CN111763620A

CN111763620A - Targeted modified high-conductivity nanoparticle enhanced cell electroporation device and method

Info

Publication number: CN111763620A
Application number: CN202010521190.7A
Authority: CN
Inventors: 米彦; 刘权; 代璐健; 吴晓; 陈嘉诚
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2020-10-13

Abstract

The invention discloses a targeted modified high-conductivity nanoparticle enhanced cell electroporation device and a method; the device comprises a nanosecond pulse generator and a nanoelectrode. The method is to enhance the electroporation effect of the cells induced by the pulse electric field by locally amplifying the electric field intensity around the cells through the nano particles, and comprises the following steps: 1) determining the type of the target cell, and acquiring a target ligand of the target cell; 2) contacting the target cell targeting ligand-bearing nano-electrode on the surface with the target cell or extending the nano-electrode into the target cell; 3) presetting pulse parameters; 4) the high-voltage direct-current power supply charges the energy storage capacitor; 5) after the charging is finished, the FPGA module controls the on-off of the MOSFET switch group based on the preset pulse parameters, so as to control the pulse parameters output by the nano electrode; 6) the nanoelectrodes perforate the target cells using pulses. The invention can realize more efficient cell electroporation under the action of lower pulse electric field intensity.

Description

Targeted modified high-conductivity nanoparticle enhanced cell electroporation device and method

Technical Field

The invention relates to the field of cell electroporation, in particular to a targeted modified high-conductivity nanoparticle enhanced cell electroporation device and a method.

Background

The pulsed electric field induced cell electroporation is a new type of biotechnology and has been widely used in the field of bioengineering. Under the action of the pulse electric field, hydrophilic micropores are generated on the cell membrane, so that molecules such as medicines, DNA and the like can enter the interior of the cell more efficiently, and the utilization rate of the substances such as the medicines, the DNA and the like is obviously improved. In addition, the generation of micropores destroys the integrity of the original cell membrane, and may induce the cells to necrose or die, which makes the electroporation technology have important application prospect in the field of tumor treatment. However, although the traditional electroporation method can induce cell perforation, the traditional electroporation method has the problems of low efficiency, poor electrical safety and the like, and the application and popularization of the electroporation technology are limited to a certain extent. Therefore, it is necessary to provide a more efficient and safe novel electroporation method to solve the technical bottleneck at present.

Disclosure of Invention

The present invention is directed to solving the problems of the prior art, namely, enhancing the piercing efficiency of the conventional electroporation method and reducing the magnitude of the electric field required to achieve piercing.

The technical scheme adopted for achieving the aim of the invention is that the targeted modified high-conductivity nanoparticle enhanced cell electroporation device comprises a nanosecond pulse generator and a nano electrode;

the nanosecond pulse generator sends excitation pulses to target cells and the nano electrodes;

the nanosecond pulse generator comprises a high-voltage direct-current power supply, an energy storage capacitor, an FPGA module and an MOSFET switch group;

the high-voltage direct-current power supply charges the energy storage capacitor;

the energy storage capacitor sends excitation pulses to the nano electrode through the MOSFET switch group;

and the FPGA module controls the on-off of the MOSFET switch group so as to control the duration and the number of the excitation pulses.

The nano electrode can realize the target of target cells;

after receiving the excitation pulse, the nano electrode enhances the electric field intensity of the excitation pulse, sends an enhanced excitation pulse signal to the target cell and enhances the electroporation effect of the target cell; the pulses are square wave pulses.

The nano-electrodes are a plurality of high-conductivity nano-particles; each highly conductive nanoparticle has a targeting ligand for the target cell on the surface.

The high-conductivity nano particles are gold nano rods.

A method of enhancing a cell electroporation device using a target-modified highly conductive nanoparticle, comprising the steps of:

1) determining the type of the target cell and obtaining a target ligand of the target cell;

2) establishing a targeted modified high-conductivity nanoparticle enhanced cell electroporation device;

3) contacting the target cell targeting ligand-bearing nano-electrode on the surface with the target cell or extending the nano-electrode into the target cell;

4) presetting pulse parameters;

5) the high-voltage direct-current power supply charges the energy storage capacitor;

6) after the charging is finished, the FPGA module controls the on-off of the MOSFET switch group based on preset pulse parameters to realize the output of pulses;

7) the pulse output by the generator flows through the nano electrode to realize pulse enhancement; the enhanced pulse is released through the tip of each highly conductive nanoparticle in the nanoelectrode, thereby realizing the perforation of the target cell.

The electric field intensity E of the tip of the high-conductivity nano particle_tipAs follows:

in the formula, E_tip、E₀The electric field intensity of the tip of the high-conductivity nano particle and the external uniform electric field intensity respectively, L, D the length and the outer diameter of the high-conductivity nano particle respectively, and α a constant.

It is worth to say that the addition of the highly conductive nanoparticles can reduce the resistivity of the surrounding environment of the cell, thereby increasing the magnitude of the pulse voltage borne by the cell and improving the utilization rate of the pulse voltage. And the conductive nanoparticles with a certain length-diameter ratio can cause the distortion of an electric field near the tip of the conductive nanoparticles, so that if the highly conductive nanoparticles are modified by a targeting ligand to realize the specific combination of target cells, the electric field strength near cell membranes can be effectively enhanced, and the electroporation effect of the cells is further enhanced.

According to the invention, a specific targeting ligand is selected for a target cell, and then the surface of the high-conductivity nanoparticle is modified by the targeting ligand, so that the modified nanoparticle has the function of identifying the target cell in a targeting manner. After the nanoparticles are combined with the cells, a pulsed electric field with corresponding parameters is applied to induce the cells to generate electroporation.

The technical result of the present invention is undoubtedly that it is possible to achieve the transmembrane voltage threshold required for electroporation to take place in a shorter time, reducing the time for electroporation to take place, and prolonging the time for the development of the electroporation during the pulsing action, which in turn leads to a stronger electroporation effect of the cells. The gold nanorods after targeted modification can more efficiently exert the characteristics of lightning rod effect and high conductivity, improve the intensity of electric field borne by cell membranes, and reduce the charging time constant of the cell membranes, thereby effectively enhancing the cell electroporation effect. The high-conductivity nano particles can enhance the electric field intensity near the tips of the nano particles, effectively improve the electric field intensity near cell membranes, and further improve the electroporation effect of cells.

Drawings

FIG. 1 is a schematic diagram of a targeted highly conductive nanoparticle in combination with a pulsed electric field to enhance the electroporation effect of cells;

FIG. 2 is a schematic diagram of an experimental platform for processing cells by nsPEFs;

FIG. 3 is a BTX shock cup;

FIG. 4 is a BTX stun cup base;

FIG. 5 is a schematic diagram of a nanosecond pulser;

FIG. 6(a) is the result of dark field imaging of control group gold nanorods and A375 cells;

FIG. 6(b) is the result of dark field imaging of GNR-PEG group gold nanorods and A375 cells;

FIG. 6(c) is the result of dark field imaging of GNR-PEG-FA group gold nanorods and A375 cells;

FIG. 7 is a graph of the effect of different electric field strengths on the proportion of PI-positive cells;

FIG. 8 is a graph of the effect of different pulse widths on the proportion of PI-positive cells;

FIG. 9 is a graph of the effect of different pulse counts on the proportion of PI-positive cells;

FIG. 10 is a schematic diagram of a five-layered cell dielectric model;

FIG. 11 is a geometric model of a gold nanorod and a spherical single cell simulation;

FIG. 12 is a diagram of a single pulse square waveform;

FIG. 13 is a diagram of a periodic pulse square waveform (when the number of pulses is changed);

FIG. 14 is the spatial electric field intensity distribution diagram near the cell membrane of the group with single action of nsPEFs;

FIG. 15 is a graph showing the distribution of the spatial electric field intensity near the cell membrane of the GNR-PEG group;

FIG. 16 is a graph showing the distribution of the spatial electric field intensity in the vicinity of the cell membrane of the GNR-PEG-FA group;

FIG. 17 is a graph showing the distribution of the electric field intensity applied to the outer membrane;

FIG. 18 is an outer membrane pore density distribution plot;

FIG. 19 is a diagram of intima pore density distribution;

FIG. 20 is a graph of the outer membrane pore radius distribution;

FIG. 21 is a diagram of the inner membrane pore radius distribution;

in the figure: an electric shocking cup 1 and a cell suspension 2.

Detailed Description

The present invention is further illustrated by the following examples, but it should not be construed that the scope of the above-described subject matter is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention and the scope of the invention is covered by the present invention according to the common technical knowledge and the conventional means in the field.

Example 1:

referring to fig. 1 to 2, a targeted modified highly conductive nanoparticle enhanced cell electroporation device includes a nanosecond pulse generator and a nanoelectrode;

the nanosecond pulse generator is connected with the nano electrode signal line;

the nanosecond pulse generator sends excitation pulses to the nanoelectrodes and the target cells;

the pulse generator releases a pulse to the mixture of cells and nanoelectrodes. The nano electrode is attached to the vicinity of a cell membrane due to targeted modification, and according to the principle of the following formula (1), the nano electrode amplifies the electric field intensity around the nano electrode, so that the electric field applied to the cell is enhanced, and the electroporation effect of the cell is improved.

The nano-electrode is in contact with or extends into a target cell;

after receiving the excitation pulse, the nano electrode enhances the electric field intensity of the excitation pulse, sends an enhanced excitation pulse signal to the target cell and enhances the electroporation effect of the target cell; the nano-electrode plays a role in amplifying the electroporation effect of the target cell as an assistant.

When the nano-electrodes around the cells receive the excitation pulse, the received pulse electric field intensity is amplified, and then the enhanced pulse is sent to the target cells,

the pulses are square wave pulses. The pulse width range of the pulse is 1ns to 1ms, the electric field intensity range is 100V/cm to 100kV/cm, and the pulse frequency is not limited.

The high-conductivity nano particles are gold nano rods.

And the target cells are perforated after receiving the excitation pulse and the enhanced excitation pulse signals.

Example 2:

4) presetting pulse parameters;

7) the pulse output by the generator flows through the nano electrode to realize pulse enhancement; the enhanced pulse is released through the tip of each highly conductive nanoparticle in the nanoelectrode, thereby realizing the perforation of the target cell. The nanoelectrodes enhance the pulsed electric field strength around the cells and perforate the target cells.

Example 3:

an experiment of using a targeting modified high-conductivity nanoparticle enhanced cell electroporation device comprises the following steps:

1) cell culture

The human A375 melanoma cell strain is obtained from the third department of basic medicine research of the military medical university, is a common human tumor cell with high malignancy degree, usually attacks on the surface of skin, is easy to observe and treat, and provides convenience for the subsequent in-vivo experimental research of the targeted gold nanorods combined with the nanosecond pulsed electric field.

1.1) cell recovery

A375 cells preserved in a-80 ℃ refrigerator or liquid nitrogen were taken out, placed in a37 ℃ water bath and gently shaken until the frozen stock solution of the cells was completely thawed, then quickly transferred to a centrifuge tube previously filled with a high-sugar modified Eagle medium (DMEM), and then centrifuged at 800 rpm, the supernatant was removed and the cells were resuspended, and finally added to a T25 flask containing 5mL of fresh DMEM medium and cultured in an incubator (5% CO2, 37 ℃). The DMEM medium used for cell culture was supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA)

1.2) cell passage

After the iron wall growth of the A375 cells reached confluence to more than 80%, the medium in the flask was aspirated and washed twice gently by adding 1mL of Phosphate Buffered Saline (PBS). After washing was complete, PBS in the medium was aspirated, 1mL of 0.25% trypsin (25200056, Gibco) was added and placed in an incubator for digestion for 1 minute, followed by addition of 1mL of medium to stop 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 to 2-3T 25 flasks for further incubation.

1.3) preparation of cell suspensions

When the cells grow and are 80% confluent, the cells are digested and centrifuged (same cell passage step), a certain amount of DMEM medium is added, counting is carried out through a blood counting chamber, finally, the cell concentration is determined to be 1 × 106 cells/mL, and the cells are placed in a centrifuge tube for pulse treatment.

1.4) cell cryopreservation

Dimethyl sulfoxide (DMSO) and fetal bovine serum were mixed at a ratio of 9: 1, and mixing to prepare the frozen liquid. And after the cells grow and converge to more than 80%, digesting and centrifuging the cells, removing supernatant, adding a prepared freezing solution in advance, filling the freezing solution into a freezing box, storing the freezing solution in a refrigerator at minus 80 ℃ overnight, taking out a freezing tube, and transferring the freezing tube into a liquid nitrogen container for storage.

2) Establishment of experimental platform

The schematic diagram of the experimental platform device constructed in this embodiment is shown in fig. 2, a375 cell suspension prepared in advance is loaded into an electric shock cup 1 (plate electrodes, interval 2mm, BTX), and then the output end of a self-made nanosecond pulse generator in a laboratory is connected to the two ends of the electric shock cup. Meanwhile, high-voltage probes (PPE5KV, Teledyne Lecroy) are connected to both ends of the electric shock cup, then a lead in a discharge loop is passed through a Pearson coil (2877, Pearson Electronics), and finally a voltage waveform and a current waveform diagram at both ends of the electric shock cup are collected through an oscilloscope. The stun cup 1 and stun cup base are shown in figures 3 and 4.

As shown in fig. 5, the schematic structural diagram of the pulse generator device used in this embodiment is that a computer PC is used for programming, a program is burned into a Field-Programmable Gate Array (FPGA) module (AX301, ALINX), and a signal output of the FPGA is transmitted to MOSFET switches (IXRFD630, IXYS) through an optical fiber to control on and off of the MOSFETs, so as to control output pulse parameters. The nanosecond pulse generator body adopts a traditional RC charging and discharging circuit structure, namely a capacitor (R75QR41004000J, KEMET) is charged through a high-voltage direct current power supply (DW-P302-35F5D, Town high-voltage power supply company), and then the action of a MOSFET switch is controlled through an output signal of an FPGA, so that the duration and the action number of pulse voltage at two ends of a load are controlled.

3) Effect of GNR-PEG-FA targeting binding to A375 cells

3.1) dark field imaging method

A375 cells were first digested from the flask with 0.25% trypsin, replated onto 6-well plates previously loaded with 24mm coverslips, and incubated in the incubator for an additional 24 hours with 3mL of media. After 24 hours, the media in the 6-well plate was aspirated, the cells were gently rinsed with PBS to remove impurities and dead cells, and then divided into three groups: GNR-PEG-FA group (with addition of targeted modified gold nanorods), GNR-PEG group (with addition of untargeted modified gold nanorods), and control group (without addition of gold nanorods), wherein the GNR-PEG-FA and GNR-PEG were both at a concentration of 0.1mg/mL (this concentration is the safe concentration determined in step 3.5). Three groups of cells were placed in an incubator for further incubation for 15 minutes, then gently washed three times with PBS to remove the gold nanorods not bound to the cells, finally fixed with paraformaldehyde, glycerol-coated, and sealed with another cover glass, and finally the prepared samples were observed under BX51 dark field microscope (Olympus, Japan) equipped with dark field condenser (U-DCW, 1.2-1.4). Scattered light from the gold nanorods and cells reached a DP72 single chip color electric coupled device camera (Olympus, Japan) through a 100X objective lens, and then a dark field image was obtained by the camera, thereby achieving positional observation of the gold nanorods.

3.2) dark field imaging results

The results of dark field imaging of the GNR-PEG-FA group and the GNR-PEG group are shown in fig. 6(a), fig. 6(b), and fig. 6(c), wherein the gold nanorods used in this example were yellow in color under dark field imaging. Comparing the results of three groups of dark field imaging, the targeting modified GNR-PEG-FA has stronger binding effect on A375 cells and is distributed around the cell membrane in a large amount. In addition, a part of gold nanorods also exist inside the cell, which is caused by folate receptor-mediated endocytosis on the cell membrane surface. The GNR-PEG group lacks targeting ligands and therefore can only bind to cells by electrostatic adsorption or endocytosis, with low and unstable binding efficiency. Therefore, the dark field imaging result intuitively proves that the GNR-PEG-FA can be combined with the A375 cells more efficiently, and lays a foundation for the development of subsequent pulsed electric field experimental study.

4) Nanosecond pulsed electric field processing scheme

The pulse parameters adopted in the suspension cell experiment in the step are shown in table 2.1, the influence mechanism of the change of the pulse frequency on the cells is complex, and the change of the frequency essentially influences the number of pulses, so that the fixed pulse frequency in the experiment is 1Hz, then 5 parameter values with different levels are respectively set for three variables of the electric field intensity (E), the pulse width (tau) and the number of pulses (N), and the parameter values are determined through the search of the early-stage pre-experiment and respectively influence the cells from weak to strong. In this embodiment, an intermediate value is first set for the parameters, i.e., E is 5kV/cm, τ is 300ns, and N is 100. When changing E, tau is fixed at 300ns, and N is 100; when τ was changed, E was fixed at 5kV/cm and N was 100. When N was changed, E was fixed at 5kV/cm and τ was 300 ns.

TABLE 1 nanosecond pulsed electric field experiment parameter table

Table 2.1Experimental parameters of nsPEFs

Cell suspension 1 was obtained by the method of step 2, and then the cell suspension was divided into three groups: the GNR-PEG-FA group, the GNR-PEG group and the nsPEFs single action group, wherein the concentrations of the gold nanorods in the GNR-PEG-FA group and the GNR-PEG group are safe concentrations, and the gold nanorods in the GNR-PEG group and the GNR-PEG group respectively have corresponding control groups without being treated by a pulsed electric field. Before the pulsed electric field treatment, about 80 μ L of the cell suspension was added to a 2mm BTX cuvette, and after waiting for about 15 minutes, the cuvette was inserted into the cuvette holder and then treated by applying a pulsed electric field of specific parameters. After the pulse electric field treatment is finished, sucking out the cell suspension in the electric shock cup, centrifuging, removing supernatant, suspending, plating the cell suspension in a 96-well plate at the cell concentration of 1 × 105/mL, adding cck-8 reagent after the incubation is continued for 8 hours in an incubator, and detecting the absorbance through a microplate reader after the incubation is continued for 1.5 hours.

5) PI staining method for detecting cell membrane permeability

5.1) PI reagent staining method

In this step, trypsin without EDTA was used to digest the cells. After pulsed electric field treatment, cells were re-seeded in 6-well plates and incubated in an incubator for 3 hours. After 3 hours the cells were digested from the 6-well plate with trypsin without ethylenediaminetetraacetic acid, centrifuged 3 times to remove the medium from the cell solution, added PBS buffer and finally brought to a volume of 200 μ L. mu.L of a mixed solution of PBS and Propidium Iodide (PI) (20: 1) was added in the dark and incubation was continued for 10 minutes, and finally detection was performed by flow cytometry (ACCURI-C6-T100, BD) under exclusion of light.

PI is a macromolecular nucleic acid dye, and when the cell membrane has an intact morphology, PI cannot penetrate through the cell membrane to enter the interior of the cell and be bound with the nucleus. When the outer cell membrane is perforated by the action of a pulsed electric field, the PI molecules can penetrate the cell membrane through micropores in the membrane, enter the interior of the cell and are combined with the cell nucleus. Therefore, the change of the membrane permeability after the cells are subjected to electroporation can be accurately reflected by using the PI staining method, and finally, the strength of the electroporation effect can be qualitatively represented by the proportion of PI positive cells.

5.2) Effect of pulse parameter variation on the proportion of PI-Positive cells

Fig. 7-9 are histograms of the percentage of PI positive cells under different pulse parameters. As shown in FIG. 7, in the control group, no pulsed electric field was applied, and the concentrations of GNR-PEG and GNR-PEG-FA were both safe, so the PI positive ratio of the three groups of cells was very low and there was no significant difference (p >0.05), indicating that no perforation occurred and the cell membrane was intact.

When the electric field intensity is changed, the PI positive proportion of the nsPEFs acting alone is increased from 3.8% of 2kV/cm to 52.7% of 8kV/cm, while the PI positive proportion of the GNR-PEG group is increased from 4.1% to 63.9%, and is improved to a certain extent compared with the nsPEFs acting alone. The PI positive proportion of the GNR-PEG-FA group is increased from 4.2% to 74.5% along with the increase of the electric field intensity, and has higher PI positive proportion than that of the GNR-PEG group and the nsPEFs acting alone. In addition, it can be seen from the figure that the enhancing effect of the GNR-PEG group is unstable, and the GNR-PEG group has no significant difference (p >0.05) compared with the nsPEFs single action group at electric field strengths of 2kV/cm, 4kV/cm and 5kV/cm, while the GNR-PEG-FA group has significant difference (p <0.05) compared with the nsPEFs single action group except for 2kV/cm, and the difference is more significant (p <0.01) particularly when the electric field strength is increased to 5kV/cm or more. Therefore, when the electric field intensity is increased to a certain degree, the addition of the GNR-PEG-FA can effectively improve the PI positive proportion of cells, and the cell electroporation effect is obviously enhanced.

When the pulse width was varied, the PI-positive ratios of the nsPEFs alone-acting group, GNR-PEG group and GNR-PEG-FA group increased from 4.1%, 5.2% and 4.9% at 100ns to 51.5%, 66.3% and 77.9% at 500ns, respectively. The GNR-PEG-FA group showed stronger a375 cell killing effect than the GNR-PEG group at the pulse width used in either experiment, and was significantly different from the nsPEFs alone (p < 0.05).

When the number of pulses was varied, 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 the number of pulses used in any experiment, and all were significantly different from the nsPEFs alone (p < 0.05).

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

In the embodiment, gold nanorods (GNR-PEG-FA) modified by folic acid, nanosecond pulsed electric fields (nsPEFs) and A375 melanoma cells are taken as examples, and research is carried out from two aspects of experiments and simulation, so that the feasibility of the method is verified together. Experimental results show that the proportion of PI positive cells of the GNR-PEG-FA group reaches 79.9% at most, and is respectively increased by 15.3% and 31.4% compared with 69.3% and 60.8% of non-targeting gold nanorod groups (GNR-PEG) and nsPEFs single action groups, the electroporation efficiency of the traditional nsPEFs is effectively improved, and the feasibility of the method is verified from the experimental point of view. In addition, single-cell finite element simulation shows that compared with the single-acting group of nsPEFs, the addition of GNR-PEG-FA increases the field strength borne by a cell membrane by 33%, the pore densities of an inner membrane and an outer membrane are respectively increased by 75.7% and 100%, and the pore flux is increased by 20%, so that an electrical mechanism of GNR-PEG-FA for enhancing cell electroporation is disclosed, and a theoretical basis is provided for the method.

Example 4:

a simulation experiment of a high-conductivity nanoparticle reinforced cell electroporation device applying targeted modification comprises the following steps:

1) establishment of gold nanorod and single cell simulation model

1.1) construction of the geometric model

As shown in FIG. 10 below, the single cell model is a conventional five-layer dielectric model, which is often used to study the electroporation effect of a pulsed electric field on the outer cell membrane and the nuclear membrane, wherein the cell radius Rm is 10 μm, the nuclear radius Rn is 5 μm, the outer membrane thickness Rm is 5nm, and the nuclear membrane thickness Rn is 40 nm. The abscissa of the subsequent experimental results takes the red base point right below the graph as a starting point (clockwise direction).

Because 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 tightly combined with the cell and is tightly attached to the periphery of the cell membrane, and the GNR-PEG is dispersedly distributed on the outer side of the cell membrane, so that different position relations between the targeted gold nanorod and the non-targeted gold nanorod and the cell are qualitatively distinguished. In addition, the size difference between the gold nanorods and the cells and the simulation space is too large, so in order to ensure the accuracy of simulation and improve the utilization rate of simulation equipment, the gold nanorods are only added near a left circle region of the cell model in fig. 11, and a subsequent simulation analysis region is also positioned on the left half side of the cells.

The simulation geometric model is shown in fig. 12 and 13, the middle is a spherical single cell model, the left and right sides are flat electrodes, and the electrode spacing is 0.2mm (in the experiment, the BTX cuvette spacing is 2mm), which is to prevent the space size and the simulation target size from being too large. The geometric models for the three cases of nsPEFs acting alone, GNR-PEG and GNR-PEG-FA are shown in the three subgraphs of FIG. 11 below. Firstly, a gold nanorod with a random angle is defined through a Matlab Monte Carlo algorithm and a random function, the size of the gold nanorod is adjusted to be 60nm in length, the outer diameter of the gold nanorod is 15nm, and the size of the gold nanorod is consistent with that of a gold nanorod used in an experiment. And repeating the method to generate a geometric model of the plurality of gold nanorods, and combining the plurality of gold nanorods with the single cell model in the Commol software through Boolean operation in the Commol software, thereby realizing the modeling of the gold nanorods and the single cell model. Since the size of folic acid molecules is much smaller than that of gold nanorods, the GNR-PEG-FA is tightly close to the cell membrane in a simulation model, and the GNR-PEG groups are randomly distributed outside the cell membrane.

1.2) creation of mathematical models

When the cell membrane is in the complete form, the outer membrane has the selective permeability, so that a plurality of macromolecular substances can be prevented from entering the cell, and the shielding and protecting effects are achieved. When a pulsed electric field of a certain intensity is applied to the cell, the voltage across the cell membrane (transmembrane voltage) rises rapidly due to the accumulation of charge. When the transmembrane voltage rises to a certain threshold, it causes the cell membrane to perforate and the integrity of the cell membrane to be destroyed, possibly causing cell death. According to the current conservation law, when a cell is placed in an electric field, the potential at any point on the cell can be represented by the following formula (1):

wherein,₀and_rrespectively the dielectric constant in vacuum and the relative dielectric constant somewhere on the cell, sigma being the electrical conductivity somewhere on the cell. Therefore, the magnitude of the transmembrane potential can be obtained by calculating the difference between the potential outside the cell membrane and inside the outer membrane of the cell.

In order to study the influence of GNR-PEG-FA in combination with nsPEFs on the cell electroporation effect, the embodiment uses a classical dynamic electroporation model for simulation, and the model can dynamically reflect the change of electroporation indexes related to intracellular and extracellular membranes during the action of the pulsed electric field, can dynamically reflect the change of relevant parameters such as pore density and pore radius in time, more accurately reflect the development process of cell electroporation, and has important significance for the mechanism study of electroporation.

The hole radius can reflect the size of the punched hole after the action of the pulse electric field, and the hole density can reflect the number of the inner holes in unit area. Thus, a larger pore radius and a higher pore density means a higher degree of disruption of the integrity of the cell membrane by the pulsed electric field.

Therefore, in order to intuitively study the effect of the combination of GNR-PEG-FA and nsPEFs on the electroporation effect of cells, the step is mainly aimed at analyzing and discussing two electroporation indexes of pore density and pore radius.

Firstly, the pore density is that under the action of a pulse electric field with certain intensity, hydrophilic pores are generated on a cell membrane, so that the conductivity of the cell membrane is increased, and based on the theory, relevant scholars provide an equation of the pore density, which is shown as the following formula (3.2):

wherein N is the pore density, U_EPFor transmembrane voltage threshold, q is the puncture formation coefficient, N₀α is a constant for the pore density size before electroporation has occurred.

After the mathematical equation of the pore density is determined, the development rule of the pore diameter needs to be determined. The widely accepted theory is that the pore radius development is believed to be a function of pore energy, which is often closely related to the force applied to the membrane surface. When no pulse electric field is applied, the stress on the surface of the cell membrane is in an equilibrium state on the whole, and the pulse electric field with certain intensity breaks the equilibrium, so that hydrophilic pores are generated and developed, and based on the theory, the related scholars obtain an equation of the pore diameter changing along with time, as shown in the following formula (3):

wherein r is_jIs the hole radius, D is the coefficient of development of the hole diameter, k is the Boltzmann constant,_effrepresents the effective tension coefficient, V, of the film_mMagnitude of transmembrane voltage, F_maxIs the magnitude of the electric field force at which the transmembrane voltage reaches the threshold at which perforation occurs.

According to the relevant literature, other specifically relevant simulation parameter values are shown in table 2 below.

TABLE 2 specific parameter values in the simulation model

Table 3.1Model parameters of simulation

Continuing with Table 2:

1.3) setting of simulation pulse waveform and parameters

When the electric field intensity is 5kV/cm, the pulse width is 300ns, and the number of pulses is 100, the level of the proportion of PI positive cells is in the middle position of the whole parameter range. Under the action of the pulse parameters, the GNR-PEG-FA group has significant difference (p is less than 0.05) compared with the nsPEFs single action group and the GNR-PEG group, and is very representative. Therefore, the parameters are selected to simulate the field intensity distribution, the pore density and the pore radius, so that not only can the particularity brought by overhigh or overlow parameter level be avoided, but also different electroporation effects among the GNR-PEG-FA, GNR-PEG group and the nsPEFs independent action group can be well contrasted.

As shown in fig. 12 below, a square wave pulse with a pulse width of 300ns is first defined by Comsol software, where the defined square wave pulse amplitude can only be 1 by default due to the software's own limitations. For the case of changing the number of pulses, it is necessary to continue to define an analytic function through Comsol software after the definition of a single square wave pulse is completed, and convert the single square wave pulse function into a periodic square wave pulse function u (t) with a frequency of 1Hz in the analytic function, as shown in fig. 13 below (for a clearer illustration, fig. 13 only shows 10 square wave pulses). Subsequently, the potential is added in the current module, the belonging domain of the potential is selected as the plate electrode in fig. 11, and then the potential is set to be 100 times of the function u (t) in volts (V), thereby completing the adjustment of the voltage amplitude. Since the spacing between the electrodes was set to 0.2mm in order to avoid an excessive size difference between the cells and the simulation space in the simulation, the uniform electric field intensity between the plates was 5kV/cm when the pulse voltage amplitude was 100V.

2) Mechanism analysis of cell electroporation effect enhanced by targeting gold nanorods

2.1) Effect of the Targeted gold nanorods on the electric field intensity applied to the cell membrane

Although the gold nanorods are only added in a partial area in the simulation, the influence of the addition of the gold nanorods on the overall conductivity is distinguished from the setting of experimental parameters, so the influence of the conductivity is actually directly included in the next simulation, and the distribution situation of the field strength is mainly related to whether the gold nanorods exist in the area, so that the simulation and analysis are mainly performed according to the magnitude of the electric field strength in the step.

The results of the electric field simulation are shown in FIGS. 14 to 17 below, in which FIGS. 14, 15 and 16 represent the local electric field distribution diagrams of the nsPEFs alone group, the GNR-PEG group and the GNR-PEG-FA group, respectively, and FIG. 17 is a graph showing the magnitude of the electric field applied to the cell membrane in three cases. It should be noted that fig. 17 is a diagram of taking a cell membrane as a two-dimensional edge, and then obtaining the magnitude of the field intensity applied to the cell membrane, and the magnitude of the field intensity does not correspond to the field intensity near the cell membrane in fig. 14, 15, and 16, and the first three diagrams are mainly used for showing the effect of locally enhancing the field intensity of the gold nanorods.

The simulation result shows that the field intensity near the tip of the gold nanorod is obviously improved, but the field intensity in the area far away from the gold nanorod cannot be influenced by the distorted electric field of the gold nanorod. The results fully show that the effect of enhancing the electric field can be more effectively exerted only when the gold nanorods are close enough to the cells. The average value of the electric field intensity applied to the cell membrane in the area can be obtained by calculation, the single action group of the nsPEFs is about 5.78kV/cm, the single action group of the GNR-PEG is about 6.01kV/cm, and the single action group of the NSPEFs is about 7.58 kV/cm. The addition of GNR-PEG-FA can increase the field intensity of the cell membrane by about 33% compared with the group acted by nsPEFs alone, while the intensity of the electric field on the cell membrane is only increased by about 4% in the GNR-PEG group without being combined with cell targeting. Therefore, the correctness and the necessity of the targeted modification of the gold nanorods are further verified through the electric field simulation result.

2.2) Effect of Targeted gold nanorods on cell electroporation Properties

In the last step, the simulation of the electric field strength proves that the GNR-PEG-FA can effectively improve the electric field strength of the cell membrane. In order to further explore the influence rule of the addition of GNR-PEG-FA on the electroporation characteristics, the step simulates the pore density and the pore radius respectively, and lays a foundation for the comparison and analysis of the subsequent pore flux and cell experiments.

FIGS. 18 and 19 are graphs showing the results of the simulation of the cell inner and outer membrane pore densities, and it can be observed from FIG. 18 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 alone, wherein the maximum pore density of the GNR-PEG-FA group reaches about 13 × 10¹⁶m^-2Higher than GNR-PEG group 8 × 10¹⁶m^-27.4 × 10 of the nsPEFs action alone group¹⁶m^-2The improvement is 60.4 percent and 75.7 percent respectively. In addition, locally significant oscillations occurred at the top of the curves for the GNR-PEG-FA group, which were mainly influenced by the gold nanorods. From equation 2, it can be known that the pore density has a close relationship with the potential difference affected by the field intensity, and it can be found from the electric field simulation result of fig. 17 that it is the non-uniform variation of the field intensity in this area, which results in the oscillation of the pore density in this area. The GNR-PEG group had a small top oscillation due to its distance from the cell, while the nsPEFs alone had little oscillation.

Comparing the membrane pore density results in FIG. 19, it can be seen that the membrane pore density in the GNR-PEG-FA group is significantly higher than that in the other two groups, wherein the maximum value of the GNR-PEG-FA group reaches 7.4 × 10¹⁷m^-2While the group GNR-PEG and nsPEFs act independentlyWith only 4.7 × 10 of each group¹⁷m^-2And 3.7 × 10¹⁷m^-2The improvement is 57.4 percent and 100 percent respectively. Therefore, the gold nanorods have the characteristics of high conductivity and distorted electric field, so that the inner and outer membranes of the cell can generate higher pore density under the same pulse parameters, and the electroporation effect of the nsPEFs on the cell is obviously improved.

FIGS. 20 and 21 show the pore size distribution of intracellular and extracellular membranes. The outer membrane pore radius results show that the GNR-PEG-FA can obviously cause the increase of the pore radius in the local area, and the outer membrane pore radius caused by the GNR-PEG-FA group is larger than the simulation results of the other two groups in the whole.

Similar rules also apply to the inner membrane pore radius experiment shown in FIG. 21, where the GNR-PEG-FA group had a higher inner membrane pore radius than the other two groups. Thus, GNR-PEG-FA was able to induce cells to generate pores with relatively larger radii, enhancing the electroporation effect of cells.

One of the most important factors of the pulsed electric field influencing the electroporation of the cell is the electric field strength, so the electric field strength borne by the cell membrane is firstly simulated and analyzed. Because the gold nanorods have a unique rod-shaped structure and have a 'lightning rod effect', a local electric field intensity enhancement effect can be generated at the tips of the gold nanorods, and the local electric field intensity enhancement effect is shown as the following formula:

in the formula, E_tip、E₀The electric field intensity of the tip of the gold nanorod and the external uniform electric field intensity are respectively shown, L, D is the length and the outer diameter of the gold nanorod respectively, and α is a constant.

In fact, in addition to the influencing factors of the field strength, the high conductivity property of the gold nanorods is also of great significance for the enhancement of the electroporation effect. Since the film can be equivalent to a capacitor, its charge time constant is:

wherein r is_cCell diameter; c_mIs the film surface capacitance; s₀Is the membrane surface conductivity; sigma_iAnd σ_mThe electrical conductivity inside the membrane and outside the membrane, respectively.

From the formula (3.5), the increase of the conductivity outside the cell can reduce the charging time constant of the cell membrane, so that under the action of the same pulse parameter, the cell containing the gold nanorods in the surrounding environment can reach the transmembrane voltage threshold required by electroporation in a shorter time, the time for electroporation formation is reduced, the time for the development of the electroporation in the pulse action period is prolonged, and a stronger cell electroporation effect is further caused.

In conclusion, the gold nanorods after targeted modification can more efficiently exert the characteristics of lightning rod effect and high conductivity, improve the intensity of electric field applied to cell membranes, and reduce the charging time constant of the cell membranes, thereby effectively enhancing the cell electroporation effect.

Claims

1. A targeted modified high-conductivity nanoparticle enhanced cell electroporation device is characterized by comprising a nanosecond pulse generator and the nanoelectrodes.

the nano-electrode is in contact with or extends into a target cell;

after receiving the excitation pulse, the nano electrode enhances the electric field intensity of the excitation pulse, sends an enhanced excitation pulse signal to the target cell and enhances the electroporation effect of the target cell;

the nano-electrodes are a plurality of high-conductivity nano-particles; each highly conductive nanoparticle has a targeting ligand of a target cell on the surface;

2. The device for electroporation of target-modified highly conductive nanoparticles as claimed in claim 1 or 2, wherein: the nanosecond pulse generator comprises a high-voltage direct-current power supply, an energy storage capacitor, an FPGA module and an MOSFET switch group;

3. The device for electroporation of cells reinforced by target-modified highly conductive nanoparticles as claimed in claim 1, wherein: the high-conductivity nano particles are gold nano rods.

4. The device for electroporation of cells reinforced by target-modified highly conductive nanoparticles as claimed in claim 1, wherein: the pulses are square wave pulses.

5. A method for enhancing a cell electroporation device using the target-modified highly conductive nanoparticles according to any one of claims 1 to 4, comprising the steps of:

3) contacting the nano-electrode with the target cell targeting ligand on the surface with the target cell or extending the nano-electrode into the target cell;

4) presetting pulse parameters;

6. The method of claim 5, wherein the highly conductive nanoparticles are targeted for modification to enhance electroporation of cells, and wherein the method comprises the steps of: the electric field intensity E of the tip of the high-conductivity nano particle_tipAs follows: