A method for regulating blood-brain barrier transcytosis mediated by magnetic nanoprobe

Abstract

The application discloses a preparation method and application of ZnFe2O4@CMD nanoparticles and a magnetic nanoprobe thereof, and discloses a method for regulating transcytosis of a blood-brain barrier (BBB). The application is verified in a BBB in-vitro model and a BBB regulation in a mouse in-vivo. The method of the application enhances the efficiency of molecules or nanoparticles crossing the BBB to enter brain tissues by regulating caveolin-mediated transcytosis of the BBB, and is expected to improve the difficulty of brain disease treatment drugs in crossing the BBB.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biology and medical nanomaterials, and particularly relates to a method for regulating blood-brain barrier transcytosis mediated by a magnetic nanoprobe. BACKGROUND

[0002] The human brain contains about 644 kilometers of blood vessels, which provide oxygen, energy metabolites, etc. for brain cells, and remove carbon dioxide and other metabolic waste from the brain to the systemic circulation. Capillaries account for about 85% of the length of brain blood vessels and are the main sites of the blood-brain barrier (BBB). The BBB is mainly composed of brain capillary endothelial cells, astrocytes, pericytes and basement membrane, etc. and is a biological interface between blood and brain cells. It blocks nearly 100% of macromolecules and more than 98% of small molecule exogenous therapeutic agents, making the treatment of central nervous system diseases such as glioma and Alzheimer's disease appear to be a bottleneck, which seriously affects people's health. The BBB is difficult to break through due to the following special structures: (1) high expression of tight junction proteins between vascular endothelial cells, with extremely high compactness, and the paracellular pathway is blocked; (2) non-specific endothelial cell transcytosis is inhibited, and the transendothelial pathway is limited; (3) the basement membrane of the vascular unit is continuous and has no window, and the carrier diffusion is blocked; (4) the peripheral cells and astrocyte processes surround the vascular unit, making the BBB structure more stable. Therefore, the development of BBB regulation technology is of great significance to the treatment of central nervous system diseases and health maintenance.

[0003] In recent years, research on BBB regulation has been emerging. Among them, the regulation of BBB tight junction proteins is expected to open the paracellular pathway. Researchers regulate the Unc5B receptor on the BBB vascular endothelial cells to regulate the permeability of the BBB. After regulation, the Wnt / β-catenin signaling in the BBB is inhibited, and the expression levels of tight junction proteins such as ZO-1 and Claudin 5 are reduced, which allows tracers with a molecular weight of less than 40 kDa to enter brain tissue through the paracellular pathway. Compared with paracellular transport, research on the vascular endothelial cells in which the transcellular transport process occurs has more target sites and a wider binding range. Among them, for the endocytosis process mediated by the high expression of large neutral amino acid transporter 1 (LAT1) on the surface of BBB vascular endothelial cells, researchers synthesized carbon quantum dots with multiple free α-amino acid groups on the surface, which were transported by specific binding with LAT1, successfully targeted to deliver anticancer drugs to glioma through the BBB with high selectivity. For the transcytosis process mediated by the low density lipoprotein receptor-related protein 1 (LRP1) highly expressed on the surface of vascular endothelial cells, researchers delivered an electric field-responsive MRI probe to the epileptic lesion through a specific ligand of LRP1, and imaged the disease in real time under abnormal brain electrical stimulation.

[0004] However, the transport of BBB transcellular pathway, most existing studies focus on the high expression of receptors on the surface of vascular endothelial cells, and transport across the BBB by simulating protein transport processes or using receptor-mediated transcytosis. Such studies are limited to the existing pathways of vascular endothelial cells, and it is difficult to efficiently transport and modularly design. Therefore, specific regulation techniques for the BBB transcellular transport process are urgently needed. SUMMARY

[0005] The technical problem solved by the present application is to provide a preparation method of ZnFe2O4@CMD nanoparticles and a magnetic nanoprobe.

[0006] The technical problem solved by the present application is to provide a preparation method of ZnFe2O4@CMD nanoparticles and a magnetic nanoprobe.

[0007] The technical problem solved by the present application is to provide a preparation method of ZnFe2O4@CMD nanoparticles and a magnetic nanoprobe.

[0008] Technical scheme: In order to solve the above technical problems, the present application provides a preparation method of ZnFe2O4@CMD nanoparticles, which comprises the following steps: ZnFe2O4 magnetic nanoparticles are added to a tetramethylammonium hydroxide solution under first stirring conditions, and second stirring is continued, and after the reaction is completed, excess oleic acid molecules are removed, and the product is dispersed in deionized water, then it is added dropwise to a CMD aqueous solution under third stirring conditions, and after the reaction is completed, it is purified to obtain the product.

[0009] Among them, the first stirring and second stirring speed is 400-600r / min, the mass concentration of the tetramethylammonium hydroxide solution is 15-30%, the second stirring time is 20-60min, the third stirring speed is 600-800r / min, and the initial concentration of the CMD aqueous solution is 1-3mg / mL.

[0010] Among them, in the preparation of the ZnFe2O4 magnetic nanoparticles, the method used is high-temperature thermal decomposition, the reaction precursor is zinc acetylacetone and iron acetylacetone, the reaction solvent is dibenzyl ether, and the reaction surfactant is oleic acid.

[0011] Among them, the amount of zinc acetylacetone is 5-6mmol, the amount of iron acetylacetone is 10-12mmol, the amount of dibenzyl ether is 20-22mL, and the amount of oleic acid is 18-22mL.

[0012] In the surface modification process of adding the CMD aqueous solution, first, the surface of ZnFe2O4 is replaced with tetramethylammonium hydroxide, and then the CMD is coated on the surface of ZnFe2O4 through the Fe-O bond between the CMD and ZnFe2O4, to obtain a ZnFe2O4@CMD magnetic nanoparticle aqueous solution.

[0013] Preferably, the mass concentration of the tetramethylammonium hydroxide solution is 20%, and the mass ratio of the CMD and ZnFe2O4 is 5:1.

[0014] The application also includes a preparation method of the magnetic nanoprobe, which comprises the following steps: adding the prepared ZnFe2O4@CMD nanoparticles into an iRGD polypeptide molecule after activation, and then performing magnetic separation and purification.

[0015] Preferably, the mass ratio of the iRGD polypeptide molecule and the ZnFe2O4@CMD nanoparticles is 0.5-1.5:10, and the reaction condition is a 100-150 r / min shaking bed reaction at room temperature for 25-60 min.

[0016] Preferably, the sequence of the iRGD polypeptide molecule is c(CRGDKGPDC), which is coupled with the terminal amino group and the carboxyl group on the surface of the ZnFe2O4@CMD through an EDC / NHS method, and the mass ratio of the iRGD and the ZnFe2O4@CMD is 1:10.

[0017] The application also includes the ZnFe2O4@CMD nanoparticles and the magnetic nanoprobe prepared by the method.

[0018] The application also includes the application of the ZnFe2O4@CMD nanoparticles or the magnetic nanoprobe in the preparation of a drug for treating diseases of the central nervous system, and preferably, the application comprises targeting the blood-brain barrier of the magnetic nanoprobe to regulate transcytosis.

[0019] The application also includes a method for regulating transcytosis of the blood-brain barrier, which comprises the following steps:

[0020] 1) the method for preparing the magnetic nanoprobe;

[0021] 2) constructing an in-vitro BBB model;

[0022] 3) placing the in-vitro BBB model in a low-frequency alternating magnetic field generator, and completing the regulation of BBB transcytosis after the magnetic field is turned on for a certain period of time.

[0023] The BBB model in step 2) is constructed by using bEnd.3 as endothelial cells, C8-D1A as astrocytes, adding 50-150 μg / magnetic nano probe per hole, and targeting for 0.5-1.5 hours.

[0024] The basic parameter range of the low-frequency alternating magnetic field controller in step 3) is as follows: magnetic field strength: 10-100 Oe, frequency: 10-50 Hz, coil parameter: 10-100 turns, diameter: 5-15 em, and circulating water cooling.

[0025] The action time of the low-frequency alternating magnetic field in step 3) is 1-30 minutes.

[0026] After step 3) of the present application, FITC-Dextran is used to detect the BBB permeability, and immunofluorescence is used to observe the expression change of caveolin.

[0027] The added FITC-Dextran has a mass of 50-150 μg per hole, the permeability detection time is 10-30 minutes, and the detection solution volume is 50-100 μL.

[0028] When the expression of caveolin is observed by immunofluorescence, the antibody used is anti-caveolin-1 antibody.

[0029] Mechanism of action: the present application prepares a magnetic nano probe with zinc-doped ferric oxide nanoparticles (ZnFe2O4) as the core, carboxymethyl dextran (CMD) as the surface hydrophilic modifier, and iRGD polypeptide as the vascular targeting molecule, then constructs a low-frequency alternating magnetic field generator composed of a controller and a coil, further constructs a BBB in vitro model, and targets the magnetic nano probe to vascular endothelial cells in the BBB model; the BBB in vitro model is placed in the low-frequency alternating magnetic field generator, and by applying a low-frequency alternating magnetic field, the oscillation of the magnetic nano probe causes the production of caveolin vesicles in the vascular endothelial cells, thereby enhancing the caveolin-mediated endocytosis of endothelial cells and controllably adjusting the BBB permeability.

[0030] Advantages: compared with the prior art, the present application has the following significant advantages:

[0031] 1、the magnetic nano probe prepared in the present application has strong brain vascular endothelial cell targeting and good biocompatibility.

[0032] 2、the BBB transcytosis regulation technology developed in the present application allows a tracer with a molecular weight of less than 2000 k to cross the BBB model.

[0033] 3、the BBB transcytosis regulation technology does not change the basic structure of the BBB model composition, and the expression of tight junction proteins also does not change, so the safety is higher.

[0034] 4、 the BBB transcytosis regulation technology has the performance of quick reversible recovery, and the BBB tightness is recovered after 3h of transcytosis enhancement.

[0035] 5、 the BBB transcytosis regulation technology can realize the directional opening of the brain BBB under the assistance of a magnet, and has strong site control performance;

[0036] 6、 the BBB transcytosis regulation technology is realized based on a magnetic field, and the excellent penetration depth of electromagnetic waves can realize the opening of the BBB of deep brain tissues. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is an electron microscope graph of ZnFe2O4 magnetic nanoparticles synthesized by a high-temperature thermal decomposition method (scale 50 nm);

[0038] Figure 2 It is a schematic diagram of the preparation process of the magnetic nano probe;

[0039] Figure 3 It is an electron microscope graph of the magnetic nano probe;

[0040] Figure 4 It is a BBB model regulation process graph;

[0041] Figure 5 It is quantitative analysis of FITC-Dextran across the BBB model;

[0042] Figure 6 It is the change of caveolin expression level before and after the regulation of the BBB model (scale 50 μm);

[0043] Figure 7 It is the change of tight junction protein expression level before and after the regulation of the BBB model (scale 50 μm)

[0044] Figure 8 It is the situation graph of Evans Blue across the BBB into brain tumor tissues after the regulation of the BBB in vivo;

[0045] Figure 9 It is the change of caveolin expression level before and after the regulation of the BBB (scale 500 μm);

[0046] Figure 10 It is the generation of caveolin vesicles before and after the regulation of the BBB;

[0047] Figure 11 It is a basic principle graph of the application. DETAILED DESCRIPTION

[0048] The technical solutions of the application will be further described below with reference to the drawings.

[0049] Example 1 Synthesis of magnetic nanoparticles by high temperature pyrolysis

[0050] The experimental apparatus was a temperature-programmed device. The reaction system included 11 mmol of iron acetylacetonate (Sigma, 44920), 5.5 mmol of zinc acetylacetonate (Sigma, 480991), 20 mL of dibenzyl ether (Sigma, 108014), and 18 mL of oleic acid (Sigma, 364525). The reaction system was heated to 290°C at a heating rate of 8°C / min and maintained for 100 min. The process was performed under nitrogen protection and condensation reflux conditions. After the reaction was completed, the ZnFe2O4 magnetic nanoparticles were obtained by magnetic separation (Macrosheng magnet, neodymium-iron-boron magnet) and washed with anhydrous ethanol three times. The nanoparticles were stored in 15 mL of chloroform, and their morphology and size were observed by transmission electron microscopy, as shown in FIG. 1. The particle size was about 20 nm, and the particle morphology was regular. Figure 1

[0051] Example 2 Preparation of magnetic nanoprobe

[0052] 40 mg of ZnFe2O4 magnetic nanoparticles prepared in Example 1 were added to a 20% mass concentration of tetramethylammonium hydroxide solution under mechanical stirring at 500 r / min, and the stirring was continued for 30 min. After the reaction was completed, the excess oleic acid molecules were removed by magnetic separation with chloroform, and the product was dispersed in 10 mL of deionized water. Then, the product was added dropwise to 50 mL of a 2 mg / mL CMD (carboxymethyl dextran, Sigma, 86524) aqueous solution under mechanical stirring at 600 r / min, and the stirring was continued overnight. After the reaction was completed, the ZnFe2O4@CMD nanoparticles were purified by dialysis with a 100 k dialysis bag for three days. 10 mg of the ZnFe2O4@CMD nanoparticle aqueous solution was activated by the EDC / NHS method to activate the carboxyl groups on the surface of the nanoparticles, and then 1 mg of iRGD polypeptide molecules (GLPBIO, 1392278-76-0) were added. The reaction was performed at room temperature on a 100 r / min shaker for 1 h, and the magnetic nanoprobe was obtained after magnetic separation and purification. The product was diluted with pure water to 10 mL and stored at 4°C (Sample 1).

[0053] Example 3 Preparation of magnetic nanoprobe under different conditions

[0054] ​Take 40 mg of ZnFe2O4 magnetic nanoparticles prepared in Example 1, under the condition of 600 r / min mechanical stirring, add it to a 30% mass concentration of tetramethylammonium hydroxide solution, continue to stir for 20 min. After the reaction is completed, remove the excess oleic acid molecules with chloroform by magnetic separation method, and disperse the product in 10 mL of deionized water. Then under the condition of 700 r / min mechanical stirring, add it dropwise to 50 mL of 3 mg / mL CMD aqueous solution, and stir at the same speed overnight. After the reaction is completed, purify with 100 k dialysis bag for three days, to obtain ZnFe2O4@CMD nanoparticles. Take 10 mg of ZnFe2O4@CMD nanoparticle aqueous solution, activate the carboxyl group on the surface of the particles by EDC / NHS method, then add 0.5 mg of iRGD polypeptide molecules, shake at room temperature for 30 min, and purify by magnetic separation to obtain a magnetic nanoprobe, and dilute to 10 mL with pure water, and store at 4°C (sample 2).

[0055] Example 4 Preparation of magnetic nanoprobe under different conditions

[0056] Take 40 mg of ZnFe2O4 magnetic nanoparticles prepared in Example 1, under the condition of 400 r / min mechanical stirring, add it to a 15% mass concentration of tetramethylammonium hydroxide solution, continue to stir for 1 h. After the reaction is completed, remove the excess oleic acid molecules with chloroform by magnetic separation method, and disperse the product in 10 mL of deionized water. Then under the condition of 800 r / min mechanical stirring, add it dropwise to 50 mL of 1 mg / mL CMD aqueous solution, and stir at the same speed overnight. After the reaction is completed, purify with 100 k dialysis bag for three days, to obtain ZnFe2O4@CMD nanoparticles. Take 10 mg of ZnFe2O4@CMD nanoparticle aqueous solution, activate the carboxyl group on the surface of the particles by EDC / NHS method, then add 1.5 mg of iRGD polypeptide molecules, shake at room temperature for 25 min, and purify by magnetic separation to obtain a magnetic nanoprobe, and dilute to 10 mL with pure water, and store at 4°C (sample 3).

[0057] The synthesis process of the magnetic nanoprobe is as shown in Figure 2 The sample 3 is observed by transmission electron microscopy, as shown in Figure 3 The magnetic nanoprobe is uniform in size and well dispersed, with a diameter of about 20 nm.

[0058] Example 5 BBB model transcytosis study

[0059] Referring to the construction schematic diagram of Figure 4 , a BBB model is constructed in vitro, and a Transwell chamber with a pore size of 4 μm is used Using a transwell chamber as the basic framework, mouse brain vascular endothelial cells bEnd.3 (purchased from ATCC) were cultured on the inner side of the bottom layer, while mouse brain astrocytes C8-D1A (purchased from ATCC) were cultured on the outer side. The BBB model was considered successfully constructed when the resistance between the inner and outer layers reached 100 ohms. BBB model regulation experiments were then conducted. 100 μg of magnetic nanoprobes (prepared in Example 2) were added to each well of the transwell chamber. After targeting for 1 hour, the BBB model was placed in the center of a low-frequency alternating magnetic field coil for 10 minutes. The parameters were: magnetic field strength 20 Oe, frequency 30 Hz, coil turns 20, and coil diameter 5 cm. After the BBB model regulation process, 50 μg of the fluorescent tracer FITC-Dextran (4k, 70k, 500k, 2000k) was added to each well of the transwell chamber. After 10 minutes, 50 μL of the culture medium outside the transwell chamber was extracted, and the BBB regulation was examined using a fluorescence spectrophotometer. The differences in the expression of caveolin and tight junction protein in endothelial cells before and after regulation were observed by immunofluorescence staining.

[0060] Example 6: Transcytosis Study using the BBB Model

[0061] See Figure 4 A schematic diagram of the construction process, showing the in vitro construction of a BBB model using Transwell chambers with a pore size of 3 μm. Using a transwell chamber as the basic framework, mouse brain vascular endothelial cells (bEnd.3) were cultured on the inner side of the bottom layer, while mouse brain astrocytes (C8-D1A) were cultured on the outer side. The BBB model was successfully established when the resistance between the inner and outer layers reached 100 ohms. BBB model regulation experiments were conducted. 150 μg of magnetic nanoprobes (prepared in Example 3) were added to each well of the transwell chamber. After targeting for 30 min, the BBB model was placed in the center of a low-frequency alternating magnetic field coil for 5 min. The parameters were: magnetic field strength 30 Oe, frequency 50 Hz, coil turns 30, and coil diameter 15 cm. After the BBB model regulation process, 100 μg of the fluorescent tracer FITC-Dextran (4k, 70k, 500k, 2000k) was added to each well of the transwell chamber. After 20 min, 100 μL of the culture medium outside the chamber was extracted, and the BBB regulation was examined using a fluorescence spectrophotometer. Immunofluorescence staining was used to observe the differences in the expression of cavernin and tight junction proteins in endothelial cells before and after regulation.

[0062] Example 7: Transcytosis Study using the BBB Model

[0063] See Figure 4 A schematic diagram of the construction process, showing the in vitro construction of a BBB model using Transwell chambers with a pore size of 1 μm. Company) as the basic framework, mouse brain vascular endothelial cells bEnd.3 were cultured in the inner side of the chamber bottom layer, and mouse brain astrocytes C8-D1A were cultured in the outer side of the bottom layer. When the inner and outer resistance values reached 100 ohms, it indicated that the BBB model was successfully constructed. The BBB model regulation experiment was carried out. 50 μg of magnetic nanoprobe (prepared in Example 4) per well was added to the transwell chamber, and after 1.5 h of targeting, the BBB model was placed in the center of the low-frequency alternating magnetic field coil for 8 min. The parameters were magnetic field strength 40 Oe, frequency 20 Hz, coil turns 50 turns, and coil diameter 10 cm. After the BBB regulation process was completed, 150 μg of fluorescent tracer FITC-Dextran (4k, 70k, 500k, 2000k) per well was added to the transwell chamber, and 80 μL of the outer side culture medium was extracted after 30 min. The BBB regulation was investigated by fluorescence spectrophotometer. The expression differences of caveolin-1 and tight junction protein in endothelial cells before and after regulation were observed by immunofluorescence staining.

[0064] The BBB model in Example 7 was regulated, and the process was as shown in Figure 4 After the low-frequency alternating magnetic field was applied, the permeability of the BBB model changed, as shown in Figure 5 The fluorescence intensity of FITC-Dextran of different molecular weights across the BBB model showed obvious enhancement. From Figure 6 immunofluorescence staining, it was found that the expression of caveolin-1 caveolin protein in the vascular endothelial cells of the regulated BBB model increased significantly. This experiment showed that the oscillation of the magnetic nanoprobe induced the high expression of caveolin protein, which further affected the permeability of the BBB. Figure 7 The expression of tight junction protein claudin 5 did not change before and after regulation, indicating that the tight junction in the basic structure of the BBB was not significantly affected.

[0065] Application of Example 8 in vivo BBB transcytosis process regulation

[0066] The mouse tail vein was injected with 20 mg / kg Fe magnetic nanoprobe (prepared in Example 4), and the auxiliary targeting magnet with a magnetic field gradient of 60 T / m was placed in the mouse brain for 10 min. Then the head of the anesthetized mouse was placed in the center of the low-frequency alternating magnetic field coil, and the magnetic field strength was 40 Oe, the frequency was 40 Hz, the coil turns were 50 turns, and the coil diameter was 15 cm. The low-frequency alternating magnetic field was applied for 8 min. After the BBB regulation process was completed, 5 mg / kg of Evans Blue solution was injected into the tail vein, and the mouse brain was observed for blue signals after half an hour. The production of caveolin protein was observed by freezing section and ultrathin section.

[0067] The BBB regulation process in Example 8 was investigated, as shown in Figure 8As shown, BBB permeability changed after the low frequency alternating magnetic field, Evans Blue signal was found in normal brain parenchyma, and the BBB permeability was significantly increased Figure 9 The immunofluorescence staining showed that the expression of caveolin-1 caveolae protein in the BBB vascular endothelial cells was significantly increased after the adjustment; from Figure 10 As can be seen, the bioelectric microscope found that a large number of caveolae protein vesicles were found in the BBB vascular endothelial cells after the adjustment, and no relevant changes were found in the unadjusted control group. It can be seen that the oscillation of the magnetic nano probe under the low frequency alternating magnetic field increases the caveolae protein-mediated transcytosis in the BBB vascular endothelial cells, thereby significantly enhancing the BBB permeability. The schematic diagram of the process principle is shown in Figure 11 .

Claims

1. A method for preparing ZnFe2O4@CMD nanoparticles, characterized in that, The method comprises the following steps: adding ZnFe2O4 magnetic nanoparticles into a tetramethylammonium hydroxide solution under first stirring, continuing second stirring, removing excess oleic acid molecules after the reaction, dispersing the product in deionized water, and then adding the product drop by drop into a CMD aqueous solution under third stirring, and obtaining the product by purification after the reaction, wherein the first stirring and the second stirring are at a speed of 400-600 r / min, the mass concentration of the tetramethylammonium hydroxide solution is 15-30%, the second stirring is continued for 20-60 min, the third stirring is at a speed of 600-800 r / min, and the initial concentration of the CMD aqueous solution is 1-3 mg / mL.

2. A method for preparing magnetic nanoprobe, characterized in that, The preparation method of the magnetic nanoprobe is that the ZnFe2O4@CMD nanoparticles prepared in claim 1 are activated, then are added into iRGD polypeptide molecules for reaction, and then are purified by magnetic separation.

3. The method for preparing the magnetic nanoprobe according to claim 2, characterized in that, The mass ratio of the iRGD polypeptide molecules to the ZnFe2O4@CMD nanoparticles is 0.5-1.5:10, and the reaction condition is that the reaction is carried out at room temperature on a 100-150 r / min shaking table for 25-60 min.

4. The ZnFe2O4@CMD nanoparticles prepared by the method of claim 1, or the magnetic nanoprobe prepared by the method of claim 2 or 3.

5. The application of the ZnFe2O4@CMD nanoparticles or the magnetic nanoprobe of claim 4 in the preparation of a drug for treating diseases of the central nervous system.

6. Use according to claim 5, characterized in that, The application comprises targeting the magnetic nanoprobe to the blood-brain barrier to regulate transcytosis.

7. A method for regulating transcytosis of the blood-brain barrier, characterized by, The method comprises the following steps: 1) preparing the magnetic nanoprobe by the method of claim 2; 2) constructing an in-vitro BBB model; 3) placing the in-vitro BBB model in a low-frequency alternating magnetic field generator, and completing the regulation of BBB transcytosis after the magnetic field is turned on for a certain period of time.

8. The method of claim 7, wherein the step of regulating comprises, In step 2), the endothelial cells used for constructing the BBB model are bEnd.3, the astrocytes are C8-D1A, the mass of the added magnetic nanoprobe is 50-150 μg / well, and the required targeting time is 0.5-1.5 h.

9. The method of claim 7, wherein the step of regulating is performed by a processor. In step 3), the basic parameter range of the low-frequency alternating magnetic field controller is as follows: magnetic field strength: 10-100 Oe, frequency: 10-50 Hz, coil parameter: number of turns: 10-100 turns, diameter: 5-15 cm, and circulating water cooling.

10. The method of claim 7, wherein the step of regulating comprises, In step 3), the action time of the low-frequency alternating magnetic field is 1-30 min.

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