Low-oxygen fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to a low-oxygen fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of biology. The novel hypoxia fluorescent probe is designed, the application mode of the hypoxia fluorescent probe in in-situ imaging in the field of neurobiology is further developed, the hypoxia fluorescent probe can effectively respond to hypoxia cells and tissues and can penetrate through a blood brain barrier, azo bonds are broken at the hypoxia brain injury position to release fluorescence, and the hypoxia fluorescent probe can be used for detecting hypoxia brain injury. The potential of clinically diagnosing the neonatal hypoxia ischemic encephalopathy is realized

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a hypoxia fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] A small-molecule fluorescent probe refers to a kind of organic small molecule capable of detecting a specific target by generating a fluorescent signal, which is usually composed of a fluorophore capable of generating a fluorescent signal and a recognition group targeting a specific target, and can be used for detecting specific biomolecules, cells or tissue structures in a living organism. The small-molecule fluorescent probe has the advantages of high sensitivity, high selectivity, multifunctionality, low toxicity, low cost, in-situ and real-time imaging capability, etc., and has excellent performance in the fields of diagnosing and treating diseases and exploring drug action mechanisms.

[0003] The blood-brain barrier (BBB) is a protective barrier located between blood vessels and brain tissue, and its main function is to limit the entry of substances in the blood into the brain tissue to protect the brain from harmful substances from the outside. The BBB is composed of brain vascular endothelial cells, basement membrane and surrounding astrocytes. The tight junction formed between brain vascular endothelial cells makes the intercellular space almost impermeable, thereby forming a difficult-to-penetrate barrier that limits the entry of most drugs and molecules into the brain. Therefore, it is an important task to study whether the small-molecule fluorescent probe can penetrate the blood-brain barrier for the research of the pathology and treatment of neurological diseases and drug delivery.

[0004] For example, for neonatal hypoxic-ischemic encephalopathy (HIE), it is a disease that causes brain dysfunction in newborns due to asphyxia during the perinatal period, leading to hypoxia and reduction or suspension of cerebral blood flow. HIE can cause a series of pathophysiological changes, including oxidative stress in the developing brain, intracellular Ca 2+ accumulation, mitochondrial dysfunction, excitotoxicity and inflammation. At present, there is still a lack of effective intervention measures. Therefore, in basic research, if a visual probe for in vivo detection of brain injury degree can be developed, it will help the research of HIE mechanism.

[0005] There are studies that have disclosed fluorescent probes that can respond to hypoxic cells, but most of them are for in vitro cell studies, and fluorescent probes that can penetrate the BBB and locate in the hypoxic brain region of the brain tissue or even in the cells are rarely reported. Therefore, the development of a fluorescent probe that can be effectively applied to imaging of hypoxic brain injury regions has important significance for the diagnosis and treatment of diseases such as HIE. SUMMARY

[0006] In response to the deficiencies of existing technologies and actual needs, the present invention provides a hypoxia fluorescent probe and its preparation method and application, designs a new hypoxia fluorescent probe that can respond to hypoxic cells and tissues, realize in situ fluorescence imaging, and can effectively cross the blood-brain barrier, providing a new method for the diagnosis of hypoxia-related diseases.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a hypoxia fluorescent probe (named BOD), which includes a probe having a molecular structure as shown in Formula I or a pharmaceutically acceptable salt, ester or solvate thereof.

[0009]

[0010] In the present invention, a new type of hypoxia fluorescent probe is designed, and its application in in situ imaging in the field of neurobiology is further developed. The hypoxia fluorescent probe can effectively respond to hypoxic cells and tissues, and can effectively penetrate the blood-brain barrier, breaking the azo bond at the site of hypoxic brain damage to release the fluorophore B, resulting in an enhancement of the red fluorescence signal at 594nm, and has the potential for clinical diagnosis of neonatal hypoxic-ischemic encephalopathy.

[0011] In a second aspect, the present invention provides a method for preparing the hypoxia fluorescent probe according to the first aspect, the preparation method comprising:

[0012] The compounds represented by formula II and formula III are used as substrates to carry out a reaction to obtain the hypoxia fluorescent probe.

[0013]

[0014] In a third aspect, the present invention provides use of the hypoxia fluorescent probe described in the first aspect in preparing a product for detecting hypoxia-related diseases.

[0015] Preferably, the hypoxia-related disease includes any one of neonatal hypoxic-ischemic encephalopathy, ischemic stroke or cancer.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] The present invention designs a new type of hypoxia fluorescent probe and further develops its application in in situ imaging in the field of neurobiology. The hypoxia fluorescent probe can effectively respond to hypoxic cells and tissues, penetrate the blood-brain barrier, break the azo bond at the site of hypoxic brain damage and release fluorescence, and has the potential for clinical diagnosis of neonatal hypoxic-ischemic encephalopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the working principle and application of the probe of the present invention.

[0019] Figure 2 For probe BOD 1 H NMR spectrum (500 MHz, DMSO-d6).

[0020] Figure 3 For probe BOD 13 C NMR spectrum (125 MHz, DMSO-d6).

[0021] Figure 4 This is the high-resolution mass spectrum (ESI-TOF) of the probe BOD.

[0022] Figure 5 This is the mass spectrum of BOD after reaction with rat liver microsomes.

[0023] Figure 6 Confocal microscopy images of BV2 cells treated with OGD and incubated with BOD probe.

[0024] Figure 7 for Figure 2 40x magnification of the Merge image.

[0025] Figure 8 This is the statistical result of fluorescence intensity of BOD channel.

[0026] Figure 9 This is the TTC staining result of mouse brain tissue 3 days after HIE modeling. The scale bar is 5 mm.

[0027] Figure 10 This is the HE staining result of mouse brain tissue 3 days after HIE modeling. The scale bar is 100 μm.

[0028] Figure 11 Confocal microscopy images of mouse brain tissue sections incubated with BOD probe 3 days after HIE modeling. The scale bar is 20 μm.

[0029] Figure 12 These are confocal microscopy images of brain tissue sections of mice injected intraperitoneally with BOD probe 6h\12h after 3 days of HIE modeling. The scale bar is 50μm. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0031] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0032] The present invention has developed a novel hypoxia fluorescent probe BOD and developed its application in the field of neurobiology for in situ imaging, such as Figure 1 As shown, the new hypoxia fluorescent probe BOD is composed of an azo bond connecting a specific fluorophore and a quencher. It can break the azo bond at the site of hypoxic brain damage to release fluorescence, thereby imaging the brain damage area. By allowing the new hypoxia fluorescent probe BOD to penetrate the BBB in the HIE model, the probe can be used to image and monitor hypoxic brain areas in situ in brain disease models. The probe can penetrate the BBB after intraperitoneal injection, and the response of the probe to the hypoxic brain area can be presented through fresh brain tissue slices. Compared with traditional in vivo imaging, it has the advantage of greater precision. While observing and recording the distribution of fluorescent probes in brain tissue at high resolution, it is more conducive to in-depth exploration of the pathological mechanism of HIE in terms of oxidative stress, as well as more precise brain region localization and changes in oxidative stress during this process. The new hypoxia fluorescent probe BOD and its application method have the potential for clinical diagnosis of neonatal hypoxic-ischemic encephalopathy.

[0033] Example 1

[0034] In this example, a novel hypoxia fluorescent probe BOD was prepared.

[0035] The synthetic route is shown below.

[0036]

[0037] Synthesis of compound 3-7: Dissolve 1,1,2-trimethyl-1H-benzo[e]indole (10.5 g, 50.2 mmol) and iodoethane (7.12 g, 50.2 mmol) in 40 mL of toluene. The mixture was reacted at 110°C overnight. After completion of the reaction, the precipitate was filtered to afford the pale purple compound 3-7 (7.3 g, 61.0% yield).

[0038]

[0039] Synthesis of compound 3-8: 4-aminobenzaldehyde (1.21 g, 10 mmol) was dissolved in distilled water (10 mL) and hydrochloric acid (4.5 mL, 37.5%), and stirred in an ice bath for 30 min. 20 mL of an aqueous sodium nitrite (NaNO2) solution (0.7 g, 10 mmol) was added dropwise to the solution, and stirred in an ice bath for 30 min. N,N-dimethylaniline (1.21 g, 0.01 mol) was dissolved in acetic acid (25 mL, 36%), and an appropriate amount of sodium acetate was added to the solution to adjust the pH of the solvent to 3-5. The prepared N,N-dimethylaniline solution was added to the reaction solution, and stirred in an ice bath for 2 h and then at room temperature for 2 h. After the reaction was completed, dilute aqueous sodium hydroxide solution was added to the reaction solution until the reaction solution became neutral, at which time a red precipitate was formed. The red solid was obtained by suction filtration, washed with water, and the product was recrystallized with ethanol and dried in a vacuum oven to obtain dark red solid compound 3-8 (2.1 g, yield 83.0%).

[0040]

[0041] Synthesis of probe BOD: Compound 3-7 (238 mg, 1 mmol) and compound 3-8 (253 mg, 1 mmol) were dissolved in 10 mL of methanol (MeOH), and piperidine (PIP, 150 μL, 1.5 mmol) was added, and the reaction was carried out at 80°C overnight under nitrogen protection. After the reaction was completed, a purple-black precipitate was formed, which was suction filtered and washed with distilled water three times to obtain the crude product. The crude product was purified by silica gel column, and the eluent was dichloromethane / methanol (DCM / MeOH, volume ratio V / V = 20 / 1). The purple-black solid obtained was probe BOD (150 mg, yield 31.7%).

[0042] The probe BOD was subjected to 1 H NMR, 13 C NMR and mass spectrometry analysis, and the results are shown in Figure 2-Figure 4 , indicating that the probe BOD was successfully prepared.

[0043] Mass spectrometry verification of BOD reaction mechanism: 10 μmol / L BOD was reacted with 320 μg / mL rat liver microsomes and 0.1 mmol / L NADPH under hypoxic conditions for 3 h, and mass spectrometry detection was performed by high-resolution mass spectrometer Agilent 1100 HPLC / TOF, and the results are shown in Figure 5As shown in the figure, after BOD (10 μmol / L) reacted with rat liver microsomes (320 μg / mL) and NADPH (0.1 mmol / L) under hypoxic conditions (vacuum) for 3 h, the peak of fluorophore B (m / z value was 341.2015, calculated result was 341.2012) could be observed by high-resolution mass spectrometry.

[0044] Prepare the probe BOD mother solution: weigh BOD (4.7 mg) and dissolve it in 5.00 mL of analytical grade dimethyl sulfoxide (DMSO) to obtain a BOD (2.0 mmol / L) mother solution.

[0045] Example 2

[0046] This example verifies the BOD probe based on a cell model.

[0047] Construction of oxygen and glucose deprivation (OGD) model: BV2 cell line was cultured at 1×10 6 The cells were seeded into a T25 cell culture flask and cultured in DMEM high glucose medium. The night before the glucose and oxygen deprivation experiment, the cell lines were digested with 0.25% trypsin and cultured at a concentration of 1×10 5 The cells were seeded at a density of 100 μg / mL in a 12-well plate (DMEM high-glucose medium). After 12 h, the DMEM high-glucose medium was replaced with serum-free medium. At the same time, the 12-well plate was placed in an hypoxic chamber and cultured in an environment of 37°C, 5% CO2 + 95% N2 for 4 h. This was designated as the OGD group. A normal control (Control) group was also set up. The cells in the Control group were cultured in normal DMEM high-glucose medium in a cell culture incubator under normal oxygen conditions without special treatment.

[0048] Fluorescent probe incubation: 0 h after the completion of the OGD experiment on BV2 cells, subsequent immunofluorescence staining was performed. The specific steps are as follows: aspirate the culture medium → wash with PBS for 5 min (wash three times) → fix with 4% paraformaldehyde for 30 min → wash with PBS for 5 min (wash three times) → block with 5% donkey serum at room temperature for 50 min → add the corresponding primary antibody (IBa-1) at 4°C overnight → wash with PBS for 5 min three times → add the fluorescently labeled secondary antibody and BOD probe complex and incubate at room temperature for 2 h → wash with PBS for 5 min (wash three times) → add DAPI fluorescent antifade agent to the slides, store at 4°C, and observe using a Zeiss confocal fluorescence microscope imaging system. The subsequent image data analysis and processing were performed using its built-in analysis software.

[0049] The results are as follows Figure 6 and Figure 7 As shown, Figure 6In the figure, IBa-1 refers to the marker of microglia, BOD refers to the BOD probe, DAPI refers to the cell nucleus marker, and Merge refers to the merge channel. Figure 7 for Figure 6 40 times magnified image of the Merge image. Figure 8 The figure shows the statistical results of the fluorescence intensity of the BOD channel. Compared with the Control group, the BV2 cells became round in shape after OGD, and the red fluorescence signal of the BOD channel was significantly enhanced, indicating that the new hypoxia fluorescent probe BOD successfully responded in the in vitro hypoxic cell model.

[0050] Example 3

[0051] In this example, an animal model was constructed and verified.

[0052] Establishment of HIE animal model: HIE group: Wild-type mice (C57BL / 6 mice) aged 9-11 days (postnatal day) were anesthetized and quickly immobilized. An incision was made in the mid-left part of the neck skin. After incision, the left anterior cervical muscles were carefully dissected with ophthalmic straight and curved forceps to fully expose the carotid sheath. The left common carotid artery was separated from the vagus nerve and ligated with a single absorbable surgical suture. The wound was sutured after blood flow was eliminated. After the newborn mice recovered for 1.5 hours, they were placed in a constant-temperature hypoxia model experimental apparatus. N2 was uniformly infused into the apparatus. The mice were incubated in a hypoxic chamber filled with a mixture of 8% O2 and 92% N2 at an ambient temperature of 34°C for 40 minutes. Sham-operated group: Newborn mice of the same littermate and age were obtained. After adequate anesthesia, the left common carotid artery was dissected without ligation, and no subsequent hypoxic incubation was performed. Three days after the model was established, the scalps of the mice in the model group were cut while they were still alive, and the damage to the left side of their brain tissue was observed. The mice in the HIE group whose damaged side brain tissue showed white liquefied infarct foci were classified as the mild injury (HIM) group; the mice in the HIE group whose damaged side brain tissue had no obvious signs were classified as the severe injury (HIS) group.

[0053] TTC (2,3,5-chlorinated triphenyl tetrazolium) staining: TTC powder reagent was weighed in physiological saline to prepare 1% TTC solution, which was prepared and used immediately. 3 mL of mixed 1% TTC solution was added to each well of the six-well plate, and the six-well plate was preheated in a water bath for half an hour in advance. The HIE group and the same age Sham group mice 3 days after modeling were anesthetized by intraperitoneal injection of 10% chloral hydrate solution (3 mL / kg), and the brain tissue was taken out directly and placed on a glass culture dish with a ruler. The brain tissue was frozen at -20°C for 13-15 min. After taking out, the culture dish containing the brain tissue was placed on crushed ice, and the forebrain was evenly cut into 1 mm thick slices along the coronal plane. The brain slices were added to the six-well plate and incubated at 37°C for 30 min in the dark. After staining, the TTC solution was aspirated, and 4% paraformaldehyde solution was used for fixation overnight. The next day, the photograph was taken for analysis.

[0054] The TTC staining results are shown in Figure 9 The yellow dashed line indicates the infarction area. Sham: sham operation group; HIM: mild injury group; HIS: severe injury group. The results show that compared with the Sham group, the HIS group mice have a large area of white infarction lesions on the left side of the brain tissue, indicating that the HIE model is successfully constructed.

[0055] HE (hematoxylin and eosin) staining: 3 days after modeling, the mice and Sham group mice were inhaled isoflurane anesthesia (30 μL per mouse), and formaldehyde heart perfusion was performed for paraffin embedding. Then paraffin section was performed, the section thickness was 5 μm, and HE staining was performed. The specific steps are as follows: the section is placed in a 65°C oven for baking (1 h) → environmental protection transparent dewaxing liquid I (45°C, 15 min) → environmental protection transparent dewaxing liquid II (45°C, 15 min) → environmental protection transparent dewaxing liquid III (45°C, 15 min) → anhydrous alcohol I (3 min) → anhydrous alcohol II (3 min) → anhydrous alcohol III (3 min) → 95% alcohol (3 min) → 85% alcohol (3 min) → 75% alcohol (3 min) → pure water (3 min) → hematoxylin (1-2 min) → pure water washing → hematoxylin differentiation liquid differentiation (15 s) → pure water washing → eosin (1-2 min) → anhydrous alcohol I (1 min) → anhydrous alcohol II (1 min) → xylene I (2 min) xylene II (2 min) → neutral balsam mounting, natural air drying, and then observed and photographed under a light microscope for image data processing and analysis.

[0056] The HE staining results are shown in Figure 10As shown in the figure, Contex refers to the cerebral cortex, Hip refers to the hippocampus, CA1 refers to the CA1 region of the hippocampus, CA2 refers to the CA1 region of the hippocampus, CA3 refers to the CA1 region of the hippocampus, and DG refers to the CA1 region of the hippocampus. The results showed that compared with the sham group, the tissue in the injury center of the HIS group mice showed obvious loosening and disorder, widened intercellular spaces, light staining, and pyknotic and fragmented cell nuclei. In the perilesional areas (hippocampal CA1, CA2, CA3, and DG regions), some cell nuclei also showed pyknotic degeneration and widened intercellular spaces. This shows that the HIE model was successfully established.

[0057] Example 4

[0058] This example performs BOD probe verification.

[0059] Direct incubation of BOD probe with brain tissue slices: Brain slices were prepared in HIE model mice and Sham group mice (see Example 3), and the new hypoxia fluorescent probe BOD was added for incubation and staining to verify whether the new hypoxia fluorescent probe BOD responds to ischemic hypoxic brain damage.

[0060] Specifically, three mice from the HIE (HIM, HIS) and sham groups of the same age were deeply anesthetized, and fresh brains were rapidly dissected, frozen, embedded, and directly cut into 5 μm slices using a cryostat. After fixation in anhydrous ethanol for 10 seconds, the sections were rinsed with 1× PBS (5 minutes / time x 3), and a solution of the novel hypoxia fluorescent probe BOD (20 μmol / L) was added dropwise. The sections were incubated in hypoxia (1% O2) in the dark for 2 hours. After washing with PBS (pH = 7.4), the sections were mounted with a fluorescence antifade mounting medium containing DAPI, air-dried, and observed under a laser confocal fluorescence microscope for images. For the control group (control), fresh brain tissue sections from HIE (HIM, HIS) and sham groups of the same age were fixed in anhydrous ethanol for 10 seconds, rinsed with 1× PBS (5 minutes / time x 3), and added dropwise with 1× PBS as a probe control. Subsequent procedures were the same as above.

[0061] The results are as follows Figure 11 As shown, compared with the Sham group, the cortical injury area of ​​the mice in the HIS group showed obvious fluorescence signals, and the HIM showed weak fluorescence signals, indicating that the BOD probe can respond to ischemic and hypoxic brain tissue and release fluorescent groups to emit light under hypoxic conditions.

[0062] Brain cryosections after intraperitoneal injection of BOD probe: Mice from the HIE model group (HIS, HIM) and the sham group (3 mice each) were deeply anesthetized and intraperitoneally injected with 5 mg / kg of a novel hypoxia fluorescent probe, BOD needle solution (20 μmol / L). Brain tissue was quickly removed 6 and 12 hours after intraperitoneal injection, completely covered with frozen embedding medium, and then rapidly frozen in a precooled (-20°C) cryostat. Sections were 5 μm thick, fixed with anhydrous ethanol for 10 seconds, washed with PBS (pH = 7.4), and mounted with a fluorescence antifade mounting medium containing DAPI, and air-dried. The sections were observed under a confocal laser scanning fluorescence microscope and images were acquired.

[0063] The results are as follows Figure 12 As shown in the figure, PWM refers to the periventricular zone, and Cortex refers to the cerebral cortex. Six hours after intraperitoneal injection of the probe solution, the periventricular zone and cortical injury areas of mice in the HIS group showed a strong positive fluorescent signal. Furthermore, 12 hours after intraperitoneal injection, the periventricular zone and cortical injury areas of mice in the HIS group still showed a weak positive signal. These results indicate that the BOD probe can enter the central nervous system (CNS) through the ventricular circulation, cross the blood-brain barrier, and respond to liquefied necrosis induced by ischemia and hypoxia. Furthermore, the fluorescence signal of the BOD probe in the HIS group was the strongest 6 hours after intraperitoneal injection, indicating that 6 hours after intraperitoneal injection is the time point when the probe has the strongest binding ability. This provides a reference time window for further combining it with therapeutic drugs and visualizing targeted drug delivery.

[0064] In summary, the present invention designs a novel hypoxia fluorescent probe that can effectively respond to the cellular hypoxic environment, effectively penetrate the blood-brain barrier, respond to hypoxic brain areas, and achieve in situ imaging of brain tissue, which has the potential for clinical diagnosis of neonatal hypoxic-ischemic encephalopathy.

[0065] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A hypoxia fluorescent probe, characterized in that The hypoxia fluorescent probe includes a probe having a molecular structure as shown in Formula I or a pharmaceutically acceptable salt, ester or solvate thereof; 2. Use of the hypoxia fluorescent probe according to claim 1 in the preparation of products for detecting hypoxia-related diseases.

3. The use according to claim 2, characterized in that The hypoxia-related diseases include any one of neonatal hypoxic-ischemic encephalopathy, ischemic stroke or cancer.

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