Near-infrared neuron tracing probe as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared neuron tracing probe as well as a preparation method and application thereof, and relates to the technical field of medical image contrast agents. In particular to a preparation method and application of a central and peripheral nerve in-vivo tracing near-infrared probe formed by coupling reaction of cholera toxin B subunit and indocyanine green-succinimide ester. The probe comprises a preparation method and an imaging application method of the near-infrared probe formed by connecting cholera toxin B subunit and indocyanine green-succinimide ester, and the probe can specifically target and indicate nerve fibers in the center and the periphery, so that in-vivo near-infrared two-region fluorescence labeling of the nerve fibers is realized; and the analysis of neuronal degenerative diseases is assisted.

Description

Technical Field

[0001] The invention belongs to the technical field of medical neurobiology and imaging contrast agents, and specifically relates to a near-infrared neuron tracing probe, a preparation method of the probe and application of the probe in research in the field of neurobiology. Background Art

[0002] Neuronal degeneration is an important cause that affects the quality of life of many patients. In the field of basic scientific research, neuronal tracing technology is one of the important means to study the structure, function and upstream and downstream relationships of neurons in the field of neurobiology, and it is also an important way to explore the mechanism behind neuronal degeneration. Currently, the commonly used neuronal tracing tools mainly include viruses (such as rabies virus (RV), pseudorabies virus (PRV), herpes simplex virus (HSV), vesicular stomatitis virus (VSV), etc.). In addition, cholera toxin subunit B (CTB), as a part of cholera toxin, is attached to the cell surface by binding to the pentose chain of ganglioside GM1, can be absorbed by the axon terminal and transported retrogradely to the cell body, and then specifically and efficiently retrogradely label nerve cells. It has become the most widely used non-toxic retrograde tracer. Its commercial products, including CTB combined with 555 nm and 488 nm fluorescent groups, have been used in neuronal tracing research.

[0003] However, due to the limitations of imaging penetration depth and resolution, traditional fluorescence in the visible light region (400-700 nm) cannot achieve in vivo imaging of neurons, especially in vivo imaging of the central nervous system. It can only be used for in vitro observation after the central nervous system is labeled and animals are dissected.

[0004] Fluorescence imaging in the near-infrared region (700-1700 nm) has a deeper penetration depth and lower biological autofluorescence background interference, which can achieve higher-quality tissue fluorescence imaging and a wider imaging range, making it possible to study neural morphology and function in vivo. So far, there has been no report on the use of near-infrared fluorescent materials and CTB to synthesize near-infrared neuronal tracing probes for in vivo tracing of peripheral and central nerves. Summary of the invention

[0005] The present invention aims to provide a near-infrared neuron tracing probe.

[0006] Another object of the present invention is to provide a method for preparing the near-infrared neuron tracing probe.

[0007] Another object of the present invention is to provide an application of the near-infrared neuron tracing probe.

[0008] The purpose of the present invention is achieved by the following scheme: a near-infrared neuron tracing probe, a near-infrared neuron tracing probe formed by combining indocyanine green (ICG) and cholera toxin B subunit (CTB), denoted as ICG-CTB, and its structural formula is Formula 1: ; Wherein CTB is Cholera Toxin subunit B, and its structural formula is Formula 2: .

[0009] The invention solves the problem of insufficient penetration and resolution of existing CTB-derived fluorescent probes.

[0010] Among them, Cholera Toxin originates from Vibrio cholerae and is composed of two subunits, A and B. Subunit A is an ADP-ribosyltransferase that causes cell dehydration by destroying G protein signal transduction. CTB is attached to the cell surface by binding to the pentose chain of ganglioside GM1, which can be absorbed by the axon terminal and transported retrogradely to the cell body, thereby specifically and efficiently retrogradely labeling nerve cells without toxic effects on cells. ICG was approved for clinical use by the U.S. Food and Drug Administration (FDA) in 2006. It has a high fluorescence quantum yield, good water solubility, and near-infrared absorption peak characteristics. At present, ICG has been widely used in the diagnosis and treatment of fundus diseases, colon cancer and other diseases.

[0011] The neuron near-infrared fluorescent probe provided by the present invention may also have the following technical feature, that is, it is synthesized by coupling indocyanine green-succinimide ester with cholera toxin B subunit.

[0012] Furthermore, the cholera toxin B subunit is selected from recombinant cholera toxin B subunit.

[0013] The present invention also provides a method for preparing the above neuronal near-infrared fluorescent probe, comprising the following steps: (1) indocyanine green-succinimide ester is dissolved in dimethyl sulfoxide to obtain an indocyanine green-succinimide ester solution; (2) dissolving the cholera toxin B subunit in a phosphate buffer adjusted with sodium hydroxide at a pH of 9.2 to obtain a cholera toxin B subunit solution; (3) adding the indocyanine green-succinimide ester solution obtained in step (1) and the cholera toxin B subunit solution obtained in step (1) to a phosphate buffer having a pH of 9.2 to obtain a mixed solution; (4) stirring the mixed solution for 4 to 6 hours to carry out a coupling reaction; (5) Purifying the reaction product obtained from the coupling reaction to obtain a nanoscale near-infrared fluorescent probe.

[0014] The invention provides a near-infrared, peripheral and central nerve tracing fluorescent probe with simple preparation process, high specificity and good photostability.

[0015] Wherein, in step (3), the molar ratio of the indocyanine green-succinimide ester solution to the cholera toxin B subunit solution is 100:1 to 20:1, preferably 50:1 to 20:1.

[0016] In step (5), the purification is performed by separation and purification using an ultrafiltration tube or by dialysis purification using a dialysis bag.

[0017] To facilitate subsequent storage, transportation and use, the prepared nanoprobes were stored at 4°C for one month.

[0018] The present invention also provides an application of a near-infrared neuron tracing probe in neural targeted drugs.

[0019] Especially used in in vivo imaging of peripheral or central nervous systems.

[0020] The applications include, but are not limited to, the study of peripheral / central nervous system structure and function under physiological and pathological conditions, and the pathological scenarios include, but are not limited to, glaucomatous optic nerve damage, optic nerve clamp injury, macular degeneration, and diabetic retinopathy; for the evaluation of central projection damage of retinal ganglion cell axons caused by optic nerve atrophy and retinal diseases; for the evaluation of central and peripheral nerve damage caused by spinal cord lesions; and for nerve biopsy and real-time monitoring of nerve activity.

[0021] The ICG-CTB prepared by the present invention can be used for peripheral nerve imaging. By local injection, nerve labeling can be completed in 3 to 5 days, and is not affected by animal perfusion and post-fixation of tissues. The imaging equipment parameters need to meet the requirements of excitation light of 808 nm and emission light greater than 1000 nm. For peripheral thick nerves, such as sciatic nerves, and peripheral micro nerves, such as cutaneous nerves with a diameter of 10 μm, the morphological characteristics of nerve fiber running, distribution, bifurcation, etc. can be clearly distinguished in situ.

[0022] The ICG-CTB prepared by the present invention can be used for central nervous imaging. It can realize forward tracing of retinal ganglion cells by intravitreal injection. The optic nerve begins to be labeled with fluorescent probes one day after injection, and is stable for 3 to 5 days, and is not affected by animal perfusion or post-fixation of tissues. It can realize reverse tracing of brain neurons by stereotaxic injection. It begins to be labeled with fluorescent probes one day after injection, and is stable for 3 to 5 days, and is also not affected by animal perfusion or post-fixation of tissues. It can realize spinal cord labeling by intrathecal injection. It begins to be labeled with fluorescent probes one day after injection, and is stable for 3 to 5 days, and is also not affected by animal perfusion or post-fixation of tissues. The parameters of the imaging device must meet the requirements of excitation light of 808 nm and emission light greater than 1000 nm. For shallow brain areas, such as cortical areas, and deep brain areas, such as superior colliculus and lateral geniculate body, in vivo imaging can be realized.

[0023] The ICG-CTB prepared by the present invention can be used to compare the damage of peripheral and central nerves under different disease states, including but not limited to the evaluation of central projection damage of retinal ganglion cell axons caused by optic nerve atrophy and retinal diseases such as glaucomatous optic nerve damage, optic nerve clamp injury, macular degeneration, and diabetic retinopathy; central and peripheral nerve damage caused by spinal cord lesions; and can be used for nerve biopsy, real-time monitoring of nerve activity, etc.

[0024] The present invention is aimed at developing near-infrared fluorescent tracing probes for peripheral and central nerves, and provides a near-infrared fluorescent probe ICG-CTB, a preparation method thereof, and an application of ICG-CTB. As a fluorescent imaging probe in a near-infrared window, the fluorescent probe has a deeper penetration depth and a higher spatial resolution, and can achieve specific targeted labeling of central and peripheral nerves. The probe preparation method is safe and simple, has good stability and repeatability, has low toxicity to animals, and is safe and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis route of the neural near-infrared tracer probe of Example 1 of the present invention; Figure 2 It is the protein electrophoresis diagram of the neural near-infrared tracing probe of the present invention; Figure 3 It is a representative image of the retinal ganglion cell body and axon labeled by the neural near-infrared tracer probe of the present invention; Figure 4 These are representative images of the spinal cord and sciatic nerve labeled with the neural near-infrared tracer probe of the embodiment of the present invention. The upper figure shows the bright field and near-infrared in vivo imaging of the back of a mouse. Five days later, the mouse was dissected and the spinal cord was taken out for imaging; the lower figure shows the exposed sciatic nerve of the mouse, which was imaged in the bright field and near-infrared regions, respectively. DETAILED DESCRIPTION

[0026] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.

[0027] The terms used in the present invention, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

[0028] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0029] The reagents used in the following examples were purchased from common commercial channels, and the experimental operations and experimental conditions not specified were referred to the conventional operations and conventional conditions in the art.

[0030] The specific implementation of the present invention is described below with reference to the accompanying drawings.

[0031] Example 1 A near-infrared neuron tracing probe, the structure of which is shown in the following formula: Among them, CTB is the cholera toxin B subunit.

[0032] A synthetic route for a near-infrared neuronal tracer probe is shown in Figure 1 As shown, follow these steps: (1) Weigh 3.55 mg of indocyanine green-succinimidyl ester ICS-NHS and dissolve it in 75 Indocyanine green-succinimide ester solution was obtained in dimethyl sulfoxide; (2) Weigh 1 mg of cholera toxin B subunit CTB and dissolve it in 1 mL of phosphate buffer adjusted with sodium hydroxide to a pH of 9.2 to obtain a cholera toxin B subunit solution; (3) 7 The above indocyanine green-succinimide ester solution and 93 The above cholera toxin B subunit solution was obtained by 100 Mixing solutions; (4) stirring the mixed solution at room temperature at a stirring speed of 100 to 300 rpm for 4 to 6 hours to carry out a coupling reaction; (5) Pass through a 3 kDa ultrafiltration tube with phosphate buffer (1 , pH: 9.2), the mixed solution was centrifuged (1000 g, 2 min, 4 °C), and the coupling reaction product was separated and purified to obtain the final product of about 100 The near-infrared neuronal tracing probe is denoted as ICG-CTB probe.

[0033] The near-infrared neural tracing probe prepared in this example was subjected to protein electrophoresis, and the results were as follows: Figure 2As shown, it can be seen that the probe band is relatively diffuse. Figure 2 In the figure, the left picture shows the Coomassie Brilliant Blue staining result, which was taken in the bright field; the right picture shows the Coomassie Brilliant Blue staining result, which was taken in the near-infrared region. The electrophoresis results show that the probes synthesized with a concentration ratio of 20:1 and 50:1 have similar protein characteristics before filtration, but after filtering to remove unbound ICG, the 50:1 concentration ratio can produce more CTB bound to ICG. Therefore, it is recommended to use a 50:1 ratio to synthesize ICG-CTB probes for neuroimaging. UF: unfiltered; F: filtered.

[0034] Example 2 In vitro and in vivo imaging of central projections of mouse retinal ganglion cells: Anesthetized mice were given 1 80 nM ICG-CTB probe, daily ex vivo tissues were taken for continuous observation, and in vivo imaging was performed at the optimal labeling time point. The results showed that injection 3 to 5 days later had a better labeling effect. Near-infrared in vivo imaging equipment 808nm external laser irradiation, laser power density of 100 mW / cm 2 ,See Figure 3 .

[0035] Figure 3 This is a representative image of the neural near-infrared tracing probe of this embodiment labeling the cell bodies and axons of retinal ganglion cells; the left figure shows retinal flat mounts and near-infrared imaging of the brain in vivo and in vitro; the right figure shows near-infrared imaging of brain slices at different levels. The display probe can well label the cell bodies and axons of retinal ganglion cells, and can penetrate the skin and skull in vivo to achieve in vivo imaging of the mouse brain. PI: post-injection; OpN: Optic nerve; OT: Optic tract; LGN: Lateral geniculate nuclei; SC, Superiorcolliculi.

[0036] Example 3 Mouse spinal cord isolated and in vivo imaging: The awake mice were given 10 80 nM ICG-CTB probe, continuous in vivo imaging every day, and ex vivo spinal cord tissue was taken for imaging at the optimal labeling time point. The results showed that injection 3 to 5 days had a good labeling effect. Near-infrared in vivo imaging equipment 808nm external laser irradiation, laser power density of 100 mW / cm 2 ,See Figure 4 .

[0037] Figure 4 The above figure shows the bright field and near-infrared in vivo imaging of the mouse back; 5 days later, the mouse was dissected and the spinal cord was taken out for imaging. It can be seen that the near-infrared fluorescence signal of the spinal cord can be seen in vivo 1 day after the probe injection, indicating the high labeling efficiency of the probe.

[0038] Example 4 In vivo imaging of mouse peripheral sciatic nerve: In anesthetized mice, 10 80 nM ICG-CTB probe, continuous in vivo imaging every day, and the sciatic nerve was isolated to observe the fluorescence signal. The results showed that injection for 3 to 5 days had a good labeling effect. Near-infrared in vivo imaging equipment 808nm external laser irradiation, laser power density of 100 mW / cm 2 ,See Figure 4 .

[0039] Figure 4 The following figure shows the mouse sciatic nerve exposed and imaged in bright field and near infrared. Two days after the peripheral sciatic nerve probe injection, the nerve fluorescence signal is visible, indicating the high labeling efficiency of the probe.

Claims

1. A near-infrared neuron tracing probe, characterized in that: The near-infrared neural tracer probe formed by combining indocyanine green (ICG) and cholera toxin B subunit (CTB) is denoted as ICG-CTB, and its structural formula is Formula 1: ； Wherein CTB is Cholera Toxin subunit B, and its structural formula is Formula 2: 。 2. The near-infrared neuron tracing probe according to claim 1, characterized in that: It is synthesized by coupling indocyanine green-succinimidyl ester with cholera toxin B subunit.

3. The near-infrared neuron tracing probe according to claim 1 or 2, characterized in that: The cholera toxin B subunit is selected from recombinant cholera toxin B subunit.

4. A method for preparing a near-infrared neuron tracing probe according to any one of claims 1 to 3, characterized in that: The steps include: (1) indocyanine green-succinimide ester is dissolved in dimethyl sulfoxide to obtain an indocyanine green-succinimide ester solution; (2) dissolving the cholera toxin B subunit in a phosphate buffer adjusted with sodium hydroxide at a pH of 9.2 to obtain a cholera toxin B subunit solution; (3) adding the indocyanine green-succinimide ester solution obtained in step (1) and the cholera toxin B subunit solution obtained in step (1) to a phosphate buffer having a pH of 9.2 to obtain a mixed solution; (4) stirring the mixed solution for 4 to 6 hours to carry out a coupling reaction; (5) Purifying the reaction product obtained from the coupling reaction to obtain a nanoscale near-infrared fluorescent probe.

5. The method for preparing the near-infrared neuron tracing probe according to claim 4, characterized in that: In step (3), the molar ratio of the indocyanine green-succinimide ester solution to the cholera toxin B subunit solution is 100:1 to 20:

1.

6. The method for preparing the near-infrared neuron tracing probe according to claim 4, characterized in that: In step (5), the purification is performed by separation and purification using an ultrafiltration tube or by dialysis purification using a dialysis bag.

7. The method for preparing the near-infrared neuron tracing probe according to claim 4, characterized in that: The prepared nanoscale near-infrared fluorescent probe was stored at 4°C.

8. Use of the near-infrared neuron tracing probe according to any one of claims 1 to 3 in neural targeted drugs.

9. The method according to claim 8, characterized in that Used in in vivo imaging of peripheral or central nervous systems.

10. The use according to claim 9, characterized in that Used for research on peripheral / central nervous system structure and function under physiological and pathological conditions, including but not limited to glaucomatous optic nerve damage, optic nerve clamp injury, macular degeneration, diabetic retinopathy; for evaluation of central projection damage of retinal ganglion cell axons caused by optic nerve atrophy and retinal diseases; for evaluation of central and peripheral nerve damage caused by spinal cord lesions; and for nerve biopsy and real-time monitoring of nerve activity.

Images