A hypoxia fluorescent probe and a preparation method and application thereof

Abstract

The present application relates to a kind of hypoxic fluorescent probe and its preparation method and application, belong to the field of biotechnology.The present application designs new type of hypoxic fluorescent probe, and further develops its in situ imaging application mode in the field of neurobiology, the hypoxic fluorescent probe can effectively respond to hypoxic cell, tissue, and can penetrate blood-brain barrier, release fluorescence at the place of hypoxic brain injury with azo bond broken, has the potential of clinical diagnosis neonatal hypoxic-ischemic encephalopathy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a hypoxia fluorescent probe, its preparation method, and its application. Background Technology

[0002] Small molecule fluorescent probes are a class of small organic molecules that can detect specific targets by generating fluorescent signals. They typically consist of a fluorophore that generates a fluorescent signal and a recognition group that targets the specific analyte. They can be used to detect specific biomolecules, cells, or tissue structures in organisms. Small molecule fluorescent probes have advantages such as high sensitivity, high selectivity, multifunctionality, low toxicity, low cost, and in-situ and real-time imaging capabilities, and have shown excellent performance in fields such as disease diagnosis and treatment and exploring drug mechanisms of action.

[0003] The blood-brain barrier (BBB) ​​is a protective barrier located between blood vessels and brain tissue. Its main function is to restrict the entry of substances from the blood into the brain, protecting the brain from harmful external substances. The BBB is composed of cerebral vascular endothelial cells, the basement membrane, and surrounding astrocytes. The tight junctions between cerebral vascular endothelial cells make intercellular communication virtually impermeable, forming a barrier that is difficult to penetrate, restricting most drugs and molecules from entering the brain. Therefore, investigating whether small molecule fluorescent probes can cross the blood-brain barrier is an important topic in the study of neurological pathology, treatment, and drug delivery.

[0004] For example, hypoxic-ischemic encephalopathy (HIE) of the newborn is a disease caused by perinatal asphyxia leading to hypoxia and reduced or stopped cerebral blood flow, resulting in neonatal brain dysfunction. HIE causes a series of pathophysiological changes, including oxidative stress in brain development and intracellular calcium deficiency. 2+ Accumulation, mitochondrial dysfunction, excitotoxicity, and inflammation are all contributing factors. Currently, effective interventions are still lacking. Therefore, developing visual probes for in vivo detection of the extent of brain damage in basic research would greatly aid in understanding the mechanisms of HIE.

[0005] Currently, there are existing studies on fluorescent probes that can respond to hypoxic cells, but these studies mainly focus on in vitro cells. There are few reports on fluorescent probes that can cross the brain block (BBB) ​​and be localized in hypoxic brain regions or even within cells. Therefore, developing fluorescent probes that can be effectively applied to imaging hypoxic brain injury areas is of great significance for the diagnosis and treatment of diseases such as hypoxic-ischemic encephalopathy (HIE). Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a hypoxia fluorescent probe, its preparation method, and its application. The novel hypoxia fluorescent probe can respond to hypoxic cells and tissues, enabling 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 objectives, the present invention adopts the following technical solution:

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

[0009]

[0010] In this invention, a novel hypoxia fluorescent probe is designed and its application in in situ imaging in the field of neurobiology is further developed. This hypoxia fluorescent probe can effectively respond to hypoxic cells and tissues and can effectively penetrate the blood-brain barrier. At the site of hypoxic brain injury, the azo bond is broken to release the fluorophore B, resulting in an enhancement of the red fluorescence signal at 594 nm. It has the potential to clinically diagnose neonatal hypoxic-ischemic encephalopathy.

[0011] In a second aspect, the present invention provides a method for preparing the hypoxic fluorescent probe described in the first aspect, the method comprising:

[0012] The hypoxic fluorescent probe was obtained by reacting the compounds shown in Formula II and Formula III as substrates.

[0013]

[0014] Thirdly, the present invention provides the application of the hypoxia fluorescent probe described in the first aspect in the preparation of products for detecting hypoxia-related diseases.

[0015] Preferably, the hypoxia-related diseases include 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] This invention designs a novel hypoxia fluorescent probe and further develops its application in in situ imaging in the field of neurobiology. This hypoxia fluorescent probe can effectively respond to hypoxic cells and tissues and can penetrate the blood-brain barrier. It breaks azo bonds and releases fluorescence at the site of hypoxic brain injury, and has the potential to clinically diagnose neonatal hypoxic-ischemic encephalopathy. Attached Figure Description

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

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

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

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

[0022] Figure 5 This is a mass spectrum of BOD reacting with rat liver microparticles.

[0023] Figure 6 This is a confocal microscopy image of BV2 cells after OGD treatment and incubation with the BOD probe.

[0024] Figure 7 for Figure 2 A 40x magnified view of the Merge plot.

[0025] Figure 8 This is a graph showing the statistical results of fluorescence intensity in the BOD channel.

[0026] Figure 9 Image showing TTC staining results of mouse brain tissue 3 days after HIE modeling, scale bar is 5mm.

[0027] Figure 10 Image showing HE staining results of mouse brain tissue 3 days after HIE modeling, scale bar is 100μm.

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

[0029] Figure 12 Confocal microscopy images of brain tissue sections from mice injected intraperitoneally with the BOD probe 6h and 12h 3 days after HIE modeling, scale bar 50μm. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and 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 is determined by the claims.

[0031] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0032] This invention develops a novel hypoxic fluorescent probe, BOD, and explores its in-situ imaging applications in neurobiology, such as... Figure 1 As shown, the novel hypoxic fluorescent probe BOD consists of an azo bond connecting a specific fluorophore and a quencher group. It can release fluorescence by breaking the azo bond at the site of hypoxic brain injury, enabling imaging of the injured brain region. By transmitting the novel hypoxic fluorescent probe BOD through the brain bulb in a HIE model, in-situ imaging and monitoring of hypoxic brain regions can be achieved. The probe can be transmitted through the BBB after intraperitoneal injection, and the response of the probe to the hypoxic brain region can be observed through fresh brain tissue slices. Compared with traditional in vivo imaging, this method offers greater precision, allowing for high-resolution observation and recording of the distribution of the fluorescent probe in brain tissue. It also facilitates a deeper understanding of the pathological mechanisms of oxidative stress in HIE, as well as the more precise localization and changes in brain regions affected by oxidative stress during this process. The novel hypoxic fluorescent probe BOD and its application method have the potential for clinical diagnosis of neonatal hypoxic-ischemic encephalopathy.

[0033] Example 1

[0034] This embodiment prepares a novel hypoxic fluorescent probe, BOD.

[0035] The synthesis route is shown below.

[0036]

[0037] Synthesis of compound 3-7: 1,1,2-trimethyl-1H-benzo[e]indole (10.5 g, 50.2 mmol) and iodoethane (7.12 g, 50.2 mmol) were dissolved in 40 mL of toluene. The reaction was carried out overnight at 110 °C. After the reaction was complete, the precipitate was filtered to give compound 3-7 (7.3 g, yield 61.0%), which is a pale purple color.

[0038]

[0039] Synthesis of Compounds 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 sodium nitrite (NaNO2) aqueous solution (0.7 g, 10 mmol) was added dropwise to this solution, and the mixture was 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 adjust the pH of the solvent to 3-5. The prepared N,N-dimethylaniline solution was added to the reaction mixture, and the mixture was stirred in an ice bath for 2 h, then cooled to room temperature and stirred for another 2 h. After the reaction was complete, diluted sodium hydroxide aqueous solution was added to the reaction mixture until the reaction mixture became neutral, at which point a red precipitate formed. The product was obtained by filtration and washed with water. The product was recrystallized with ethanol and dried in a vacuum oven to give a dark red solid compound 3-8 (2.1 g, yield 83.0%).

[0040]

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

[0042] Perform probe BOD 1 H NMR, 13 C NMR and mass spectrometry analyses, results are as follows Figures 2-4 As shown, this indicates that the probe BOD was successfully prepared.

[0043] Mass spectrometry verification of the BOD reaction mechanism: 10 μmol / L BOD was reacted with 320 μg / mL rat liver microparticles and 0.1 mmol / L NADPH under hypoxic conditions for 3 h. The reaction was then detected by mass spectrometry using an Agilent 1100 HPLC / TOF instrument. The results are as follows: Figure 5As shown, 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 could be observed by high-resolution mass spectrometry (m / z value 341.2015, calculated result 341.2012).

[0044] Preparation of probe BOD stock 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) stock solution.

[0045] Example 2

[0046] This embodiment validates the BOD probe based on a cell model.

[0047] Constructing a cellular oxygen and glucose deprivation (OGD) model: BV2 cell lines were constructed at a rate of 1×10⁻⁶ cells / year. 6 Cells were seeded in a T25 cell culture flask and cultured in DMEM high-glucose medium. The night before the glucose-oxygen deprivation experiment, the cell line was digested with 0.25% trypsin and cultured at a concentration of 1 × 10⁻⁶ cells / flask. 5 Cells were seeded at the specified density in 12-well plates (DMEM high glucose medium). After 12 hours, the DMEM high glucose medium was replaced with serum-free medium, and the 12-well plates were placed in an anoxic chamber and cultured under anoxic conditions of 37°C, 5% CO2, and 95% N2 for 4 hours. This group was designated as the OGD group. A normal control group was also set up. The cells in the control group were cultured in normal DMEM high glucose medium under normal oxygen conditions in a cell culture incubator without any special treatment.

[0048] Fluorescent probe incubation: 0 h after the completion of the BV2 cell OGD experiment, subsequent immunofluorescence staining was performed. The specific steps were as follows: aspirate the culture medium → wash with PBS for 5 min (3 washes) → fix with 4% paraformaldehyde for 30 min → wash with PBS for 5 min (3 washes) → block with 5% donkey serum at room temperature for 50 min → add the corresponding primary antibody and incubate overnight at 4℃ (IBa-1) → wash with PBS for 5 min (3 washes) → add the complex of the fluorescently tagged secondary antibody and BOD probe and incubate at room temperature for 2 h → wash with PBS for 5 min (3 washes) → add DAPI fluorescent anti-quencher and mount the slide, store at 4℃, observe using a Zeiss confocal fluorescence microscope imaging system, and perform subsequent image data analysis and processing using its built-in analysis software.

[0049] The results are as follows Figure 6 and Figure 7 As shown, Figure 6IBa-1 refers to a marker for microglia, BOD refers to the BOD probe, DAPI refers to a nuclear marker, and Merge refers to the merging channel. Figure 7 for Figure 6 A 40x magnified view of the Merge plot. Figure 8 The graph shows the statistical results of the fluorescence intensity of the BOD channel. Compared with the Control group, BV2 cells were round after OGD treatment, and the red fluorescence signal of the BOD channel was significantly enhanced, indicating that the novel hypoxia fluorescent probe BOD responded successfully in the in vitro hypoxic cell model.

[0050] Example 3

[0051] This embodiment constructs and validates an animal model.

[0052] Establishing a HIE animal model: HIE group: Wild-type mice (C57BL / 6 mice) aged 9-11 days (d) were anesthetized and quickly fixed. An incision was made slightly to the left of the mid-neck skin. After opening, the left anterior cervical muscles were meticulously dissected using 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 absorbable surgical needle-and-suture sutures. After no blood flow was observed, the wound was sutured. After 1.5 hours of recovery, the newborn mice were placed in a hypoxic model experimental device. N2 was injected into the device at a uniform rate, and the mice were cultured for 40 minutes in a hypoxic chamber filled with a mixture of 8% O2 and 92% N2 at an ambient temperature of 34℃. Sham-operated group (Sham): Newborn mice from the same litter and of the same age were fully anesthetized. Only the left common carotid artery was dissected without ligation, and no subsequent hypoxic culture treatment was performed. Three days after the model was established, the scalp of the mice in the model group was cut open in vivo to observe the damage to the left brain tissue. Mice in the HIE group with white liquefied infarct foci in the damaged side of the brain tissue were classified into the mild injury (HIM) group; mice in the HIE group with no obvious characteristics in the damaged side of the brain tissue were classified into the severe injury (HIS) group.

[0053] TTC (2,3,5-triphenyltetrazolium chloride) staining: Prepare a 1% TTC solution by dissolving TTC powder in physiological saline in the dark. Add 3 mL of the mixed 1% TTC solution to each well of a six-well plate and preheat the plate in a water bath for half an hour beforehand. Three days after modeling, HIE group and age-matched Sham group mice were anesthetized with an intraperitoneal injection of 10% chloral hydrate solution (3 mL / kg). The brain tissue was completely removed and placed directly on a glass culture dish with a ruler. The brain tissue was frozen at -20℃ for 13–15 min. After removal, the culture dish containing the brain tissue was placed on crushed ice. The forebrain was evenly sliced ​​into 1 mm thick sections along the coronal plane. The brain sections were added to the six-well plates and incubated at 37℃ for 30 min in the dark. After staining, the TTC solution was aspirated, and the tissue was fixed overnight with 4% paraformaldehyde solution. Images were taken and analyzed the following day.

[0054] TTC staining results are as follows Figure 9 As shown, the area within the yellow dashed line represents the infarct region. Sham: sham-operated group; HIM: mild injury group; HIS: severe injury group. The results indicate that compared with the Sham group, the HIS group mice had a large area of ​​white infarct lesions on the left side of the brain tissue, indicating that the HIE model was successfully constructed.

[0055] Eosin (HE) staining: Mice from the model group and the Sham group were anesthetized with isoflurane (30 μL / mouse) 3 days after model establishment. After formaldehyde heart perfusion, the mice were embedded in paraffin and then sectioned with paraffin to a thickness of 5 μm. HE staining was performed. The specific steps are as follows: Place the sections in a 65℃ oven for baking (1 h) → Environmentally friendly transparent dewaxing solution I (45℃, 15 min) → Environmentally friendly transparent dewaxing solution II (45℃, 15 min) → Environmentally friendly transparent dewaxing solution III (45℃, 15 min) → Anhydrous ethanol I (3 min) → Anhydrous ethanol II (3 min) (min) → Anhydrous ethanol III (3min) → 95% ethanol (3min) → 85% ethanol (3min) → 75% ethanol (3min) → Pure water (3min) → Hematoxylin (1-2min) → Rinse with pure water → Differentiate with hematoxylin differentiation solution (15s) → Rinse with pure water → Eosin (1-2min) → Anhydrous ethanol I (1min) → Anhydrous ethanol II (1min) → Xylene I (2min) → Xylene II (2min) → Mount with neutral resin, air dry naturally, observe and photograph under a light microscope, and perform image data processing and analysis.

[0056] HE staining results are as follows Figure 10As shown, Contex refers to the cerebral cortex, Hip to the hippocampus, CA1 to the hippocampal CA1 region, CA2 to the hippocampal CA1 region, CA3 to the hippocampal CA1 region, and DG to the hippocampal CA1 region. The results indicate that, compared to the Sham group mice, the HIS group mice showed significantly loose and disordered tissue arrangement in the central injury area, widened intercellular spaces, lighter staining, and pyknosis and fragmentation of cell nuclei. The surrounding injury areas (hippocampal CA1, CA2, CA3, and DG regions) also showed partial nuclear pyknosis and widened intercellular spaces. This demonstrates the successful construction of the HIE model.

[0057] Example 4

[0058] This embodiment 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 (refer to Example 3), and the novel hypoxic fluorescent probe BOD was added for incubation and staining to verify whether the novel hypoxic fluorescent probe BOD responds in ischemic hypoxic brain injury.

[0060] Specifically, the procedure involved deep anesthesia of three mice from the HIE group (HIM, HIS) and the age-matched Sham group. Fresh brain tissue sections were rapidly dissected, frozen, embedded, and directly sliced ​​into 5μm sections using a cryostat. After fixation in anhydrous ethanol for 10 seconds, the sections were rinsed with 1×PBS (5 min / time × 3 times), and then incubated with a novel hypoxic fluorescent probe BOD solution (20 μmol / L) for 2 hours in the dark under hypoxic (1% O2) conditions. After washing with PBS (pH = 7.4), the sections were mounted with a DAPI-containing fluorescent anti-quenching mounting medium, air-dried, and observed under a laser confocal fluorescence microscope. The control group consisted of fresh brain tissue sections from the HIE group (HIM, HIS) and the age-matched Sham group. These sections were fixed in anhydrous ethanol for 10 seconds, rinsed with 1×PBS (5 min / time × 3 times), and then incubated with 1×PBS solution 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 HIS group mice showed obvious fluorescence signals in the cortical injury area, while 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 frozen sections were performed after intraperitoneal injection of the BOD probe: Three mice from each of the HIE model group (HIS, HIM) and the Sham group were deeply anesthetized and injected intraperitoneally with 5 mg / kg of the novel hypoxic fluorescent probe BOD needle solution (20 μmol / L). Brain tissue was rapidly removed 6 h and 12 h after injection, completely covered with cryopreservation medium, and then rapidly frozen in a pre-cooled (-20℃) cryostat. Sections were 5 μm thick, fixed with anhydrous ethanol for 10 s, washed with PBS (pH = 7.4), mounted with DAPI-containing fluorescent anti-quenching mounting medium, and air-dried. Images were captured under a laser confocal fluorescence microscope.

[0063] The results are as follows Figure 12 As shown, PWM refers to the periventricular region, and Cortex refers to the cerebral cortex. Six hours after intraperitoneal injection of the probe solution, the periventricular region and cortical injury areas of the HIS group mice showed significant strong positive fluorescence signals; furthermore, 12 hours after intraperitoneal injection, the periventricular region and cortical injury areas of the HIS group mice still showed weak positive signals. These results indicate that the BOD probe can cross the blood-brain barrier via ventricular circulation to enter the central nervous system (CNS) and respond to ischemia-hypoxia-induced liquefactive necrotic tissue. Moreover, the strongest fluorescence signal of the BOD probe in the HIS group was observed at 6 hours after intraperitoneal injection, suggesting that 6 hours after intraperitoneal injection is the time point with the strongest binding ability of the probe, providing a reference time window for further combining it with therapeutic drugs and visualizing targeted drug delivery.

[0064] In summary, this invention designs a novel hypoxic fluorescent probe that can effectively respond to cellular hypoxic environments and can effectively penetrate the blood-brain barrier to respond to hypoxic brain regions, enabling in situ imaging of brain tissue and possessing the potential for clinical diagnosis of neonatal hypoxic-ischemic encephalopathy.

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

Claims

1. A hypoxic fluorescent probe, characterized in that, The hypoxia fluorescent probe is a probe with the molecular structure shown in Formula I or a pharmaceutically acceptable salt thereof. Formula I.

2. The application of the hypoxia fluorescent probe according to claim 1 in the preparation of products for detecting hypoxia-related diseases, characterized in that, The hypoxia-related diseases mentioned are neonatal hypoxic-ischemic encephalopathy or ischemic stroke.

Images