Application of heptamethine indocyanine compound in heart optical mapping

Abstract

The invention discloses an application of a heptamethine indocyanine compound in heart optical mapping. The compound can make fluorescence response to cell transmembrane potential change, and can reflect cell action potential and electric signal conduction of the whole heart when being applied to heart optical mapping as a near-infrared voltage sensitive dye. The dye has strong absorption and emission in a near-infrared region, has the advantages of weak tissue autofluorescence, small scattering, deep tissue penetration depth and the like, and ensures the definition and reliability of cardiac structure imaging; the polymer can be positioned on the surface of a cell membrane, has excellent light stability after being combined with a lipid bilayer, and can perform long-time stable imaging; the hydrogel is high in biocompatibility, low in toxicity and good in affinity to heart tissue, and the use safety is guaranteed. By applying the voltage-sensitive dye, the accuracy, sensitivity and resolution of heart optical mapping are greatly improved, and the voltage-sensitive dye has wide application prospects and important practical value in the field of heart optical mapping.

Description

Technical Field

[0001] The present invention relates to the technical field of optical imaging, and particularly to an application of a heptamethine indocyanine compound in cardiac optical mapping. Background Art

[0002] Optical Mapping of cardiac electrical activity is a new functional imaging technique that converts changes in myocardial cell membrane potential into optical signals for recording. It can study the conduction of cardiac electrical excitation at the cellular level through the action potential of myocardial cells and its electrical properties of propagation between cells. This technique is of great significance for studying the pathogenesis of heart diseases such as arrhythmia. Voltage sensitive dyes (VSDs), also known as molecular probes or optical probes, can bind to cell membranes and change their own fluorescence or absorbance characteristics with the change of the local electric field, and this change has a linear relationship with the membrane potential change recorded by electrodes. Therefore, they can be applied to optical mapping techniques. Currently, more than 2,000 voltage sensitive dyes have been discovered, which can be divided into fast-response dyes and slow-response dyes according to the speed of the dye's response to the change of the electric field. Fast-response dyes are mostly used in electrophysiological studies of nerve cells and myocardial cells, including Di-8-ANEPPS, Di-4-ANEPPS, RH421 dyes, etc. that produce large fluorescence changes. The response time of these dyes is less than one millisecond, so they can record the electrical signals of various biological cells without distortion. However, most of the current fast-response voltage sensitive dyes have problems such as short wavelength, low brightness, insufficient photostability, unsatisfactory tissue affinity, or potential toxicity.

[0003] Therefore, actively exploring and providing voltage sensitive dyes with excellent optical properties, good tissue affinity, non-toxic, and safe has become an urgent problem to be solved. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention proposes an application of a heptamethine indocyanine compound in cardiac optical mapping, aiming to explore voltage sensitive dyes with excellent optical properties, good tissue affinity, non-toxic, and safe, and provide strong support for cardiac optical mapping.

[0005] An embodiment of the first aspect of the present invention provides an application of a heptamethine indocyanine compound in cardiac optical mapping, and the application is for non-diagnostic or therapeutic purposes.

[0006] The application according to the embodiments of the first aspect of the present invention has at least the following beneficial effects: The heptamethine indocyanine compounds provided by the present invention are a class of near-infrared fluorescent dyes with special optical properties. These dyes have strong absorption and emission in the near-infrared region, strong tissue penetration ability, high fluorescence brightness, and have good biocompatibility, low toxicity, and the characteristic of rapid clearance from the body. Moreover, the heptamethine indocyanine compounds can bind to myocardial cell membrane proteins or lipids and make a rapid fluorescence response to changes in cell transmembrane potential, and have the function of reflecting cardiac electrical excitation conduction. The present invention applies the heptamethine indocyanine compounds as new near-infrared voltage-sensitive dyes to cardiac optical mapping and discovers a new use of the heptamethine indocyanine dyes.

[0007] When the present invention applies the heptamethine indocyanine compounds as voltage-sensitive dyes to cardiac optical mapping, it can reflect cell action potentials and the electrical signal conduction of the entire heart, has a high dynamic range, and rapidly responds to changes in membrane potential. It can not only achieve multi-site, synchronous, and non-invasive recording of membrane potential changes in myocardial cell populations, but also record the electrical activities of the entire heart and convert them into color images such as cell action potential maps and action potential duration maps. The above-mentioned heptamethine indocyanine compound dyes have near-infrared absorption and emission, and near-infrared light has the advantages of weak tissue autofluorescence, small scattering, and deep tissue penetration depth, which ensure the clarity and reliability of cardiac structure imaging; moreover, the heptamethine indocyanine dyes can be localized on the cell membrane surface and have excellent photostability after binding to the lipid bilayer, enabling long-term stable imaging; furthermore, the heptamethine indocyanine dyes have high biocompatibility, low toxicity, and good affinity for cardiac tissue, ensuring the safety of use. The application of the above-mentioned voltage-sensitive dyes greatly improves the accuracy, sensitivity, and resolution of cardiac optical mapping and has broad application prospects and important practical value in the field of cardiac optical mapping.

[0008] In some embodiments of the present invention, the core structure of the heptamethine indocyanine compounds includes two atomic nitrogen centers, and the two atomic nitrogen centers are connected by a heptamethine chain to form a conjugated system; one of indolyl or benzindolyl is also connected to each end of the heptamethine chain respectively.

[0009] In some embodiments of the present invention, the core structure of the heptamethine indocyanine compounds is selected from

[0010]

[0011] any one of them; wherein, R 1 、R 2 each time it appears, is independently selected from a straight-chain alkyl group of C 1 ~C 5 and C1 ~C 5 alkyl carboxyl groups of C 1 ~C 5 alkyl sulfonic acid groups of, or carboxybenzyl groups.

[0012] The core structure of the heptamethine indocyanine compounds described in the present invention is formed by connecting two atomic nitrogen centers through a heptamethine chain to form a conjugated system with a uniform bond length distribution. The two ends of the heptamethine chain are connected to indolyl groups or benzindolyl groups. The structural formula of this type of compound can be simplified to the above four core structures (Core 1, Core 2, Core 3, Core 3), where R 1 and R 2 groups can be the same substitution groups or different substitution groups.

[0013] The heptamethine indocyanine compounds provided by the present invention have the property of binding to the plasma membrane and localizing on the cell membrane surface. The principle of action is as follows: First, the flexible chain molecular structure of the heptamethine indocyanine compound dye can easily bind to proteins or lipids. In the absence of blood, it will preferentially bind to the cell membrane surface proteins, and the stability and fluorescence intensity of this type of dye are greatly enhanced after binding to proteins. Second, the structure of the heptamethine indocyanine compound usually consists of a lipophilic part and a hydrophilic part, enabling the molecule to be embedded in the lipid bilayer of the cell membrane, thus avoiding the process of most dyes being internalized by cells. When the dye has no hydrophilic part, due to its strong lipophilicity, the dye will first bind to the membrane proteins or lipids, but will be internalized by cells in a short time and cannot be embedded in the cell membrane for a long time.

[0014] The heptamethine indocyanine compounds provided by the present invention have membrane potential response properties, can rapidly respond to changes in cell transmembrane potential, have the function of reflecting cardiac electrical excitation conduction, and can be used as new near-infrared voltage-sensitive dyes. After these dyes are successfully loaded into cells and excited by a light source with a specific wavelength, the optical imaging system can successfully detect the changes in fluorescence on the cell membrane surface. The principle of its membrane potential response is as follows: First, the heptamethine indocyanine compounds can rapidly bind to the plasma membrane after injection, and the molecular orientation is usually perpendicular to the cell membrane surface without changing any "normal" functions or structural characteristics of the cell membrane; Second, when the surface is irradiated with a specific light source, the change in membrane potential will cause a change in the direction and / or position of the charges within the dye molecule, thereby causing a synchronous change in the light absorption or fluorescence of the dye; Finally, the fluorescence emission spectrum of the heptamethine indocyanine dye is dome-shaped, and according to the position of the measured fluorescence emission relative to the dome, the change in fluorescence can be positive or negative. The above-mentioned heptamethine indocyanine compounds can be used as new near-infrared voltage-sensitive dyes in cardiac optical mapping, have a high dynamic range, and rapidly respond to changes in membrane potential. They can not only achieve multi-site, synchronous, and non-invasive recording of membrane potential changes in myocardial cell populations, but also record the electrical activity of the entire heart and convert it into color images such as cell action potential maps and action potential duration maps.

[0015] In some embodiments of the present invention, the structure of the heptamethine indocyanine compound is selected from

[0016]

[0017]

[0018] any one of

[0019] The heptamethine indocyanine compounds are a large class with a relatively large number of compounds. For example, Compounds 1-7 with the structural formulas shown above, where:

[0020] Compound 1 is indocyanine green (ICG), with a maximum absorption wavelength of 780 nm and a maximum emission wavelength of 810 nm. Its core structure is Core 1, and R 1 and R 2 are both sulfobutyl groups.

[0021] Compound 2 is Cy7.5-COOH, with a maximum absorption wavelength of 788 nm and a maximum emission wavelength of 808 nm. Its core structure is Core 1, R 1 is a carboxypentyl group, and R 2 is a methyl group.

[0022] Compound 3 is Cy7-diacid, with a maximum absorption wavelength of 754 nm and a maximum emission wavelength of 778 nm. Its core structure is Core 3, R1 and R 2 are both pentyl carboxylic acids.

[0023] Compound 4 is IR825, with a maximum absorption wavelength of 720 nm and a maximum emission wavelength of 825 nm. Its core structure is Core 2, and R 1 and R 2 are both benzyl carboxylic acids.

[0024] Compound 5 is IR783, with a maximum absorption wavelength of 779 nm and a maximum emission wavelength of 784 nm. Its core structure is Core 4, and R 1 and R 2 are both butyl sulfonic acids.

[0025] Compound 6 is IR820, with a maximum absorption wavelength of 710 nm and a maximum emission wavelength of 820 nm. Its core structure is Core 2, and R 1 and R 2 are both butyl sulfonic acids.

[0026] Compound 7 is IR780, with a maximum absorption wavelength of 780 nm and a maximum emission wavelength of 823 nm. Its core structure is Core 4, and R 1 and R 2 are both propyl groups.

[0027] The above Compounds 1 - 7 are only examples for better illustrating the properties of the heptamethine indocyanine compounds. The heptamethine indocyanine dyes are a class of near-infrared fluorescent dyes with special optical properties. These dyes have strong absorption and emission in the near-infrared region, strong tissue penetration ability, high fluorescence brightness and good photostability, and are very suitable for imaging of deep tissues in biomedicine. Among them, Indocyanine green (ICG) is a typical representative, which can be excited in the wavelength range of 650 - 850 nm and emit near-infrared fluorescence with a wavelength of 750 - 950 nm. Moreover, due to its good biocompatibility, low toxicity and the characteristic of rapid clearance from the body, it has been widely used in clinical fluorescence angiography, cardiac output measurement, liver function monitoring and tumor imaging. However, its property of responding to changes in cell membrane potential has not been reported, and its application in cardiac optical mapping has not been fully explored. The application of the heptamethine indocyanine dyes provided by the present invention as voltage-sensitive dyes in cardiac optical mapping will provide new possibilities for the development of cardiac optical mapping technology, and improve the accuracy, sensitivity and resolution of cardiac optical mapping. In fact, in addition to the above Compounds 1 - 7, as long as the heptamethine indocyanine compounds have the property of membrane potential response, can rapidly respond to changes in cell transmembrane potential, and have the function of reflecting cardiac electrical excitation conduction, they can all be used as new near-infrared voltage-sensitive dyes and have potential uses in cardiac optical mapping.

[0028] The heptamethine indocyanine compound provided by the present invention can be directly purchased or synthesized by using the existing technologies in the art. For example, unsubstituted heptamethine cyanine with a methine chain is usually prepared by reacting 2,4-dinitrophenylpyridine (Zincke salt) with aniline and then reacting with a heterocyclic quaternary ammonium salt containing an active methyl group under the action of a basic catalyst (Route 1). The reaction formula is as follows:

[0029]

[0030] The heptamethine cyanine dye with a cyclized methine chain uses cycloalkyl ketone as a raw material, and under the DMF / POCl 3 system, a chloroacrolein is obtained through a Vilsmeier-Haack reaction, and then reacted with aniline to obtain a meso-chloro condensing agent, and finally condensed with a heterocyclic quaternary ammonium salt to obtain a meso-chloro heptamethine cyanine dye (Route 2). The reaction formula is as follows:

[0031]

[0032] An embodiment of the second aspect of the present invention provides a method for applying a heptamethine indocyanine compound in cardiac optical mapping, including the steps of diluting the above-mentioned heptamethine indocyanine compound in Tyrode's solution, perfusing an isolated heart with the obtained dilution for a predetermined time, and detecting the change in the fluorescence signal on the surface of the myocardial cell membrane under the excitation of a light source to obtain a fluorescence change fraction.

[0033] According to the application method of the embodiment of the second aspect of the present invention, it has at least the following beneficial effects: The present invention dilutes the dye in Tyrode's solution and then performs Langendorff perfusion on the isolated heart, which can uniformly stain the heart surface. Under the excitation of a light source, the optical imaging system can successfully detect the change in the fluorescence signal on the cell membrane surface. The principle of its action is as follows: The flexible chain molecular structure of the heptamethine indocyanine dye can easily bind to proteins or lipids. In the absence of blood, it will preferentially bind to the proteins on the cell membrane surface, and the stability and fluorescence intensity of this type of dye are greatly enhanced after binding to proteins; secondly, the structure of the heptamethine indocyanine dye usually consists of a lipophilic part and a hydrophilic part, enabling the molecule to be located on the cell membrane and perfectly embedded in the lipid bilayer of the cell membrane, avoiding the process of most dyes being internalized by cells. Then, it is excited by an LED lamp with a specific wavelength, and after filtering out the excitation light through an optical filter, the optical imaging system can successfully detect the change in the fluorescence on the cell membrane surface. This method is simple to operate and convenient to apply, providing a practical possibility for the wide application of heptamethine indocyanine compounds in cardiac optical mapping.

[0034] In some embodiments of the present invention, the calculation method of the fluorescence change fraction is as follows: subtract the fluorescence signal F1 detected on the surface of the myocardial cell membrane during depolarization from the fluorescence signal F0 on the surface of the myocardial cell membrane in the resting state to obtain the fluorescence change value ΔF; the ratio ΔF / F0 of the fluorescence change value ΔF to the fluorescence signal F0 on the surface of the myocardial cell membrane in the resting state is the fluorescence change fraction.

[0035] A common problem with dye loading is that the resulting fluorescence may show spatiotemporal variations. Non-uniform distribution of the probe within the tissue, non-uniform tissue structure, non-uniform excitation light intensity, probe bleaching, motion artifacts, and local changes in the cell membrane structure can all cause the detection signal to vary over time. Therefore, it is necessary to scale the signal at each pixel by calculating the transient fluorescence change fraction (ΔF / F) so that it has the same range, thereby eliminating these variations. The calculation process of the fluorescence change fraction is as follows: record the fluorescence signal on the surface of the heart with a high-resolution camera at a shooting speed of 300 fps, and the data acquired at one time is 2 s. Under the same concentration of dye staining, subtract the fluorescence signal (F1) recorded during depolarization from the fluorescence signal (F0) in the resting state to obtain the fluorescence change value ΔF. The ratio of the fluorescence change value (ΔF) to the fluorescence signal (F0) in the resting state is the fluorescence change fraction (ΔF / F).

[0036] In some embodiments of the present invention, the range of the fluorescence change fraction is 0.1 - 3.0%.

[0037] In some preferred embodiments of the present invention, the range of the fluorescence change fraction is 0.18 - 2.17%.

[0038] It has been verified that the fluorescence changes of the seven dyes, compound 1 - 7, provided by the present invention all reflect the action potential changes. When the membrane potential changes, the range of the fluorescence change fraction (ΔF / F) is between 0.18 - 2.17%, further verifying that the heptamethine indocyanine dyes have good membrane potential response performance. Among them, indocyanine green (ICG) has the highest fluorescence change fraction, reaching 2.17%, and can be used as an excellent voltage-sensitive dye for cardiac optical mapping.

[0039] In some embodiments of the present invention, after obtaining the fluorescence change fraction, the following steps are further included: according to the fluorescence change fraction, calculate the membrane potential change of the myocardial cells and convert it into a cell action potential map or an action potential duration map of the myocardial cells.

[0040] The heptamethine indocyanine dyes provided by the present invention can be used as new near-infrared voltage-sensitive dyes in cardiac optical mapping, have a high dynamic range, and respond rapidly to membrane potential changes. They can not only achieve multi-site, synchronous, and non-invasive recording of membrane potential changes in myocardial cell populations, but also record the electrical activities of the entire heart and convert them into color images such as cell action potential maps and action potential duration maps.

[0041] In some embodiments of the present invention, the heptamethine indocyanine compound is added to Tyrode's solution for dilution. After perfusing the isolated heart with the obtained dilution for 5 minutes, the changes in fluorescence signals on the surface of the myocardial cell membrane are detected under light source excitation to obtain the fluorescence change fraction.

[0042] In some preferred embodiments of the present invention, the concentration of the heptamethine indocyanine compound in the dilution is 3 - 6 μM, more preferably about 5 μM.

[0043] An embodiment of the third aspect of the present invention provides an application of a heptamethine indocyanine compound in myocardial cell optical mapping or myocardial cell electrophysiological research. The heptamethine indocyanine compound includes indocyanine green, and the structural formula of the indocyanine green is

[0044] The application is for non-diagnostic or therapeutic purposes.

[0045] The application according to the embodiment of the third aspect of the present invention has at least the following beneficial effects: The present invention provides the application of indocyanine green (ICG) as a new near-infrared voltage-sensitive dye in myocardial cell optical mapping. It is first discovered that the heptamethine indocyanine dye ICG can bind to myocardial cell membrane proteins or lipids and make a fluorescence response to changes in the cell transmembrane potential. When the membrane potential changes, the fluorescence change fraction (ΔF / F) reaches 2.17%. The present invention proves that ICG can reflect cell action potentials and the electrical signal conduction of the entire heart. Moreover, ICG has near-infrared absorption and emission. Near-infrared light has the advantages of weak tissue autofluorescence, small scattering, and deep tissue penetration depth, ensuring the clarity and reliability of cardiac structure imaging; and it has excellent photostability after binding to the lipid bilayer and can perform long-term stable imaging. In addition, ICG has high biocompatibility, low toxicity, and good affinity for cardiac tissue. It is the only near-infrared dye approved by the US FDA for clinical use, ensuring the safety of use and having broad application prospects and important practical value in the field of cardiac optical mapping.

[0046] An embodiment of the fourth aspect of the present invention provides an application of a heptamethine indocyanine compound in studying the pathogenesis of arrhythmia. The structure of the heptamethine indocyanine compound is selected from

[0047]

[0048] Any one of the above; the application is for non-diagnostic or non-therapeutic purposes.

[0049] The application according to the embodiment of the fourth aspect of the present invention has at least the following beneficial effects: The heptamethine indocyanine compound provided by the present invention has the technical effects described above. When applied to cardiac optical mapping, it can quickly respond to changes in cell transmembrane potential and has the function of reflecting cardiac electrical excitation conduction, so as to solve the deficiencies in the prior art and improve the accuracy, sensitivity and resolution of cardiac optical mapping. This technology is of great significance for the research of arrhythmia and the prevention / treatment of heart diseases.

[0050] An embodiment of the fifth aspect of the present invention provides an application of a heptamethine indocyanine compound in the preparation of a molecular probe for cardiac optical mapping. The structure of the heptamethine indocyanine compound is selected from

[0051]

[0052] Any one of the above; the application is for non-diagnostic or non-therapeutic purposes.

[0053] The application according to the embodiment of the fifth aspect of the present invention has at least the following beneficial effects: The heptamethine indocyanine compound provided by the present invention has the technical effects described above. When applied to the development of new molecular probes for cardiac optical mapping, it can help explore other voltage-sensitive dyes with excellent optical properties, good tissue affinity, non-toxic and safe, providing strong support for clinical cardiac optical mapping.

[0054] Other features and advantages of the present invention will be described in the following specification, and part of them will become obvious from the specification or be understood by implementing the present invention. The objectives and other advantages of the present invention can be realized and obtained through the structures specifically pointed out in the specification, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a schematic diagram of the image after zebrafish heart staining and the corresponding optical signal change diagram provided by the embodiment of the present invention;

[0056] Figure 2 It is a schematic diagram of the image after guinea pig isolated heart staining and the corresponding fluorescence signal change diagram provided by the embodiment of the present invention;

[0057] Figure 3 It is a fluorescence change fraction diagram of seven dyes provided by the embodiment of the present invention;

[0058] Figure 4 It is a schematic diagram of an ICG-loaded rabbit heart (isolated), an optical action potential diagram and an excitation conduction diagram of an isolated rabbit heart provided by the embodiment of the present invention;

[0059] Figure 5 are the schematic diagram of ICG-loaded rabbit heart (in vivo), the optical action potential map, and the excitation conduction map of the in vivo rabbit heart provided by the embodiments of the present invention;

[0060] Figure 6 are the schematic diagram of ICG-loaded pig heart (ex vivo), the optical action potential map, and the excitation conduction map of the ex vivo pig heart provided by the embodiments of the present invention;

[0061] Figure 7 are the schematic diagram of ICG-loaded pig heart (in vivo), the optical action potential map, and the excitation conduction map of the pig heart provided by the embodiments of the present invention. Detailed implementation manners

[0062] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0063] In the description of the present invention, the description with reference to terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0064] In the description of the present invention, unless otherwise specified, the numerical range "a~b" represents the abbreviated representation of any real number combination between a and b, where a and b are both real numbers. Unless otherwise specified, each reaction or operation step can be carried out in sequence or not in sequence. Preferably, the reaction method in the present invention is carried out in sequence.

[0065] In the following embodiments, those without specific technologies or conditions indicated are carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. All reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0066] Example 1 Cell membrane staining experiment of heptamethine indocyanine dyes

[0067] This example is the qualitative experiment of indocyanine green (ICG) on zebrafish.

[0068] 1. Experimental materials:

[0069] (1) Experimental animals: Zebrafish (purchased from Aquatic Pet World (Shenzhen) Technology Co., Ltd.);

[0070] (2) Experimental instruments: Small scissors, small forceps (toothed and non-toothed), instrument trays, disposable culture dishes (30 mm and 60 mm), sponges, insect pins, disposable droppers;

[0071] (3) Main reagents: Tyrode's solution (prepared freshly before use), Pluronic F127, dimethyl sulfoxide, indocyanine green (ICG), Blebbistation, dimethylglyoxime (BDM), ice water;

[0072] (4) Main instruments: Electron microscope, an optical imaging system independently built in the laboratory (parameters are adjusted using different light sources, dichroic mirrors and filters);

[0073] 2. Experimental methods:

[0074] (1) Heart extraction: First, place the zebrafish in a beaker filled with ice water and anesthetize it at 0 - 4 °C for 10 min. After anesthesia, use forceps to pick up the zebrafish and place it in the sponge groove containing ice water, fix its head and body, and magnify it under an electron microscope. Then, gently hold the head with forceps, make a longitudinal cut at the gill area to expose the red heart of the zebrafish, carefully take it out and place it in a culture dish containing 2 mL of Tyrode's solution, and observe that the heart survives normally after being removed from the body;

[0075] (2) Staining: First, add 0.04% Pluronic F127 to the culture dish containing the heart and incubate for 25 min to increase the cell membrane permeability. Secondly, weigh a certain amount of the dye ICG (1.0 mg) solid to prepare a 1.0 mM DMSO mother solution, and then dilute the mother solution concentration to 5.0 μM with Tyrode's solution. Take 2.0 mL and add it to the culture dish containing the zebrafish heart for dye loading, and at the same time add the uncoupler Blebbistation (20.0 μM) and BDM (5.0 mM) to inhibit contraction. Imaging can be carried out after about 20 min;

[0076] (3) Imaging: Use a disposable dropper to wash away the free dye in the culture dish, then place the zebrafish heart in the center of the optical imaging platform, directly image after adjusting the optical path, and keep the whole process light - shielded during the operation.

[0077] Example 2 - 7 Cell Membrane Staining Experiment of Heptamethine Indocyanine Dyes

[0078] Examples 2-7 are different from Example 1 in that the heptamethine indocyanine dyes are different, and the light source and filter parameters are different. Other steps and parameters are the same as those in Example 1.

[0079] Among them, the heptamethine indocyanine dyes, light sources, and filter parameters of Examples 1-7 are shown in Table 1:

[0080] Table 1

[0081]

[0082]

[0083] ICG was purchased from Shanghai Maokang Biotechnology Co., Ltd., and IR780, IR783, IR820, IR825, Cy7.5-COOH, and Cy-diacid were all purchased from Shanghai Macklin Biochemical Co., Ltd.

[0084] In the zebrafish qualitative experiments of Examples 1-7, the imaging schematic diagrams of zebrafish hearts after staining and the corresponding optical signal change diagrams are as Figure 1 shown. As can be seen from the figure, after incubating with zebrafish isolated hearts for a period of time with a certain concentration (5.0 μM) of heptamethine indocyanine dyes (ICG, IR780, IR783, IR820, IR825, Cy7.5-COOH, Cy7-diacid), the heptamethine indocyanine dyes will bind to the myocardial cell plasma membrane of zebrafish. After using a specific light source and filter combination, it was observed that the cell membrane of zebrafish was illuminated, and strong fluorescence signals were shown in the ventricular and atrial tissue regions. When using ImageJ software to process the fluorescence change signals at any position of the ventricle, it was found that the dyes loaded on the cell membrane would undergo reversible fluorescence changes with the change of membrane potential, thus reflecting the sinus rhythm and optical action potential of zebrafish.

[0085] Example 8 Test on the membrane potential response properties of heptamethine indocyanine dyes

[0086] This example is a static in vitro guinea pig heart experiment with the dye ICG.

[0087] 1. Experimental materials:

[0088] (1) Experimental animals: Ordinary guinea pigs (purchased from Guangdong Provincial Medical Experimental Animal Center);

[0089] (2) Experimental instruments: Four-limb fixing ropes, hair clippers, hair suckers, thoracotomes (for small animals), needle holders, hemostats, scissors (one large and one small), suture threads, toothed forceps, non-toothed forceps (2 pieces), instrument trays (2 pieces), fixing frames (for guinea pigs), and surgical pads;

[0090] (3) Experimental reagents and instruments: Tyrode's solution (prepared freshly before use), dimethyl sulfoxide, absolute ethanol, heparin sodium, Zoletil, atropine, isoflurane, xylazine, indocyanine green; Langendorff isolated heart perfusion system, electrophysiological stimulator, electrocardiogram monitor ECG, pH meter, manometer, thermometer, constant temperature heating water tank, peristaltic pump, bipolar silver electrodes;

[0091] (4) Optical conditions: LED light sources (780 nm and 660 nm), various filters, Kowa near-infrared lens, high-speed sensitive Kinetix camera (shooting speed is 300 fps, resolution is 500×500 pixels, data acquired each time is for 2 s);

[0092] 2. Experimental methods:

[0093] (1) Thoracotomy to obtain the heart: After weighing the guinea pigs, inject atropine subcutaneously and heparin sodium (3125 U / kg) intraperitoneally. After 15 min, inject a mixture of Zoletil and xylazine intramuscularly for anesthesia and detect the muscle strength response. Then open the chest cavity, quickly take out the heart, install it on the Langendorff perfusion system with a constant pressure of ~60 mmHg and a constant temperature of 37℃±0.5℃, and perfuse with Tyrode's solution containing oxygen (95% O 2 +5% CO 2 )(in mmol / L: 135 NaCl, 5.4 KCl, 1.8 CaCl 2 , 0.9 MgCl 2 , 0.33 NaH 2 PO 4 , 10 glucose, 10 HEPES; pH 7.35). The coronary perfusion rate is 10 mL / min and the pressure is 3.0 - 7.0 Pa. After equilibration, the perfusion is switched to the recirculation mode. To reduce movement, Blebbistatin (BB, 20.0 μmol / L) and 2,3-butanedione monoxime (BDM, 5.0 mmol / L) are added to the perfusion fluid to eliminate cardiac contraction.

[0094] (2) Dye loading: Slowly inject indocyanine green (5.0 μmol / L) into the perfusion fluid to stain the heart. After 5 min of circulatory perfusion, an isolated guinea pig heart with dye loading is obtained.

[0095] (3) Imaging: Image the anterior surface of the heart. The bipolar silver electrodes contact the apex of the heart, and electrical stimulation is performed through an electrophysiological stimulator (frequency 180 - 210 beats / min). Under the excitation of the light source, white light and fluorescence imaging of the heart surface are performed through a high-speed sensitive camera (Kinetix) to obtain fluorescence and action potential signals.

[0096] Test on the membrane potential response properties of heptamethine indocyanine dyes in Examples 9 - 14

[0097] The differences between Examples 9 - 14 and Example 8 are as follows: the heptamethine indocyanine dyes are different, and the parameters of the light source and filter are different. Other steps and parameters are the same as those in Example 8.

[0098] Among them, the heptamethine indocyanine dyes, light source, and filter parameters of Examples 8 - 14 are shown in Table 2:

[0099] Table 2

[0100] Example Dye Concentration Light source Emission filter Example 8 ICG 5.0 μM 660 BP808 Example 9 Cy7.5-COOH 5.0 μM 660 LP700 Example 10 Cy7-diacid 5.0 μM 780 LP800 Example 11 IR825 5.0 μM 660 LP800 Example 12 IR783 5.0 μM 780 BP840 Example 13 IR820 5.0 μM 660 BP720 Example 14 IR780 5.0 μM 780 BP808

[0101] ICG was purchased from Shanghai Maokang Biotechnology Co., Ltd., and IR780, IR783, IR820, IR825, Cy7.5 - COOH, and Cy7 - diacid were all purchased from Shanghai Macklin Biochemical Co., Ltd.

[0102] In the verification of isolated guinea - pig hearts in Examples 8 - 14, the imaging schematic diagram of the isolated guinea - pig hearts after staining and the corresponding fluorescence signal change diagram are as Figure 2 shown. It can be seen from the figure that dyes (ICG, IR780, IR783, IR820, IR825, Cy7.5 - COOH, Cy7 - diacid) at a certain concentration (5.0 μM) will rapidly bind to the cell membrane during perfusion. After using a specific combination of light source and filter, it can be observed that the surface of the guinea - pig heart is evenly stained and has a high fluorescence brightness. When using Image J software to process the signals at any position of the ventricle or atrium, it is found that the fluorescence intensity of these dyes changes with the change of membrane potential and shows voltage - dependent fluorescence changes in optical mapping. This shows that these dyes all have membrane potential response properties and can be used as voltage - indicating dyes in optical mapping.

[0103] In the verification of isolated guinea - pig hearts in Examples 8 - 14, the fluorescence change fraction (ΔF / F) of the seven dyes was calculated, and the results are as Figure 3 shown.

[0104] The fluorescence generated during optical imaging may show spatiotemporal changes. Uneven distribution of dyes in tissues, uneven excitation light intensity, photobleaching effects, motion artifacts, etc. may all cause the detected fluorescence signal to change with time. By calculating the transient fluorescence change fraction (ΔF / F), the signal at each pixel can be scaled to have the same range, thereby eliminating spatiotemporal fluorescence changes. Specifically, the fluorescence signal (F1) recorded during depolarization can be subtracted from the background fluorescence signal (F0) at rest to obtain the fluorescence change value ΔF. The ratio of the fluorescence change value (ΔF) to the background fluorescence signal (F0) at rest is the fluorescence change fraction (ΔF / F).

[0105] In the verification of the isolated guinea pig hearts in Examples 8-14, the optical images were preprocessed using Image J software. The OS for analysis was taken from a 5×5 pixel region, and the background fluorescence was subtracted from each recorded frame. To reduce noise, five consecutively recorded OSs were averaged, and then the average signal was normalized using the background fluorescence to obtain the fractional change in the voltage-sensitive fluorescence signal.

[0106] Figure 3 It is a fractional change map of the fluorescence of seven dyes, as Figure 3 shown. The fractional changes in the fluorescence of these seven dyes are consistent with the optical action potentials of the heart, and the fractional change range is between 0.18% and 2.17%, further verifying that the heptamethine indocyanine dyes have good membrane potential response performance. Among them, ICG has the highest fractional change in fluorescence, which favorably supports the new use of ICG for optical mapping of the living heart.

[0107] Example 15 Static Isolated Rabbit Heart Experiment of Indocyanine Green (ICG)

[0108] 1. Experimental materials: Ordinary rabbits (purchased from Guangdong Provincial Medical Experimental Animal Center), and the rest are the same as in Example 8.

[0109] 2. Experimental methods:

[0110] (1) Open the chest to remove the heart: Weigh the New Zealand rabbits, inject atropine and sodium heparin after induction with isoflurane and respiratory anesthesia. Then open the chest cavity, quickly remove the heart, install it on a Langendorff perfusion system with a constant pressure of -60 mmHg and a constant temperature of 37°C ± 0.5°C, and perfuse with oxygenated (95% O 2 + 5% CO 2 ) Tyrode's solution (in mmol / L: 135 NaCl, 5.4 KCl, 1.8 CaCl 2 , 0.9 MgCl 2 , 0.33 NaH 2 PO 4 , 10 glucose, 10 HEPES; pH 7.35). The coronary perfusion rate is 20 mL / min, and the pressure is 3.0 - 7.0 Pa. After equilibration, the perfusion is switched to the recirculation mode. To reduce movement, Blebbistatin (BB, 20 μmol / L) and 2,3-butanedione monoxime (BDM, 5 mmol / L) are added to the perfusion fluid to eliminate cardiac contraction.

[0111] (2) Dye loading: Slowly inject indocyanine green (5.0 μmol / L) into the perfusion fluid to stain the heart, and an isolated rabbit heart with dye loading is obtained after 5 min of circulating perfusion.

[0112] (3) Imaging: Image the anterior surface of the heart. The bipolar silver electrode contacts the apex of the heart, and electrical stimulation is performed using an electrophysiological stimulator (frequency 150 - 210 beats per minute). Under the excitation of the light source, white light and fluorescence imaging of the heart surface are performed using a high-speed sensitive camera (Kinetix) to obtain fluorescence and action potential signals.

[0113] Figure 4 It is a schematic diagram of an ICG-loaded rabbit heart (ex vivo), an optical action potential map, and an activation conduction map of the ex vivo rabbit heart. It can be seen from the figure that indocyanine green can uniformly stain the ex vivo rabbit heart tissue and has good fluorescence brightness. After signal processing of the local area of the left ventricle (LV) of the ex vivo rabbit heart using Image J software, it is found that under 780 nm excitation, when the membrane depolarizes, the ICG fluorescence intensity decreases, and when the membrane repolarizes, the ICG fluorescence intensity increases, reflecting the optical action potential of the ex vivo rabbit heart. In addition, by recording the changes in the optical signals of the whole heart, an activation conduction map of the ex vivo rabbit heart in the normal state is obtained, which conducts from the apex position (pacemaker) to the ventricle - atrium, realizing cardiac optical mapping of the static ex vivo rabbit heart.

[0114] Example 16 In Vivo Experiment of Indocyanine Green (ICG) on Rabbit Heart

[0115] 1. Experimental materials: The same as in Example 15.

[0116] 2. Experimental method: Induce anesthesia in New Zealand rabbits and place them in an induction anesthesia box. After 5 minutes of anesthesia, clean the chest hair of the rabbit and prepare the skin, then perform intubation respiratory anesthesia. The concentration of isoflurane is 3%, the tidal volume of the ventilator is 5 mL, the respiratory ratio is 1:2, and the respiratory frequency is 30 times / min. Then make a median thoracotomy with an anatomical scissors to expose the rabbit heart, and then perform intracoronary injection of ICG outside the heart, slowly injecting 1.0 - 4.0 mL of ICG (5.0 μmol / L) once. After injection, use two 780 nm light sources to excite the anterior surface of the heart, and the Kinetix camera takes pictures and collects data directly above the heart to obtain data.

[0117] Figure 5Schematic diagram of ICG-loaded rabbit heart (in vivo), optical action potential map and excitation conduction map of in vivo rabbit heart. It can be seen from the figure that indocyanine green can be evenly loaded on the in vivo rabbit heart and has good fluorescence brightness. First, the movement and contraction of the rabbit heart were tracked by an algorithm. After registration, the Image J software was used to process the signal of the local area of the left ventricle (LV) of the in vivo rabbit heart. It was found that when the membrane was depolarized, the ICG fluorescence intensity decreased under 780 nm excitation, and when the membrane was repolarized, the ICG fluorescence intensity increased, reflecting the optical action potential of the in vivo rabbit heart. In addition, by recording the changes in the optical signals of the whole heart, we obtained the excitation conduction map of the in vivo rabbit heart in the normal state, which conducted from the apex position (pacemaker) to the ventricle-atrium, realizing the cardiac optical mapping of the dynamic in vivo rabbit heart.

[0118] Example 17 Static ex vivo porcine heart experiment of indocyanine green (ICG)

[0119] 1. Experimental materials: Experimental white porcine heart (purchased from the slaughterhouse), and the rest is the same as in Example 8.

[0120] 2. Experimental method:

[0121] (1) Obtain the porcine heart: After obtaining the ex vivo heart of a white pig from the slaughterhouse, it was cryopreserved with a cardiac preservation solution. A perfusion experimental platform was established. Through aortic cannulation, it was installed on a Langendorff perfusion system with a constant pressure of ~85 mmHg and a constant temperature of 37 °C ± 0.5 °C, and perfused with oxygenated (95% O 2 + 5% CO 2 ) Tyrode's solution (in mmol / L: 135 NaCl, 5.4 KCl, 1.8 CaCl 2 , 0.9 MgCl 2 , 0.33 NaH 2 PO 4 , 10 glucose, 10 HEPES; pH 7.35), and the coronary perfusion rate was 400 mL / min. After equilibration, the perfusion was switched to the recirculation mode. To reduce movement, Blebbistatin (BB, 20.0 μmol / L) and 2,3-butanedione monoxime (BDM, 5.0 mmol / L) were added to the perfusion solution to eliminate cardiac contraction.

[0122] (2) Dye loading: Indocyanine green (5.0 μmol / L) was slowly injected into the perfusion solution to stain the heart, and the ex vivo porcine heart with dye loading was obtained after 5 min of circulating perfusion.

[0123] (3) Imaging: Image the anterior surface of the heart. The bipolar silver electrode contacts the apex of the heart, and electrical stimulation is performed using an electrophysiological stimulator (frequency 120 beats per minute). Under the excitation of the light source, white light and fluorescence imaging of the heart surface are performed using a high-speed sensitive camera (Kinetix) to obtain fluorescence and action potential signals.

[0124] Figure 6 It is a schematic diagram of an ICG-loaded porcine heart (ex vivo), an optical action potential map, and an activation conduction map of an ex vivo porcine heart. As can be seen from the figure, indocyanine green can uniformly stain the ex vivo porcine heart and has good fluorescence brightness. After signal processing of a local area of the ex vivo porcine heart using Image J software, it was found that when the membrane depolarizes, the ICG fluorescence intensity decreases, and when the membrane repolarizes, the ICG fluorescence intensity increases under 780 nm excitation, reflecting the optical action potential of the ex vivo rabbit heart. In addition, by recording the changes in optical signals in the local area, we obtained the activation conduction map of the ex vivo porcine heart in the normal state, which conducts from the apex position (pacemaker) to the ventricle-atrium, realizing cardiac optical mapping of the static ex vivo porcine heart.

[0125] Example 18 In vivo experiment of indocyanine green (ICG) on porcine heart

[0126] 1. Experimental materials: 45 - 60 kg experimental white pigs (purchased from Boshi (Guangzhou) Medical Technology Co., Ltd.).

[0127] 2. Experimental method: First, induce anesthesia by intravenous injection of 150 mg of Zoletil 50, 50 mg of propofol, and 37,500 U of heparin sodium, and then perform intubation respiratory anesthesia. Perform median sternotomy to expose most of the anterior area of the heart, insert a cannula into the posterior femoral artery of the hind limb, and establish a channel for coronary angiography. After establishment, test the correctness of the coronary angiography channel and observe whether the dye is correctly injected under DSA. Then perform ICG staining, continuously stain 60 mL of ICG (3.0 μmol / L), and then take pictures to obtain data.

[0128] Figure 7 It is a schematic diagram of an ICG-loaded porcine heart (in vivo), an optical action potential map, and an activation conduction map of the porcine heart. As can be seen from the figure, indocyanine green can be uniformly loaded on the in vivo porcine heart and has good fluorescence brightness. First, algorithmically track the movement and contraction of the porcine heart. After registration, use Image J software to perform signal processing on a local area of the in vivo porcine heart. It was found that when the membrane depolarizes, the ICG fluorescence intensity decreases, and when the membrane repolarizes, the ICG fluorescence intensity increases under 780 nm excitation, reflecting the optical action potential of the in vivo porcine heart. In addition, by recording the changes in optical signals in the local area, we obtained the activation conduction map of the in vivo porcine heart in the normal state, which conducts from the apex position (pacemaker) to the ventricle-atrium, realizing cardiac optical mapping of the in vivo porcine heart and laying a foundation for subsequent clinical cardiac optical mapping experiments.

[0129] The above is a specific description of the preferred embodiment of the present application. However, the present application is not limited to the above-mentioned embodiments. Those skilled in the art can make various equivalent deformations or substitutions without departing from the spirit of the present application, and these equivalent deformations or substitutions are all included within the scope defined by the claims of the present application.

Claims

1. A use of a heptamethine indocyanine compound in cardiac optical mapping, wherein the use is for non-diagnostic or therapeutic purposes.

2. The use according to claim 1, characterized in that: The core structure of the heptamethine indole cyanine compound includes two atomic nitrogen centers, which are connected by a heptamethine chain to form a conjugated system; the two ends of the heptamethine chain are also connected to one of an indole group or a benzindole group.

3. The use according to claim 2, characterized in that: The core structure of the heptamethine indocyanine compound is selected from Any one of; wherein, each occurrence of R1 and R2 is independently selected from a C1-C5 straight-chain alkyl group, a C1-C5 alkyl carboxyl group, a C1-C5 alkyl sulfonic acid group, or a carboxylic acid benzyl group.

4. The use according to claim 1, characterized in that: The structure of the heptamethine indole cyanine compound is selected from Any of .

5. A method for using a heptamethine indocyanine compound in cardiac optical mapping, characterized in that: The method comprises the following steps: adding the heptamethine indocyanine compound as described in any one of claims 1 to 4 into Tyrode's solution for dilution, perfusing the isolated heart with the obtained diluted solution for a predetermined time, detecting the change of the fluorescence signal on the surface of the myocardial cell membrane under the excitation of a light source, and obtaining the fluorescence change score.

6. The application method according to claim 5, characterized in that: The calculation method of the fluorescence change score is: subtract the fluorescence signal F0 on the surface of the myocardial cell membrane in the resting state from the fluorescence signal F1 on the surface of the myocardial cell membrane detected during depolarization to obtain the fluorescence change value ΔF; the ratio ΔF / F0 of the fluorescence change value ΔF to the fluorescence signal F0 on the surface of the myocardial cell membrane in the resting state is the fluorescence change score.

7. The application method according to claim 5, characterized in that: The fluorescence change fraction ranges from 0.1 to 3.0%. Preferably, the fluorescence change fraction ranges from 0.18 to 2.17%.

8. The application method according to claim 5, characterized in that: After obtaining the fluorescence change score, the method further includes the steps of: calculating the membrane potential change of the cardiomyocytes according to the fluorescence change score, and converting it into a cell action potential diagram or an action potential time course diagram of the cardiomyocytes.

9. A use of a heptamethine indocyanine compound in optical mapping of cardiomyocytes or electrophysiological research of cardiomyocytes, characterized in that: The heptamethine indocyanine compounds include indocyanine green, and the structural formula of the indocyanine green is The use described is not for diagnostic or therapeutic purposes.

10. A use of a heptamethine indocyanine compound in the preparation of a molecular probe for optical mapping of the heart or in the study of the pathogenesis of arrhythmias, characterized in that: The structure of the heptamethine indole cyanine compound is selected from Any of the following; the application is not for diagnostic or therapeutic purposes.

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