Nanometer plasma photo-thermal treatment method and device capable of regulating and controlling simulated medicine

Abstract

The invention discloses a nano plasma photo-thermal treatment method and device capable of regulating and controlling a simulated drug. The method comprises the steps of microelectrode array device preparation, chronic arrhythmia model establishment and nano plasma photo-thermal treatment. A parameter-adjustable photo-thermal therapy system is established by combining a microelectrode array device, a gold nanorod, an electrophysiological recording instrument and a near-infrared laser, electrophysiological signals of myocardial cells are monitored synchronously, and the photo-thermal therapy effect is evaluated. The size and concentration of the gold nanorods and the intensity and duration of near-infrared laser irradiation are regulated and controlled, efficient and long-time chronic arrhythmia treatment is achieved, and an effective and simple method and system are provided for research of cardiovascular diseases.

Description

Technical Field

[0001] The present invention relates to a photothermal therapy method, and in particular to a nanoplasma photothermal therapy method and a device capable of regulating and simulating drugs. Background Art

[0002] Chronic arrhythmias are characterized by slow heart rates and may be caused by a variety of pathological or physiological factors, including coronary heart disease, cardiomyopathy, conduction system disorders, and drug effects. Approximately 1 in 1,000 people worldwide suffer from chronic arrhythmias, with symptoms ranging from mild dizziness and fatigue to severe heart failure and sudden cardiac death, which imposes a significant psychological and economic burden on patients. With the aging population, chronic arrhythmias are increasingly putting pressure on the medical system, and effective prevention and treatment strategies are urgently needed to alleviate this growing threat of disease. Currently, the treatment strategies for chronic arrhythmias are mainly divided into drug therapy and interventional therapy. Drug options include atropine, which increases heart rate by antagonizing vagal nerve activity, and isoproterenol, which enhances myocardial contractility and conduction velocity through β-receptor activation. Drug therapy is non-invasive and simple to operate, but has limited efficacy, and long-term use may lead to drug resistance or adverse reactions. Interventional therapy usually involves the implantation of a pacemaker, which monitors the heartbeat and delivers electrical impulses to maintain a normal heart rate. This method is stable, durable, and suitable for long-term treatment, but it carries surgical risks, potential equipment failure, and high maintenance costs.

[0003] In recent years, the potential of photothermal therapy in regulating cardiomyocyte activity and treating heart disease has received increasing attention. Photothermal therapy uses light absorbers (such as gold nanoparticles, carbon nanotubes, or organic dyes) to convert light energy into heat energy under irradiation of specific wavelengths. This process can precisely control the local temperature, thereby affecting the electrophysiological activity and rhythm of cardiomyocytes. Among them, gold nanorods have become a prominent choice for photothermal therapy due to their chemical inertness and good biocompatibility. When near-infrared lasers match their main absorption spectrum, gold nanorods increase the local temperature through the plasma photothermal effect. The increased temperature can stimulate proteins in cardiomyocytes and regulate the excitability and conduction properties of cardiomyocytes. However, research on the regulation of cardiomyocyte rhythm by photothermal therapy is still in its early stages, and its long-term efficacy and potential mechanisms still need to be further verified and explored. Summary of the invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a nanoplasma photothermal therapy method and device that can regulate and simulate drugs, aiming to address the adverse reactions of existing chronic arrhythmia treatment strategies. The system can not only perform photothermal regulation on myocardial cells with chronic arrhythmia, but also can synchronously monitor the electrophysiological signals of myocardial cells in real time.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention is a method for controlling the photothermal treatment of a nanoplasma that simulates drugs, the method comprising the following steps:

[0007] (1) preparing a microelectrode array by microfabrication technology and assembling it with a customized printed circuit board and a glass culture chamber into a microelectrode array device;

[0008] (2) Primary cardiomyocytes were isolated and extracted from neonatal rats and cultured on microelectrode array devices. A chronic arrhythmia model was established after 6-8 days of culture.

[0009] (3) Adding gold nanorods to the cell culture medium and irradiating with near-infrared laser to utilize the nanoplasmonic photothermal effect to treat myocardial cells with chronic arrhythmia.

[0010] Furthermore, the step (1) prepares the microelectrode array, specifically: using a 6-inch quartz wafer with a thickness of 0.5 mm, ultrasonically cleaned with acetone and ethanol in sequence as a substrate, spin-coating AZ nLOF 2035 photoresist, then baking, ultraviolet exposure, and developing; and using magnetron sputtering to deposit a 10-nanometer titanium layer and a 100-nanometer gold layer, and then peeling in acetone to prepare a metal layer electrode; then, using photolithography SU-8 as an insulating layer and defining the effective site of the electrode; and then cutting the quartz wafer into 25 pieces, each with a size of 2×2 cm 2 , and a single microelectrode array is fixed on a printed circuit board for electrophysiological detection, and a glass culture chamber is bonded on top for cell culture.

[0011] Specifically, the substrate is a glass, silicon oxide or aluminum oxide substrate, the number of electrodes is 32 or 64, the thickness of the insulating layer is 3-5 μm, and the diameter of the electrode site is 10-30 μm.

[0012] Furthermore, the step (2) of establishing a chronic arrhythmia model is specifically as follows:

[0013] First, the microelectrode array device was sterilized, and then 10 μg / mL fibronectin solution was added to enhance cell adhesion. One-day-old SD rats were sterilized with 75% ethanol, and the ventricular muscle tissue was removed. The removed ventricular muscle tissue was rinsed in DMEM and then chopped into 1 mm pieces in HBSS. 3 The tissue fragments were repeatedly digested in HBSS containing 0.07% trypsin and 0.05% type II collagenase; the digested tissue was blown off, centrifuged, and resuspended; then filtered through a 70 μm cell sieve and subjected to two 45-minute differential adhesions in a 37°C, 5% CO2 incubator; finally, the purified cardiomyocytes were cultured at approximately 3×10 5The cells were seeded at a density of 100 / mL on each device, and the culture medium was replaced every 48 hours. After 7 days of culture, chronic arrhythmic cardiomyocytes were obtained.

[0014] Specifically, the culture density of the obtained cardiomyocytes on the microelectrode array device is 3×10 5 ~4×10 5 Pieces / mL.

[0015] Furthermore, the step (3) of treating myocardial cells with chronic arrhythmia by nanoplasma photothermal effect is specifically as follows:

[0016] Gold nanorods with adjustable size and concentration and near-infrared lasers with adjustable intensity and duration are used for photothermal therapy of cardiomyocytes with chronic arrhythmia. First, a gold nanorod solution is added to the cell culture medium, and the amount of the gold nanorod solution added is 1 / 9 of the cell culture medium. Then, an 808nm near-infrared laser is placed above the sample solution to vertically irradiate the culture solution. The temperature change during the irradiation process is monitored by a visual thermal imager, and the electrophysiological signals of the cardiomyocytes are detected in real time by an electrophysiological recording instrument. The aspect ratio and concentration of the gold nanorods are adjusted respectively, and laser irradiation is used to complete the plasma photothermal therapy.

[0017] Specifically, when the nanoplasma photothermal effect is used to treat myocardial cells with chronic arrhythmia, the aspect ratio of the gold nanorods is 8:1 to 8:4, and the concentration is 10 to 40 μg / mL.

[0018] Specifically, the intensity of the near infrared is 0.5 to 1.5 W / cm 2 The irradiation time is 3-7 minutes.

[0019] The second aspect of the present invention is a nanoplasma photothermal therapy device that can regulate and simulate drugs, and the device includes the following modules:

[0020] Preparation of microelectrode array modules: They are prepared by microfabrication technology and assembled into microelectrode array devices with customized printed circuit boards and glass culture chambers;

[0021] Establishment of chronic arrhythmia model module: primary cardiomyocytes were isolated and extracted from newborn rats and cultured on microelectrode array devices. After 6-8 days of culture, a chronic arrhythmia model was established.

[0022] Nanoplasma photothermal effect module: Add gold nanorods to the cell culture medium and use near-infrared laser irradiation to treat myocardial cells with chronic arrhythmia using the nanoplasma photothermal effect.

[0023] The beneficial effects of the present invention are that the treatment method of the present invention is simple, and the adjustable gold nanorods and near-infrared laser irradiation are conducive to optimizing the photothermal treatment effect. The adjustable drug-simulating nanoplasma photothermal treatment method and system of the present invention are an effective and non-invasive chronic arrhythmia treatment strategy, which is of great significance to the study of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0025] Figure 1 is a microscope image of the microelectrode array of the present invention;

[0026] Figure 2 is a schematic diagram of a microelectrode array device of the present invention;

[0027] Figure 3 It is a signal diagram corresponding to the chronic arrhythmia model constructed by the present invention;

[0028] Figure 4 It is a transmission electron microscope image of three aspect ratios of gold nanorods of the present invention;

[0029] Figure 5 is a schematic diagram of a photothermal therapy system of the present invention;

[0030] Figure 6 It is the electrophysiological signal diagram of myocardial cells corresponding to before, during and after the nanoplasma photothermal treatment of the present invention.

[0031] Reference numerals: microelectrode array-1, glass culture chamber-2, printed circuit board adapter-3, near-infrared laser-4, electrophysiological recording instrument-5. DETAILED DESCRIPTION

[0032] Here, exemplary embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application.

[0033] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0034] The present invention provides a nanoplasma photothermal treatment method that can regulate and simulate drugs, including preparation of a microelectrode array device, establishment of a chronic arrhythmia model and nanoplasma photothermal treatment; specifically, the following steps are included:

[0035] (1) preparing a microelectrode array by microfabrication technology and assembling it with a customized printed circuit board and a glass culture chamber into a microelectrode array device;

[0036] (2) Primary cardiomyocytes were isolated and extracted from neonatal rats and cultured on microelectrode array devices. A chronic arrhythmia model was established after 6-8 days of culture.

[0037] (3) Adding gold nanorods to the cell culture medium and irradiating with near-infrared laser to utilize the nanoplasmonic photothermal effect to treat myocardial cells with chronic arrhythmia.

[0038] In addition, the present invention also provides a nanoplasma photothermal therapy device that can regulate and simulate drugs, and the device includes the following modules:

[0039] Preparation of microelectrode array modules: They are prepared by microfabrication technology and assembled into microelectrode array devices with customized printed circuit boards and glass culture chambers;

[0040] Establishment of chronic arrhythmia model module: primary cardiomyocytes were isolated and extracted from newborn rats and cultured on microelectrode array devices. After 6-8 days of culture, a chronic arrhythmia model was established.

[0041] Nanoplasma photothermal effect module: Add gold nanorods to the cell culture medium and use near-infrared laser irradiation to treat myocardial cells with chronic arrhythmia using the nanoplasma photothermal effect.

[0042] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0043] Example 1

[0044] The preparation method of the microelectrode array device comprises the following steps:

[0045] A 6-inch quartz wafer (0.5 mm thick) was ultrasonically cleaned with acetone and ethanol in sequence and used as a substrate. AZnLOF 2035 photoresist was spin-coated, then baked, UV-exposed, and developed to prepare metal layer electrodes. The titanium layer (10 nm) and gold layer (100 nm) were deposited by magnetron sputtering and then peeled off in acetone. Subsequently, photolithographic SU-8 was used as an insulating layer and to define the effective sites of the electrodes. The quartz wafer was then cut into 25 pieces, each with a size of 2×2 cm 2 , Figure 1 The electrode arrangement of a single microelectrode array is shown. Figure 2As shown, a single microelectrode array 1 is fixed on a printed circuit board 3 for electrophysiological detection, and a glass culture chamber 2 is bonded on top for cell culture.

[0046] Example 2

[0047] The establishment of chronic arrhythmia model includes the following steps:

[0048] The microelectrode array device was sterilized, and then fibronectin solution was added to enhance cell adhesion. One-day-old SD rats were sterilized with 75% ethanol, and ventricular muscle tissue was removed. These tissues were rinsed in ice-cold DMEM and then minced into small pieces in ice-cold HBSS. The tissue fragments were repeatedly digested in HBSS containing 0.07% trypsin and 0.05% type II collagenase. The digested tissue was blown off, centrifuged, and resuspended. It was then filtered using a 70μm cell sieve and subjected to two 45-minute differential adhesions in a 37°C, 5% CO2 incubator. Finally, the purified cardiomyocytes were cultured at approximately 3×10 5 The density of cells / mL was sown on each device, and the culture medium was replaced every 48 hours. Figure 3 As shown, after 7 days of culture, cardiomyocytes developed chronic arrhythmias.

[0049] Example 3

[0050] Nanoplasma photothermal therapy method includes the following steps:

[0051] Gold nanorods with adjustable size and concentration and near-infrared lasers with adjustable intensity and duration are used for photothermal therapy of cardiomyocytes with chronic arrhythmias. Figure 4 Shown are gold nanorods with three different aspect ratios. Figure 5 The system shown is for photothermal therapy, including a near-infrared laser 4 and an electrophysiological recording instrument 5. 0.3 mL of gold nanorod solution is added to the cell culture medium, and the amount of the gold nanorod solution added is 1 / 9 of the cell culture medium; then a 700-950 nm near-infrared laser 4 is placed 20.0 cm above the sample solution to vertically irradiate the culture solution; the 808 nm near-infrared laser has the best effect. The temperature change during irradiation is monitored by a visual thermal imager, and the electrophysiological signals of cardiomyocytes are detected in real time by an electrophysiological recording instrument 5. The aspect ratio (8:4, 8:2, 8:1) and concentration (10, 20, 40 μg / mL) of the gold nanorods, as well as the laser irradiation intensity (0.5, 1.0, 1.5 W / cm 2 ) and irradiation time (3, 5, 7min) to optimize the plasma photothermal therapy effect.

[0052] Figure 6This is the effect of nanoplasma photothermal therapy. Before irradiation, the electrophysiological signal emission rate of cardiomyocytes was slow and arrhythmic. During irradiation, the electrophysiological signal emission rate gradually increased, and the cardiomyocytes returned to normal rhythm after irradiation, indicating that the plasma photothermal effect of gold nanorods under near-infrared laser irradiation can effectively treat chronic arrhythmias of cardiomyocytes.

[0053] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A nanoplasma photothermal therapy method that can regulate and simulate drugs, characterized in that: The method comprises the following steps: (1) preparing a microelectrode array by microfabrication technology and assembling it with a customized printed circuit board and a glass culture chamber into a microelectrode array device; (2) Primary cardiomyocytes were isolated and extracted from neonatal rats and cultured on microelectrode array devices. A chronic arrhythmia model was established after 6-8 days of culture. (3) Adding gold nanorods to the cell culture medium and irradiating with near-infrared laser to utilize the nanoplasmonic photothermal effect to treat myocardial cells with chronic arrhythmia.

2. The nanoplasma photothermal therapy method of adjustable simulated drug according to claim 1 is characterized in that: The step (1) prepares the microelectrode array, specifically: using a 6-inch quartz wafer with a thickness of 0.5 mm and ultrasonically cleaned with acetone and ethanol in sequence as a substrate, spin-coating AZ nLOF 2035 photoresist, then baking, ultraviolet exposure, and developing; and using magnetron sputtering to deposit a 10-nanometer titanium layer and a 100-nanometer gold layer, and then peeling them in acetone to prepare a metal layer electrode; then, using photolithography SU-8 as an insulating layer and defining the effective site of the electrode; and then cutting the quartz wafer into 25 pieces, each with a size of 2×2 cm 2 , and a single microelectrode array is fixed on a printed circuit board for electrophysiological detection, and a glass culture chamber is bonded on top for cell culture.

3. The nanoplasma photothermal therapy method of adjustable simulated drug according to claim 2 is characterized in that: The substrate is a glass, silicon oxide or aluminum oxide substrate, the number of electrodes is 32 or 64, the thickness of the insulating layer is 3-5 μm, and the diameter of the electrode site is 10-30 μm.

4. The nanoplasma photothermal therapy method of adjustable drug simulation according to claim 1 is characterized in that: The step (2) of establishing a chronic arrhythmia model is specifically as follows: First, the microelectrode array device was sterilized, and then 10 μg / mL fibronectin solution was added to enhance cell adhesion. One-day-old SD rats were sterilized with 75% ethanol, and the ventricular muscle tissue was removed. The removed ventricular muscle tissue was rinsed in DMEM and then chopped into 1 mm pieces in HBSS. 3 The tissue fragments were repeatedly digested in HBSS containing 0.07% trypsin and 0.05% type II collagenase. The digested tissue was blown off, centrifuged, and resuspended. Then, it was filtered through a 70 μm cell sieve and subjected to two 45-minute differential adhesion tests in a 37°C, 5% CO2 incubator. Finally, the purified cardiomyocytes were cultured at a rate of approximately 3 × 10 5 The cells were seeded at a density of 100 / mL on each device, and the culture medium was replaced every 48 hours. After 7 days of culture, chronic arrhythmic cardiomyocytes were obtained.

5. The nanoplasma photothermal therapy method of adjustable drug simulation according to claim 1 is characterized in that: The culture density of the obtained cardiomyocytes on the microelectrode array device is 3×10 5 ~4×10 5 Pieces / mL.

6. The adjustable drug-simulating nanoplasma photothermal therapy method according to claim 1, characterized in that: The step (3) of treating myocardial cells with chronic arrhythmia by using the nanoplasma photothermal effect is specifically as follows: Gold nanorods with adjustable size and concentration and near-infrared lasers with adjustable intensity and duration are used for photothermal therapy of cardiomyocytes with chronic arrhythmia. First, a gold nanorod solution is added to the cell culture medium, and the amount of the gold nanorod solution added is 1 / 9 of the cell culture medium. Then, an 808nm near-infrared laser is placed above the sample solution to vertically irradiate the culture solution. The temperature change during the irradiation process is monitored by a visual thermal imager, and the electrophysiological signals of the cardiomyocytes are detected in real time by an electrophysiological recording instrument. The aspect ratio and concentration of the gold nanorods are adjusted respectively, and laser irradiation is used to complete the plasma photothermal therapy.

7. The adjustable drug-simulating nanoplasma photothermal therapy method according to claim 1, characterized in that: When the nanoplasma photothermal effect is used to treat myocardial cells with chronic arrhythmia, the aspect ratio of the gold nanorods is 8:1 to 8:4, and the concentration is 10 to 40 μg / mL.

8. The adjustable drug-simulating nanoplasma photothermal therapy method according to claim 1, characterized in that: The intensity of the near infrared is 0.5 to 1.5 W / cm 2 The irradiation time is 3-7 minutes.

9. A nanoplasma photothermal therapy device capable of regulating simulated drugs, characterized in that: The device includes the following modules: Preparation of microelectrode array modules: They are prepared by microfabrication technology and assembled into microelectrode array devices with customized printed circuit boards and glass culture chambers; Establishment of chronic arrhythmia model module: primary cardiomyocytes were isolated and extracted from newborn rats and cultured on microelectrode array devices. After 6-8 days of culture, a chronic arrhythmia model was established. Nanoplasma photothermal effect module: Add gold nanorods to the cell culture medium and use near-infrared laser irradiation to treat myocardial cells with chronic arrhythmia using the nanoplasma photothermal effect.

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