Method for improving electrophysiological activity of myocardial cells based on molecular thermal vibration

Abstract

The invention discloses a method for improving electrophysiological activity of myocardial cells based on molecular thermal vibration. The method comprises the following steps: culturing purified animal myocardial cells on a cell electrophysiological sensor so as to establish a chronic arrhythmia model of an in-vitro myocardial cell type, and constructing a photo-thermal sensing regulation and control platform for improving the electrophysiological activity of the myocardial cells on the basis of the chronic arrhythmia model. After the small organic molecules with the photothermal effect are added in a bionic drug mode, light energy can be quickly converted into heat energy under irradiation of near-infrared light, so that the temperature of a myocardial cell culture system is increased, myocardial cells are stimulated to react, and then the electrophysiological activity of the myocardial cells is improved. The myocardial cell electrophysiological signals before irradiation, during irradiation and after irradiation are recorded, and the change of the characteristic parameters of the electrophysiological signals is analyzed, so that the regulation and control effect is evaluated; and a brand new research thought is provided for bionic medicines and precise biomedical treatment.

Description

Technical Field

[0001] The invention relates to photothermal sensing medical technology, and in particular to a method for improving the electrophysiological activity of myocardial cells based on molecular thermal vibration. Background Art

[0002] Slow arrhythmias are the leading cause of morbidity and mortality from cardiovascular diseases, and are characterized by being hidden in the early stages, gradually worsening in the middle stages, and potential complications in the late stages. In traditional clinical treatment, non-invasive drug therapy can effectively intervene in slow arrhythmias in the early stages, but potential drug side effects (such as nausea, insomnia, and depression) limit the long-term use of this therapy. For patients in the middle and late stages, invasive surgical treatments such as implantable cardioverter-defibrillators and radiofrequency catheter ablation can effectively treat slow arrhythmias. However, invasive surgery, potential allergic reactions, and complications limit the widespread application of this strategy. Therefore, there is an urgent need to explore a universally effective strategy for the treatment of chronic arrhythmias.

[0003] In recent years, photothermal therapy has broad application prospects in the precision treatment of diseases due to its low toxicity, strong specificity, low invasiveness, and fast recovery. Especially for excitable cells, their electrophysiological activities can be regulated by regulating the type and structure of photothermal agents. Among the many photothermal agents, gold-based nanomaterials, as representatives of metal structures, are one of the most promising drugs in photothermal technology due to their unique physical and chemical properties, such as structural specificity and adjustable absorption peaks.

[0004] However, various treatment strategies based on photothermal technology to improve the electrophysiological activity of cardiomyocytes in the existing technologies still have various shortcomings. For example, gold nanoparticles have variable shapes and uncertain toxicity, making it difficult for them to be approved as clinically safe drugs by the U.S. Food and Drug Administration (FDA); existing experimental operations usually need to be based on animal experiments, with high R&D costs and low efficiency; cardiomyocytes maintain normal electrophysiological activity for a short time after photothermal treatment; and it is difficult to monitor the treatment effect in real time, etc.

[0005] Therefore, compared with directly testing gold nanomaterials on animals, it is obviously in line with actual needs to develop an experimental technology with low overall cost, simple operation, and stable efficacy, and on this basis conduct experiments on using photothermal technology to improve the electrophysiological activity of cardiomyocytes. Summary of the invention

[0006] The purpose of the present invention is to provide an experimental method for improving the electrophysiological activity of cardiomyocytes based on molecular thermal vibration in view of the potential toxicity and low therapeutic efficiency of photothermal agents in existing photothermal technologies. This method can not only effectively regulate the electrophysiological activity of cardiomyocytes, but also maintain the restored normal activity for a longer period of time.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for improving the electrophysiological activity of cardiomyocytes based on molecular thermal vibrations. The method is based on a platform for improving the electrophysiological activity of cardiomyocytes based on molecular thermal vibrations. The platform includes a cardiomyocyte biosensor system based on a microelectrode array and a photothermal control device; wherein:

[0009] The myocardial cell biosensor system based on microelectrode array comprises a cell electrophysiological sensor device, a signal acquisition module and a host computer; the cell electrophysiological sensor device comprises a hollow glass culture chamber, a microelectrode array chip and a PCB adapter; the signal acquisition module is connected to the host computer through a signal line;

[0010] The photothermal control device comprises a power supply, a near-infrared laser and a near-infrared laser output end; the near-infrared laser output end is fixed directly above the cell electrophysiological sensor device, and the two maintain a distance; the power supply is connected to the near-infrared laser through a cable, thereby regulating the laser power of the laser output end;

[0011] The method comprises the following steps:

[0012] (1) Build a photothermal sensing control platform:

[0013] The cardiomyocyte biosensor system based on the microelectrode array is connected to the photothermal control device, and the near-infrared laser is connected to the near-infrared laser output end; the laser light source output end is fixed above the cell electrophysiological sensor device, and the laser spot is aligned with the central position of the cell electrophysiological sensor device;

[0014] (2) Cultivation of primary cardiomyocytes:

[0015] The cell electrophysiological sensor device is sterilized by ultraviolet light; the isolated and purified primary cardiomyocytes are planted in the culture chamber of the sensor device in the form of a cell suspension, and then continue to be cultured in an incubator;

[0016] (3) Analysis of the photothermal conversion effect of molecular thermal vibration:

[0017] The photothermal agent is prepared into solutions of different concentrations, and the photothermal performance is continuously optimized by adjusting the energy of the laser under near-infrared laser irradiation;

[0018] (4) Evaluation of electrophysiological activity of cardiomyocytes:

[0019] The culture medium containing the photothermal agent is added to the cell culture chamber; the power and irradiation time of the near-infrared laser are adjusted, and the electrophysiological signals detected by the sensor device are recorded in real time; after collecting sufficient signals, the characteristic parameters of the signals within a certain period of time are extracted, and their emission frequency and amplitude are calculated and statistical curves are drawn to evaluate the effects of molecular thermal vibrations under different conditions on the electrophysiological activities of cardiomyocytes.

[0020] Furthermore, in the photothermal sensing and control platform, the distance between the light source output end of the near-infrared laser and the cell electrophysiological sensor device is in the range of 15 to 20 cm.

[0021] Furthermore, the ultraviolet sterilization treatment in step (2) includes: first sterilizing with 75% alcohol, and then irradiating with ultraviolet light for 60 to 120 minutes.

[0022] Specifically, the planting density of primary cardiomyocytes in step (2) is 3.0×10 5 ~5.0×10 5 cells / cm 2 The culture conditions of the cells in the incubator were: temperature 37°C, culture atmosphere 5.0% (v / v) CO2, and the culture medium was replaced every 48 hours.

[0023] Specifically, the photothermal agent in step (3) is indocyanine green which utilizes molecular thermal vibration to perform photothermal conversion; its concentration range is 10 to 40 μg / mL.

[0024] Specifically, the power of the near-infrared laser in step (3) is 0.0 to 1.5 W / cm 2 ; The irradiation time is 30s to 120s.

[0025] Specifically, in the step (4), after collecting a sufficient amount of signals, characteristic parameters of the signals within a certain period of time are extracted, and the characteristic parameters are the emission frequency and amplitude.

[0026] Specifically, in step (4), a culture medium containing a photothermal agent is added to the cell culture chamber. After the photothermal agent is added and the treatment begins, the electrophysiological signals of the cardiomyocytes are recorded continuously for 0 to 600 minutes, characteristic parameters in the signals are extracted and counted at set intervals, and the emission frequency and amplitude of the electrical signals are calculated.

[0027] The beneficial effect of the present invention is that the experiment of the present invention is simple and efficient, can be reproduced in large quantities, and the experimental effect can be quickly and efficiently evaluated in the form of experimental model simulation. The present invention selects small molecules with good biocompatibility and high photothermal conversion efficiency as photothermal agents, reduces the potential toxicity of photothermal agents, and improves the implementation efficiency of molecular thermal vibrations, thereby achieving effective regulation of the electrophysiological activity of cardiomyocytes. By analyzing the changes in the electrophysiological signals of cardiomyocytes before and after photothermal treatment, the applicant found that the longest time for the rhythmicity of its electrophysiological signals can reach 600 minutes, which is 2 to 3 times the results achieved by the applicant's research team in previous work (gold nanoelectrode arrays and gold nanorods of different lengths). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0029] Figure 1 It is a structural schematic diagram of the photothermal sensing control platform of the present invention;

[0030] Figure 2 This is a diagram showing changes in electrophysiological signals of myocardial cells before and after the molecular thermal vibration treatment of the present invention;

[0031] Figure 3 It is a statistical diagram of characteristic parameters of electrophysiological signals of myocardial cells of the present invention;

[0032] Figure 4 It is a long-term recording diagram of electrophysiological signals of myocardial cells that restore rhythmicity according to the present invention;

[0033] Figure 5 It is a statistical diagram of the maintenance time of the rhythmic electrophysiological signal of the present invention.

[0034] Figure numerals: cell electrophysiological sensor device-1, signal acquisition module-2, host computer-3, photothermal control device-4. DETAILED DESCRIPTION

[0035] The structure of the cell electrophysiological sensor device described in the present invention and its preparation and use methods are all based on the applicant's previous work results. For details, please refer to the disclosure of the patent document "A sensor detection method and system for myocardial cell excitation-contraction coupling" (CN115078466A) for the description of "nano-microelectrode array device". Those skilled in the art can make detailed adjustments according to the specific experimental scheme design when reproducing the technical solution of the present invention, and the present invention will not repeat them.

[0036] The implementation of the present invention is described in detail below in conjunction with the accompanying drawings, but the present invention is not limited thereto.

[0037] 1. Experimental device of the present invention

[0038] like Figure 1As shown, the present invention provides a platform for improving the electrophysiological activity of cardiomyocytes based on molecular thermal vibration, including a cardiomyocyte biosensor system based on a microelectrode array and a photothermal control device 4; wherein, the cell electrophysiological sensor device 1 is a technology recorded in existing public documents, including a hollow glass culture chamber, a microelectrode array chip and a PCB adapter; the cell electrophysiological sensor device is above the signal acquisition module 2, and the two are connected through a PCB adapter; the acquisition module and the host computer 3 are connected through a USB cable. The structure and processing method of the sensor device can be specifically referred to the patent disclosure CN115078466A. The cardiomyocyte biosensor system based on a microelectrode array includes a signal acquisition module 2 and a host computer 3; the signal acquisition module 2 is connected to the host computer 3 through a signal line. The photothermal control device 4 includes a power supply, a near-infrared laser and a laser output end; the light source output end is fixed above the glass culture chamber of the cell electrophysiological sensor device 1, and the two maintain a distance; the power supply is connected to the near-infrared laser in the photothermal control device 4 through a cable, and the laser power at the collimator output end is regulated by providing an adjustable constant current.

[0039] 2. Experimental Methods of the Present Invention

[0040] Based on the above experimental platform, the present invention realizes a method for improving the electrophysiological activity of cardiomyocytes based on molecular thermal vibration, which specifically includes the following steps:

[0041] (1) Build a photothermal sensing experimental device

[0042] The cardiomyocyte biosensor system based on the microelectrode array is connected to the photothermal control device 4, and the near-infrared laser is connected to the light source output end; the laser light source output end is fixed 15 to 20 cm above the cell electrophysiological sensor device 1, and the laser spot is aligned with the central position of the cell electrophysiological sensor device 1.

[0043] (2) Cultivation of primary cardiomyocytes

[0044] The cell electrophysiological sensor device 1 is sterilized by ultraviolet light; the isolated and purified primary cardiomyocytes are planted in the culture chamber of the sensor device in the form of a cell suspension, and then continue to be cultured in an incubator.

[0045] As an example, the sterilization treatment is first sterilized with 75% ethanol solution, and then irradiated with ultraviolet light for 60 to 120 minutes. The planting density of the cardiomyocyte suspension in the culture chamber is 3.0×10 5 ~5.0×10 5 cells / cm 2 During the implantation process, ensure that the cardiomyocytes are evenly covered on the surface of the nano-microelectrode array chip and can fuse into a single layer; at the same time, replace the culture medium every 48 hours to ensure that the cells obtain sufficient nutrition.

[0046] (3) Analysis of the photothermal conversion effect of molecular thermal vibration

[0047] The photothermal agent is prepared into solutions of different concentrations (the higher the concentration of the photothermal agent, the stronger the energy of the laser, the more heat generated, and the better the photothermal performance of the photothermal agent). Under near-infrared laser irradiation, its photothermal performance is optimized by adjusting the energy of the laser.

[0048] As an example, the concentration of the photothermal agent is 10 μg / mL and the power of the near-infrared laser is 1.0 W / cm 2 , use a near-infrared thermal imager to record its temperature changes every 10 seconds to facilitate the evaluation of photothermal performance.

[0049] As an example, the concentration of the photothermal agent is 20 μg / mL and the power of the near-infrared laser is 1.0 W / cm 2 , use a near-infrared thermal imager to record its temperature changes every 10 seconds to facilitate the evaluation of photothermal performance.

[0050] As an example, the concentration of the photothermal agent is 40 μg / mL and the power of the near-infrared laser is 1.0 W / cm 2 , use a near-infrared thermal imager to record its temperature changes every 10 seconds to facilitate the evaluation of photothermal performance.

[0051] (4) Evaluation of electrophysiological activity of cardiomyocytes

[0052] According to the experimental design, organic small molecule solutions of different concentrations are added to the cardiomyocyte culture chamber. As an example, the organic small molecule is indocyanine green, and the concentration range is 10-40 μg / mL.

[0053] The irradiation time and laser power of the near-infrared laser are adjusted, and the electrophysiological signals are recorded in real time. As an example, the irradiation time of the near-infrared laser is 30 to 90 seconds; the laser power is 0.0 to 1.5 W / cm 2 .

[0054] After the illumination experiment, the electrophysiological signals of cardiomyocytes were recorded continuously for 0 to 600 minutes, and the characteristic parameters in the signals were extracted at set intervals. After recording enough signals, the characteristic parameters (i.e., the emission frequency and amplitude) were extracted and a correlation curve was drawn to evaluate the effect of molecular thermal vibration on improving the electrophysiological activity of cardiomyocytes.

[0055] Example

[0056] 1. Fabrication and assembly of cell electrophysiological sensor devices

[0057] A 4-inch quartz glass substrate was used as the substrate, and the positive photoresist was spin-coated to determine the conductive pattern. The Au / Ti (100 / 10 nm) layer was deposited as the electrode material by magnetron sputtering, and then a 2 μm insulating layer was formed by SU-8. The processed glass substrate was cut into 25 2×2 cm 2 For device assembly, a chip containing 32 microelectrodes was fixed on a custom printed circuit board (PCB) adapter with polydimethylsiloxane (PDMS, Sylgard 184), and the electrode pads on the chip were electrically connected to the PCB pads via wire bonding. A glass ring (diameter: 1.5 cm; height: 1.0 cm) was then glued to the chip with PDMS to culture cells, and a pin header connector was soldered on the above PCB to match the cell biosensing system. After rinsing with 75% ethanol and UV sterilization for 2 h, it was coated with fibronectin (10 μg / mL) and placed at 37 °C, 5.0% CO 2 The cells were cultured in an incubator for 6 h before myocardial cell culture.

[0058] 2. Isolation and culture of primary cardiomyocytes

[0059] Primary cardiomyocytes were obtained from 1- to 3-day-old newborn rats. After disinfection with 75% ethanol, the rat ventricular tissue was quickly removed and then rinsed three times in ice-cold Dulbecco's Modified Eagle Medium (DMEM). The obtained tissue was cut into approximately 1 mm pieces in Hanks' Balanced Salt Solution (HBSS). 3 Subsequently, incubate at 37°C, 5.0% CO 2 The cells were then redispersed in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cell suspension was centrifuged at 800 rpm for 5 min, purified with a 70 μm cell sieve, and adhered to the wall at a differential speed for 45 min twice. Finally, the cells were plated at 3.2 × 10 5 cells / cm 2 The density of the culture medium was 37℃ and 5.0% CO. 2 The cell culture medium was renewed every 48 hours to ensure sufficient energy.

[0060] 3. Evaluation of molecular thermal vibration regulation effect

[0061] When the firing frequency drops significantly by 10% to 40% of the highest firing frequency, the electrophysiological activity of cardiomyocytes can be recorded and evaluated through a photothermal sensing control platform based on the molecular thermal vibration of organic small molecules. The specific steps include the following:

[0062] 3.1 Record the electrophysiological signals of myocardial cells whose firing frequency decreased significantly for 3 minutes as a control;

[0063] 3.2 Preheat 20 μg / mL ICG solution to 37°C in a water bath and add it to the cell culture chamber. Let it stand for 2 minutes to allow the two to mix thoroughly.

[0064] 3.3 In the photothermal sensing platform, adjust the power of the near-infrared laser to 1.0 W / cm 2 , laser irradiation time was 60s, and the changes of electrophysiological signals during and after treatment were recorded ( Figure 2 ), extract the characteristic parameters of electrophysiological signals ( Figure 3 ), which is convenient for analyzing the treatment effect;

[0065] 3.4 After the myocardial cells resume rhythmic beating, the electrophysiological signals of the myocardial cells were recorded every 10 minutes, with each recording lasting 40 seconds, to facilitate long-term recording and evaluation of the electrophysiological activity of myocardial cells (e.g. Figure 4 and 5 shown).

[0066] 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 method for improving the electrophysiological activity of cardiomyocytes based on molecular thermal vibration, characterized in that: The method is based on a platform for improving the electrophysiological activity of cardiomyocytes based on molecular thermal vibrations, which includes a cardiomyocyte biosensor system based on a microelectrode array and a photothermal control device; The myocardial cell biosensor system based on microelectrode array comprises a cell electrophysiological sensor device, a signal acquisition module and a host computer; the cell electrophysiological sensor device comprises a hollow glass culture chamber, a microelectrode array chip and a PCB adapter; the signal acquisition module is connected to the host computer through a signal line; The photothermal control device comprises a power supply, a near-infrared laser and a near-infrared laser output end; the near-infrared laser output end is fixed directly above the cell electrophysiological sensor device, and the two maintain a distance; the power supply is connected to the near-infrared laser through a cable, thereby regulating the laser power of the laser output end; The method comprises the following steps: (1) Build a photothermal sensing control platform: The cardiomyocyte biosensor system based on the microelectrode array is connected to the photothermal control device, and the near-infrared laser is connected to the near-infrared laser output end; the laser light source output end is fixed above the cell electrophysiological sensor device, and the laser spot is aligned with the central position of the cell electrophysiological sensor device; (2) Cultivation of primary cardiomyocytes: The cell electrophysiological sensor device is sterilized by ultraviolet light; the isolated and purified primary cardiomyocytes are planted in the culture chamber of the sensor device in the form of a cell suspension, and then continue to be cultured in an incubator; (3) Analysis of the photothermal conversion effect of molecular thermal vibration: The photothermal agent is prepared into solutions of different concentrations, and the photothermal performance is continuously optimized by adjusting the energy of the laser under near-infrared laser irradiation; (4) Evaluation of electrophysiological activity of cardiomyocytes: The culture medium containing the photothermal agent is added to the cell culture chamber; the power and irradiation time of the near-infrared laser are adjusted, and the electrophysiological signals detected by the sensor device are recorded in real time; after collecting sufficient signals, the characteristic parameters of the signals within a certain period of time are extracted, and their emission frequency and amplitude are calculated and statistical curves are drawn to evaluate the effects of molecular thermal vibrations under different conditions on the electrophysiological activities of cardiomyocytes.

2. The method according to claim 1, characterized in that In the photothermal sensing and control platform, the distance between the light source output end of the near-infrared laser and the cell electrophysiological sensor device is in the range of 15 to 20 cm.

3. The method according to claim 1, characterized in that The ultraviolet sterilization treatment in step (2) includes: first sterilizing with 75% alcohol, and then irradiating with ultraviolet light for 60 to 120 minutes.

4. The method according to claim 1, characterized in that: The planting density of primary cardiomyocytes in step (2) is 3.0×10 5 ~5.0×10 5 cells / cm 2 The culture conditions of the cells in the incubator were: temperature 37°C, culture atmosphere 5.0% (v / v) CO2, and the culture medium was replaced every 48 hours.

5. The method according to claim 1, characterized in that The photothermal agent in step (3) is indocyanine green which utilizes molecular thermal vibration to perform photothermal conversion; its concentration range is 10 to 40 μg / mL.

6. The method according to claim 1, characterized in that The power of the near-infrared laser in step (3) is 0.0 to 1.5 W / cm 2 ; The irradiation time is 30s to 120s.

7. The method according to claim 1, characterized in that In the step (4), after collecting a sufficient amount of signals, characteristic parameters of the signals within a certain period of time are extracted, and the characteristic parameters are the emission frequency and amplitude.

8. The method according to claim 1, characterized in that In the step (4), a culture medium containing a photothermal agent is added to the cell culture chamber. After the photothermal agent is added and the treatment begins, the electrophysiological signals of the cardiomyocytes are recorded continuously for 0 to 600 minutes, characteristic parameters in the signals are extracted and counted at set intervals, and the emission frequency and amplitude of the electrical signals are calculated.

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