A targeted modified highly conductive nanoparticle enhanced cell electroporation device and method

Abstract

The application discloses a kind of targeted modification high-conductivity nanoparticle enhanced cell electroporation device and method;Device includes nanosecond pulse generator and nano electrode.The method is by local amplification of electric field intensity around cell through nanoparticle, to enhance the cell electroporation effect induced by pulsed electric field, steps are as follows: 1) determine the type of target cell, obtain the target ligand of target cell;2) the surface has target cell targeting ligand nano electrode and target cell are contacted or nano electrode is extended into target cell;3) preset pulse parameter;4) high-voltage DC power supply charges storage capacitor;5) after charging, FPGA module controls the on-off of MOSFET switch group based on preset pulse parameter, to control the pulse parameter output by nano electrode;6) nano electrode utilizes pulse to carry out perforation to target cell.The application can realize more efficient cell electroporation under the action of lower pulsed electric field intensity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell electroporation, in particular to a kind of target modification high-conductive nanoparticle enhanced cell electroporation device and method. BACKGROUND

[0002] Pulse electric field induced cell electroporation as a new type of biotechnology has been widely used in the field of biological engineering. Under the action of pulse electric field, hydrophilic micropore is generated on cell membrane, so that drugs, DNA and other molecules can enter the cell interior more efficiently, which significantly improves the utilization rate of drugs, DNA and other substances. In addition, the generation of micropore destroys the integrity of the original cell membrane, which may induce cell necrosis or apoptosis, which makes the electroporation technology also has important application prospect in the field of tumor treatment. However, although the traditional electroporation method can induce cell perforation, it has low efficiency and poor electrical safety, which limits the application and promotion of electroporation technology to some extent. Therefore, it is an inevitable demand to solve the current technical bottleneck to propose a new type of more efficient and safe electroporation method. SUMMARY

[0003] The purpose of the present application is to solve the problems in the prior art, that is, to enhance the perforation efficiency of the traditional electroporation method and reduce the required electric field strength.

[0004] The technical scheme adopted to achieve the purpose of the present application is as follows: a kind of target modification high-conductive nanoparticle enhanced cell electroporation device, including nanosecond pulse generator and nano electrode;

[0005] The nanosecond pulse generator sends excitation pulse to target cell and nano electrode;

[0006] The nanosecond pulse generator includes high-voltage DC power supply, energy storage capacitor, FPGA module and MOSFET switch group;

[0007] The high-voltage DC power supply charges the energy storage capacitor;

[0008] The energy storage capacitor sends excitation pulse to nano electrode through MOSFET switch group;

[0009] The FPGA module controls the on-off of MOSFET switch group, and further controls the duration and number of excitation pulse.

[0010] The nano electrode can realize the targeting of target cell;

[0011] When the nano electrode receives the excitation pulse, the excitation pulse electric field intensity is enhanced, and the enhanced excitation pulse signal is sent to the target cell, and the target cell electroporation effect is enhanced; The pulse is square wave pulse.

[0012] The nanoelectrode is several high-conductivity nanoparticles; each high-conductivity nanoparticle has a targeting ligand of the target cell on the surface.

[0013] The high-conductivity nanoparticle is a gold nanorod.

[0014] A method for enhancing cell electroporation by using a high-conductivity nanoparticle with targeted modification, comprising the following steps:

[0015] 1) Determine the type of target cell, and obtain the targeting ligand of the target cell;

[0016] 2) Establish a high-conductivity nanoparticle with targeted modification to enhance cell electroporation;

[0017] 3) Contact the nanoelectrode with the target cell or extend the nanoelectrode into the target cell, wherein the nanoelectrode has the targeting ligand of the target cell on the surface;

[0018] 4) Pre-set pulse parameters;

[0019] 5) Charge the storage capacitor by using a high-voltage DC power supply;

[0020] 6) After charging, the FPGA module controls the on-off of the MOSFET switch group based on the pre-set pulse parameters to realize the output of the pulse;

[0021] 7) The pulse output by the generator flows through the nanoelectrode to realize pulse enhancement; the enhanced pulse is released through the tip of each high-conductivity nanoparticle in the nanoelectrode to realize the perforation of the target cell.

[0022] The electric field intensity E of the tip of the high-conductivity nanoparticle tip As shown below:

[0023]

[0024] In the formula, E tip , E0 are the electric field intensity of the tip of the high-conductivity nanoparticle and the intensity of the applied uniform electric field, respectively; L and D are the length and outer diameter of the high-conductivity nanoparticle, respectively; and a is a constant.

[0025] It is worth noting that the addition of high-conductivity nanoparticles can reduce the resistivity of the environment around the cell, thereby increasing the pulse voltage that the cell can withstand and improving the utilization rate of the pulse voltage. The electrically conductive nanoparticles with a certain length-diameter ratio can cause distortion of the electric field near the tip, and therefore, if the high-conductivity nanoparticles are modified with a targeting ligand to achieve specific binding to the target cell, the electric field intensity near the cell membrane can be effectively enhanced, thereby further enhancing the electroporation effect of the cell.

[0026] Firstly, the present application needs to select a specific targeting ligand for target cells, and then modify the targeting ligand on the surface of the high-conductivity nanoparticle, so that the modified nanoparticle has the function of target recognition of target cells. When the nanoparticle is combined with the cell, a pulse electric field with corresponding parameters is applied to induce cell electroporation.

[0027] The technical effect of the present application is self-evident. The present application can reach the transmembrane voltage threshold required for electroporation in a shorter time, reduce the time for electroporation formation, prolong the time for the development of the pulse effect, and further cause stronger cell electroporation effect. The gold nanorod after targeted modification can more efficiently play its "lightning rod effect" and high conductivity characteristics, improve the electric field strength of the cell membrane, reduce the charging time constant of the cell membrane, and effectively enhance the cell electroporation effect. The high-conductivity nanoparticle can enhance the electric field strength near its tip, effectively improve the electric field strength near the cell membrane, and thus improve the cell electroporation effect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Schematic diagram of targeted high-conductivity nanoparticle combined with pulse electric field to enhance cell electroporation effect;

[0029] Figure 2 Schematic diagram of experimental platform device for nsPEFs treatment of cells;

[0030] Figure 3 BTX shock cup;

[0031] Figure 4 BTX shock cup base;

[0032] Figure 5 Schematic diagram of nanosecond pulse generator structure;

[0033] Figure 6 (a) is the dark field imaging result of gold nanorod and A375 cells in the control group;

[0034] Figure 6 (b) is the dark field imaging result of gold nanorod and A375 cells in the GNR-PEG group;

[0035] Figure 6 (c) is the dark field imaging result of gold nanorod and A375 cells in the GNR-PEG-FA group;

[0036] Figure 7 Effect of different electric field strengths on the proportion of PI positive cells;

[0037] Figure 8 Effect of different pulse widths on the proportion of PI positive cells;

[0038] Figure 9 The effect of different pulse numbers on the proportion of PI positive cells;

[0039] Figure 10 The schematic diagram of five-layer cell dielectric model;

[0040] Figure 11 The simulation geometric model of gold nanorod and spherical single cell;

[0041] Figure 12 The single pulse square wave form diagram;

[0042] Figure 13 The periodic pulse square wave form diagram (when the pulse number is changed);

[0043] Figure 14 The spatial electric field intensity distribution diagram near the cell membrane of the nsPEFs single action group;

[0044] Figure 15 The spatial electric field intensity distribution diagram near the cell membrane of the GNR-PEG group;

[0045] Figure 16 The spatial electric field intensity distribution diagram near the cell membrane of the GNR-PEG-FA group;

[0046] Figure 17 The distribution diagram of the electric field intensity on the cell outer membrane;

[0047] Figure 18 The outer membrane pore density distribution diagram;

[0048] Figure 19 The inner membrane pore density distribution diagram;

[0049] Figure 20 The outer membrane pore radius distribution diagram;

[0050] Figure 21 The inner membrane pore radius distribution diagram;

[0051] In the figure: electric shock cup 1, cell suspension 2. DETAILED DESCRIPTION

[0052] The present application will be further described below in conjunction with examples, but should not be understood as limiting the above-mentioned subject matter of the present application to the following examples. According to ordinary technical knowledge and conventional means in the art, various substitutions and modifications can be made without departing from the above-mentioned technical idea of the present application, and all of them should be included in the protection scope of the present application.

[0053] Example 1:

[0054] Reference Figures 1 to 2A high-conductivity nanoparticle enhanced cell electroporation device with targeted modification, comprising a nanosecond pulse generator and a nano electrode;

[0055] The nanosecond pulse generator and the nano electrode signal line are connected;

[0056] The nanosecond pulse generator sends excitation pulses to the nano electrode and the target cell;

[0057] The pulse generator releases pulses to the mixture of cells and nano electrodes. The nano electrode is attached near the cell membrane due to the targeted modification. According to the principle of formula (1) below, the nano electrode will amplify the electric field intensity around it, so the electric field experienced by the cell is also enhanced, so the cell electroporation effect is improved.

[0058] The nanosecond pulse generator comprises a high-voltage DC power supply, an energy storage capacitor, an FPGA module and a MOSFET switch group;

[0059] The high-voltage DC power supply charges the energy storage capacitor;

[0060] The energy storage capacitor sends excitation pulses to the nano electrode through the MOSFET switch group;

[0061] The FPGA module controls the on-off of the MOSFET switch group, and further controls the duration and number of excitation pulses.

[0062] The nano electrode is in contact with or extends into the target cell;

[0063] When the nano electrode receives the excitation pulse, it enhances the excitation pulse electric field intensity and sends the enhanced excitation pulse signal to the target cell, enhancing the target cell electroporation effect; the nano electrode acts as an auxiliary and amplifies the target cell electroporation effect.

[0064] When the nano electrode around the cell receives the excitation pulse, it amplifies the received pulse electric field intensity and then sends the enhanced pulse to the target cell,

[0065] The pulse is a square wave pulse. The pulse width of the pulse ranges from 1 ns to 1 ms, the electric field intensity ranges from 100 V / cm to 100 kV / cm, and the pulse frequency is not limited.

[0066] The nano electrode is a plurality of high-conductivity nanoparticles; each high-conductivity nanoparticle has a target cell targeting ligand on its surface.

[0067] The high-conductivity nanoparticles are gold nanorods.

[0068] After the target cell receives the excitation pulse and the enhanced excitation pulse signal, it is perforated.

[0069] Example 2

[0070] A method for enhancing a cell electroporation device using a targeted modification of high-conductivity nanoparticles, comprising the following steps:

[0071] 1) Determine the type of target cells, and obtain the target ligand of the target cells;

[0072] 2) Establish a targeted modification of high-conductivity nanoparticle-enhanced cell electroporation device;

[0073] 3) Contact the nanoelectrode with the target cell ligand on the surface with the target cell or extend the nanoelectrode into the target cell;

[0074] 4) Pre-set pulse parameters;

[0075] 5) High-voltage DC power supply charges the energy storage capacitor;

[0076] 6) After charging, the FPGA module controls the on-off of the MOSFET switch group based on the pre-set pulse parameters to realize the output of the pulse;

[0077] 7) The pulse stream output by the generator flows through the nanoelectrode to realize pulse enhancement; the enhanced pulse is released through the tip of each high-conductivity nanoparticle in the nanoelectrode to realize the perforation of the target cell. The nanoelectrode enhances the pulse electric field intensity around the cell and perforates the target cell.

[0078] The electric field intensity E of the tip of the high-conductivity nanoparticle tip As shown below:

[0079]

[0080] In the formula, E tip , E0are the electric field intensity of the tip of the high-conductivity nanoparticle and the applied uniform electric field intensity, respectively; L, D are the length and outer diameter of the high-conductivity nanoparticle, respectively; and a is a constant.

[0081] Example 3

[0082] An experiment of applying a targeted modification of high-conductivity nanoparticles to enhance a cell electroporation device, comprising the following steps:

[0083] 1) Cell culture

[0084] Human A375 melanoma cell line is obtained from the Department of Basic Medical Research, Third Military Medical University, which is a common human tumor cell with high malignancy, usually occurring on the surface of the skin, easy to observe and handle, providing convenience for subsequent in vivo experimental research of targeted gold nanorod combined with nanosecond pulse electric field.

[0085] 1.1) Cell recovery

[0086] A375 cells stored in -80 °C refrigerator or liquid nitrogen were taken out and placed in a 37 °C water bath for gentle shaking until the cell cryopreservation solution was completely melted, then it was quickly transferred to a centrifuge tube containing high glucose modified Dulbecco's Eagle medium (DMEM) in advance, and then centrifuged at 800 rpm, the supernatant was removed and the cells were resuspended, finally added to a T25 culture flask containing 5 mL of fresh DMEM medium, and cultured in an incubator (5% CO2, 37 °C). The DMEM medium used for cell culture was added with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA)

[0087] 1.2) Cell passage

[0088] When the A375 cells reached more than 80% confluence, the medium in the culture flask was aspirated, and 1 mL of phosphate buffered saline (PBS) was added for gentle washing twice. After washing, the PBS in the medium was aspirated, 1 mL of 0.25% trypsin (25200056, Gibco) was added and placed in the incubator for 1 minute, then 1 mL of medium was added to stop the digestion. The digested cells were transferred to a centrifuge tube and centrifuged (800 rpm), the supernatant was removed, and finally the cells were evenly distributed into 2-3 T25 culture flasks for further incubation.

[0089] 1.3) Preparation of cell suspension

[0090] When the cells reached 80% confluence, the cells were digested and centrifuged (same as the cell passage step), a certain amount of DMEM medium was added, then counted by a hemocytometer, and finally the cell concentration was determined at 1 × 106 / mL, placed in a centrifuge tube and waited for pulse treatment.

[0091] 1.4) Cell cryopreservation

[0092] Dimethyl sulfoxide (DMSO) and fetal bovine serum were mixed at a volume ratio of 9:1 to prepare a cryopreservation solution. When the cells reached more than 80% confluence, the cells were digested and centrifuged, the supernatant was removed, then the prepared cryopreservation solution was added, placed in a cryopreservation box and stored in a -80 °C refrigerator overnight, finally the cryopreservation tube was taken out and placed in a liquid nitrogen container for storage.

[0093] 2) Construction of experimental platform

[0094] The schematic diagram of the experimental platform device constructed in this example is as followsFigure 2 As shown, the pre-prepared A375 cell suspension was placed into electroporation cup 1 (flat electrode, 2mm spacing, BTX), and the output of a self-made nanosecond pulse generator was connected to both ends of the electroporation cup. Simultaneously, a high-voltage probe (PPE 5KV, Teledyne Lecroy) was connected to both ends of the electroporation cup, and the wires in the discharge circuit were passed through a Pearson coil (2877, Pearson Electronics). Finally, the voltage and current waveforms at both ends of the electroporation cup were acquired using an oscilloscope. Electroporation cup 1 and its base are shown in the diagram. Figure 3 and Figure 4 As shown.

[0095] The schematic diagram of the pulse generator device used in this embodiment is shown below. Figure 5 As shown, programming is performed via a PC, and the program is burned into a Field-Programmable Gate Array (FPGA) module (AX301, ALINX). The FPGA's signal output is transmitted via optical fiber to a MOSFET switch (IXRFD630, IXYS) to control the MOSFET's on / off state, thereby controlling the output pulse parameters. The nanosecond pulse generator itself uses a traditional RC charging and discharging circuit structure. A high-voltage DC power supply (DW-P302-35F5D, Dongwen High Voltage Power Company) charges a capacitor (R75QR41004000J, KEMET), and the FPGA's output signal controls the MOSFET switch's operation, thus controlling the duration and number of pulses across the load.

[0096] 3) The effect of GNR-PEG-FA targeting and binding to A375 cells

[0097] 3.1) Dark-field imaging method

[0098] Firstly, A375 cells were digested from culture flask with 0.25% trypsin, and then the cells were re-plated on 6-well plates with 24 mm coverslips in advance, and 3 mL medium was added to continue incubation in the incubator for 24 hours. After 24 hours, the medium in the 6-well plate was aspirated, and the cells were gently washed with PBS to remove impurities and dead cells, and then the cells were divided into three groups: GNR-PEG-FA group (adding targeted modified gold nanorods), GNR-PEG group (adding non-targeted modified gold nanorods) and control group (without adding gold nanorods), wherein the concentration of GNR-PEG-FA and GNR-PEG was 0.1 mg / mL (this concentration is the safe concentration determined in step 3.5). The three groups of cells were placed in the incubator for continued incubation for 15 minutes, and then gently washed with PBS for three times to remove the gold nanorods not combined with the cells, and finally fixed with paraformaldehyde, glycerol coated, and sealed with another coverslip. Finally, the prepared sample was observed under a BX51 dark field microscope (Olympus, Japan) equipped with a dark field condenser (U-DCW, 1.2-1.4). The scattered light from the gold nanorods and the cells reached the DP72 single-chip color charge-coupled device camera (Olympus, Japan) through the 100X objective lens, and then the dark field image was obtained through the camera, thereby realizing the observation of the position of the gold nanorods.

[0099] 3.2) Dark field imaging results

[0100] The dark field imaging results of the GNR-PEG-FA group and the GNR-PEG group are shown in Figure 6 (a), Figure 6 (b), Figure 6 (c), wherein the gold nanorods used in this embodiment appear yellow under dark field imaging. By comparing the dark field imaging results of the three groups, it can be found that the targeted modified GNR-PEG-FA has stronger binding effect on A375 cells, and a large number of them are distributed around the cell membrane. In addition, there is also a part of gold nanorods inside the cell, which is caused by the folate receptor-mediated endocytosis on the cell membrane surface. The GNR-PEG group lacks a targeting ligand, so it can only combine with the cells through electrostatic adsorption or endocytosis, and the binding efficiency is low and unstable. Therefore, the dark field imaging results directly prove that GNR-PEG-FA can more efficiently bind A375 cells, which lays a foundation for the subsequent pulsed electric field experimental research.

[0101] 4) Nanosecond pulsed electric field treatment scheme

[0102] The pulse parameters used in the cell suspension experiment of this step are shown in Table 2.1. Since the effect of pulse frequency change on cells is more complex, and the change in frequency mainly affects the number of pulses in nature, the pulse frequency is fixed at 1 Hz in this experiment. Then, five different levels of parameter values are set for the three variables of electric field strength (E), pulse width (τ) and pulse number (N). These parameter values are determined through preliminary experiments, and have weak to strong effects on cells. In this embodiment, an intermediate value of the parameters is first set, i.e. E is 5 kV / cm, τ is 300 ns, and N is 100. When E is changed, τ is fixed at 300 ns and N is 100. When τ is changed, E is fixed at 5 kV / cm and N is 100. When N is changed, E is fixed at 5 kV / cm and τ is 300 ns.

[0103] Table 1 Nanosecond pulse electric field experiment parameter table

[0104]

[0105] The cell suspension 2 was obtained by the method of step 2, and then the cell suspension was divided into three groups: GNR-PEG-FA group, GNR-PEG group and nsPEFs alone group, wherein the concentration of gold nanorods in the GNR-PEG-FA group and the GNR-PEG group is a safe concentration, and each has a corresponding control group without pulse electric field treatment. Before pulse electric field treatment, about 80 μL of cell suspension was added to a 2 mm BTX electric shock cup, and after waiting for about 15 minutes, the electric shock cup was inserted into the electric shock cup base, and then the pulse electric field with specific parameters was applied for treatment. After the pulse electric field treatment, the cell suspension in the electric shock cup was sucked out, then centrifuged, the supernatant was removed and resuspended, and then plated in a 96-well plate at a cell concentration of 1 × 105 / mL. After 8 hours of incubation in the incubator, cck-8 reagent was added, and after 1.5 hours of incubation, the absorbance was detected by an enzyme marker.

[0106] 5) PI staining method for detecting cell membrane permeability

[0107] 5.1) PI reagent staining method

[0108] In this step, the cells were digested by trypsin without EDTA. After the pulse electric field treatment, the cells were reseeded in 6-well plates and incubated in the incubator for 3 hours. After 3 hours, the cells were digested from the 6-well plates by trypsin without EDTA, centrifuged 3 times to remove the culture medium in the cell solution, added PBS buffer and finally made up to 200 μL. Add 200 μL of mixed solution of PBS and propidium iodide (PI) (20: 1) in the dark environment and continue to incubate for 10 minutes, and finally detect by flow cytometry (ACCURI-C6-T100, BD) under light-proof conditions.

[0109] PI is a macromolecular nucleic acid dye. When the cell membrane has an intact form, PI cannot penetrate the cell membrane into the cell and bind to the nucleus. When the cell membrane is perforated by the pulse electric field, PI molecules can penetrate the cell membrane through the micropores on the membrane, thereby entering the cell and binding to the nucleus. Therefore, the use of PI staining method can accurately reflect the change of membrane permeability after cell electroporation, and finally the proportion of PI positive cells is used to qualitatively characterize the strength of electroporation effect.

[0110] 5.2) Effect of pulse parameter change on the proportion of PI positive cells

[0111] Figures 7 to 9 The percentage of PI positive cells under different pulse parameters is shown in the bar chart. As shown in Figure 7 The control group did not apply pulse electric field, and the concentration of GNR-PEG and GNR-PEG-FA was safe concentration, so the proportion of PI positive cells in the three groups was low and there was no significant difference (p>0.05), indicating that no perforation occurred at this time, and the cell membrane was in an intact state.

[0112] When the electric field intensity was changed, the PI positive proportion of the nsPEFs alone group increased from 3.8% at 2 kV / cm to 52.7% at 8 kV / cm, while the PI positive proportion of the GNR-PEG group increased from 4.1% to 63.9%, which was improved to a certain extent compared with the nsPEFs alone group. The PI positive proportion of the GNR-PEG-FA group increased from 4.2% to 74.5%, which had a higher PI positive proportion than the GNR-PEG group and the nsPEFs alone group. In addition, it can be found from the figure that the enhancement effect of the GNR-PEG group is unstable. At the electric field intensity of 2 kV / cm, 4 kV / cm and 5 kV / cm, the GNR-PEG group had no significant difference compared with the nsPEFs alone group (p>0.05), while the GNR-PEG-FA group had significant difference with the nsPEFs alone group except at 2 kV / cm (*p<0.05), especially when the electric field intensity increased to more than 5 kV / cm, the difference was more significant (**p<0.01). Therefore, when the electric field intensity increases to a certain extent, the addition of GNR-PEG-FA can effectively improve the PI positive proportion of cells and significantly enhance the cell electroporation effect.

[0113] When the pulse width was changed, the PI positive proportion of the nsPEFs alone group, the GNR-PEG group and the GNR-PEG-FA group increased from 4.1%, 5.2% and 4.9% at 100 ns to 51.5%, 66.3% and 77.9% at 500 ns, respectively. The GNR-PEG-FA group showed stronger A375 cell killing effect than the GNR-PEG group at any pulse width used in the experiment, and had significant difference with the nsPEFs alone group (*p<0.05).

[0114] When the pulse number was changed, the PI positive proportion of the nsPEFs alone group, the GNR-PEG group and the GNR-PEG-FA group increased from 3.9%, 4.1% and 3.8% at 15 pulses to 60.8%, 69.3% and 79.9% at 260 pulses, respectively. The GNR-PEG-FA group showed stronger A375 cell killing effect than the GNR-PEG group at any pulse number used in the experiment, and had significant difference with the nsPEFs alone group (*p<0.05).

[0115] Therefore, based on the experimental results above, it can be found that the GNR-PEG-FA group has a higher PI positive cell proportion under the same pulse parameters, which preliminarily proves that the addition of GNR-PEG-FA can significantly improve the cell electroporation effect.

[0116] This embodiment takes folate modified gold nanorods (GNR-PEG-FA), nanosecond pulsed electric fields (nsPEFs) and A375 melanoma cells as examples to verify the feasibility of this method from both experimental and simulation aspects. The experimental results show that the PI positive cell ratio of the GNR-PEG-FA group reaches up to 79.9%, which is increased by 15.3% and 31.4% compared with the non-targeted gold nanorod group (GNR-PEG) and the nsPEFs alone group of 69.3% and 60.8% respectively, effectively improving the electroporation efficiency of traditional nsPEFs, which verifies the feasibility of this method from the experimental point of view. In addition, through single cell finite element simulation, compared with the nsPEFs alone group, the addition of GNR-PEG-FA makes the field strength of the cell membrane rise by 33%, the inner and outer membrane pore density increase by 75.7% and 100% respectively, and the pore flux increase by 20%, which reveals the electrical mechanism of GNR-PEG-FA enhancing cell electroporation and provides a theoretical basis for this method.

[0117] Example 4:

[0118] A simulation experiment of a cell electroporation device enhanced by targeted modified high-conductivity nanoparticles includes the following steps:

[0119] 1) Establishment of gold nanorod and single cell simulation model

[0120] 1.1) Establishment of geometric model

[0121] As shown in the following Figure 10 , the single cell model adopts the commonly used five-layer dielectric model, which is often used to study the electroporation effect of pulse electric field on cell outer membrane and nuclear membrane, wherein the cell radius Rm is 10 μm, the cell nucleus radius Rn is 5 μm, the outer membrane thickness rm is 5 nm, and the nuclear membrane thickness rn is 40 nm. The abscissa of the subsequent experimental results is taken as the starting point (clockwise direction) of the red base point in the lower right of the figure.

[0122] Since the actual position of the gold nanorod cannot be accurately evaluated in the experiment, in the establishment of the geometric model, the GNR-PEG-FA group is closely combined with the cell and closely adheres to the cell membrane, while the GNR-PEG is scattered and distributed outside the cell membrane, so as to qualitatively distinguish the different position relationships of the targeted gold nanorod and the non-targeted gold nanorod with the cell. In addition, the size gap between the gold nanorod and the cell, the simulation space is too large, therefore, in order to ensure the accuracy of the simulation and improve the utilization rate of the simulation equipment, only the gold nanorod is added near the left circle area of the cell model, and the subsequent simulation analysis area is also located on the left half of the cell. Figure 11

[0123] The simulation geometric model is as shown in the following Figure 12 ​and Figure 13 As shown in the middle, the left and right sides are plate electrodes, the electrode spacing is 0.2mm (the BTX shock cup spacing is 2mm in the experiment), which is to prevent the gap between the space size and the simulation target size. As follows Figure 11 As shown in the middle, the left and right sides are plate electrodes, the electrode spacing is 0.2mm (the BTX shock cup spacing is 2mm in the experiment), which is to prevent the gap between the space size and the simulation target size. As follows

[0124] 1.2) Establishment of mathematical model

[0125] When the cell membrane is in the form of integrity, the outer membrane has selective permeability, which can prevent many macromolecular substances from entering the cell interior, and plays a shielding and protection role. When a certain intensity of pulsed electric field is applied to the cell, the voltage at both ends of the cell membrane (transmembrane voltage) rises rapidly due to the accumulation of electric charge. When the transmembrane voltage rises to a certain threshold, it will cause the cell membrane to be perforated, and the integrity of the cell membrane is destroyed, which may cause cell death. According to the current conservation law, the potential at any point on the cell when the cell is in the electric field can be represented by the following formula (1):

[0126]

[0127] Where ε0and ε r are the vacuum dielectric constant and the relative dielectric constant at some point on the cell, respectively, and σ is the conductivity at some point on the cell. Therefore, the size of the transmembrane potential can be obtained by calculating the difference between the potential outside the cell membrane and the potential inside the cell membrane.

[0128] In order to study the effect of GNR-PEG-FA combined with nsPEFs on the cell electroporation effect, the classical dynamic electroporation model was used for simulation in this embodiment. This model can dynamically reflect the changes of the cell membrane related to the electroporation index during the action of the pulsed electric field, and can dynamically reflect the changes of pore density, pore radius and other related parameters in time, which can more accurately reflect the development process of cell electroporation and has important significance for the mechanism research of electroporation.

[0129] The pore radius can reflect the size of the perforation after the action of the pulsed electric field, and the pore density can reflect the number of pores per unit area. Therefore, the larger the pore radius and the higher the pore density, the higher the degree of damage to the cell membrane integrity by the pulsed electric field.

[0130] Therefore, in order to intuitively study the effect of GNR-PEG-FA combined with nsPEFs on the cell electroporation effect, this step mainly analyzes and discusses the pore density and pore radius, two electroporation indicators.

[0131] First, the pore density, under the action of a certain intensity of pulsed electric field, hydrophilic pores will be generated on the cell membrane, resulting in an increase in the conductivity of the cell membrane. Based on this theory, relevant scholars have proposed the equation of pore density, as shown in the following formula (3.2):

[0132]

[0133] Where N is the pore density, U EP is the transmembrane voltage threshold, q is the pore formation coefficient, N0 is the pore density before electroporation, and a is a constant.

[0134] After determining the mathematical equation of the pore density, the development law of the pore diameter needs to be determined. The widely recognized theory is that the development of the pore radius changes with the change of the pore energy, and the pore energy is closely related to the stress on the membrane surface. When there is no pulsed electric field, the stress on the cell membrane surface is in a state of overall balance, and a certain intensity of pulsed electric field will break this balance, leading to the generation and development of hydrophilic pores. Based on this theory, relevant scholars have obtained the equation of the change of the pore diameter with time, as shown in the following formula (3):

[0135]

[0136] Where r j is the pore radius, D is the development coefficient of the pore diameter, k is the Boltzmann constant, δ eff represents the effective tension coefficient of the membrane, V m is the transmembrane voltage, and F max is the electric field force when the transmembrane voltage reaches the pore formation threshold.

[0137] According to relevant literature, other specific simulation parameter values are shown in Table 2.

[0138] Table 2 Specific parameter values in the simulation model

[0139]

[0140]

[0141] Table 2 (Continued):

[0142]

[0143] 1.3) Setting of the simulation pulse waveform and parameters

[0144] When the electric field intensity is 5 kV / cm, the pulse width is 300 ns, and the number of pulses is 100, the average proportion of PI positive cells is at the middle position in the entire parameter range. And under the action of this pulse parameter, the GNR-PEG-FA group has a significant difference compared with the nsPEFs alone group and the GNR-PEG group (*p<0.05), which is very representative. Therefore, this step selects this parameter to simulate the field intensity distribution, pore density and pore radius, which can not only avoid the particularity brought by too high or too low parameter level, but also can well compare the different electroporation effects between GNR-PEG-FA, GNR-PEG group and nsPEFs alone group.

[0145] As shown in the following Figure 12 , first of all, a square wave pulse with a pulse width of 300 ns is defined by Comsol software. Due to the limitation of the software itself, the amplitude of the square wave pulse defined at this place can only be 1 by default. For the case of changing the number of pulses, after the definition of a single square wave pulse is completed, an analytical function is defined by Comsol software, which converts the above single square wave pulse function into a periodic square wave pulse function U(t) with a frequency of 1 Hz, as shown in the following Figure 13 (To show more clearly, Figure 13 only 10 square wave pulses are shown). Subsequently, the potential is added in the current module, and the domain of the potential is selected as the plate electrode in Figure 11 , and then the potential size is set to 100 times the function U(t) with the unit of volt (V), thereby completing the adjustment of the voltage amplitude. Because in the simulation, in order to avoid the large size difference between the cell and the simulation space, the spacing of the electrode is set to 0.2 mm, so when the pulse voltage amplitude is 100 V, the uniform electric field intensity between the plates is 5 kV / cm.

[0146] 2) Mechanism analysis of the targeted gold nanorod enhancing cell electroporation effect

[0147] 2.1) Influence of targeted gold nanorod on electric field intensity on cell membrane

[0148] Although gold nanorods are only added in part of the area in simulation, the influence of gold nanorods on the overall conductivity has been distinguished from the setting of experimental parameters, so the influence of conductivity has actually been directly included in the following simulation, and the distribution of field strength is closely related to whether gold nanorods exist in the area, so this step mainly simulates and analyzes the electric field intensity.

[0149] The electric field simulation results are as follows Figures 14 to 17 as shown in the figure Figure 14 , 15 , 16 respectively represent the local electric field distribution diagrams of nsPEFs alone group, GNR-PEG group and GNR-PEG-FA group, and Figure 17 is the curve diagram of the electric field intensity on the cell membrane in the three cases. It should be particularly pointed out that Figure 17 is to take the cell membrane as a two-dimensional side, and then get the field strength on the cell membrane, which is not corresponding to the field strength near the cell membrane Figure 14 , 15 , 16, the first three figures are mainly to show the effect diagram of local field strength enhancement of gold nanorods.

[0150] From the simulation results, it can be seen that the field strength near the tip of gold nanorods is obviously improved, but in the area far away from gold nanorods, it cannot be affected by the distorted electric field of gold nanorods. The results fully show that only when gold nanorods are close enough to the cell, can it more effectively play the effect of enhancing the electric field. By calculation, the average value of the electric field intensity on the cell membrane in the area can be obtained, which is about 5.78 kV / cm in the nsPEFs alone group, about 6.01 kV / cm in the GNR-PEG group, and about 7.58 kV / cm in the GNR-PEG-FA group. The addition of GNR-PEG-FA can make the field strength on the cell membrane increase by about 33% compared with the nsPEFs alone group, and the GNR-PEG group without cell targeting combination only increases the electric field intensity on the membrane by about 4%. Therefore, the correctness and necessity of the targeted modification of gold nanorods are further verified by the electric field simulation results.

[0151] 2.2) Influence of targeted gold nanorods on cell electroporation characteristics

[0152] The previous step verifies that GNR-PEG-FA can more effectively improve the electric field intensity on the cell membrane through electric field intensity simulation. In order to further explore the influence of the addition of GNR-PEG-FA on the electroporation characteristics, this step will simulate the pore density and pore radius respectively, which lays a foundation for the comparison and analysis of pore flux and cell experiments in the following step.

[0153] Figure 18 ,19 This is a simulation result diagram of the pore density of the inner and outer cell membranes. From... Figure 18 It can be observed that the outer membrane pore density of the GNR-PEG-FA group is significantly higher than that of the GNR-PEG group and the nsPEFs-only group, with the highest pore density in the GNR-PEG-FA group reaching approximately 13 × 10⁻⁶. 16 m -2 8×10 higher than the GNR-PEG group 16 m -2 The combined effect of nsPEFs alone resulted in 7.4 × 10⁻⁶. 16 m -2 These figures represent increases of 60.4% and 75.7%, respectively. Furthermore, the curves for the GNR-PEG-FA group exhibited noticeable local oscillations at the top, primarily due to the influence of gold nanorods. Equation 2 shows a close relationship between pore density and the potential difference influenced by the electric field strength, and also... Figure 17 The electric field simulation results also show that it is the non-uniform change in the field strength in this region that causes the oscillation of the pore density in this region. The oscillation at the top of the GNR-PEG group is very small because it is at a certain distance from the cell, while the oscillation is almost invisible in the nsPEFs alone group.

[0154] contrast Figure 19 The intima pore density results show that the intima pore density of the GNR-PEG-FA group was significantly higher than that of the other two groups, with the maximum value of 7.4 × 10⁻⁶ in the GNR-PEG-FA group. 17 m -2 The GNR-PEG group and the nsPEFs-only group only had 4.7 × 10⁻⁶ cells / mL, respectively. 17 m -2 and 3.7×10 17 m -2 The results showed that the electroporation effects of nsPEFs on cells were increased by 57.4% and 100%, respectively. This demonstrates that, due to the high conductivity and distorted electric field properties of gold nanorods, they can induce higher pore densities in the inner and outer membranes of cells under the same pulse parameters, significantly enhancing the electroporation effect of nsPEFs on cells.

[0155] as follows Figure 20 , 21 The diagram shows the distribution of pore sizes in the inner and outer membranes of the cell. The results of the outer membrane pore radius analysis indicate that GNR-PEG-FA can significantly increase the pore radius in local areas, and the overall outer membrane pore radius caused by the GNR-PEG-FA group is larger than that of the other two groups in the simulation results.

[0156] A similar pattern has also appeared. Figure 21The intima pore radius of GNR-PEG-FA group was higher than that of the other two groups. Therefore, GNR-PEG-FA can induce cells to produce pores with a relatively larger radius, enhancing the electroporation effect of cells.

[0157] One of the most important factors affecting cell electroporation by pulsed electric field is the electric field strength, so the electric field strength on the cell membrane is simulated and analyzed first. Because gold nanorods have a unique rod structure, they have a "lightning rod effect", which enhances the local electric field strength at their tips, as shown in the following formula:

[0158]

[0159] In the formula, E tip , E0are the electric field strength at the tip of the gold nanorod and the applied uniform electric field strength, respectively; L, D are the length and outer diameter of the gold nanorod, respectively; and α is a constant.

[0160] In fact, in addition to the influence of field strength, the high conductivity of gold nanorods also plays an important role in enhancing the electroporation effect. Since the membrane can be equivalent to a capacitor, its charging time constant is:

[0161]

[0162] where r c is the cell diameter; C m is the membrane surface capacitance; S0is the membrane surface conductivity; σ i and σ m are the conductivities inside and outside the membrane, respectively.

[0163] As can be seen from equation (3.5), the increase in extracellular conductivity will reduce the charging time constant of the cell membrane, so under the same pulse parameters, cells containing gold nanorods in the surrounding environment can reach the transmembrane voltage threshold required for electroporation in a shorter time, reducing the time for electroporation to develop, extending the time for the development of the pulse effect, and thus causing a stronger cell electroporation effect.

[0164] In summary, the targeted modification of gold nanorods can more efficiently exert their "lightning rod effect" and high conductivity characteristics, increasing the electric field strength on the cell membrane and reducing the cell membrane charging time constant, thereby effectively enhancing the cell electroporation effect.

Claims

1. A targeted modified highly conductive nanoparticle-enhanced cell electroporation device, characterized in that, Including nanosecond pulse generators and nanoelectrodes; The nanosecond pulse generator and the nanoelectrode signal line are connected; The nanosecond pulse generator sends excitation pulses to the target cells and nanoelectrodes; The nanoelectrode is in contact with or extends into the target cell; The target cells were human A375 melanoma cells. When the nanoelectrode receives the excitation pulse, it enhances the electric field strength of the excitation pulse and sends the enhanced excitation pulse signal to the target cell to enhance the electroporation effect of the target cell. The nanoelectrode comprises several highly conductive nanoparticles; each highly conductive nanoparticle has a target ligand for the target cell on its surface; the target ligand for the target cell is folic acid. The highly conductive nanoparticles are gold nanorods; The target cell perforates after receiving the excitation pulse and the enhanced excitation pulse signal; The nanosecond pulse generator includes a high-voltage DC power supply, an energy storage capacitor, an FPGA module, and a MOSFET switch group; The high-voltage DC power supply charges the energy storage capacitor. The energy storage capacitor sends excitation pulses to the nanoelectrode through a MOSFET switching group; The FPGA module controls the switching on and off of the MOSFET switching group, thereby controlling the duration and number of excitation pulses.

2. The targeted modified highly conductive nanoparticle-enhanced cell electroporation device according to claim 1, characterized in that: The pulse is a square wave pulse.

3. A method for using a cell electroporation device enhanced with targeted modified highly conductive nanoparticles as described in any one of claims 1 to 2, characterized in that, Includes the following steps: 1) Determine the type of target cells and obtain the target ligands for those cells; 2) Establish a cell electroporation device using targeted modified highly conductive nanoparticles; 3) Contact the nanoelectrode with the target cell targeting ligand on its surface with the target cell or extend the nanoelectrode into the target cell; 4) Preset pulse parameters; 5) The high-voltage DC power supply charges the energy storage capacitor; 6) After charging is complete, the FPGA module controls the switching of the MOSFET switch group based on preset pulse parameters to achieve pulse output; 7) The pulses output by the generator flow through the nanoelectrodes, achieving pulse enhancement; The enhanced pulse is released through the tip of each highly conductive nanoparticle in the nanoelectrode, achieving perforation of the target cell.

4. The method according to claim 3, characterized in that: Electric field strength at the tip of the highly conductive nanoparticles E tip As shown below: （1） In the formula, E tip , E 0 represents the electric field strength at the tip of the highly conductive nanoparticle and the applied uniform electric field strength, respectively; L and D represent the length and outer diameter of the highly conductive nanoparticle, respectively; α is a constant.

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