CN112980674A - Device and method for enhancing high-frequency pulse magnetic field to induce magnetic perforation of cells based on targeted modified magnetic nanoparticles - Google Patents
Device and method for enhancing high-frequency pulse magnetic field to induce magnetic perforation of cells based on targeted modified magnetic nanoparticles Download PDFInfo
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Abstract
The invention discloses a device and a method for enhancing high-frequency pulse magnetic field induced cell magnetic perforation based on targeted modified magnetic nanoparticles. The system comprises a high-frequency pulse magnetic field generator and magnetic nanoparticles; the method comprises the following steps: 1) determining the type of the target cell, and acquiring a target ligand of the target cell; 2) contacting the magnetic nanoparticles with a target cell through a receptor on the surface of the target cell membrane or extending the magnetic nanoparticles into the target cell; 3) placing a pore plate containing cell solution in a magnetic field coil target area of a magnetic perforation device; 4) presetting pulse parameters; 5) the high-voltage direct-current power supply charges the energy storage capacitor; 6) the FPGA module controls the on-off of the IGBT switch group to realize the output of the pulse magnetic field; 7) the pulse magnetic field output by the generator is enhanced by the magnetic nanoparticles to realize perforation of target cells. The invention can reach the parameter condition needed by magnetic perforation in shorter time, and reduces the time for forming the magnetic perforation, thereby causing stronger magnetic perforation effect of cells.
Description
Technical Field
The invention relates to the field of cell perforation, in particular to a device and a method for enhancing high-frequency pulse magnetic field induction cell magnetic perforation based on targeted modification magnetic nanoparticles.
Background
Pulsed magnetic field induced cell magnetic perforation is widely researched by scholars at home and abroad as a novel biotechnology. The technology of cell perforation caused by the pulsed magnetic field has the outstanding advantages of non-contact, non-heat and the like, and is a research direction with great prospect in the field of cell perforation. Although the traditional magnetic perforation method can induce cells to be perforated, the traditional magnetic perforation method has the problems of low efficiency, poor electrical safety and the like, and the application and popularization of the magnetic perforation technology are limited to a certain extent. Therefore, a more efficient new magnetic perforation method is proposed to solve the inevitable need of the current technical bottleneck.
Disclosure of Invention
The invention aims to provide a device for enhancing high-frequency pulse magnetic field induced cell magnetic perforation based on targeted modified magnetic nanoparticles, which comprises a high-frequency pulse magnetic field generator and magnetic nanoparticles.
The high-frequency pulse magnetic field generator sends an excitation pulse magnetic field to the magnetic nanoparticles.
The high-frequency pulse magnetic field generator comprises a high-voltage direct-current power supply, a pulse capacitor, a solid-state switch group, a switch driving module, a magnetic field coil and a discharge resistor for protecting the stable work of a circuit.
The high-voltage direct-current power supply charges the pulse capacitor.
The pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group.
The switch drive controls the on-off of the solid-state switch group, so that the parameter of the high-frequency pulse current is controlled.
The magnetic field coil generates a pulse magnetic field after receiving the high-frequency pulse current.
And the high-voltage direct current power supply is connected with the pulse capacitor to form a charging loop.
The solid-state switch group, the magnetic field coil and the discharge resistor form an RLC pulse discharge loop.
The switch driving module comprises an FPGA module. And the FPGA module sends a switch control signal to the solid-state switch group.
The magnetic nanoparticles comprise a plurality of highly magnetically permeable nanoparticles. The surface of each high magnetic conductive nano particle is provided with a targeting ligand of a target cell.
The magnetic nanoparticles are in contact with a target cell.
Preferably, the magnetic nanoparticles extend into the target cells.
And after receiving the excitation pulse magnetic field, the magnetic nanoparticles send a pulse magnetic field to the target cells to perforate the target cells.
The high magnetic permeability nano particles are nano iron oxide.
And after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
A method for enhancing high-frequency pulse magnetic field induced cell magnetic perforation by using the high-permeability magnetic nanoparticles based on targeted modification comprises the following steps:
1) determining the type of the target cell, and obtaining the target ligand of the target cell.
2) The magnetic nanoparticles with the target cell targeting ligand on the surface are contacted with the target cells through receptors on the surface of target cell membranes.
3) Target cells are placed in the magnetic field coil target region of action. The target action area of the magnetic field coil is the area where the magnetic field generated by the magnetic field coil is located.
4) And presetting pulse parameters.
5) The high-voltage direct-current power supply charges the energy storage capacitor.
6) After charging is finished, the FPGA module controls the on-off of the IGBT switch group based on preset pulse parameters.
The pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group.
7) And after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
And the high-permeability nano particles send a pulse magnetic field to the target cells to perforate the target cells.
It is worth to be noted that the addition of the high magnetic permeability nanoparticles can cause the distortion of the magnetic field in the vicinity of the high magnetic permeability nanoparticles, so that if the high magnetic permeability nanoparticles are modified by the targeting ligand to realize the specific binding to the target cells, the magnetic field amplitude in the vicinity of the cell membrane can be effectively enhanced, thereby further enhancing the magnetic perforation effect of the cells.
According to the invention, specific targeting ligands are selected for target cells, and then the surface of the high-permeability nano-particles is modified by the targeting ligands, so that the modified nano-particles have the function of identifying the target cells in a targeted manner. After the nanoparticles are combined with the cells, a pulsed magnetic field with corresponding parameters is applied to induce the cells to generate magnetic perforation.
The technical effect of the invention is undoubtedly that the invention can reach the parameter conditions needed for generating magnetic perforation in shorter time, reduce the time for forming the magnetic perforation and further cause stronger magnetic perforation effect of cells. The nano iron oxide after targeted modification can more efficiently exert the characteristic of high magnetic permeability, and the amplitude of a magnetic field borne by a cell membrane is improved, so that the magnetic perforation effect of the cell is effectively enhanced.
Drawings
FIG. 1 is a schematic diagram of a targeted modified magnetic nanoparticle for enhancing a high-frequency pulsed magnetic field induced cell magnetic perforation effect;
FIG. 2 is a schematic diagram of an experimental platform for processing cells by nsPMFs;
FIG. 3 is a schematic diagram of a high frequency pulse generator;
FIG. 4 is a graph showing the effect of different magnetic field amplitudes on the proportion of PI-positive cells;
FIG. 5 is a graph showing the effect of different pulse counts on the proportion of PI-positive cells;
FIG. 6 is a graph showing the effect of different magnetic field amplitudes on the proportion of cells undergoing perforation;
FIG. 7 is a graph showing the effect of different pulse counts on the proportion of cells undergoing perforation;
FIG. 8(a) is a scanning electron microscope imaging of human red blood cells without any treatment;
FIG. 8(b) is the result of scanning electron microscope imaging of human red blood cells under the action of high-frequency pulsed magnetic field;
in the figure: adherent cells 1, 48-well plate 2 and magnetic field coil 3.
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 3, the device for enhancing high-frequency pulsed magnetic field induced cell magnetic perforation based on targeted modified magnetic nanoparticles comprises a high-frequency pulsed magnetic field generator and magnetic nanoparticles.
The high-frequency pulse magnetic field generator sends an excitation pulse magnetic field to the magnetic nanoparticles.
The high-frequency pulse magnetic field generator comprises a high-voltage direct-current power supply, a pulse capacitor, a solid-state switch group, a switch driving module, a magnetic field coil and a discharge resistor for protecting the stable work of a circuit.
The high-voltage direct-current power supply charges the pulse capacitor.
The pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group.
The switch drive controls the on-off of the solid-state switch group, so that the parameter of the high-frequency pulse current is controlled.
The magnetic field coil generates a pulse magnetic field after receiving the high-frequency pulse current.
And the high-voltage direct current power supply is connected with the pulse capacitor to form a charging loop.
The solid-state switch group, the magnetic field coil and the discharge resistor form an RLC pulse discharge loop.
The switch driving module comprises an FPGA module. And the FPGA module sends a switch control signal to the solid-state switch group.
The magnetic nanoparticles comprise a plurality of highly magnetically permeable nanoparticles. The surface of each high magnetic conductive nano particle is provided with a targeting ligand of a target cell.
The magnetic nanoparticles are in contact with the target cells, and the magnetic nanoparticles do not extend into the target cells.
And after receiving the excitation pulse magnetic field, the magnetic nanoparticles send a pulse magnetic field to the target cells to perforate the target cells.
The high magnetic permeability nano particles are nano iron oxide.
And after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
Example 2:
a device for enhancing high-frequency pulse magnetic field to induce cell magnetic perforation based on targeted modification magnetic nanoparticles comprises a high-frequency pulse magnetic field generator and magnetic nanoparticles.
The high-frequency pulse magnetic field generator sends an excitation pulse magnetic field to the magnetic nanoparticles.
The high-frequency pulse magnetic field generator comprises a high-voltage direct-current power supply, a pulse capacitor, a solid-state switch group, a switch driving module, a magnetic field coil and a discharge resistor for protecting the stable work of a circuit.
The high-voltage direct-current power supply charges the pulse capacitor.
The pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group.
The switch drive controls the on-off of the solid-state switch group, so that the parameter of the high-frequency pulse current is controlled.
The magnetic field coil generates a pulse magnetic field after receiving the high-frequency pulse current.
And the high-voltage direct current power supply is connected with the pulse capacitor to form a charging loop.
The solid-state switch group, the magnetic field coil and the discharge resistor form an RLC pulse discharge loop.
The switch driving module comprises an FPGA module. And the FPGA module sends a switch control signal to the solid-state switch group.
The magnetic nanoparticles comprise a plurality of highly magnetically permeable nanoparticles. The surface of each high magnetic conductive nano particle is provided with a targeting ligand of a target cell.
The magnetic nanoparticles extend into the target cell.
And after receiving the excitation pulse magnetic field, the magnetic nanoparticles send a pulse magnetic field to the target cells to perforate the target cells.
The high magnetic permeability nano particles are nano iron oxide.
And after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
Example 3:
referring to fig. 1 to 3, the device for enhancing high-frequency pulsed magnetic field induced cell magnetic perforation based on targeted modified magnetic nanoparticles comprises a high-frequency pulsed magnetic field generator and magnetic nanoparticles.
The high-frequency pulse magnetic field generator sends an excitation pulse magnetic field to the magnetic nanoparticles.
The high-frequency pulse magnetic field generator comprises a high-voltage direct-current power supply, a pulse capacitor, a solid-state switch group, a switch driving module, a magnetic field coil and a discharge resistor for protecting the stable work of a circuit.
The high-voltage direct-current power supply charges the pulse capacitor.
The pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group.
The switch drive controls the on-off of the solid-state switch group, so that the parameter of the high-frequency pulse current is controlled.
The magnetic field coil generates a pulse magnetic field after receiving the high-frequency pulse current.
And the high-voltage direct current power supply is connected with the pulse capacitor to form a charging loop.
The solid-state switch group, the magnetic field coil and the discharge resistor form an RLC pulse discharge loop.
The switch driving module comprises an FPGA module. And the FPGA module sends a switch control signal to the solid-state switch group.
The magnetic nanoparticles comprise a plurality of highly magnetically permeable nanoparticles. The surface of each high magnetic conductive nano particle is provided with a targeting ligand of a target cell.
The magnetic nanoparticles are in contact with the target cells, and the magnetic nanoparticles do not extend into the target cells.
And after receiving the excitation pulse magnetic field, the magnetic nanoparticles send a pulse magnetic field to the target cells to perforate the target cells.
The high magnetic permeability nano particles are nano iron oxide.
And after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
The method for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells by using the high-permeability nano particles based on the targeted modification comprises the following steps:
1) determining the type of the target cell, and obtaining the target ligand of the target cell.
2) The magnetic nanoparticles with the target cell targeting ligand on the surface are contacted with the target cells through receptors on the surface of target cell membranes.
3) Target cells are placed in the magnetic field coil target region of action. The target action area of the magnetic field coil is the area where the magnetic field generated by the magnetic field coil is located.
4) And presetting pulse parameters.
5) The high-voltage direct-current power supply charges the energy storage capacitor.
6) After charging is finished, the FPGA module controls the on-off of the IGBT switch group based on preset pulse parameters.
The pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group.
7) And after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
And the high-permeability nano particles send a pulse magnetic field to the target cells to perforate the target cells.
Example 4:
a method for enhancing high-frequency pulse magnetic field induced cell magnetic perforation by using the high-permeability magnetic nanoparticles based on targeted modification comprises the following steps:
1) determining the type of the target cell, and obtaining the target ligand of the target cell.
2) The magnetic nanoparticles with the target cell targeting ligand on the surface are contacted with the target cells through receptors on the surface of target cell membranes.
3) Target cells are placed in the magnetic field coil target region of action. The target action area of the magnetic field coil is the area where the magnetic field generated by the magnetic field coil is located.
4) And presetting pulse parameters.
5) The high-voltage direct-current power supply charges the energy storage capacitor.
6) After charging is finished, the FPGA module controls the on-off of the IGBT switch group based on preset pulse parameters.
The pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group.
7) And after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
And the high-permeability nano particles send a pulse magnetic field to the target cells to perforate the target cells.
Example 5:
referring to fig. 1 to 2, a highly conductive nanoparticle enhanced cell magnetic perforation device based on targeted modification comprises a nanosecond pulse generator and a nano electrode;
the high-frequency pulse generator sends excitation pulses to the magnetic nanoparticles;
the high-frequency pulse generator sends excitation pulses to target cells;
the high-frequency pulse generator comprises a high-voltage direct-current power supply, a pulse capacitor, a solid-state switch group, a magnetic field coil and a discharge resistor;
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 IGBT switch group;
and the FPGA module controls the on-off of the IGBT switch group so as to control the duration and the number of the excitation pulses.
The magnetic nanoparticles can achieve targeting of target cells;
the magnetic nanoparticles are in contact with or extend into a target cell;
after receiving the excitation pulse, the magnetic nanoparticles around the cells send enhanced pulses to the target cells to perforate the target cells; the pulse is a triangular wave pulse.
The magnetic nano-particles are a plurality of high-permeability nano-particles; the surface of each high magnetic conductive nano particle is provided with a targeting ligand of a target cell.
The high magnetic permeability nano particles are nano iron oxide.
Example 6:
a method for enhancing magnetic perforation of cells using highly conductive nanoparticles based on targeted modification, comprising the steps of:
1) determining the type of the target cell, and acquiring a target ligand of the target cell;
2) contacting the magnetic nanoparticles with the target cell targeting ligand on the surface with target cells through a receptor on the surface of a target cell membrane or extending the magnetic nanoparticles into the target cells, and enabling the target cells to adhere to the wall in a pore plate;
3) placing a pore plate containing cell solution in a magnetic field coil target area of a magnetic perforation device;
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 IGBT switch group based on preset pulse parameters to realize the output of a pulse magnetic field;
7) the pulse magnetic field output by the generator is enhanced by the magnetic nanoparticles to realize perforation of target cells.
Example 7:
an experiment of using a high-permeability nanoparticle enhanced high-frequency pulse magnetic field based on targeted modification to induce cell magnetic perforation device comprises the following steps:
1) cell culture
Experiments were performed in this section using a375 human melanoma cells and human red blood cells.
1.1) 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.
1.2) preparation of adherent cells 1
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, and finally the cell concentration is determined to be 2.5X 105And each/mL, placing the prepared cell suspension in a 48-well plate 2 for 24 hours, and performing pulse treatment after the cells are completely attached to the wall.
2) Establishment of experimental platform
The schematic diagram of the experimental platform device constructed in this embodiment is shown in fig. 2, and a375 cell suspension prepared in advance is filled into a 48-well plate (diameter of each well is 1cm), and is kept stand for 24h to wait for cell adhesion. After the cells are attached to the wall, the 48-hole plate is placed at the output end of a self-made high-frequency magnetic field pulse generator in a laboratory, and the holes containing the cells are correspondingly placed right above the magnetic field coil 3. Meanwhile, a high-voltage probe (connected to two ends of a resistor connected in parallel with a coil and finally acquiring voltage waveforms at two ends of the resistor through an oscilloscope, wherein the voltage waveform obtained by testing is considered to be the voltage waveform received by the magnetic field coil because the resistor and the coil generating the magnetic field are in a parallel connection relation.
The schematic structural diagram of the pulse generator device used in this embodiment is shown in fig. 3, and a computer PC is used for programming, and a program is burned into a Field-Programmable Gate Array (FPGA) module, and a signal output of the FPGA is transmitted to an IGBT switch through an optical fiber to control on and off of the IGBT, thereby controlling an output pulse parameter. The nanosecond pulse generator body adopts a traditional RC charge-discharge circuit structure, namely, a capacitor is charged through a high-voltage direct-current power supply, and then the action of an IGBT switch is controlled through an output signal of an FPGA, so that the duration time and the action number of pulse voltage at two ends of a load are controlled.
3) A375 cell adherence Effect assay
The same volume of cells as the concentration of the cells used in the experiment was placed in a 48-well plate, the cells and the plate were allowed to stand together in an incubator for 24 hours, and after 24 hours, the cells were observed using a microscope, at which time the cells were fusiform. And then, using a PBS solution which is prepared in advance in a laboratory and has no influence on the cell state to slightly wash the cells, and placing the washed cells under an optical microscope again for observation to obtain the cells which are still fusiform, wherein the cell number is basically consistent with that of the cells before washing, which indicates that the cells can be completely attached to the wall on a pore plate after 24 hours. Because only living cells can adhere to the wall, the cells adhere to the wall and dead cells can be screened out when the subsequent experiment treatment is facilitated, and the cells after adhering to the wall can approach the magnetic field coil to a greater extent and are beneficial to obtaining better experiment effect under the condition of smaller magnetic field amplitude.
4) High-frequency pulse magnetic field treatment scheme
The pulse parameters used in the adherent cell experiment of this step are shown in table 1. In the experiment, the pulse frequency in the string is fixed to be 100kHz, the pulse width is 800ns, the pulse frequency outside the string is fixed to be 1Hz, and the average value (B) and the pulse of the magnetic field amplitude are determinedThe two variables of the punching number (N) are respectively provided with 5 parameter values with different levels. The present embodiment first sets an intermediate value for the parameters, i.e., B is 310.3mT and N is 2 × 104And (4) respectively. When B is changed, N is fixed to 2X 104A plurality of; when changing N, then B is fixed at 310.3 mT.
TABLE 1Experimental parameter Table of high frequency pulsed magnetic field Table 1Experimental parameters of nsPMFs
And (3) obtaining adherent cells in the pore plate by the method in the step (2), wherein the adherent cells in each experiment are divided into an experiment group which is added with a magnetic field for treatment, an experiment group which is added with nano ferric oxide particles and then treated with the magnetic field, and a blank group which is respectively contrasted with different experiment groups and is not added with any treatment. The 48-hole plate which is paved in advance is placed right above the magnetic field generator, and then a pulse magnetic field with specific parameters is applied for treatment. And after the pulse magnetic field treatment is finished, placing the orifice plate in an incubator for 48 hours. After 48 hours of incubation, cck-8 reagent was added, and after 1.5 hours of incubation, absorbance was measured by a microplate reader.
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 the pulsed magnetic field treatment, the treated cells and the well plate were directly placed in an incubator and incubated for 3 hours. After 3 hours the cells were digested from the 48-well plates 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 in the dark.
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 under the action of a pulse magnetic field, PI molecules can penetrate through the cell membrane through micropores in the membrane, enter the interior of the cell and are combined with the cell nucleus. Therefore, the PI staining method can accurately reflect the change of the membrane permeability after the cells are subjected to magnetic perforation, and finally qualitatively characterize the strength of the magnetic perforation effect through the proportion of PI positive cells.
5.2) Effect of pulse parameter variation on the proportion of PI-Positive cells
FIGS. 4 and 5 are histograms of the percentage of PI-positive cells under different pulse parameters. As shown in FIG. 4, in the control group, no pulse magnetic field is applied, and the concentrations of the folate-modified targeted superparamagnetic Iron Oxide nanoparticles (Super-small particles of Iron Oxide nanoparticles-FA, SPIONs-FA) are safe concentrations, so that the PI positive ratio of the two groups of cells is very low and has no significant difference (p >0.05), which indicates that perforation does not occur basically and the cell membrane is in an intact state.
When the Magnetic field amplitude is changed, the PI positive proportion of Nanosecond Pulsed Magnetic Fields (nsPMFs) acting alone is increased from 1.8% of 103.4mT to 35.6% of 517.1mT, while the PI positive proportion of the SPIONS-FA group is increased from 2.1% to 53.3% along with the increase of the Magnetic field amplitude, has higher PI positive proportion than the nsPMFs acting alone, and has a very significant difference (p <0.01) compared with the nsPMFs acting alone. Therefore, it is demonstrated that the A375 melanoma cells used in the experiment were perforated after treatment with the pulsed magnetic field, and the addition of SPIONs-FA enhanced the effect of magnetic perforation of the cells.
When the number of pulses is changed, the group of nsPMFs acting alone and the group of SPIONs-FA act from 1 × 10 respectively42.1% and 4.3% rise to 3X 10 under one pulse438.9% and 47.1% under each pulse. The SPIONs-FA group at the number of pulses used in any experiment showed a stronger A375 cell perforation effect than the nsPMFs alone acting group, and all had significant differences from the nsPMFs alone acting group (. p)<0.05, representing the size of the difference in the statistical analysis) or the most significant difference (. about.p)<0.01). Therefore, this indicates where the addition of a pulsed magnetic field is proceedingAfter that, the A375 melanoma cells used in the experiment were perforated, and the addition of SPIONs-FA enhanced the magnetic perforation effect of the cells.
Therefore, based on the above experimental results, it can be found that under the pulse parameters used in the experiment, the melanoma cells of the target cell a375 can be magnetically perforated, the perforation degree is enhanced along with the enhancement of the pulse parameters, and the effect of the high-frequency pulse magnetic field on the magnetic perforation of the cells can be effectively enhanced after the SPIONs-FA nanoparticles are added.
6) PI staining method for detecting proportion of cells generating perforation
6.1) PI reagent staining method
In this step, trypsin without EDTA was used to digest the cells. After the pulsed magnetic field treatment, the treated cells and the well plate were directly placed in an incubator and incubated for 30 minutes. After 30 minutes, the cells were gently rinsed 2 times with pre-prepared PBS to ensure that the cell surface media was washed clean. Then 200. mu.L of a mixed solution of PBS and Propidium Iodide (PI) (100: 1) was added in the dark and incubation was continued for 10 minutes, and finally detection was performed by fluorescence microscope (DMi8, Leica) under dark conditions.
In step 5, the change of the membrane permeability after the magnetic perforation of the cells can be accurately reflected by using a PI staining method. The ratio of stained cells to total cells in the field of view observed by statistical fluorescence microscopy can be used to determine the ratio of cells undergoing magnetic perforation in relation to different pulse parameters.
6.2) Effect of pulse parameter variation on the proportion of cells undergoing perforation
FIGS. 6 and 7 are bar graphs of the percentage of stained cells generated by different pulse parameters. As shown in fig. 6, in the control group, since no pulsed magnetic field was applied, staining of the cells was not observed, indicating that perforation did not occur substantially and the cell membrane was intact.
When the magnetic field amplitude is changed, the proportion of stained cells in the case of single action of nsPMFs is increased from 1.7% of 103.4mT to 20.2% of 517.1mT, while the proportion of stained cells in the SPIONs-FA group is increased from 1.7% to 62.5% with the increase of the magnetic field amplitude, and has a higher proportion of stained cells than the nsPMFs single action group, and has a very significant difference compared with the nsPMFs single action experimental group (p <0.01) and a very significant difference compared with the blank control group (p < 0.01). This shows that the A375 melanoma cells used in the experiment are actually perforated after the pulsed magnetic field is applied for treatment, the number of the perforated cells is increased along with the increase of the amplitude of the pulsed magnetic field, and the effect of magnetic perforation of the cells is enhanced by the addition of the SPIONs-FA.
When the number of pulses was changed, the ratio of stained cells was from 1X 10 in the case where the nsPMFs acted alone42.0% rise to 3X 10 under one pulse410.9% under each pulse. While the proportion of stained cells in the SPIONS-FA group increased from 3.1% to 39.0% with the increase in the number of pulses, and when the number of pulses was 1.5X 104And 2 x 104At one time, the experimental group with single action of nsPMFs has significant difference (p) compared with the SPIONs-FA group<0.05); when the number of pulses is 2.5 multiplied by 104And 3 x 104At one time, the nsPMFs alone acted on the experimental group, and the SPIONs-FA group had very significant difference (p)<0.01). The above experimental results all show that the A375 melanoma cells used in the experiment are actually perforated after the pulsed magnetic field is added for treatment, the number of the perforated cells is increased along with the increase of the amplitude of the pulsed magnetic field, and the addition of the SPIONs-FA enhances the magnetic perforation effect of the cells.
Therefore, based on the above experimental results, it was found that the melanoma cells of the target cell a375 were able to be magnetically perforated under the pulse parameters used in the experiment, and the proportion of the cells where perforation occurred increased with the increase of the pulse parameters. Based on the experimental results, the SPIONs-FA group can be found to have higher proportion of the cells which are stained under the same pulse parameters, and the preliminary proof that the addition of the SPIONs-FA can obviously improve the magnetic perforation effect of the cells.
7) Scanning electron microscope imaging of cells after high-frequency pulse magnetic field treatment
7.1) sample preparation method of scanning electron microscope
Human whole blood was obtained and placed in a centrifuge tube and centrifuged at 3000 rpm for 2 minutes, after which the supernatant liquid was aspirated using a pipette gun. The red blood cells precipitated in the lower part of the centrifuge tube were resuspended by adding the previously prepared PBS solution, and centrifuged at 3000 rpm for 2 minutes. Repeating the above operations until the supernatant is clear and transparent, sucking the supernatant by using a pipette gun, adding the PBS again to resuspend the red blood cells, and waiting for subsequent treatment.
Adding SPIONs-FA with safe concentration into the human erythrocyte suspension extracted at the early stage, and putting the mixed cell solution into a 48-pore plate. After a period of time, the 48-hole plate is placed above a high-frequency pulse magnetic field generator to carry out corresponding parameters (the magnetic field amplitude is 310.3mT, the number of pulses is 2 multiplied by 10)4One) and a control group without high-frequency pulsed magnetic field treatment is set up.
After the pulsed magnetic field treatment is finished, the human red blood cells in the 48-well plate are lightly blown by using a pipette gun, the cell solution is placed in a centrifuge tube and centrifuged at the speed of 1200 rpm for 3 minutes, then the cells are resuspended by using a PBS solution, and the centrifugation is carried out again at the same rotating speed, and the operation is repeated for 3 times until the supernatant is clear and transparent.
After the supernatant was aspirated, the treated erythrocytes were resuspended with PBS, and the resuspended cells were cultured in 6-well plates previously placed on 18mm coverslips. After three hours of standing, the PBS solution in the 6-well plate was gently aspirated along the walls of the wells. After the PBS was aspirated, the fixative was gently added along the plate wall and left for 30 minutes. After fixing the sample, the sample is dehydrated by 50%, 75% and 90% alcohol for 15 minutes each time, and then dehydrated by anhydrous alcohol for 30 minutes, and then naturally air-dried. And observing the air-dried sample by using a scanning electron microscope after the gold spraying treatment.
7.2) scanning Electron microscope imaging results
The results of scanning electron microscopy imaging of the cells without any treatment and the cells under the action of the high-frequency pulsed magnetic field are shown in FIGS. 8(a) and 8(b), respectively, in which the cells used in the preparation of the sample are human erythrocytes. The scanning electron microscope imaging result shows that the cell surface without any treatment is smooth and regular, the cell surface is rough under the action of certain magnetic pulse parameters, and the phenomena of cell membrane rupture and perforation can be seen. The scanning electron microscope imaging result of the cell shows that the cell has the morphology change under the combined action of the SPIONs-FA and the high-frequency pulse magnetic field, and the fact that the cell can generate the magnetic perforation phenomenon under the combined action of the high-frequency pulse magnetic field and the targeted magnetic nanoparticles is proved.
Claims (10)
1. Device based on target modification magnetic nanoparticle reinforcing high frequency pulse magnetic field induction cell magnetic perforation, its characterized in that: comprises the high-frequency pulse magnetic field generator and magnetic nano-particles.
The high-frequency pulse magnetic field generator sends an excitation pulse magnetic field to the magnetic nanoparticles;
the magnetic nanoparticles comprise a plurality of high permeability magnetic nanoparticles; the surface of each high magnetic permeability nano particle is provided with a targeting ligand of a target cell;
the magnetic nanoparticles are in contact with target cells;
and after receiving the excitation pulse magnetic field, the magnetic nanoparticles send a pulse magnetic field to the target cells to perforate the target cells.
2. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 1, is characterized in that: the high-frequency pulse magnetic field generator comprises a high-voltage direct-current power supply, a pulse capacitor, a solid-state switch group, a switch driving module and a magnetic field coil;
the high-voltage direct-current power supply charges a pulse capacitor;
the pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group;
the switch drive controls the on-off of the solid-state switch group, so as to control the parameter of the high-frequency pulse current;
the magnetic field coil generates a pulse magnetic field after receiving the high-frequency pulse current.
3. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 2, is characterized in that: the protection circuit also comprises a discharge resistor used for protecting the stable operation of the circuit.
4. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 2, is characterized in that: and the high-voltage direct current power supply is connected with the pulse capacitor to form a charging loop.
5. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 4, is characterized in that: the solid-state switch group, the magnetic field coil and the discharge resistor form an RLC pulse discharge loop.
6. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 1, is characterized in that: the switch driving module comprises an FPGA module; and the FPGA module sends a switch control signal to the solid-state switch group.
7. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 1, is characterized in that: the high magnetic permeability nano particles are nano iron oxide.
8. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 1, is characterized in that: and after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, so that the magnetic field distribution of the magnetic field area is enhanced.
9. The device for enhancing the high-frequency pulse magnetic field to induce the magnetic perforation of the cells based on the targeted modified magnetic nanoparticles as claimed in claim 1, is characterized in that: the magnetic nanoparticles extend into the target cell.
10. A method for enhancing high-frequency pulse magnetic field to induce cell magnetic perforation by using the high-permeability magnetic nanoparticles based on targeted modification according to any one of claims 1 to 10, which is characterized by comprising the following steps:
1) determining the type of the target cell, and acquiring a target ligand of the target cell;
2) contacting the magnetic nanoparticles with the target cell targeting ligand on the surface with the target cell through a receptor on the surface of a target cell membrane;
3) placing the target cells in the magnetic field coil target action area; the target action area of the magnetic field coil is the area where the magnetic field generated by the magnetic field coil is located;
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 IGBT switch group based on preset pulse parameters;
the pulse capacitor sends high-frequency pulse current with adjustable parameters to the magnetic field coil through the solid-state switch group;
7) after receiving the excitation pulse magnetic field, the high-permeability nano particles are magnetized, and the magnetic field distribution of the magnetic field area is enhanced;
and the high-permeability nano particles send a pulse magnetic field to the target cells to perforate the target cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118147127A (en) * | 2024-05-10 | 2024-06-07 | 深圳市赛特罗生物医疗技术有限公司 | Large-scale continuous electrotransfection method and system |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102341500A (en) * | 2009-03-03 | 2012-02-01 | 埃因霍温科技大学 | Device and method for treating cells |
| US20120070376A1 (en) * | 2010-08-14 | 2012-03-22 | University Of Massachusetts | Yeast cell wall particles for receptor-targeted nanoparticle delivery |
| CN106573069A (en) * | 2014-08-14 | 2017-04-19 | 梁平 | Methods for killing cancer cells and cellular imaging using magneto-electric nano-particles and external magnetic field |
| WO2020198328A1 (en) * | 2019-03-25 | 2020-10-01 | Kansas State University Research Foundation | Synergist therapy for enhanced drug delivery: magnetic field facilitated nanoparticle microporation |
-
2021
- 2021-02-01 CN CN202110133602.4A patent/CN112980674A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102341500A (en) * | 2009-03-03 | 2012-02-01 | 埃因霍温科技大学 | Device and method for treating cells |
| US20120070376A1 (en) * | 2010-08-14 | 2012-03-22 | University Of Massachusetts | Yeast cell wall particles for receptor-targeted nanoparticle delivery |
| CN106573069A (en) * | 2014-08-14 | 2017-04-19 | 梁平 | Methods for killing cancer cells and cellular imaging using magneto-electric nano-particles and external magnetic field |
| WO2020198328A1 (en) * | 2019-03-25 | 2020-10-01 | Kansas State University Research Foundation | Synergist therapy for enhanced drug delivery: magnetic field facilitated nanoparticle microporation |
Non-Patent Citations (3)
| Title |
|---|
| VITALIJ NOVICKIJ等: "Irreversible magnetoporation of micro-organisms in high pulsed magnetic fields", 《IET NANOBIOTECHNOL》 * |
| YAN MI等: "Viability inhibition of A375 melanoma cells in vitro by a high-frequency nanosecond-pulsed magnetic field combined with targeted iron oxide nanoparticles via membrane magnetoporation", 《NANOTECHNOLOGY》 * |
| 米彦等: "高频纳秒脉冲串对皮肤癌细胞的杀伤效果实验研究", 《高电压技术》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118147127A (en) * | 2024-05-10 | 2024-06-07 | 深圳市赛特罗生物医疗技术有限公司 | Large-scale continuous electrotransfection method and system |
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