction method using this principle is called electroporation. If this electroporation is continuously induced in cells, apoptosis may also be induced.

Among the non-thermal resection methods, irreversible electroporation (IRE) is a new technology to remove malignant tissues including various cancers, especially perivascular tumors, which are difficult to treat. It is a procedure that induces irreversible electroporation by altering the permeability. Targeted tissue removal can be performed by coagulating and necrosis of malignant cells and cancer cells whose homeostasis is disrupted due to electroporation. Even in IRE, a non-thermal excision method, excessive stimulation, which is one of the problems of the existing interventional medical devices for cancer treatment, is a factor that causes side effects. Excessive electrical stimulation may generate heat within the tissue and cause damage to surrounding normal tissues of the target tissue. For the optimal IRE procedure that minimizes damage and side effects of surrounding normal tissues, electricity must be applied by adjusting the voltage, output pulse, and interval between electrodes according to characteristics such as the resistance value and size of the target tissue.

Recently, electroporation has been widely used even for cancer treatment using a relatively high voltage using a high voltage. In particular, it is widely used as an alternative therapy to radiation therapy to minimize the side effects of radiation during cancer treatment. However, high-voltage electroporation has a problem in that perforation occurs not only in cancer tissue, which is the target of perforation, but also in normal cells around the cancer tissue, and there is a high risk of damaging important tissues such as blood vessels or nerves. Also, a limitation of general irreversible electroporation is that the ablation area is relatively small. Relatively small tumors less than 3 cm can be treated, but as the size of the tumor increases, the likelihood of treatment decreases.

Hsiao CY, et al., “Irreversible Electroporation: a novel ultrasound-guided modality for non-thermal tumor ablation”, Journal of Medical Ultrasound, 25(4), 195-200, 2017

The present invention is to overcome the limitations of cancer treatment using the conventional electroporation using a high voltage, to provide a device and method using the same, which is more efficient than the conventional irreversible electroporation in apoptosis, controlled by various pulse parameters for that technical task.

According to one aspect of the present invention, there is provided an apparatus for simultaneously generating a pulse of electroporation and a pulse of electrolysis to promote apoptosis.

According to one embodiment of the present invention, the device comprises

a power supply unit for supplying power;

pulse providing a user interface and setting values of the amplitude, pulse width, pulse width interval and pulse tail time of the pulses of the electroporation and the values of the amplitude, pulse width, pulse width interval and pulse tail time of the pulses of the electrolysis setting unit; and

It is configured to include at least one capacitor and at least one switching element, and based on the values of the amplitude, pulse width, pulse width interval, and pulse tail time set by the pulse setting unit by receiving the output voltage of the power supply unit, the It may include; a pulse generator for controlling at least one or more switching elements to generate the pulse of the electroporation or the pulse of the electrolysis.

According to an embodiment of the present invention, the apparatus may further include an electrode unit for applying the pulse generated by the pulse generating unit to the target sample.

According to an embodiment of the present invention, the pulse width in the pulse of the electroporation may be 1 ~ 300 μs, more preferably 50 ~ 200 μs.

According to an embodiment of the present invention, the pulse interval in the pulse of the electroporation (pulse interval) may be 1 ~ 4 ms.

According to an embodiment of the present invention, in the pulse of the electrolysis, a pulse tail time may be 100 μs or more and less than 2 ms, and more preferably 400 to 2000 μs.

According to an embodiment of the present invention, the relay capacitor may be 100 to 400 μF.

According to an embodiment of the present invention, the electrolysis pulse: the electroporation pulse may be 1:1 to 1:10. Preferably, it is 1: 6 to 1: 10.

According to an embodiment of the present invention, the device may be for treating cancer.

According to another aspect of the present invention, there is provided a method for promoting apoptosis by simultaneously applying a pulse of electroporation and a pulse of electrolysis. According to an embodiment, an animal other than a human may be a subject.

According to an embodiment of the present invention, the method may be for treating cancer.

Significantly higher apoptosis can be obtained when the electroporation pulse and the electrolysis pulse are sequentially applied than when the conventional electroporation pulse is applied alone. This can exert the effect of reducing the burden on the patient by lowering the voltage in application to cancer treatment.

1 is A block diagram of a slew rate adjustable high voltage pulse.
Fig. 2 is a falling edge time constant adjustable pulse (E2) circuit in which electroporation and electrolysis are combined.
3 shows the pulses of conventional irreversible electroporation (IRE).
Figure 4 shows a falling edge time constant adjustable pulse (E2) combined with electroporation and electrolysis.
5 shows a falling edge time constant adjustable pulse (E2) sequence in which electroporation and electrolysis are combined.
6 is a graph showing the result of variously changing the value of the capacitor connected to the electronic switch.
Figure 7 shows the pulses of conventional irreversible electroporation (IRE) used in the apoptosis test.
8 shows the pulses of the combination of electroporation and electrolysis (E2) used in the apoptosis test.
9 shows a cross section stained with triphenyl tetrazorium chloride (TTC) after applying the conventional irreversible electroporation pulse (IRE) to potatoes.
10 shows a cross section stained with TTC after applying a pulse (E2 800 μs) in which electroporation and electrolysis combined with a pulse tail time of 800 μs (E2 800 μs) was applied.
11 is a graph comparing the area of apoptosis 2 hours and 4 hours after applying the conventional IRE and E2 800 μs pulses to potatoes.
12 is a graph of impedance before, after, and 1 hour, 3 hours, and 6 hours after the experiment in which the conventional IRE pulse is applied.
13 is a graph of impedances before, after, and 1 hour, 3 hours, and 6 hours after the experiment in which a pulse of E2 800 μs is applied.
14 shows a pulse graph of E2_A and a cross section stained with TTC when it is applied to potatoes.
15 shows a pulse graph of E2_B and a cross section stained with TTC when it is applied to potatoes.
16 shows a pulse graph of E2_C and a cross section stained with TTC when it is applied to potatoes.
17 shows a pulse graph of E2_D and a cross-section stained with TTC when it is applied to potatoes.
18 shows a pulse graph of E2_D2 and a cross-section stained with TTC when it is applied to potatoes.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto. Rather, the content introduced herein is thorough and complete, and is provided to sufficiently convey the spirit of the present invention to those of ordinary skill in the art.

1. Experimental method

1) Implementation of E2 high voltage pulse generator

The parameters of the existing square wave pulse type consist of amplitude, pulse width, and pulse width interval. As shown in FIG. 1 , a pulse whose falling edge time constant can be adjusted is composed of five parameters: amplitude, pulse width, pulse width interval, tail end voltage (TEV), and pulse tail time (PTT).

The step-up module boosts the voltage with a flyback structure using the UC3845D device to charge the capacitor connected to the CAP BANK and the electronic switch. An electronic switch is used to output a pulse. The time constant of the falling edge of the pulse is controlled by controlling the number of capacitors connected to the electronic switch and controlling the pulse tail time. Electronic switch control through the user interface changes the capacitor value. The tail voltage can be set by adjusting the number of capacitors connected to the electronic switch. The load performed in this study was fixed to 1 kΩ and the experiment was performed. Referring to Equation (1), when the adjustable capacitor values are 0, 1000, 2000, and 3000, the respective time constant values are 0, 1, 2, 3 seconds.

Formula (1) τ = RC

The user interface is composed of LCD using MCU, encoder, switch, and buzzer. The encoder and switch are designed to control amplitude, pulse width, pulse width interval, pulse tail time, and capacitor value change connected to the electronic switch, and the LCD is configured so that the previously set values can be viewed. The buzzer is set to sound when charging is complete and when pulse output is output. The pulse driving sequence consists of a total of five steps as shown in FIG. 2 .

i) Set the output voltage, pulse width, pulse width interval, pulse tail time, number of electronic switches through the user interface. The total number of cases of the electronic switch is four. 0, 1000, 2000, 3000.

ii) First, the electronic switch 1 is driven, and then the CAP BANK and the capacitor connected to the electronic switch are charged by the set output voltage.

iii) Drive the electronic switch2. By driving electronic switch 2, the output goes out from the voltage charged in the Cap Bank and the capacitor connected to the electronic switch. Drive electronic switch 1 and electronic switch 2 up to the pulse width.

iv) If the driving of electronic switch 1 is stopped at the end of the pulse width, the voltage stored in the CAP BANK cannot be output to the output, so the voltage of the capacitor connected to the electronic switch is output. Since the capacitor connected to the electronic switch is relatively smaller than the capacitor value of CAP BANK, the output voltage drops rapidly. If the electronic switch 1 is not driven until the pulse tail time and only the electronic switch 2 is driven, a pulse in the shape of a time constant is generated.

v) When the pulse tail time is over, stop the electronic switch 2 so that the output voltage becomes 0 Volt.

A pulse circuit diagram implementing E2 is shown in FIG. 2 .

2) Measurement of apoptosis using potatoes

Potatoes were cut to make a section and the degree of apoptosis was measured by inserting an electrode with a width of 1 mm and a length of 10 cm. measured. For the TTC (triphenyl tetrazorium chloride) solution, 5 g of TTC powder dissolved in 1 L of purified water was used. After the pulse was applied to the potato, the electrode was pulled out, and the area reflecting apoptosis was measured by immersing it in a petri dish containing TTC solution for a certain period of time.

2. Results

1) E2 high voltage pulse generation

Conventional irreversible electroporation (IRE) and the pulse of the combination of electroporation and electrolysis (E2 800 μs) with a pulse tail time of 800 μs were the same as in the forms of FIGS. 3 and 4 , respectively. Table 1 shows the electronic parameters of IRE and E2 800 μs.

Table 1

Figure 5 shows the pulse circuit of the combination of electroporation and electrolysis (E2 800 μs). 6 shows the result of the operation check experiment with the value of the capacitor connected to the electronic switch at 0 μF, 1000 μF, 2000 μF, and 3000 μF. The pulse amplitude was 500 V, the pulse width time was 600 ms, and the pulse tail time was 400 ms. As a result, the tail voltage was 0 V, 270 V, 360 V and 380 V. It can be seen that the edge time constant is adjusted according to the value of the capacitor.

7 shows a pulse of IRE applied to the potato, and FIG. 8 shows a pulse of E2 800 μs.

2) cell death

As a result of comparing FIGS. 9 and 10, it can be seen that the death area increases when a pulse (E2 800 μs) that combines electroporation and electrolysis is applied. 11 shows this at the same time. When 800 μs of E2 was applied to potatoes for 2 hours, it was 1.62 times significantly higher than that of IRE, and when 800 μs of E2 was applied for 4 hours, it was significantly higher by 1.58 times.

The impedance results of the conventional IRE and pulses (E2 800 μs) combining electroporation and electrolysis are shown in Table 2, Table 3, and FIGS. 12 and 13 . This suggests that electroporation was significantly performed according to the pulse application.

Table 2

Table 3

3) Combination pulse of electroporation and electrolysis by several parameters

As shown in Table 4, pulses were generated with different parameters, and the degree of apoptosis was measured by applying this to the potato. The TTC area is the area of a compartment that is differentiated from normal cells due to TTC staining, and is a result reflecting the quantitative measurement of apoptosis. In the case of E2_D and E2_D2, it was confirmed that the potato was damaged and thus the pulse could not be applied to the living body. Each of the pulse graphs and cross sections stained with TTC when applied to potatoes are shown in FIGS. 14 to 18 . Table 5 shows the impedance results of E2_A to E2_D2.

Table 4

Table 5

Claims (8)

As a device for promoting apoptosis
Electroporation pulses and electrolysis pulses are sequentially generated,
The electroporation pulse is controlled by parameters of a pulse width and a pulse interval,
The electrolysis pulse is controlled by parameters of a pulse width, a pulse interval, a pulse tail time and a relay capacitor (TEV).
The apparatus of claim 1 , wherein the device
a power supply unit for supplying power;
pulse providing a user interface and setting values of the amplitude, pulse width, pulse width interval and pulse tail time of the pulses of the electroporation and the values of the amplitude, pulse width, pulse width interval and pulse tail time of the pulses of the electrolysis setting unit; and
It is configured to include at least one capacitor and at least one or more switching elements, and based on the values of the amplitude, pulse width, pulse width interval, and pulse tail time set by the pulse setting unit by receiving the output voltage of the power supply unit, the a pulse generator controlling at least one switching element to generate the electroporation pulse or the electrolysis pulse;
A device for promoting apoptosis, comprising a.
The device for promoting apoptosis according to claim 1, wherein the pulse width is 1 to 300 μs.
According to claim 1, wherein the pulse interval is characterized in that 1 ~ 4 ms, the device for promoting apoptosis.
The device for promoting apoptosis according to claim 1, characterized in that the pulse tail time of the electrolysis is 100 μs or more and less than 2 ms.
The device for promoting apoptosis according to claim 1, wherein the device is for treating cancer.
A method for promoting apoptosis in animals other than humans by applying a pulse generated from the device of claim 1 .
8. The method of claim 7, wherein the method for promoting apoptosis is for treating cancer.

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