Method for improving myocardial cell killing through high-frequency irreversible electroporation pulse parameters

Abstract

The invention provides a method for improving myocardial cell killing through high-frequency irreversible electroporation pulse parameters, which comprises the following steps: setting pulse parameters of H-FIRE, the pulse parameters comprising inter-phase delay and inter-pulse delay in H-FIRE, and the selected pulse parameters being within a non-stress time range of myocardial cells; and putting the cell suspension into an electric shock cup, and carrying out pulsed electric field treatment according to set pulse parameters. Within the pulse parameter research range, stronger cell killing can be generated by increasing the inter-phase delay and / or the inter-pulse delay in the H-FIRE.

Description

Technical Field

[0001] The present invention relates to the technical field of high-frequency ablation, and in particular to a method for achieving a highly efficient cell killing effect by changing parameters of a high-frequency irreversible electroporation ablation technique. Background Art

[0002] High-frequency irreversible electroporation (H-FIRE) is a new ablation technique for the treatment of atrial fibrillation (AF) and is currently receiving widespread attention. Compared with irreversible electroporation (IRE), it has some advantages. First, it reduces muscle contraction and minimizes the formation of bubbles. Second, it ensures a more uniform distribution of the pulsed electric field, resulting in more thorough ablation. Currently, clinical trials have verified the safety and effectiveness of this technology.

[0003] In the process of studying pulsed electric fields, pulse parameters are one of the most important factors affecting ablation, and different combinations of pulse parameters will produce different killing effects on cells. H-FIRE has several modifiable pulse parameters, such as pulse width, voltage amplitude, number of pulses, pulse frequency, phase delay, and inter-pulse delay. Many studies have explored the biological effects, ablation effects, and potential mechanisms of different pulse parameters. For example, the results showed that compared with IRE with a long pulse width, H-FIRE with a pulse width of 2μs during treatment can effectively reduce the production of muscle contraction. Miklovcic et al. studied the effects of pulse width and pulse number on tissue model ablation in H-FIRE, and the results showed that longer pulses can produce more significant ablation effects; in addition, when the pulse width, voltage, etc. remain unchanged, the ablation area does not increase significantly when 40-200 pulse trains are applied, which indicates that there is a saturation effect on the number of pulses. Sano et al. conducted experiments on cells by applying bipolar pulsed electric fields, and found that for pulses with shorter pulse widths, higher electric field strengths need to be applied to achieve the same killing effect as IRE. Dong et al. investigated the effect of electric field intensity in H-FIRE pulses on tissues using an animal model. The study found that with the increase of electric field intensity, the observed tissue damage increased significantly.

[0004] Although the biological effects related to H-FIRE parameters have been extensively studied and these research results play an important guiding role in its application, how to determine the optimized pulse parameters for pulsed electric field ablation is still a very challenging task. Compared with other parameters such as pulse width, voltage and number of pulses, the exact effect of the combination of phase delay and interpulse delay on cardiomyocyte killing is still unclear. By studying the interphase delay and interpulse delay, the results can provide some references for the optimal design of H-FIRE parameters in pulsed electric field therapy. Summary of the invention

[0005] The present invention proposes a method for improving myocardial cell killing by using high-frequency irreversible electroporation pulse parameters. Within the pulse parameter research range, increasing the interphase delay and / or interpulse delay in H-FIRE can produce stronger cell killing.

[0006] The technical solution of the present invention is implemented as follows: a method for improving myocardial cell killing by high-frequency irreversible electroporation pulse parameters, comprising the following steps:

[0007] (1) Setting the pulse parameters of H-FIRE, including the interphase delay and interpulse delay in H-FIRE, and the selected pulse parameters are within the refractory period of myocardial cells;

[0008] (2) Place the cell suspension in an electric shock cup and perform pulse electric field treatment according to the pulse parameters set in step (1).

[0009] Furthermore, in step (1), the pulse parameters set include: phase delay 0.2-10 μs, pulse delay 1-500 μs.

[0010] Furthermore, in step (1), the pulse parameters set also include: pulse width 2 μs, pulse number 50×4, frequency 15 Hz, electrode spacing 1 mm, and electric field strength 3 kV / cm-6 kV / cm.

[0011] Furthermore, the electric shock cup is cleaned before use, and the cleaning method is as follows: inject sterile pure water into the electric shock cup for rinsing, repeat twice and absorb dry, inject 75% alcohol for rinsing twice or soak for a while and absorb dry;

[0012] The electric shock cup was sterilized by ultraviolet light, rinsed with sterile pure water three times and then dried. The electric shock cup was placed on a clean bench and air was blown on the clean bench to assist in drying the electric shock cup.

[0013] Furthermore, the shock cup is cleaned after use to remove the residual cell solution in the shock cup. The cleaning method is as follows: rinse the shock cup with sterile pure water for 3 times, and blow it slowly with a straw for the next 1 to 2 times; add an appropriate amount of 75% alcohol to the electrode cup and soak it for more than 5 minutes;

[0014] Remove the alcohol and rinse twice with pure water; ultrasonically clean the electric shock cup for 30 seconds, then rinse again with pure water, and finally place the electric shock cup in a clean bench to air dry.

[0015] Furthermore, in step (2), 100 μL of cell suspension containing about 2×10 5 The cells were placed in a 1 mm electroporation cuvette.

[0016] Beneficial effects of the present invention:

[0017] In the present invention, within the range of pulse parameter research, increasing the interphase delay and / or interpulse delay in H-FIRE can produce stronger cell killing. However, when the interpulse delay reaches 100μs and above, the killing effect continues to increase, but the change in the interphase delay will not affect the killing effect of the pulsed electric field on cells. In addition, cell membrane damage detection found that short-term high cell membrane permeability does not necessarily lead to low cell survival rate, and cell death is affected by multiple factors. Cell apoptosis detection found that there are two ways of cell death after treatment with different high-frequency pulsed electric fields, namely, early cell necrosis and late cell apoptosis. In the study of other factors, it was found that after treatment with different high-frequency pulsed electric fields, Ca 2+ concentration, mitochondrial membrane potential, etc., which indicates that Ca 2+ , mitochondria, etc. also play an important role in cell death. In addition, the study found that the experimental phenomena caused by changing the interphase delay and interpulse delay were less correlated with the thermal effects caused by Joule heat. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Flowchart for pulsed electric field treatment of cardiomyocytes;

[0020] Figure 2 The effect of pulse delay on cardiomyocyte survival under different electric field strengths: (a) the effect of pulse delay on cardiomyocytes when the electric field strength is 3 kV / cm; (b) the effect of pulse delay on cardiomyocytes when the electric field strength is 6 kV / cm;

[0021] Figure 3 The effect of different electric field strengths on the viability of cardiomyocytes;

[0022] Figure 4Effect of inter-pulse delay on the survival of cardiomyocytes AC16, (a) Schematic diagram of pulse waveform, inter-pulse delay is fixed, inter-pulse delay varies with 1, 10, 100, 500 μs; (b) Cell survival rate after 24 h of treatment when inter-pulse delay is 0.2 μs; (c) Cell survival rate after 24 h of treatment when inter-pulse delay is 1 μs; (d) Cell survival rate after 24 h of treatment when inter-pulse delay is 10 μs;

[0023] Figure 5 Effect of interphase delay on the survival of cardiomyocytes AC16, (a) Schematic diagram of pulse waveform, the interpulse delay is fixed at 1μs and 10μs, and the interphase delay changes to 0.2, 1, and 10μs; (b) Cell survival rate after 24h treatment when the interpulse delay is 1μs; (c) Cell survival rate after 24h treatment when the interpulse delay is 10μs;

[0024] Figure 6 Effect of interphase delay on the survival of cardiomyocytes AC16, (a) Schematic diagram of pulse waveform, the interpulse delay was fixed at 100μs and 500μs, and the interphase delay varied at 0.2, 1, and 10μs; (b) Cell survival rate after 24h treatment when the interpulse delay was 100μs; (c) Cell survival rate after 24h treatment when the interpulse delay was 500μs;

[0025] Figure 7 Effect of inter-pulse delay on the cell membrane permeability of cardiomyocyte AC16 cells, (a) Schematic diagram of pulse waveform, the inter-pulse delay is fixed, and the inter-pulse delay changes to 0.2, 1, and 10 μs; (b) Changes in cell membrane permeability after pulse electric field treatment when the inter-pulse delay is 1 μs; (c) Changes in cell membrane permeability after pulse electric field treatment when the inter-pulse delay is 10 μs; (d) Changes in cell membrane permeability after pulse electric field treatment when the inter-pulse delay is 100 μs; (e) Changes in cell membrane permeability after pulse electric field treatment when the inter-pulse delay is 500 μs.

[0026] Figure 8 Figure 3 Effect of the inter-pulse delay on the cell membrane permeability of cardiomyocyte AC16 cells. (a) Schematic diagram of pulse waveform, with fixed inter-pulse delay and variable inter-pulse delay; the variation values ​​are 1, 10, 100, and 500 μs; (b) Changes in cell membrane permeability after pulse electric field treatment when the inter-pulse delay is 0.2 μs; (c) Changes in cell membrane permeability after pulse electric field treatment when the inter-pulse delay is 1 μs; (d) Changes in cell membrane permeability after pulse electric field treatment when the inter-pulse delay is 10 μs.

[0027] Fig. 9 Schematic diagram of H-FIRE pulse waveform. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention selects normal human cardiomyocytes AC16 as the research object. The cell line was purchased from China Beina Biotechnology (Beijing, China), and the cell number is BNCC339980.

[0030] A method for improving myocardial cell killing by high-frequency irreversible electroporation pulse parameters, comprising the following steps:

[0031] (1) Setting the pulse parameters of H-FIRE. The selection of parameters is mainly based on clinical data. In addition, as the parameter size increases, the duration of the pulse will be extended. In this process, the synchronization with the clinical electrocardiogram needs to be considered. Given that the refractory period of myocardial cells is 250ms to 300ms, the relevant value can be calculated according to the formula. According to the calculation, the parameters we selected are within the refractory period of myocardial cells.

[0032] T b =N(2T p +d 1+ d2)-d2#(3.1)

[0033] Where, T b is the pulse duration, N is the number of bipolar pulses, T p is the pulse width, d1 is the phase delay, and d2 is the pulse delay.

[0034] The pulse parameter settings of H-FIRE are shown in the following table:

[0035] In Table 1, the voltage is selected at 600V, that is, the electric field strength is 6kV / cm, while keeping the pulse width, pulse number, frequency, and electrode spacing unchanged. Then, the phase delay and pulse delay are changed. S1~S3, S4~S6, S7~S9 explore the influence of different pulse delays; at the same time, the parameter pulse delay is kept consistent to explore the influence of different phase delays.

[0036] Table 1 Electroporation protocol 1

[0037]

[0038] When the electric field strength is 6 kV / cm, the pulsed electric field has a significant killing effect on cardiomyocytes. The cell survival rates of the 9 groups of parameters S1-S9 are all below 10%. The effects of phase delay and pulse delay on cells cannot be observed. Therefore, further exploration of parameters such as electric field strength is needed.

[0039] In Table 2, we changed the electric field strength and inter-pulse delay while keeping the pulse width, inter-pulse delay, number of pulses, frequency, and electrode spacing constant. T1 to T6 explored the effect of inter-pulse delay on cells when the electric field strength was 3 kV / cm and 6 kV / cm.

[0040] Table 2 Electroporation protocol 2

[0041]

[0042]

[0043] In Table 3, while keeping the pulse width, phase delay, inter-pulse delay, pulse number, frequency, and electrode spacing unchanged, we changed the electric field strength in the range of 4 kV / cm to 5.6 kV / cm to observe the effect of the electric field strength.

[0044] Table 3 Electroporation protocol 3

[0045]

[0046] In Table 4, in order to further explore the effect of longer delays on cell killing, the interpulse delay in Table 1 was increased to 500 μs. G1-G4, G5-G8, G9-G12 explored the effect of different interpulse delays; at the same time, the interpulse delay parameter was kept consistent to explore the effect of different phase delays.

[0047] Table 4 Electroporation protocol 4

[0048]

[0049]

[0050] (2) Use an electric shock cup to treat cells with pulsed electric fields to further observe changes in cells. Before pulsed electric field treatment, the pulse equipment should be checked to ensure that the waveform output on the oscilloscope is correct. Figure 1 As shown in the figure, the specific process of pulsed electric field treatment of myocardial cells is as follows:

[0051] 1) Cultivate and collect cells, and centrifuge at 1000 rpm for 5 minutes;

[0052] 2) Transfer the centrifuge tube to the clean bench, discard the supernatant, take an appropriate amount of complete culture medium and add it to the centrifuge tube to mix the cells, then count the cell suspension, repeat three times, calculate the average value, and finally adjust the cell concentration to 2.0×10 6 mL -1 ;

[0053] 3) Wash the electroporation cup and then take 100 μL of cell suspension containing about 2×10 5 The cells were placed in a 1mm electric shock cup and subjected to pulsed electric field treatment using a high-frequency microsecond pulse electric field generator according to the parameter plan. The cells in the negative control group were also placed in the electric shock cup, but were not subjected to pulsed electric field treatment. The high-voltage probe was connected to an oscilloscope to monitor the applied pulse waveform and voltage. The bandwidth of the high-voltage probe and oscilloscope used were 220MHz and 200MHz, respectively.

[0054] The method of cleaning the electric shock cup is as follows:

[0055] Take out the dried and stored electric shock cup and place it in the clean bench. Pour sterile pure water into the electric shock cup, then rinse it with a pipette, repeat twice to dry it, pour 75% alcohol and rinse it with a pipette twice or soak it for a while to dry it;

[0056] Arrange the electric shock cups upright in the clean bench, turn on the ultraviolet light of the clean bench, and sterilize the electric shock cups with ultraviolet light;

[0057] Inject sterile pure water and rinse with a pipette three times, then suck dry. Place the electric shock cup on the clean bench and keep the clean bench blown to assist the electric shock cup in drying, and then enter the subsequent pulsed electric field treatment process.

[0058] After the experiment, the electric shock cup needs to be cleaned to remove the residual cell solution in the electric shock cup to prevent the residual solution from affecting subsequent experiments and to increase the repeated service life of the electric shock cup. The cleaning method is as follows:

[0059] After use, rinse the electric shock cup with sterile pure water for 3 times, and blow it slowly with a straw for the next 1-2 times; especially for the 1mm electrode cup, ensure that most of the cells are removed during the cleaning process; add an appropriate amount of 75% alcohol to the electrode cup and soak it for more than 5 minutes;

[0060] Absorb the alcohol and rinse with pure water for about 2 times. If conditions permit, put the electric shock cup into a small ultrasonic cleaner for about 30 seconds to clean the slit surface of the electric shock cup, then rinse it again with pure water to rinse away the substances after the ultrasonic treatment; place the electric shock cup in a clean bench to air dry.

[0061] like Figure 2As shown in the figure, when the electric field strength is 3kV / cm, compared with the T0 group, the pulse electric field has no obvious killing effect on cells by changing the delay between pulses, and there is no significant difference between the experimental group and the control group. When the electric field strength is 6kV / cm, the pulse electric field with the change of the delay between pulses has a strong killing ability on cells, and the survival rate is less than 10%, which is not enough to see the effect of the change of the delay between pulses on cells. According to the above results, it can be seen that there is a suitable electric field strength between 3kV / cm and 6kV / cm, and this suitable electric field strength can be used to observe the effect of different delays on cells.

[0062] like Figure 3 As shown in the figure, when the electric field strength is less than 4.5kV / cm, the pulse electric field treatment of cardiomyocytes has no significant effect on the vitality of the cells; and when the electric field strength gradually increases, the survival rate of cardiomyocytes decreases. When the electric field strength reaches 5kV / cm~5.3kV / cm, the survival rate of cardiomyocytes is in the range of 50%~60%; and when the electric field strength continues to increase to 5.6kV / cm, the survival rate of cardiomyocytes drops to about 20%. From this result, it can be seen that when the pulse width of the bipolar pulse is 2μs, the phase delay is 10μs, the pulse delay is 1μs, the number of pulses is 4 strings, each string is 50, the frequency is 15Hz, and the electrode spacing is 1mm, the electric field strength is changed in the range of 4.5kV / cm~5.6kV / cm, and the pulse can effectively kill the cells. Within this range, the cell survival rate drops from 100% to about 20%. Based on this result, in order to facilitate further observation of the effect of delay in subsequent experiments, the electric field strength was fixed at 5.25 kV / cm.

[0063] like Figure 4 As shown in the figure, when the inter-phase delay remains unchanged, the killing ability of the pulsed electric field on cardiomyocytes is significantly enhanced with the increase of the inter-pulse delay (P<0.05).

[0064] like Figure 5 As shown in the data, when the inter-pulse delay remains unchanged (1μs or 10μs), the killing ability of the pulsed electric field on cells is significantly enhanced with the increase of the inter-pulse delay. That is, when the inter-pulse delay changes within the range of 10μs, the cell killing ability of the pulsed electric field is dose-dependent in terms of the inter-pulse delay (P<0.05).

[0065] like Figure 6 As shown in the figure, further extending the inter-pulse delay to 100μs and 500μs further significantly improved the killing ability of the pulsed electric field on AC16 cells. However, it is worth noting that when the inter-pulse delay remains unchanged (100μs or 500μs), the survival rate of cells will not decrease significantly with the increase of the inter-phase delay.

[0066] like Figure 7As shown in the figure, when the interpulse delay was fixed and within the range of 100μs, the permeability of the cell membrane showed obvious dose dependence as the interpulse delay increased. However, as the interpulse delay continued to increase, when the interpulse delay reached 500μs, there was no significant difference among the three treatment groups (P>0.05).

[0067] like Figure 8 As shown, when the inter-phase delay remains unchanged, the change of cell membrane permeability has a clear dose-dependency as the inter-pulse delay increases.

[0068] like Fig. 9 As shown in the figure, the H-FIRE pulse mainly includes pulse width, voltage amplitude, number of pulses, phase delay, pulse delay and pulse frequency. Phase delay refers to the time interval between positive and negative pulses in a bipolar pulse. Pulse delay refers to the time interval between two consecutive adjacent bipolar pulses.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving myocardial cell killing by high-frequency irreversible electroporation pulse parameters, characterized in that: The following steps are involved: (1) Setting the pulse parameters of H-FIRE, including the interphase delay and interpulse delay in H-FIRE, and the selected pulse parameters are within the refractory period of myocardial cells; (2) Place the cell suspension in an electric shock cup and perform pulsed electric field treatment according to the pulse parameters set in step (1).

2. A method for improving myocardial cell killing by high-frequency irreversible electroporation pulse parameters according to claim 1, characterized in that: In step (1), the pulse parameters to be set include: phase delay 0.2-10 μs, pulse delay 1-500 μs.

3. The method of improving myocardial cell killing by high-frequency irreversible electroporation pulse parameters according to claim 2, characterized in that: In step (1), the set pulse parameters also include: pulse width 2 μs, pulse number 50×4, frequency 15 Hz, electrode spacing 1 mm, and electric field strength 3 kV / cm-6 kV / cm.

4. A method for improving myocardial cell killing by using high-frequency irreversible electroporation pulse parameters according to any one of claims 1 to 3, characterized in that: The electric shock cup should be cleaned before use. The cleaning method is as follows: inject sterile pure water into the electric shock cup to rinse, repeat twice to dry, inject 75% alcohol to rinse twice or soak for a while to dry; The electric shock cup was sterilized by ultraviolet light, rinsed with sterile pure water three times and then dried. The electric shock cup was placed on a clean bench and air was blown on the clean bench to assist in drying the electric shock cup.

5. A method for improving myocardial cell killing by using high-frequency irreversible electroporation pulse parameters according to any one of claims 1 to 3, characterized in that: After use, the shock cup should be cleaned to remove the residual cell solution in the shock cup. The cleaning method is as follows: rinse the shock cup with sterile pure water for 3 times, and blow it slowly with a straw for the last 1-2 times; add an appropriate amount of 75% alcohol to the electrode cup and soak it for more than 5 minutes; Remove the alcohol and rinse twice with pure water; ultrasonically clean the electric shock cup for 30 seconds, then rinse again with pure water, and finally place the electric shock cup in a clean bench to air dry.

6. The method of claim 1 for improving myocardial cell killing by using high-frequency irreversible electroporation pulse parameters, characterized in that: In step (2), 100 μL of cell suspension containing approximately 2×10 5 The cells were placed in a 1 mm electroporation cuvette.

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