Application of near-infrared hypochlorous acid fluorescent dye in ultrasensitive urine diagnosis of acute kidney injury

Abstract

The invention provides an application of a near-infrared hypochlorous acid fluorescent dye in ultra-sensitive urine diagnosis of acute kidney injury. Phenyl isothiocyanate (PITC) and heptamethylamine-cyanine fluorophore of a piperazine unit are integrated to form a fluorescent dye capable of specifically responding to hypochlorous acid, urine of normal people and urine of patients with acute kidney injury can be distinguished, the fluorescent dye is used for diagnosis of acute kidney injury detection, and the fluorescent dye has the advantages of ultrafast response time, ultralow detection limit, long emission wavelength, high sensitivity and the like. And the background signal is low.

Description

Technical Field

[0001] The invention relates to the technical field of near-infrared probes, and in particular to application of a near-infrared hypochlorous acid fluorescent dye in ultrasensitive urine diagnosis of acute kidney injury. Background Art

[0002] Kidney injury, especially acute kidney injury (AKI), remains a difficult problem to be solved in clinical practice. Although a lot of research has been done on its pathogenesis and early detection in recent years, diagnostic methods still face many challenges. Traditional renal function test methods such as serum creatinine and urea nitrogen levels, although widely used, have obvious lags and usually change only hours or even days after renal injury has occurred. In addition, these indicators are not specific markers and are easily affected by individual factors such as age, weight, diet, etc., resulting in insufficient sensitivity and specificity. Ideal biomarkers or imaging probes need to have good kidney targeting, low toxicity and high resolution, but there are still limited tools that can meet all these criteria. An important mechanism of acute kidney injury is the generation of reactive oxygen species and subsequent damaging oxidative stress, and hypochlorous acid is one of the components of reactive oxygen species and is produced and released into the urine during the process of acute kidney injury. Excessive production of hypochlorous acid in acute kidney injury may reflect the state of inflammatory response, oxidative stress and impaired renal function, so the detection of hypochlorous acid in urine is expected to become an effective tool for diagnosing AKI.

[0003] There are many methods for detecting hypochlorous acid, such as starch-potassium iodide method, N,N-diethyl-p-phenylenediamine colorimetry, chemiluminescence method, spectrophotometry, etc. However, the amount of hypochlorous acid produced in normal cells of organisms is very small, about 0.47 nmol hypochlorous acid molecules can be produced per minute in every 10 neutrophils. The above methods for detecting hypochlorous acid are complicated to operate and many require to be carried out in organic media, which is not conducive to large-scale use. Small molecule fluorescent probes have unique advantages, including high cost-effectiveness, high sensitivity, simple implementation, real-time and non-invasive fluorescence imaging, and excellent spatiotemporal resolution, and are indispensable diagnostic tools. Imaging has the characteristics of instantaneous response and high sensitivity, and is an effective method for monitoring substances in complex biological systems. Near-infrared fluorescence (650~900 nm) is particularly suitable for biological imaging because it has some outstanding advantages, such as low level of photodamage, minimal interference from the organism's own fluorescence, weak light scattering and deep tissue penetration. However, many existing hypochlorous acid probes based on naphthylimide, rhodamine, imide or other fluorophores are affected by background signals to some extent, and cannot distinguish hypochlorous acid from other reactive oxygen species or reactive nitrogen species, especially ONOO, or have high detection limits and low sensitivity. Therefore, it is urgent to provide a small molecule fluorescent probe for acute kidney injury detection with high sensitivity, high specificity and simple operation method. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes an application of near-infrared hypochlorous acid fluorescent dye in ultra-sensitive urine diagnosis of acute kidney injury.

[0005] The present invention provides an application of a near-infrared hypochlorous acid fluorescent dye in the preparation of an ultrasensitive urine diagnostic preparation for acute kidney injury. The structural formula of the near-infrared hypochlorous acid fluorescent dye is shown in (I): (I).

[0006] The present invention integrates phenyl isothiocyanate (PITC) with the heptamethylamine-cyanine fluorophore of the piperazine unit to form a molecular probe that can specifically respond to hypochlorous acid. PITC has the property of being easy to react with nucleophilic reagents, and combined with the design of the piperazine unit, the molecular response to ClO can be increased. − In the response mechanism of the molecular silver light probe of the present application, ClO − / HClO can rapidly oxidize and attack the C––S bonds in the probe structure. ClO − / HClO (hypochlorite / hypochlorous acid) will react quickly with the C–S group in the probe molecule. − It preferentially attacks the C atom in the C–S group, oxidizing the C—S bond, leading to the transformation of the sulfamide region, and selectively oxidizing sulfamide to amide. The ClO captured during the reaction − is released, thus inducing structural changes, which further affect the electronic state of the fluorophore. Before the reaction, the photoinduced electron transfer effect of the fluorophore causes the fluorescence to be in a quenched state, showing weak or no fluorescence. Once the C–S group is replaced by ClO − / HClO oxidation and conversion to amide, the photoinduced electron transfer effect is suppressed, thereby restoring or enhancing the fluorescence signal.

[0007] In some embodiments, the preparation method of the near-infrared hypochlorous acid fluorescent dye comprises the following steps: (i) under inert gas conditions, mixing compound 1 represented by the structural formula (II), phenyl isothiocyanate and anhydrous ethanol, and stirring at 0°C to 10°C to obtain a mixed solution; (ii) removing the solvent from the mixed solution of (i) and purifying it to obtain a near-infrared hypochlorous acid fluorescent dye; (II).

[0008] In some embodiments, in step (i), the molar ratio of compound 1 to phenyl isothiocyanate is (2.0-2.5): (2.5-3).

[0009] In some embodiments, in step (i), the amount of anhydrous ethanol added is 20-25 mL.

[0010] In some embodiments, the purification method in step (ii) is: purification by silica gel column chromatography using dichloromethane and methanol as eluent to obtain a dark blue solid.

[0011] In some embodiments, the volume ratio of dichloromethane to methanol is (55-60): (1-1.5).

[0012] In some embodiments, the preparation method of compound 1 is as follows: S1: Under inert gas conditions, compound 2 represented by the structural formula (III), anhydrous dimethylformamide and piperazine are mixed and stirred to obtain a mixed solution; S2: removing the solvent from the mixed solution in S1, eluting and purifying to obtain the compound 1; (III).

[0013] In some embodiments, in step S1, the molar ratio of compound 2 to piperazine is (1.8-2.0):1.

[0014] In summary, compared with the prior art, the present invention achieves the following technical effects: The near-infrared fluorescent dye of the present invention can distinguish urine from normal people and patients with acute kidney injury, and can be used for rapid clinical diagnosis of acute kidney injury. It has an ultrafast response time (0.3 s), a long emission wavelength, and a low background signal. When applied to acute kidney injury detection, the near-infrared fluorescent dye of the present invention is not only highly sensitive, but also can realize real-time and visual monitoring of hypochlorous acid concentration changes, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 The mass spectrum of Cy-P of the present invention is: Figure 2 is the nuclear magnetic hydrogen spectrum of Cy-PITC of the present invention; Figure 3 is the nuclear magnetic carbon spectrum of Cy-PITC of the present invention; Figure 4 The ultraviolet-visible absorption and fluorescence spectra of Cy-PITC of the present invention; Figure 5 is a response time diagram of Cy-PITC of the present invention; Figure 6 This is the Cy-PITC cell imaging diagram of the present invention; Figure 7 The mouse urine imaging results of Cy-PITC of the present invention; (a) is the imaging process of mouse urine; (b) is the fluorescence imaging diagram of urine; (c) is the quantitative analysis of urine fluorescence imaging; Figure 8 The human urine imaging results of Cy-PITC of the present invention; (a) is the imaging process of human urine; (b) is the fluorescence imaging diagram of urine; (c) is the quantitative analysis of urine fluorescence imaging. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0018] The present invention provides a near-infrared hypochlorous acid fluorescent dye that can detect acute kidney injury with high sensitivity, and can be used for urine detection of acute kidney injury. The first part of the present invention provides a method for preparing a near-infrared hypochlorous acid fluorescent dye, by integrating PITC with a heptamethylamine-cyanine fluorophore of a piperazine unit. The response mechanism includes capturing analyte molecules (oxidative desulfurization, in which ClO − / HClO can attack the C atom of the C––S group ultrafast), inducing the regioselective conversion of thioamide to amide, releasing the trapped ClO − The second part verifies the effect of near-infrared fluorescent molecular probes on in vitro imaging of acute kidney injury in mice. The third part verifies the effect of near-infrared hypochlorous acid fluorescent dyes on in vitro imaging of clinical detection of acute kidney injury in humans, with ultrafast response time and ultra-low detection limit, and has broad application prospects.

[0019] Example 1 Synthesis of fluorescent probe compound Cy-PITC (1) Synthesis of Cy-P: a. Place IR-780 iodine (0.5 g, 0.75 mmol) in a dry three-necked flask. Add anhydrous dimethylformamide (DMF, 20 mL) to the flask to ensure that there is no interference from moisture. DMF can dissolve IR-780 iodine well. Connect the flask to a nitrogen atmosphere device, purge with nitrogen to remove oxygen and moisture in the air, maintain an anhydrous and inert environment, and stir the solution until IR-780 iodine is completely dissolved.

[0020] b. Accurately weigh piperazine (258 mg, 3 mmol) and slowly add it to the above IR-780 iodine DMF solution. Seal the container under nitrogen atmosphere and continue stirring. Place the flask in a water bath and heat to 90°C. Maintain this temperature and continue stirring the reactants for about 2 hours to promote the reaction between piperazine and IR-780 iodine.

[0021] c. After 2 hours, remove the reaction mixture from the water bath and cool to room temperature. Use a rotary evaporator to evaporate the solvent (DMF) under reduced pressure. This process needs to be controlled slowly to avoid overheating and product decomposition.

[0022] d. Dissolve the obtained residue with a small amount of dichloromethane / methanol (60:1) mixed solution. This ratio of solvent system has moderate polarity and can help dissolve the unreacted substances and by-products in the reaction mixture.

[0023] e. Use silica gel column chromatography for separation and purification. When packing the column, pay attention to the appropriate column height so that the sample can be effectively separated.

[0024] f. The collected blue solid residue containing the target product is dissolved in a mixed solvent of hexane / DCM (5:1) for recrystallization purification. The specific ratio of hexane to DCM helps to obtain the target product Cy-P with high purity.

[0025] g. After purification, pure Cy-P (yield 430 g, 76%) was obtained. The molecular formula was C 40 H 53 N4 + Calculated molecular weight [MI] + :m / z 589.4265 Da, measured value 589.40 Da. This indicates that the product quality is in line with expectations. The mass spectrum of Cy-P is shown in Figure 1 shown.

[0026] The synthesis route of Cy-P is as follows: .

[0027] (2) Synthesis of Cy-PITC a. Weigh Cy-P (143 mg, 0.2 mmol) and place it in a dry three-necked flask and connect it to a stirring device.

[0028] b. Add anhydrous ethanol (20 mL) to the flask to ensure an anhydrous environment. Ethanol, as a solvent, can effectively dissolve Cy-P and provide a uniform medium for the reaction. Under stirring, Cy-P will gradually dissolve to form a uniform dark blue solution.

[0029] c. Accurately measure phenyl isothiocyanate (30 μL, 0.25 mmol) and slowly add it to the ethanol solution in which Cy-P has been dissolved under a nitrogen atmosphere.

[0030] d. Place the three-necked flask in an ice bath and continue stirring the mixture for 4 h. Maintaining the temperature between 0°C and 10°C will help control the reaction rate, reduce the formation of by-products, and inhibit over-reaction.

[0031] e. After the reaction was completed for 4 hours, the mixture was removed from the ice bath, returned to room temperature, and the solvent (anhydrous ethanol) was evaporated under reduced pressure using a rotary evaporator. After removing the solvent, a crude product residue was obtained.

[0032] f. Prepare a silica gel column, select an appropriate column size, and use DCM / MeOH (50:1, v / v) as the eluent. Separate by silica gel column chromatography and collect the fractions containing Cy-PITC to obtain dark blue solid Cy-PITC (120 mg, yield 70.6%).

[0033] g. Structural characterization of purified Cy-PITC.

[0034] The synthesis route of Cy-PITC is as follows: .

[0035] The nuclear magnetic hydrogen spectrum of Cy-PITC is shown in Figure 2 As shown, the NMR carbon spectrum is as follows Figure 3 shown.

[0036] The optical properties of Cy-PITC were investigated by UV-Vis absorption spectroscopy (UV-Vis) and fluorescence spectroscopy. Figure 4 As shown, the maximum absorption peak of Cy-PITC is at 700 nm, and the maximum emission peak is at 796 nm.

[0037] The response time of Cy-PITC was then measured as follows: At room temperature, prepare 3 mL of 10 μM HClO solution with PBS, add it to a quartz dish, turn on the fluorescence time scan, add 3 μL of Cy-PITC, and measure the relationship between the fluorescence intensity change and time of the system. When the plateau phase is reached, it means that the reaction is basically completed.

[0038] like Figure 5 It is shown that the response time of the Cy-PITC prepared in this example is 0.3 s, indicating that the Cy-PITC prepared in this application can quickly interact with the target molecule, react quickly and generate a fluorescent signal, and the detection accuracy and sensitivity are high.

[0039] Example 2 Cy-PITC cell imaging Experimental cells: HK-2 cells; Chemical reagents: Cisplatin, prepared at a concentration of 1 mg / mL; Cy-PITC solution: 2 mM.

[0040] The imaging steps are as follows: (1) HK-2 cells were plated in an eight-well chamber (10,000 cells / well) and 200 μL of DMEM / F12 medium was added.

[0041] (2) After 24 h, add cisplatin solution to each well to make the final concentration of cisplatin 4 μg / mL and continue culturing for 24 h.

[0042] (3) Remove the culture medium containing cisplatin and wash three times with PBS.

[0043] (4) Add culture medium containing Cy-PITC to each well to make the final concentration of Cy-PITC 2 μM and incubate for 20 min.

[0044] (5) Remove the culture medium containing Cy-PITC, wash the cells three times with PBS, and image the cells using a Leica confocal microscope (λex = 635 nm, λem = 710~800 nm).

[0045] The results are as follows Figure 6 As shown, it can be seen that there is a strong fluorescence signal in the HK-2 cells after cisplatin treatment, and the fluorescence signal is mainly distributed in the cytoplasm.

[0046] Example 3 In vitro imaging of acute kidney injury in mice Experimental animals: Healthy C57BL / 6 adult mice; Chemical reagents: Cisplatin, prepared at a concentration of 20 mg / kg; Cy-PITC solution: 2 mM.

[0047] The mice were divided into a normal group and an experimental group. The experimental group was used to establish an acute kidney injury model. The urine of mice in each group was collected and fluorescent imaging was performed using Cy-PITC solution. Figure 7 As shown in (a), the specific steps are as follows: (1) Prepare cisplatin solution, dilute it to a dose of 20 mg / kg, and load it into a syringe under sterile conditions. Perform an intraperitoneal injection in the lower left abdomen of the mouse, ensuring that the injection needle penetrates the peritoneum but does not damage the internal organs. After the injection, observe the condition of the mouse to ensure that there are no obvious complications.

[0048] (2) 48 hours after the establishment of the acute kidney injury model, the mice were placed in metabolic cages and allowed to move freely in the metabolic cages to collect urine samples.

[0049] (3) Transfer 1 mL of the collected urine sample to a clean centrifuge tube, add 1 μL of Cy-PITC solution to each urine sample, and mix the urine sample and Cy-PITC to ensure thorough mixing.

[0050] (4) Preheat the IVIS imaging system and calibrate the device to ensure image quality. Place the urine sample containing Cy-PITC on the sample tray of the IVIS imaging system and take a fluorescent image of the urine sample.

[0051] (5) Imaging results are as follows Figure 7 As shown in (b) and (c), the fluorescence signal in the urine of the control group mice was lower. Compared with the control group, the fluorescence signal in the urine of the AKI group mice was significantly increased, indicating that Cy-PITC can be successfully used for the diagnosis of AKI mice.

[0052] Example 4 In vitro imaging of human acute kidney injury This example uses clinical verification of the in vitro imaging effect of the Cy-PITC probe. The specific steps are as follows: Figure 8 (a) shown.

[0053] Recruit healthy volunteers and patients with acute kidney injury, and ensure that participants have not received any treatment or drug intervention that may affect urine composition before the experiment. Instruct participants to collect urine in the morning before the experiment. Collect urine samples using a sterile urine collection container, aliquot 1 mL into a clean centrifuge tube, add 1 μL Cy-PITC, and mix the urine sample and Cy-PITC to ensure even distribution.

[0054] Referring to Example 3, a fluorescence image of the urine sample was taken and quantitative analysis was performed.

[0055] The results are as follows Figure 8As shown in (b) and (c), the fluorescence signal in urine of normal people is low. Compared with the control group, the urine fluorescence signal of patients in the AKI group was significantly increased, indicating that Cy-PITC can be successfully used in clinical AKI diagnosis.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of a near-infrared hypochlorous acid fluorescent dye in the preparation of an ultrasensitive urine diagnostic preparation for acute kidney injury, characterized in that: The structural formula of the near-infrared hypochlorous acid fluorescent dye is shown in (I): （I）。 2. The use according to claim 1, characterized in that: The preparation method of the near-infrared hypochlorous acid fluorescent dye comprises the following steps: (i) under inert gas conditions, mixing compound 1 represented by the structural formula (II), phenyl isothiocyanate and anhydrous ethanol, and stirring at 0°C to 10°C to obtain a mixed solution; (ii) removing the solvent from the mixed solution of (i) and purifying it to obtain the near-infrared hypochlorous acid fluorescent dye; (II)。 3. The use according to claim 2, characterized in that: In step (i), the molar ratio of compound 1 to phenyl isothiocyanate is (2.0-2.5): (2.5-3).

4. The use according to claim 2, characterized in that: In step (i), the amount of anhydrous ethanol added is 20-25 mL.

5. The use according to claim 2, characterized in that: The purification method in step (ii) is: purification by silica gel column chromatography using dichloromethane and methanol as eluent to obtain a dark blue solid.

6. The use according to claim 5, characterized in that: The volume ratio of dichloromethane to methanol is (55-60): (1-1.5).

7. The use according to claim 2, characterized in that: The preparation method of compound 1 is as follows: S1: Under inert gas conditions, compound 2 represented by the structural formula (III), anhydrous dimethylformamide and piperazine are mixed and stirred to obtain a mixed solution; S2: removing the solvent from the mixed solution in S1, eluting and purifying to obtain the compound 1; (III)。 8. The use according to claim 7, characterized in that: In step S1, the molar ratio of compound 2 to piperazine is (1.8-2.0):1.

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