CN107681916A - One kind collaboration irreversible electroporation device of pulse - Google Patents

Abstract

The invention discloses a coordinated pulse irreversible electroporation device, which mainly includes a power supply system, a coordinated pulse forming system, a pulse measurement system, a control system and a signal conversion system. The synergistic pulse irreversible electroporation device outputs synergistic pulses to the electrode needles, and the electrodes ablate tumor cells. The invention adopts the synergistic pulse to effectively improve the killing effect of tumor cells and expand the ablation area of biological tissues.

Description

A Cooperative Pulse Irreversible Electroporation Device

technical field

The invention relates to the field of biological tissue ablation, in particular to a coordinated pulse irreversible electroporation device.

Background technique

Cancer is one of the major diseases that endanger human health. Traditional tumor therapy and the newly developed thermal ablation physical therapy characterized by minimally invasive ablation are limited by factors such as indications, contraindications, treatment side effects, and heat depression. Therefore, the above methods have certain limitations in clinical application. In recent years, with the continuous development of pulsed bioelectrics, electric field pulses have attracted extensive attention from researchers in the field of bioelectromagnetics in many countries due to their non-thermal and minimally invasive biomedical effects. Among them, the irreversible electroporation treatment of tumors has attracted extensive attention from researchers in the field of bioelectricity at home and abroad due to its advantages and characteristics of fast, controllable, visible, selective and non-thermal mechanism, and has been gradually applied in the clinical treatment of tumors. .

At present, irreversible electroporation technology (typical pulse parameters are: field strength 1500-3000V/cm, pulse width 100μs, repetition frequency 1Hz, pulse number 90-120) has been applied to clinical tumor treatment and achieved very good results . Clinical trials have proved that this technology has a good effect on the treatment of pancreatic cancer, liver cancer, kidney cancer, prostate cancer and other tumors.

Angio Dynamics of the United States invested in the production of NanoKnife, a commercialized irreversible electroporation tumor treatment instrument, and obtained the clinical trial license of the US FDA in 2009. In 2010, the company carried out the world's first clinical trial of irreversible electroporation ablation of prostate cancer, and in 2012 it was approved by the US FDA for clinical application. In June 2015, the company obtained the clinical application license in mainland China, and carried out the first clinical application treatment in China at Guangzhou Fuda Cancer Hospital. As of early 2017, the therapeutic device has successfully cured hundreds of tumor patients in mainland China.

It can be seen that irreversible electroporation technology, as a safe and feasible local solid tumor treatment method, has shown good development prospects in the treatment of tumors.

Scheffer et al. analyzed the effectiveness and safety of IRE in the treatment of 221 patients with liver cancer, pancreatic cancer, lung cancer, lymphatic cancer, etc., and found that IRE can safely ablate small-sized tumors near blood vessels or bile ducts.

Although the irreversible electroporation technology based on electric field pulses has achieved exciting therapeutic effects in clinical applications at home and abroad, there are still obvious problems in clinical applications.

For example, for tumor ablation, the optimization of pulse parameters has always attracted extensive attention of researchers in the field of bioelectricity at home and abroad.

Generally speaking, the physical therapy method of irreversible electroporation induced by electric field pulse is only effective for solid tumors with a size less than 3 cm, and the effectiveness of its irreversible electroporation technology gradually decreases with the growth of tumor size. On the one hand, although large-sized tumors can be completely ablated by increasing the intensity of electric field pulses (such as voltage, pulse width, etc.), high-intensity electric field pulses will cause thermal effects and cause irreversible damage to normal tissues such as blood vessels. Athermal Therapeutic Principles of Irreversible Electroporation. On the other hand, although increasing and optimizing the number of electrodes can effectively ablate large-sized tumors, it will increase the complexity and medical risks of the treatment, and even increase the invasiveness of the treatment.

Starting from the mechanism of tumor research, for tumor cells, the greater the damage to the cell membrane, the greater the possibility of cell death.

Where E is the applied pulse field strength, r is the radius of the cell membrane, θ is the angle between the direction of the field strength and the radial direction of the cell membrane, t is the pulse width, C is the capacitance of the cell membrane, se is the conductivity of the extracellular fluid, and si is the cytoplasm conductivity.

It can be concluded from formula (1) that the higher the field strength, the larger the perforated area on the cell membrane will be.

Although high voltage and narrow pulse can produce a large perforation area on the cell membrane, due to the narrow pulse width, the resulting micropore size is small and the micropore is easy to recover, so it is difficult for irreversible electroporation to occur. Weaver et al. found that low-voltage, broad pulses can generate large-sized pores in cell membranes. However, due to the limitation of the threshold field strength, the damage to the cell membrane by low voltage and wide pulse is relatively small. The synergistic pulse combines the respective advantages of high voltage, narrow pulse and low voltage, wide pulse, that is: high voltage, narrow pulse produces a large perforation area on the cell membrane, and the subsequent low voltage, wide pulse has no limit of threshold field strength , so large-sized micropores can be created in the existing cell membrane perforation area, which can greatly damage the cell membrane, resulting in cell death.

For biological tissues, high voltage and narrow pulses can produce a large perforation area on the tissue, but due to the short pulse width, the ablation area only exists near the electrode. Due to the limitation of the threshold field strength, the low voltage and wide pulse can only produce a small perforation area, but due to the longer pulse width, the perforation area can basically be transformed into an ablation area.

Contents of the invention

The purpose of the present invention is to provide a device and method for synergistic pulse ablation of biological tissue, which is used for the ablation of tumor tissue and other biological tissues, in view of the problem that the ablation area of the existing irreversible electroporation therapy is small in clinical trials.

The invention divides the traditional irreversible electroporation electric field pulse waveform into high voltage, narrow pulse and low voltage, wide pulse form. High voltage and narrow pulse can produce a large perforation area on the tissue, but due to the short pulse width, the ablation area only exists near the electrode, and low voltage and wide pulse can only produce small-scale perforation due to the limitation of the threshold field strength Due to the longer pulse width, the perforation area can basically be turned into an ablation area. The synergistic electric field pulse is used to act on tumors or other biological tissues to induce irreversible electroporation and death of cells, so as to achieve the purpose of expanding the tumor ablation area and other biological tissues, and can effectively solve the limitation of tumor volume in irreversible electroporation therapy, and can be applied in the treatment of human and animal tumors.

The technical solution adopted to realize the object of the present invention is as follows: a cooperative pulse irreversible electroporation device mainly includes a power supply system, a cooperative pulse forming system, a pulse measurement system, a control system and a signal conversion system.

The power system supplies power to the cooperating pulse forming system, pulse measurement system, control system and signal conversion system.

The input terminals of the coordinated pulse forming system include an input terminal A1, an input terminal A2, an input terminal B1 and an input terminal B2.

The output terminals of the coordinated pulse forming system include an output terminal E1 and an output terminal E2.

The power supply system is connected between the input terminal A1 and the input terminal A2.

The input terminal A1 is connected to the input terminal A2 after the charging resistor R1 and the capacitor C1 are serially connected in series.

The input terminal A1 is connected in series with a resistor R1 and an inductance L1 in series, and then connected in series with the D pole of the semiconductor switch MOSFET/IGBT S1.

The S pole of the semiconductor switch MOSFET/IGBT S1 is connected in series with the anode of the diode D1. The cathode of the diode D1 is connected to the input terminal A2 after being loaded in series. The cathode of the diode D1 is connected in series with the load to the input terminal B2.

The power supply system is connected between the input terminal B1 and the input terminal B2.

The input terminal B1 is connected to the input terminal B2 after serially connecting a resistor R2 and a capacitor C2 in series.

The input terminal B1 is connected in series with the inductor L2 and the resistor R2 in sequence, and then connected to the D pole of the semiconductor switch MOSFET/IGBT S2.

The S pole of the semiconductor switch MOSFET/IGBT S2 is connected in series with the anode of the diode D2. The cathode of the diode D2 is connected to the input terminal A2 after being loaded in series. The cathode of the diode D2 is connected to the input terminal B2 after being loaded in series.

The load is connected between the output terminal E1 and the output terminal E2.

Further, the pulse measurement system mainly includes a voltage divider, a current sensor and a processing circuit.

The voltage divider measures the voltage at the output of the coordinated pulse forming system.

The current sensor measures the current at the output of the coordinated pulse forming system.

The processing circuit receives the voltage signal measured by the voltage divider. The processing circuit receives the current signal measured by the current sensor.

Further, the control system mainly includes an FPGA module, a switch control module and the single-chip microcomputer module.

The FPGA module receives the voltage signal and the current signal at the output terminal of the processing circuit. The voltage signal and the current signal are processed for data exchange with the single-chip microcomputer module.

The single-chip microcomputer module controls the cooperative pulse irreversible electroporation device through the switch control module.

The characteristic parameters of the pulse can be set in the control system. The control system converts the set parameters into electrical signals through an algorithm. The control system monitors the voltage signal and current signal in the signal conversion system in real time to ensure the accuracy of output pulse parameters.

The signal conversion system mainly includes the optical/electrical converter K1, the optical/electrical converter K2, the electrical/optical converter J1, and the electrical/optical converter J2.

The electrical signal is respectively transmitted to the power supply system, the coordinated pulse forming system and the pulse measurement system through the signal conversion system.

The electrical/optical converter J1 converts the electrical signal received by the FPGA module into an optical signal.

The optical/electrical converter K1 converts the optical signal of the electrical/optical converter J1 into an electrical signal. The optical/electrical converter K1 transmits the converted electrical signal to the power supply system.

The electrical/optical converter J2 converts the electrical signal received by the FPGA module into an optical signal.

The optical/electrical converter K2 converts the optical signal of the electrical/optical converter J2 into an electrical signal. The optical/electrical converter K2 transmits the converted electrical signal to the coordinated pulse forming system.

Further, the power supply system mainly includes a power supply, a power filter device, a high-voltage direct current module, and a switching power supply module.

The power supply is 220V alternating current.

The switching power supply module converts 220V alternating current into 12V direct current.

The power filter device filters the signal of the switching power supply module to obtain a power signal with a specific frequency.

The ground terminal of the power filter device is directly grounded. The power filter device provides the obtained power signal to the high voltage direct current module.

The high voltage direct current module supplies power to the input of the coordinated pulse forming system.

Further, the device also includes a PC. The power supply system supplies power to the PC. The PC monitors the voltage signal and current signal received by the control system in real time.

The high-voltage pulse amplitude of the coordinated pulse is continuously adjustable between 0 and 3kV. The interval time of the high-voltage pulse is between 1 and 200s. The low-voltage pulse amplitude of the cooperative pulse is continuously adjustable between 0 and 3kV. The interval time of the low-voltage pulse is between 1 and 200s.

The sub-pulse width is continuously adjustable between 0.2 and 100μs. The pulse period is adjustable between 0.1 and 10s. The rise time of the coordinated pulse is 30ns. The fall time of the coordinated pulse is 30ns.

The capacitance of the capacitor C1 and the capacitor C2 is determined by the total pulse width, the output pulse voltage amplitude, the allowable drop value of the output pulse voltage, the load resistance value and the discharge time constant.

Assuming that the maximum total pulse width is τ, the output pulse voltage amplitude is V ₀ , the output pulse voltage allowable drop value is △V _d , and the load resistance value is R _L , then the minimum capacitance of the capacitor C1 and the capacitor C2 is based on The following formula is used for calculation:

After each pulse train is discharged, the voltages of the capacitors C1 and C2 decrease by at most 5%.

The withstand voltage values of the capacitor C1 and the capacitor C2 are determined by the maximum amplitude of the required pulse.

The technical effect of the present invention is unquestionable. The device of the present invention can accurately and reliably generate synergistic pulses, which can induce the transmembrane potential of tumor cell membranes to be greater than the perforation threshold, so that irreversible electroporation occurs in the cell membranes, thereby leading to tumor cell death. At the same time, using The synergistic pulsed electric field can effectively break through the limitation of the threshold field strength in irreversible electroporation, realize the expansion of the effective electric field range of the irreversible electroporation dose in the target tumor tissue area, and solve the problem of small ablation area in the clinical application of irreversible electroporation.

At the same time, the new electric field pulse application method proposed by the present invention is to apply high voltage and narrow pulse before the traditional low voltage and wide pulse irreversible electroporation parameters to reduce and eliminate the influence of threshold field strength, so as to further expand the tumor ablation area, that is, The high-voltage, narrow pulse produces a larger perforation area on the tissue, and the subsequent low-voltage, wide pulse has no limit of threshold field strength, and produces a larger ablation area in the existing perforation area.

The present invention can induce irreversible electroporation in the cell membrane by applying high-strength electric field pulses to biological tissues, thereby leading to cell death, and under the action of high-intensity pulses, cells undergo irreversible electroporation, and can achieve therapeutic effects without applying chemotherapeutic drugs , to avoid the side effects of chemotherapy drugs. At the same time, the present invention is quick (the treatment pulse time is only tens of seconds, and the whole process only takes a few minutes), controllable (the treatment parameters can be calculated and obtained by three-dimensional modeling electric field, and the treatment range is accurate and safe), visual ( The treatment process can be completed under the guidance of ultrasound/CT/MRI, and the curative effect can be evaluated by ultrasound/CT/MRI), selectivity (no damage to the bile duct, blood vessels and nerves, etc. in the ablation area) and non-thermal mechanism (no thermal effect, can be overcome Advantages of 'heat damage' and 'heat sink') brought about by thermotherapy.

Description of drawings

Fig. 1 is the functional block diagram of the synergistic pulse irreversible electroporation device of the inventive method;

Fig. 2 is the circuit block diagram of the cooperative pulse forming system of the inventive method;

Fig. 3 is the synergistic pulse cell and tissue experiment diagram (pulse application method and experimental platform) of the method of the present invention;

Figure 4 is a synergistic pulse cell killing rate figure (*p<0.05) of the method of the present invention;

Fig. 5 is the electrode needle diagram of the animal tissue experiment used in the method of the present invention;

Figure 6 is a diagram of the tissue ablation results of the method of the present invention (*p<0.05, **p<0.01, ***p<0.001);

Fig. 7 is the result figure of tissue H&E staining of the method of the present invention;

In the figure: power supply system 1, cooperative pulse forming system 2, pulse measurement system 3, control system 4 and signal conversion system 5, power supply 11, power filter device 12, high voltage DC module 13, switching power supply module 14, PC 6, branch A voltage regulator 31, a current sensor 32, a processing circuit 33, an FPGA module 41, a switch control module 42 and the single-chip microcomputer module 43.

Detailed ways

The present invention will be further described below in conjunction with the examples, but it should not be understood that the scope of the subject of the present invention is limited to the following examples. Without departing from the above-mentioned technical ideas of the present invention, various replacements and changes made according to common technical knowledge and conventional means in this field shall be included in the protection scope of the present invention.

Example 1:

A coordinated pulse irreversible electroporation device, see FIG. 1 , the coordinated pulse irreversible electroporation device mainly includes a power supply system 1 , a coordinated pulse forming system 2 , a pulse measurement system 3 , a control system 4 and a signal conversion system 5 .

The power supply system 1 supplies power to the coordinated pulse forming system 2 , pulse measurement system 3 , control system 4 and signal conversion system 5 .

Further, the power supply system 1 mainly includes a power supply 11 , a power supply filtering device 12 , a high-voltage direct current module 13 , and a switching power supply module 14 .

The power supply 11 is 220V alternating current.

The switching power supply module 14 converts 220V AC power into 12V DC power.

The power filtering device 12 filters the signal of the switching power supply module 14 to obtain a power signal with a specific frequency.

The ground terminal of the power filter device 12 is directly grounded. The power filtering device 12 provides the obtained power signal to the high voltage DC module 13 .

Further, the power filtering device 12 is a passive bidirectional network. The greater the impedance adaptation between the input end and the output end of the power filter device and the power supply and load, the more effective the signal filtering will be.

The high voltage direct current module 13 supplies power to the input end of the cooperative pulse forming system 2 .

Further, the pulse measurement system 3 mainly includes a voltage divider 31 , a current sensor 32 and a processing circuit 33 .

The voltage divider 31 measures the voltage at the output of the coordinated pulse forming system 2 .

The current sensor 32 measures the current at the output of the coordinated pulse forming system 2 .

The processing circuit 33 receives the voltage signal measured by the voltage divider 31 . The processing circuit 33 receives the current signal measured by the current sensor 32 .

Further, the control system 4 mainly includes an FPGA module 41 , a switch control module 42 and the single-chip microcomputer module 43 .

The FPGA module 41 receives the voltage signal and current signal from the output terminal of the processing circuit 33 . The voltage signal and the current signal exchange data with the single-chip microcomputer module 43 after being processed.

The single-chip microcomputer module 43 controls the coordinated pulse irreversible electroporation device through the switch control module 42 .

The characteristic parameters of pulses can be set in the control system 4 . The control system 4 converts the set parameters into electrical signals through an algorithm. The control system 4 monitors the voltage signal and current signal in the signal conversion system 5 in real time to ensure the accuracy of output pulse parameters.

The signal conversion system 5 mainly includes the optical/electrical converter K1, the optical/electrical converter K2, the electrical/optical converter J1, and the electrical/optical converter J2.

The electrical signal is respectively transmitted to the power supply system 1 , the coordinated pulse forming system 2 and the pulse measurement system 3 through the signal conversion system 5 .

The electrical/optical converter J1 converts the electrical signal received by the FPGA module 41 into an optical signal.

The optical/electrical converter K1 converts the optical signal of the electrical/optical converter J1 into an electrical signal. The optical/electrical converter K1 transmits the converted electrical signal to the power supply system 1 .

The electrical/optical converter J2 converts the electrical signal received by the FPGA module 41 into an optical signal.

The optical/electrical converter K2 converts the optical signal of the electrical/optical converter J2 into an electrical signal. The optical/electrical converter K2 transmits the converted electrical signal to the coordinated pulse forming system 2 .

Further, the coordinated pulse irreversible electroporation device also includes a PC 6 .

The power supply system 1 supplies power to the PC 6 . The PC 6 monitors the voltage signal and current signal received by the control system 4 in real time.

Adjusting the pulse is accomplished by adjusting the output voltage of the power supply system 1 and the conduction time, sequence of break time and break times of the solid-state switches in the high-voltage and low-voltage circuits.

The high-voltage pulse amplitude of the coordinated pulse is continuously adjustable between 0 and 3kV. The interval time of the high-voltage pulse is between 1 and 200s. The low-voltage pulse amplitude of the cooperative pulse is continuously adjustable between 0 and 3kV. The interval time of the low-voltage pulse is between 1 and 200s.

The sub-pulse width is continuously adjustable between 0.2 and 100μs. The pulse period is adjustable between 0.1 and 10s. The rise time of the coordinated pulse is 30ns. The fall time of the coordinated pulse is 30ns.

The capacitance of the capacitor C1 and the capacitor C2 is determined by the total pulse width, the output pulse voltage amplitude, the allowable drop value of the output pulse voltage, the load resistance value and the discharge time constant.

Assuming that the maximum total pulse width is τ, the output pulse voltage amplitude is V ₀ , the output pulse voltage allowable drop value is △V _d , and the load resistance value is R _L , then the minimum capacitance of the capacitor C1 and the capacitor C2 is based on The following formula is used for calculation:

After each pulse train is discharged, the voltages of the capacitors C1 and C2 decrease by at most 5%.

The withstand voltage values of the capacitor C1 and the capacitor C2 are determined by the maximum amplitude of the required pulse.

Example 2:

A coordinated pulse irreversible electroporation device, referring to FIG. 2 , the input terminal of the coordinated pulse forming system 2 includes an input terminal A1 , an input terminal A2 , an input terminal B1 and an input terminal B2 .

The output terminals of the coordinated pulse forming system 2 include an output terminal E1 and an output terminal E2.

The power supply system 1 is connected between the input terminal A1 and the input terminal A2.

The input terminal A1 is connected to the input terminal A2 after the charging resistor R1 and the capacitor C1 are serially connected in series.

Further, the high-voltage DC module 13 charges the energy storage capacitor C1 through the charging resistor R1 according to the set pulse amplitude. After charging is completed, the energy storage capacitor C1 releases energy to the load.

The input terminal A1 is connected in series with a resistor R1 and an inductance L1 in series, and then connected in series with the D pole of the semiconductor switch MOSFET/IGBT S1.

The S pole of the semiconductor switch MOSFET/IGBT S1 is connected in series with the anode of the diode D1. The cathode of the diode D1 is connected to the input terminal A2 after being loaded in series. The cathode of the diode D1 is connected in series with the load to the input terminal B2.

The power supply system 1 is connected between the input terminal B1 and the input terminal B2.

The input terminal B1 is connected to the input terminal B2 after serially connecting a resistor R2 and a capacitor C2 in series.

Further, the high voltage direct current module 13 charges the energy storage capacitor C2 through the charging resistor R2 according to the set pulse amplitude. After charging is completed, the energy storage capacitor C2 releases energy to the load.

The input terminal B1 is connected in series with the inductor L2 and the resistor R2 in sequence, and then connected to the D pole of the semiconductor switch MOSFET/IGBT S2.

The S pole of the semiconductor switch MOSFET/IGBT S2 is connected in series with the anode of the diode D2. The cathode of the diode D2 is connected to the input terminal A2 after being loaded in series. The cathode of the diode D2 is connected to the input terminal B2 after being loaded in series.

The load is connected between the output terminal E1 and the output terminal E2.

Further, the coordinated pulse forming system 2 forms high voltage and narrow pulses, and then forms low voltage and wide pulses. High voltage and narrow pulse, low voltage and wide pulse appear in sequence.

Through the cooperative pulse forming system 2, a new type of electric field pulse application method can be formed, that is, a high voltage and a narrow pulse are applied before the traditional low voltage and wide pulse irreversible electroporation parameters to reduce and eliminate the influence of the threshold field strength, thereby further expanding the tumor Ablation area. That is to say, the high-voltage, narrow pulse produces a larger perforation area on the tissue, and the subsequent low-voltage, wide pulse has no limit of threshold field strength, and can generate a larger ablation area in the existing perforation area.

Example 3:

The steps for using the synergistic pulse irreversible electroporation device are as follows:

1) Initialize the device.

2) Determine the form and application method of the electrode, as well as the characteristic parameters of the coordinated pulse, so as to ensure the effective coverage of the electric field area.

3) Set pulse width, pulse interval and pulse number.

4) Adjust the pulse parameters of the coordinated pulse device and the application method of electrodes according to the characteristics of the patient and the specific conditions of the tumor tissue. It is worth noting that splint electrodes or adsorption electrodes are used for body surface tumor tissues. Needle electrodes are used for tumors in the body, the insertion position of the needle electrodes is determined by the position of the tumor tissue, and the depth of the needle electrodes is determined by the size of the tumor tissue. Commonly used electrode needles for applying pulses are combined into two-needle electrodes.

5) The characteristic parameters of the cooperative pulse determined by the control system setting. Pulse width, pulse interval and number of pulses set by the user.

6) Perform corresponding switching actions on the coordinated pulse irreversible electroporation device to control the output pulse width, number, pulse interval, etc.

7) The electrodes are applied to the patient's tumor tissue, and the coordinated pulse required by the patient is generated by the coordinated pulse irreversible electroporation device and applied to the electrode to stimulate the patient's tumor tissue with a pulsed electric field to induce irreversible electroporation of the tumor tissue, thereby Effectively kill tumor cells.

8) During the electrode application, the user monitors the voltage signal and current signal in real time through the control system to ensure the accuracy of the output pulse parameters.

9) After the treatment is over, the user withdraws the electrodes from the patient's tumor tissue.

Example 4:

When using the high voltage and narrow pulse output by the coordinated pulse irreversible electroporation device to ablate cells and tissues, the following devices are mainly required: oscilloscope, coordinated pulse irreversible electroporation device, temperature sensor and electrode cup.

The circuit of the coordinated pulse irreversible electroporation device outputs high voltage and narrow pulse.

The current probe of the oscilloscope detects the current signal of the positive load of the coordinated pulse irreversible electroporation device, and the voltage probe of the oscilloscope detects the voltage signal connected to the positive load of the coordinated pulse irreversible electroporation device; the oscilloscope converts the electrical signals detected by the current probe and the voltage probe into random Time-varying waveform curve.

The electrode needles have a pitch of 5 mm.

The fiber optic probe of the temperature sensor detects the temperature of the electrode needle; the temperature sensor converts the temperature detected by the fiber optic probe into an available output signal.

One end of the electrode cup is connected to the negative pole of the cooperative pulse forming system 2 of the cooperative pulse irreversible electroporation device, and the other end is connected to the positive pole of the cooperative pulse forming system 2 of the cooperative pulse generating device.

The electrode cup receives the pulse signal output by the coordinated pulse irreversible electroporation device.

Example 5:

When using the low-voltage, wide-pulse ablation of cells and tissues output by the coordinated pulse irreversible electroporation device, the following devices are mainly required: an oscilloscope, a coordinated pulse irreversible electroporation device, a temperature sensor, and an electrode cup.

The circuit of the coordinated pulse irreversible electroporation device outputs low voltage and wide pulse.

The current probe of the oscilloscope detects the current signal at both ends of the load of the coordinated pulse irreversible electroporation device, and the voltage probe of the oscilloscope detects the voltage signal at both ends of the load connected to the coordinated pulse irreversible electroporation device; the oscilloscope converts the electrical signals detected by the current probe and the voltage probe into random Time-varying waveform curve.

The electrode needles have a pitch of 5 mm.

The fiber optic probe of the temperature sensor detects the temperature of the electrode needle; the temperature sensor converts the temperature detected by the fiber optic probe into an available output signal.

One end of the electrode cup is connected to the negative pole of the cooperative pulse forming system 2 of the cooperative pulse irreversible electroporation device, and the other end is connected to the positive pole of the cooperative pulse forming system 2 of the cooperative pulse generating device.

The electrode cup receives the pulse signal output by the coordinated pulse irreversible electroporation device.

Embodiment 6:

In this example, when using the synergistic pulse irreversible electroporation device, human ovarian cancer cell SKOV-3 was used as the experimental object, and an orthogonal experiment and CCK-8 activity detection means were adopted. The test steps are as follows:

1) First prepare the improved RPMI-1640 medium (Hyaline Company) and the corresponding 1640 complete culture medium, which contains 10% standard fetal bovine serum (Shanghai Yikesai Biological Products Co., Ltd.) and 1% double Anti (penicillin, streptomycin) (Genview). And prepare a BTX electrode cup, the electrode portion of which is 10mm long, 4mm wide, and 20mm high.

2) Human ovarian cancer cell SKOV-3 (provided by Chongqing Medical University) was grown adherently, and the modified RPMI-1640 medium was placed in a T25 cell culture flask (BeaverBio).

3) Place the T25 cell culture flask in a 37°C, 5% CO2 cell culture incubator (Thermo).

4) Use a glass pipette to suck out the modified RPMI-1640 medium in the T25 cell culture flask full of cells in the ultra-clean workbench (Suzhou Purification Equipment Co., Ltd.).

5) Add 1-2 mL of PBS buffer solution (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) to the T25 cell culture flask. The PBS buffer solution infiltrates and washes the cells, and then sucks out the cells and the PBS buffer solution.

6) Add 1mL of 0.25% trypsin to a T25 cell culture flask, that is, weigh 0.25g of trypsin (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) powder and 0.033g of EDTA (domestic analytical grade), and then add PBS buffer solution until 100 mL of trypsin solution was prepared in a T25 cell culture flask.

7) Trypsin solution was used to digest the cells (SKOV-3) in the culture flask, and the trypsin was aspirated after about 1 minute. Digestion was terminated by adding trypsin to the medium.

8) Add 5 mL of 1640 complete medium to the medium to prepare a cell suspension, thereby diluting the cells to 5×10 ⁵ cells/mL.

9) During the experiment, the pulse signal was added to the electrode cup, and 100 μL of cell suspension was added to the electrode cup for each experiment to perform corresponding electrical stimulation.

10) Research the different parameters of the cooperative pulse respectively, the specific parameters are shown in Table 1. Untreated cell suspension and blank group served as controls. Each experiment was repeated three times.

Table 1

11) After the experiment, the survival rate of the cells was detected by the CCK-8 method, that is, the cells after the experimental treatment were added to a 96-well plate, and placed in an incubator for 24 hours for CCK-8 measurement. Among them, 5 replicate holes were set for each set of parameters.

12) The medium was removed, and the cells were washed with PBS, and 20 μL of CCK-8 (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) reagent was added to each well of the 96-well plate. Shake the serum-free medium well and incubate at 37°C for 2-4 hours in the dark.

13) Carefully suck out the medium, add dimethyl sulfoxide (DMSO, Beijing Dingguo Changsheng Biotechnology Co., Ltd.) Ward Biomedical Instruments Inc.) for 20 minutes.

14) Measure the light absorption value of each group of parameter wells on an enzyme-linked immunoassay detector (BIO-RAD) with a wavelength of 450 nm. Record the results and calculate the killing rate of the cells. The experimental data were expressed as mean ± standard deviation (x ± s), and were analyzed using GraphPadPrism 5 software, and compared by one-way analysis of variance.

Through this embodiment, the following results can be drawn:

Referring to Figure 4, when high voltage and narrow pulse are applied alone, the cell survival rate is 62.4%, and when low voltage and wide pulse width are applied alone, the cell survival rate is 68.8%.

However, the cell viability was only 19.0% when synergistic pulses were applied (first high voltage, narrow pulse, then low voltage, wide pulse). Compared with the high voltage, narrow pulse and low voltage, wide pulse when the synergistic pulse is applied separately, the cell killing rate has a significant difference, and the survival rate is 3.28 times that of the high voltage and narrow pulse, and 3.62 times that of the low voltage and wide pulse. times.

Therefore, the results show that the synergistic pulse can increase the killing rate of cells, which indirectly proves the correctness of the method of the present invention, that is, the high voltage and narrow pulse produce a large perforation area on the cell membrane, while the subsequent low voltage and wide pulse have no threshold and field strength Therefore, the synergistic pulse can generate large-sized micropores in the existing cell membrane perforation area and then greatly damage the cell membrane, resulting in cell death.

And it can be found from the figure that if the low voltage and wide pulse are applied first and then the high voltage and narrow pulse are applied, the cell survival rate is 56.1%. The rate decreased slightly, but there was no significant difference. This suggests that the order of application can also affect its killing effect.

On the other hand, the application interval of high voltage, narrow pulse and low voltage wide pulse will also affect the survival rate of cancer cells. When the application interval of high voltage, narrow pulse and low voltage wide pulse is extended to 100s, the degree of cell damage caused by the synergistic pulse is more serious, and the survival rate of the cells is only 7.9%. Significant difference, which shows that increasing the interval time can further improve the inhibition rate of cells.

Embodiment 7:

The steps to study the law of synergistic pulse, high-voltage narrow pulse and low-voltage wide pulse and tissue ablation effect by using the cooperative pulse irreversible electroporation device based on FPGA control are as follows:

1) Prepare 8 New Zealand white rabbits (female, 6 months old, body weight 2.5kg±0.2kg), which are provided by Animal Experiment Center of Chongqing Medical University. And 8 New Zealand white rabbits were raised in a clean constant temperature animal breeding laboratory. The experiment in this case strictly complied with the relevant regulations in the "Regulations on the Administration of Experimental Animals" of the People's Republic of China.

2) 10 minutes before the pulse treatment, New Zealand white rabbits were anesthetized by injecting 3% pentobarbital sodium solution into the marginal ear vein (1 mL/kg). The anesthesia time is about 60 minutes or more, which is enough for the experiment. The experiment was carried out through surgical laparotomy. During the experiment, the rabbit was fixed on the operating table in a lying position, and a 50mm opening was opened in the upper part of the abdominal cavity so that the electrode needle could be directly inserted into the liver tissue. The experimental scene is shown in Figure 5 .

3) Spacer is used for the electrode needles, and the distance between them is fixed at 5 mm. The electrode needles are fixed with a bracket and located directly above the abdominal cavity in the figure, and synergistic pulse electric fields with different parameters are applied to the electrode needles. The specific applied pulse parameters are shown in Table 2.

Table 2

4) After the pulse treatment, the abdominal wound of the New Zealand white rabbit was sutured with medical suture. And the sutured New Zealand white rabbits were raised in a sterile animal experiment room for three days.

5) After being raised in the animal experiment room for 3 days, the New Zealand white rabbits were anesthetized with 3% sodium pentobarbital solution, and the vital signs of the rabbits were monitored in real time before euthanasia. After euthanasia, the rabbit liver tissue was removed by laparotomy. After sampling, the samples were soaked in 10% formalin solution for 24 hours, then embedded in paraffin for fixation, cut and stained by H&E to make tissue sections.

6) Aperio LV1 () numerical case slide scanner is used to scan the slices, so as to obtain color scanned images of the tissue slices.

The experimental results are shown in Figure 6. Taking 20 high voltage, narrow pulse parameters, 1600V, 2μs pulses and 60 low voltage, narrow pulse parameters, 360V, 100μs pulses as examples, when high voltage and narrow pulses are applied alone, the rabbit liver The tissue ablation area is 21.7mm2; when low voltage and wide pulse width are applied alone, the ablation area of rabbit liver tissue is 23.8mm2.

However, the ablation area of rabbit liver tissue was 50.7mm2 when applying synergistic pulses (high voltage, narrow pulse first, then low voltage, wide pulse). Moreover, there are significant differences in the ablation area between the high voltage and narrow pulse and the low voltage and wide pulse of the synergistic pulse when applied separately. Compared with high voltage and narrow pulse, the tissue ablation area increased by 133.6%, while compared with high voltage and narrow pulse, the tissue ablation area increased by 113.0%.

Therefore, the results show that the synergistic pulse can effectively increase the ablation area of the tissue, which indirectly proves the correctness of the method of the present invention, that is: high voltage, narrow pulse produces a larger perforation area on biological tissue, and then the threshold field of low voltage, wide pulse The intensity is reduced, so the ablation area of liver tissue can be further expanded.

On the other hand, in this case, it is found that the coordinated pulse can increase the ablation area by adjusting the voltage of low voltage and wide pulse, that is, the ablation area becomes larger and larger as the voltage of low voltage and wide pulse increases. When the voltage of low voltage and wide pulse is applied to 480V, the ablation area reaches 86.0mm2; when low voltage and wide pulse are applied alone, the ablation area is only 59.8mm2; The ablation area increased by 43.8% when applied.

Embodiment 8:

The H&E staining image is shown in Figure 7. After scanning the liver tissue with H&E staining, the boundary between the ablation area of the liver tissue and the normal tissue can be observed more clearly and accurately. As shown in Figure 5, taking coordinated pulses (20 high voltage, narrow pulse parameters, 1600V, 2μs pulses and low voltage, 60 narrow pulse parameters, 480V, 100μs pulses) as an example, in the actual liver tissue due to the hepatic lobule, The composition of blood vessels and bile ducts, the heterogeneity of their structure leads to the anisotropy of their electrical parameters, so the actual electric field distribution is not a standard dumbbell shape or ellipse. Figure 7 shows that the ablation boundary is very clear, reaching the μm level The ablation borders of the bile duct and blood vessels were completely ablated, and no residual liver cells appeared.

Claims (5)

1. The coordinated pulse irreversible electroporation device is characterized by mainly comprising a power supply system (1), a coordinated pulse forming system (2), a pulse measuring system (3), a control system (4) and a signal conversion system (5);

the power supply system (1) supplies power to the cooperative pulse forming system (2), the pulse measuring system (3), the control system (4) and the signal conversion system (5);

the input end of the collaborative pulse forming system (2) comprises an input terminal A1, an input terminal A2, an input terminal B1 and an input terminal B2;

the output end of the cooperative pulse forming system (2) comprises an output terminal E1 and an output terminal E2;

the power supply system (1) is connected between the input terminal A1 and the input terminal A2;

the input terminal A1 is connected with the input terminal A2 after being sequentially connected with a charging resistor R1 and a capacitor C1 in series;

the input terminal A1 is sequentially connected with a resistor R1 and an inductor L1 in series and then connected with a D pole of a semiconductor switch MOSFET/IGBT S1 in series;

the S pole of the semiconductor switch MOSFET/IGBT S1 is connected with the anode of the diode D1 in series; the negative electrode of the diode D1 is connected with a load in series and then is connected with the input terminal A2; the negative electrode of the diode D1 is connected with a load in series and then is connected with the input terminal B2;

the power supply system (1) is connected between the input terminal B1 and the input terminal B2;

the input terminal B1 is connected with the input terminal B2 after being sequentially connected with a resistor R2 and a capacitor C2 in series;

the input terminal B1 is connected with the D pole of a semiconductor switch MOSFET/IGBT S2 after being sequentially connected with an inductor L2 and a resistor R2 in series;

the S pole of the semiconductor switch MOSFET/IGBT S2 is connected with the anode of the diode D2 in series; the negative electrode of the diode D2 is connected with a load in series and then is connected with the input terminal A2; the negative electrode of the diode D2 is connected with a load in series and then is connected with the input terminal B2;

the load is connected in series between the output terminal E1 and the output terminal E2;

the pulse measurement system (3) mainly comprises a voltage divider (31), a current sensor (32) and a processing circuit (33);

the voltage divider (31) measures the voltage at the output of the co-pulse forming system (2);

the current sensor (32) measures the current at the output of the co-pulse forming system (2);

-the processing circuit (33) receives the voltage signal measured by the voltage divider (31); -the processing circuit (33) receives the current signal measured by the current sensor (32);

the control system (4) mainly comprises an FPGA module (41), a switch control module (42) and the single chip microcomputer module (43);

the FPGA module (41) receives a voltage signal and a current signal at the output end of the processing circuit (33); the voltage signal and the current signal are subjected to operation processing and then exchange data with the singlechip module (43);

the singlechip module (43) controls the cooperative pulse irreversible electroporation device through the switch control module (42);

characteristic parameters of the pulse can be set in the control system (4); the control system (4) converts the set parameters into electric signals through an algorithm; the control system (4) monitors the voltage signal and the current signal in the signal conversion system (5) in real time to ensure the accuracy of the output pulse parameters;

the signal conversion system (5) mainly comprises the optical/electrical converter K1, the optical/electrical converter K2, the electrical/optical converter J1 and the electrical/optical converter J2;

the electric signals are respectively transmitted to the power supply system (1), the cooperative pulse forming system (2) and the pulse measuring system (3) through the signal conversion system (5);

the electric/optical converter J1 converts the electric signal received by the FPGA module (41) into an optical signal;

the optical/electrical converter K1 converts an optical signal of the electrical/optical converter J1 into an electrical signal; the optical/electrical converter K1 transmits the converted electrical signal to the power supply system (1);

the electric/optical converter J2 converts the electric signal received by the FPGA module (41) into an optical signal;

the optical/electrical converter K2 converts the optical signal of the electrical/optical converter J2 into an electrical signal; the optical/electrical converter K2 transmits the converted electrical signal to the cooperative pulse forming system (2).

2. A collaborative pulsed irreversible puncturing device according to claim 1, wherein: the power supply system (1) mainly comprises a power supply (11), a power supply filtering device (12), a high-voltage direct current module (13) and a switch power supply module (14);

the power supply (11) is 220V alternating current;

the switching power supply module (14) converts 220V alternating current into 12V direct current;

the power supply filtering device (12) filters the signal of the switching power supply module (14) to obtain a power supply signal with specific frequency;

the grounding end of the power supply filtering device (12) is directly grounded; the power supply filtering device (12) provides the obtained power supply signal to the high-voltage direct current module (13);

the high-voltage direct current module (13) supplies power to the input end of the collaborative pulse forming system (2).

3. The co-pulsing irreversible electroporation device according to claim 1, wherein: the system also comprises a PC (6); the power supply system (1) supplies power to the PC (6); and the PC (6) monitors the voltage signal and the current signal received by the control system (4) in real time.

4. A collaborative pulsed irreversible puncturing device according to claim 1, wherein:

the high-voltage pulse amplitude of the cooperative pulse is continuously adjustable between 0 and 3 kV; the interval time of the high-voltage pulse is between 1 and 200 s; the low-voltage pulse amplitude of the cooperative pulse is continuously adjustable between 0 and 3 kV; the interval time of the low-voltage pulse is between 1 and 200 s;

the sub-pulse width is continuously adjustable between 0.2 and 100 mu s; the pulse period is adjustable between 0.1 and 10 s; the rise time of the cooperative pulse is 30ns; the falling time of the co-pulse is 30ns.

5. A collaborative pulsed irreversible puncturing device according to claim 1, wherein: the capacitance of the capacitor C1 and the capacitance of the capacitor C2 are determined by the total pulse width, the amplitude of the output pulse voltage, the allowable drop value of the output pulse voltage, the load resistance value and the discharge time constant;

let the maximum total pulse width be tau and the output pulse voltage amplitude be V ₀ The allowable drop value of the output pulse voltage is DeltaV _d Load resistance value of R _L Then the minimum capacitance of the capacitor C1 and the capacitor C2 is calculated according to the following formula:

after the discharge of each pulse train is finished, the voltage of the capacitor C1 and the capacitor C2 is reduced by 5% at most;

the withstand voltage values of the capacitor C1 and the capacitor C2 are determined by the maximum amplitude of the required pulse.