KR102277440B1 - Method for detecting electroporated area using ttc on potato model - Google Patents

Abstract

The present invention relates to an electroporation area detection method. The electroporation area detection method according to an embodiment of the present invention comprises: an electrical stimulation step of applying electrical stimulation by inserting an electrode into a potato; a dyeing step of dyeing the potato on which the electrical stimulation step was performed using 2,3,5-triphenyltetrazolium chloride (TTC); and an evaluation step of dividing the dyed area in the potato where the dyeing step is completed into first and second areas, wherein the first region is a region dyed darker than the second region, and evaluating the dyed first area and an undyed third area as the electroporation area. The purpose is to provide the electroporation region detection method that can distinguish and detect a reversible electroporation region and an irreversible electroporation region in the potato model.

Description

Electroporation area detection method using TTC in demagnetization model {METHOD FOR DETECTING ELECTROPORATED AREA USING TTC ON POTATO MODEL}

The present invention relates to a method for detecting an electroporation region, and more particularly, to a method for detecting an electroporation region using 2,3,5-triphenyltetrazolium chloride (TTC) in a potato model.

Electroporation is a technique for piercing a cell by applying electrical stimulation, and is generally divided into a reversible electroporation method and an irreversible electroporation method. Among them, the reversible electroporation method is a technology that increases the permeability of the plasma membrane by applying an electric field to the cells so that drugs such as anticancer drugs or DNA are introduced into the cells. The irreversible electroporation method is a non-thermal tissue ablation technique that locally induces apoptosis by applying a high-pressure, strong microwave electric field to the cells compared to the reversible electroporation method. The irreversible electroporation method can minimize the destruction of surrounding tissues other than cancer cells compared to the thermal local treatment method using high frequency or microwave, and thus can be used for local cancer treatment to replace it.

Potatoes are generally used as a tissue phantom for an experiment to evaluate the irreversible electroporation area. In an experiment to evaluate the conventional irreversible electroporation area, the area stained black with melanin generated due to the oxidation of polyphenols in cells after applying electrical stimulation to the potato and left at room temperature for more than 12 hours is evaluated as the electroporation area. .

However, when using this method of detecting the electroporation region using melanin production, a long time of 12 hours or more is required to leave the potatoes subjected to electrical stimulation, and it is difficult to distinguish between the reversible electroporation region and the irreversible electroporation region. In addition, an inaccurate electroporation area may be detected due to the inhomogeneity of the potato tissue.

In order to solve this problem, a method of detecting an electroporation area of a potato subjected to electrical stimulation using magnetic resonance imaging (MRI) is used. However, the method using magnetic resonance imaging can shorten the time, but it is difficult to distinguish between the reversible electroporation region and the irreversible electroporation region. In addition, there is a problem that the cost is high.

Patent Publication No. 10-2021-0018228 (2021. 02. 17.)

The present invention is to solve the above problems, an object of the present invention is to provide an electroporation region detection method that can reduce the time for detecting the electroporation region in the demagnetization model. In addition, it is an object of the present invention to provide an electroporation region detection method capable of distinguishing and detecting a reversible electroporation region and an irreversible electroporation region in a demagnetization model. It is also an object of the present invention to provide a method for detecting an electroporated area that is not affected by the heterogeneity of potato tissue.

Electroporation area detection method according to an embodiment of the present invention, an electrical stimulation step of applying electrical stimulation by inserting an electrode into the potato; A dyeing step of dyeing the potato on which the electrical stimulation step has been performed using 2,3,5-triphenyltetrazolium chloride (TTC); and dividing the dyed region in the potato where the dyeing step is completed into first and second regions, the first region is a region dyed darker than the second region, and the dyed first region and undyed third region and evaluating the area as an electroporation area.

In the evaluation step, the first region may be evaluated as a reversible electroporation region, and the third region may be evaluated as an irreversible electroporation region.

The staining step may be performed within 5 minutes after the electrical stimulation step is completed.

The TTC solution is provided with physiological saline as a solvent at a concentration of 0.5% (w/v), and in the dyeing step, the potatoes subjected to the electrical stimulation step may be immersed in the TTC solution for 1 hour or more.

The electroporation region detection method according to the present invention can reduce the time for detecting the electroporation region in the demagnetization model.

In addition, the electroporation region detection method according to the present invention can be detected by distinguishing the reversible electroporation region and the irreversible electroporation region in the demagnetization model.

In addition, the electroporated area detection method according to the present invention is not affected by the heterogeneity of the potato tissue.

1 is a flowchart illustrating an electroporation region detection method according to an embodiment of the present invention.
FIG. 2 is a view showing an example of a cut surface of a potato in which an area is divided by the electroporation area detection method of FIG. 1 .
3 is a view showing the conditions during the experiment of the present invention.
4 is a view showing the appearance of potatoes dyed in Experiment 1.
5 is a view showing the appearance of the potatoes dyed in Experiment 2 and a graph analyzing the same.
6 is a view showing a graph and a heat map analyzed in the area stained by different staining methods in Experiment 3;
7 is a view showing the appearance of potatoes dyed in Experiment 4.
8 is a view showing the appearance of the dyed potatoes according to the dyeing time and leaving time in Experiment 5.

Hereinafter, embodiments of the present invention will be described clearly and in detail to the extent that those skilled in the art can easily practice the present invention. The electroporation region detection method according to the present invention detects the electroporation region using TTC (2,3,5-triphenyltetrazolium chloride) in a potato model.

1 is a flowchart illustrating an electroporation region detection method according to an embodiment of the present invention. FIG. 2 is a view showing an example of a cut surface of a potato in which an area is divided by the electroporation area detection method of FIG. 1 . 1 and 2 , the electroporation region detection method may include an electrical stimulation step (S10), a dyeing step (S20) and an evaluation step (S30). The electrical stimulation step (S10), the staining step (S20) and the evaluation step (S30) are sequentially performed.

In the electrical stimulation step (S10), an electrode is inserted into the potato 10 to apply electrical stimulation. In the electrical stimulation step (S10), it is possible to perform irreversible electroporation on the potato (10).

In the dyeing step (S20), the potato 10, on which the electrical stimulation step (S10) is performed, is dyed using TTC. In the dyeing step (S20), the potato may be dyed by TTC in a cut state so that the inside to which electrical stimulation is applied is visible. At this time, the dyeing may proceed by immersing the potato 10 in the TTC solution. After that, the TTC staining time means the time in which the potato is immersed in the TTC solution. At this time, living cells are stained red by a reduction reaction by mitochondrial dehydrogenase, and dead cells are not stained.

As shown in FIG. 2, the cut surface of the potato on which the dyeing step (S20) is completed is an undyed area 13 surrounding the two electrodes to which electrical stimulation is applied, and a dyed area 11 surrounding the undyed area 13, 12) can be distinguished. The dyed regions 11 and 12 may be divided into a relatively dark first region 11 adjacent to the undyed region 13 and a relatively light second region 12 surrounding the first region 11 . . The dyeing step (S20) may be performed within a predetermined time after the electrical stimulation step (S10) is completed.

In the evaluation step (S30), the relatively dark first area 11 and the undyed third area 13 among the dyed areas 11 and 12 in the potato where the dyeing step S20 is completed is evaluated as an electroporation area. . At this time, the relatively soft second region 12 among the dyed regions 11 and 12 is evaluated as a non-electroporated region that is not electroporated. In addition, the relatively dark first region 11 among the dyed regions 11 and 12 of the potato is evaluated as a reversible electroporation region, and the undyed third region 13 is evaluated as an irreversible electroporation region.

Unlike the case shown in FIG. 2 , when all regions of the potato are dyed, the relatively dark first region 11 among the dyed regions 11 and 12 is evaluated as a reversible electroporation region, and the dyed region 11 is evaluated as a reversible electroporation region. , 12), the relatively soft second region 12 is evaluated as a non-electroporated region, and an irreversible electroporated region is evaluated as not formed.

Experiments to support the above-described electroporation region detection method were conducted as follows. However, the following experimental examples are merely illustrative of the present invention, and the claims of the present invention are not limited thereto.

3 is a view showing the conditions during the experiment of the present invention. Referring to FIG. 3 , in these experiments, irreversible electroporation is performed on potatoes using an EPO-S1 generator, which is a prototype pulse generator for irreversible electroporation (IRE) as shown in FIG. 3A . This can be assumed to apply electrical stimulation to the potato in the electrical stimulation step (S10) of the electroporation area detection method of the present invention.

At this time, as shown in FIG. 3B, a pair of electrodes inserted into the potato to apply electrical stimulation is provided as a needle electrode made of stainless steel, respectively. Each electrode is provided with an exposed length from the sheath of about 15 mm, each having a diameter of about 1 mm.

The electrodes are spaced apart so that the horizontal distance between the centers of each other is about 10 mm when viewed along the longitudinal direction, and are inserted into the potato to be parallel to each other. At this time, the exposed portion of each electrode is completely inserted into the potato. In the case of these experiments, the insertion length of the deepest electrode among the electrodes according to the curvature of the potato is about 25 mm. In addition, the ends of the electrodes are inserted into the potato so as to be placed at the same position with respect to the longitudinal direction of the electrode. Each electrode is inserted into the potato so as to be perpendicular to the cut plane to be cut for dyeing the potato.

A plurality of potatoes of a certain size are prepared. For example, each potato may be provided in a size of about 240 g in mass, about 80 mm in length, and about 60 mm in longest width with respect to a direction perpendicular to the longitudinal direction of the potato.

In these experiments, a square wave (Square pulse) is applied to the magnetic field using the pulse generator and electrode as described above. At this time, impedance measurement was performed using a 4192A LF impedance analyzer (Yokogawa-Hewlett-Packard Ltd., Japan). A frequency of 10 Hz was chosen to evaluate the change in conductivity after irreversible electroporation. A square pulse of 100 μs pulse width and 2000 μs pulse delay is applied to the demagnetization multiple times.

Results measured during electrical stimulation in these experiments are expressed as mean ± standard deviation (S.D.). One-way analysis of variance (ANOVA) was used to calculate major differences between mean values. In addition, the correlation between electrical properties and puncture area in different staining methods after electrical stimulation for irreversible electroporation was calculated by Pearson correlation coefficient statistics. A p-value of less than 0.05 was considered statistically significant. Statistical analysis was performed using Microsoft Excel 2013 (Microsoft Co., Ltd., USA).

As described above, the potatoes to which electrical stimulation is applied in these experiments are cut in a direction perpendicular to the electrode so as to be bisected based on the exposed area of the electrode. At this time, one side of the divided potato is stained by TTC, and the other side is left for melanin production as a comparative example. In these experiments, TTC staining is performed by immersing the potato after electrical stimulation in a TTC solution in which TTC (Kanto Chemical Co., Ltd., Japan) is dissolved in physiological saline at a concentration of 0.5% (w/v). This may correspond to the dyeing step (S20) of the present invention.

As described above, ImageJ 1.53c (Wayne Rasband, National Institutes of Health) was used to evaluate the stained and unstained areas of potatoes subjected to TTC staining or melanin staining. Since it is possible to visually distinguish the dyed areas and the unstained areas of the potato on which TTC staining or melanin staining has been performed, a ruler may be used when measuring the range of the stained area. On the other hand, in order to clearly distinguish the relatively dark first region 11 or melanin-stained region among the regions 11 and 12 stained in TTC staining from other regions, a learnable Weka segmentation is performed to obtain a binary image. can make This may correspond to the evaluation step (S30) of the present invention.

Equipment and conditions other than those separately described below among the experiments below are as described above.

<Experiment 1>

In this experiment, TTC staining and melanin staining according to the staining time were compared. According to this experiment, under the device and conditions as described above, a square pulse of an electric field strength of 1000V/cm was applied to the potato 100 times. After that, under the same conditions as described above, potatoes were cut and one side of the potato was TTC stained. At this time, TTC staining was performed within 5 minutes after electrical stimulation was applied to prevent the perforated cells from sealing over time. In addition, the other side of the potato was left at room temperature for melanin production. After dyeing or leaving the cut side of one side and the other side of the potato, respectively, 2 hours passed and 18 hours had passed.

In the simulation of the applied electric field in this experiment, the external electric field strength applied between the electrodes is determined by the Laplace equation. The electric field distribution in the steady state is obtained by solving the following equation.

∇ ² φ = 0

Here, φ is the potential applied from the outside, assuming that the length of the electrodes is greater than the distance between the electrodes. In this case, the surface effect of the potato is neglected. The electric field strength is given as the slope of the external potential.

E = ∇φ

The boundary condition of the tissue applied here _{is defined as φ=V 0} or φ=0. Resting boundaries were assumed to be electrically insulated (dφ/dn = 0). The electric field was calculated using ^{EPO code TM} developed with OpenFOAM (The Standard Co. Ltd., Korea), an open source software.

4 is a view showing the appearance of potatoes dyed in Experiment 1. Referring to FIG. 4A , in the case of a cross-section on which TTC staining is performed, the white perforated area inside the dark red area is clearly distinguished after the staining time has progressed for 2 hours. On the other hand, the cut surface after 2 hours of being left unattended is somewhat darkened, but it is difficult to distinguish a clear perforation area.

Referring to FIG. 4B , in the case of the cross section on which TTC staining is performed, an unstained dead cell area is observed in the central area even after 18 hours of staining. The cut area after 18 hours of standing still clearly shows the perforated area in dark black. Therefore, it can be seen that when TTC staining is performed, the electroporation region can be detected faster than when melanogenesis is used.

<Experiment 2>

In this experiment, TTC staining and melanin staining according to electric field strength were compared. In this experiment, a square wave (Square pulse) having different electric field strengths of 300, 600, 900, 1200 and 1500V/cm, respectively, was applied to the magnetic field 32 times by using the pulse generator and electrode as described above. Then, under the same conditions as described above, each potato was cut and one side of each potato was TTC stained. At this time, TTC staining was performed within 5 minutes after electrical stimulation was applied to prevent the perforated cells from sealing over time. In addition, the other side of each potato was left at room temperature for melanin production. The cut side of one side and the other side of the potato was observed at a time point 2 hours after dyeing or leaving, respectively.

5 is a view showing the appearance of the potatoes dyed in Experiment 2 and a graph analyzing the same. Referring to P of FIG. 5 , in both dyeing methods, the area of the perforated area increases according to the intensity of the voltage. In general, it is observed that the unstained areas of TTC-stained potatoes and the black areas of melanin-stained potatoes are similar. In the case of TTC staining, the unstained area was not significantly different from the black area of the melanin-stained potato according to the voltage conditions (P>0.005). Referring to Q of FIG. 5 , it can be seen that the correlation between the regions of the two staining methods is high with a Pearson correlation coefficient of 0.9789 (P<0.0001).

However, the results of TTC staining are closer to simulations that predict the puncture area by electric field intensity in irreversible electroporation. Referring to FIG. 5B , in the cut surface of the potato on which TTC staining was performed at 600 V/cm, an unstained area surrounded by a relatively darkly stained area was separated into two and observed.

This is similar to the area surrounded by the 500 V/cm line (indicated by the white arrow) of the simulation shown in K to O of FIG. 5 . 600 V/cm (L in Fig. 5) is known as the Liver threshold for irreversible electroporation in previous studies. On the other hand, in the case of melanin staining, as shown in Fig. 5G, unlike the simulation, the black-stained area was observed as one combined area, and it can be seen that it has a difference from the simulation showing the area surrounded by two separate lines. . Therefore, the unstained region of the cut surface of the potato subjected to TTC staining is expected to be an irreversibly electroporated region.

In addition, as shown in FIG. 5A to E, the cut surface of the potato on which TTC staining is performed is divided into a relatively dark area in which the dyed area surrounds the undyed area and a relatively light area outside the area, and have. In contrast, the cut surface of the potato on which melanin staining was performed, as shown in F to J of FIG. 5, was divided only into a black-stained portion and an unstained region, and other distinct regions are not observed.

At this time, in the case of TTC staining, the relatively dark staining area shows a similar tendency to the 200 V/cm line of the simulation result (indicated by yellow arrows in K to O in FIG. 5 ). In particular, referring to FIG. 5B , in the case of TTC staining, a relatively dark staining area has a dumbbell shape as shown in the simulation result of FIG. 3L . Therefore, considering that 200 V/cm is the threshold of Liver tissue for reversible electroporation, a relatively dark staining area for TTC staining is predicted as a reversibly electroporated area. In addition, the reversible electroporated region is a region where perforated but surviving cells are present. Due to the perforation, more mitochondrial dehydrogenase is secreted to the outside, and it is predicted to be stained darker than the region in which non-perforated surviving cells exist. .

<Experiment 3>

In this experiment, TTC staining and melanin staining according to the current and conductance change rates calculated or measured in Experiment 2 were compared. Here, the current is the current flowing when the electrical stimulation pulse is applied, and the conductance change rate is the conductance change rate at 10 Hz, which indicates the degree of cell damage in the electrically stimulated tissue.

6 is a view showing a graph and a heat map analyzed in the area stained by different staining methods in Experiment 3; Referring to FIG. 6A , the correlation between the current and the unstained area (region predicted as an irreversibly electroporated area, TTC IRE area) during TTC staining has a Pearson correlation coefficient of 0.9566 (P<0.0001). On the other hand, referring to FIG. 6C, the Pearson correlation coefficient between the current and the melanin staining region is 0.9493 (P<0.0001). Therefore, in the case of the TTC staining method, it can be seen that the unstained area has a slightly higher correlation with the current than in the case of the melanin staining method.

In addition, referring to FIG. 6B , there is a correlation of a Pearson correlation coefficient of 0.9209 (P<0.0001) between the conductance change rate of 10 Hz and the unstained region during TTC staining. On the other hand, referring to D of FIG. 6 , there is a correlation between the conductance change rate and the melanin staining region with a Pearson correlation coefficient of 0.9086 (P<0.0001). Therefore, it can be seen that the unstained area in the case of the TTC staining method has a slightly higher correlation with the conductance change rate than in the case of the melanin staining method.

The result of the heat map E of FIG. 6 shows correlation coefficient values between variables. Referring to the heat map of FIG. 6E , it can be seen that the Pearson correlation coefficient value is higher than 0.85 among other variables except for the relatively darkly stained area (TTC RE) during TTC staining, which is predicted to be a reversible electroporation area. can In addition, it can be seen that the non-stained region during TTC staining has a higher Pearson correlation coefficient value between the current and the conductance change rate compared to the melanin-stained region.

<Experiment 4>

In this experiment, the time between irreversible electroporation treatment and TTC staining was compared between TTC staining. In this experiment, a square pulse having an electric field strength of 1000V/cm, respectively, was applied to two magnetic fields 100 times by using the pulse generator and electrode as described above. Then, under the same conditions as described above, each potato was cut, and one side of one potato and one side of the other potato were subjected to TTC staining within 5 minutes after electrical stimulation, and the other side of one potato and the other The other side of one potato was subjected to TTC staining 21 hours after electrical stimulation.

7 is a view showing the appearance of potatoes dyed in Experiment 4. Referring to FIG. 7 , in the case of potatoes in which TTC staining is performed within 5 minutes after electrical stimulation for irreversible electroporation is completed, a region that is relatively darkly stained is generated as shown in A and B of FIG. 7 . As the irreversibly electroporated region is surrounded by the reversibly electroporated region, such a relatively heavily stained region is predicted to be a reversibly electroporated region.

On the other hand, in the case of potatoes that have been TTC-stained 21 hours after the completion of the electrical stimulation, as shown in C and D of FIG. 7 , a relatively darkly stained region is not generated. In general, it takes more than 5 minutes for the cell membrane to be re-sealed in the case of living cells after electroporation, so that, before TTC staining, it takes 21 hours, which is sufficient time to seal the cells, in the case of C and D of FIG. 7 , in the case of mitochondria. It is predicted that this is because the dehydrogenase is not distributed in a large amount and the dye is not darkly colored. In addition, in the case of TTC staining after 21 hours of electrical stimulation, a light-dyed area and a non-stained area are clearly distinguished. Therefore, these results indirectly show that the relatively darkly stained region is the reversible electroporation region.

<Experiment 5>

In this experiment, TTC staining and melanin staining were performed by varying the electric field intensity as in Experiment 2, but the dyeing areas of potatoes according to the TTC staining time and the time left at room temperature were compared.

8 is a view showing the appearance of the dyed potatoes according to the dyeing time and leaving time in Experiment 5. Referring to FIG. 8, in the case of a method using dyeing by melanin generated by leaving it at room temperature, a long standing time of 7 hours or more is required to distinguish the dyeing area even at a low electric field intensity of 200 V/cm, and at all electric field strengths It can be seen that 12 hours or more are required for clearer region division. On the other hand, in the case of the TTC staining method, it is possible to distinguish the stained area even when TTC staining has been performed for 1 hour, and it can be seen that a staining time of 2 hours or more is required for clearer area division in all electric field strength areas. have.

Therefore, in the case of using the TTC staining method as in the electroporation area detection method of the present invention, staining is started within 5 minutes after applying the electrical stimulation and preferably, if the staining is performed for 2 hours or more, the area can be distinguished, so the conventional melanin staining method The electroporation region can be detected in a shorter time compared to

In addition, as shown in the previous experiments, in the case of the electroporation region detection method of the present invention, not only the irreversible puncture region but also the reversible puncture region can be detected by performing staining using TTC. Therefore, it is possible to apply the demagnetization model not only to the experiments on the irreversible electroporation method, but also to the experiments on the reversible electroporation method.

In addition, referring to C and D of FIG. 3 showing each piece of the same potato cut in two, in the case of the conventional melanin staining method, the area stained with melanin depending on the potato is affected by the medulla tissue inside the potato. It may not properly represent the perforated area. On the other hand, as shown in D of FIG. 3, when using TTC as in the electroporation area detection method of the present invention, the water quality inside the potato is not affected. Therefore, this result can detect the perforated area more accurately than when using melanin when using TTC as in the electroporation area detection method of the present invention.

The above are specific embodiments for carrying out the present invention. In addition to the above-described embodiments, the present invention may include simple design changes or easily changeable embodiments. In addition, the present invention will include techniques that can be easily modified and implemented using the embodiments. Therefore, the scope of the present invention should not be limited to the above-described embodiments, and should be defined by the claims and equivalents of the claims of the present invention as well as the claims to be described later.

10: Potatoes
11: First region relatively heavily stained by TTC
12: second region relatively lightly stained by TTC
13: third area not colored by TTC

Claims (4)

An electrical stimulation step of applying electrical stimulation by inserting a pair of electrodes into a potato having a mass of 240 g, a length of 80 mm, and the longest width of 60 mm in a direction perpendicular to the longitudinal direction;
A dyeing step of dyeing the potato on which the electrical stimulation step has been performed using 2,3,5-triphenyltetrazolium chloride (TTC); and
In the potato on which the dyeing step is completed, the dyed region is divided into first and second regions, wherein the first region is a region dyed darker than the second region, and the first region and the undyed third region are an evaluation step of evaluating as a perforated region, wherein the first region is evaluated as a reversible electroporation region, and wherein the third region is evaluated as an irreversible electroporation region,
In the electrical stimulation step, the electrode, the area exposed from the sheath is provided with a needle electrode made of stainless steel with a length of 15 mm from the tip and a diameter of 1 mm, parallel to each other and the tip to each other in the longitudinal direction of the needle electrode. A square wave of 100 μs pulse width and 2000 μs pulse delay is applied multiple times between the electrodes, which is inserted into the potato so that it is placed in the same position and the entire exposed area from the sheath is located within the potato,
In the evaluation step, in order to clearly distinguish the first region from other regions, a learnable Weka segmentation is performed to make a binary image, and the first region is the electric field strength when electrical stimulation is applied between the electrodes. is evaluated as a region of 200V/cm or more and less than 500V/cm, and the third region is an electroporation region detection method in which an electric field strength is evaluated as a region of 500V/cm or more when electrical stimulation is applied between the electrodes. delete The method of claim 1,
The staining step is an electroporation area detection method that is performed within 5 minutes after the electrical stimulation step is completed. The method of claim 1,
The TTC solution is provided at a concentration of 0.5% (w/v) in physiological saline as a solvent,
In the dyeing step, the electroporation area detection method of immersing the potato on which the electrical stimulation step is performed in the TTC solution for at least 1 hour.

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