Methylene blue-based esterase-activated nanocluster probe and method of making same
Patent Information
- Application Number
- CN202610945894.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-29
- Publication Date
- 2026-08-21
AI Technical Summary
尽管它们能够实现灵敏检测，但静脉注射不仅限制了真正无创诊断的实现，还引发了患者依从性低和注射相关风险的担忧，给临床转化带来了重大挑战，尤其是在社区或家庭自检场景中
1、本发明首次建立了基于口服-尿液分析的急性肾损伤无创诊断新方法，完全摆脱了穿刺程序，实现了真正意义上的无创检测；
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Figure CN122604973A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical detection technology, specifically to a methylene blue-based esterase-activated nanoaggregating probe, its preparation method, and its application. Background Technology
[0002] Acute kidney injury (AKI) is a common clinical syndrome characterized by a rapid decline in renal function, with high morbidity and mortality. Early and accurate diagnosis and timely protective intervention are crucial to preventing the progression of AKI. Currently, the clinical diagnosis of AKI still mainly relies on the measurement of serum creatinine (sCr) and blood urea nitrogen (BUN). However, these biomarkers only show significant changes after significant loss of renal function, making early diagnosis difficult. Therefore, developing non-invasive methods suitable for the early diagnosis of AKI is of great importance. An ideal non-invasive diagnostic method should have the advantages of simple operation, high patient compliance, and accurate and reliable results. Urine analysis, as a classic clinical test, plays an important role in disease screening and monitoring due to its advantages such as convenient sample collection, convenient operation, and suitability for repeated testing. In recent years, a variety of molecular probes with high renal clearance rates have been developed for urine analysis-based diagnosis. However, most of these probes rely on intravenous injection. While intravenous administration enables sensitive detection, it not only limits the realization of truly non-invasive diagnosis but also raises concerns about low patient compliance and injection-related risks, posing a significant challenge to clinical translation, especially in community or home self-testing scenarios. In contrast, oral administration, which requires no invasive procedures, is an ideal approach for achieving completely non-invasive diagnosis. The strategy of combining oral administration with urine analysis—i.e., "oral-urine analysis"—holds the promise of integrating the non-invasive advantages of oral administration with the convenience of urine testing, thereby establishing a highly promising new paradigm for disease diagnosis. Therefore, this application aims to develop a probe for early non-invasive diagnosis of AKI via an oral-urine analysis modality. Summary of the Invention
[0003] The purpose of this invention is to provide a methylene blue-based esterase-activated nanoaggregate probe and its preparation method, as well as its application in the early non-invasive diagnosis of acute kidney injury.
[0004] This invention provides a methylene blue-based esterase-activating nanoparticle probe, MB-ES, whose Chinese name is 4-(benzoyloxy)benzyl-3,7-bis(dimethylamino)-10 H 4-Phenthiazine-10-carboxylic acid ester, its English name is 4-(benzoyloxy)benzyl-3,7-bis(dimethylamino-10) H-phenothiazine-10-carboxylate. Its structure is: .
[0005] The synthesis method of MB-ES includes the following steps: (1) Synthesis of compound 1: 1 mmol methylene blue, 4 mmol anhydrous sodium carbonate, distilled water and dichloromethane were mixed in a two-necked flask and stirred at 40°C under nitrogen protection; then, 4 mmol of sodium dithionite aqueous solution was injected and the mixture was stirred for 30 minutes to obtain compound 1. (2) Synthesis of compound 2: The reaction solution of compound 1 was cooled to room temperature and placed in an ice-water bath. Under nitrogen protection, 0.6 mmol of triphosgene in dichloromethane solution was added dropwise. The reaction was stirred at room temperature for 2 hours to obtain a yellow-brown solution. After the reaction was completed, the solution was extracted with saturated sodium chloride solution and ethyl acetate. The organic phase was collected and the solvent was removed by vacuum evaporation. Compound 2 was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluent. (3) Synthesis of compound 3: 5 mmol of p-hydroxybenzyl alcohol was dissolved in dichloromethane and stirred in an ice-water bath. After 5 minutes, 15 mmol of triethylamine was added, and stirring was continued for 30 minutes. Then, 5 mmol of benzoyl chloride dissolved in dichloromethane was added stepwise, and the mixture was stirred overnight. The organic phase was separated, washed three times with 50 mL of saturated sodium chloride solution, and then the aqueous phase was extracted three times with 50 mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The compound 3 was purified by column chromatography using ethyl acetate / petroleum ether (1 / 3) as the eluent to obtain a white solid compound 3.
[0006] (4) Synthesis of MB-ES: 1 mmol of compound 2, 1 mmol of compound 3, 1 mmol of 4-dimethylaminopyridine and 3 mmol of anhydrous sodium carbonate were dissolved in anhydrous dichloromethane. The reaction mixture was stirred at room temperature for 3 hours, and the reaction progress was monitored by thin-layer chromatography. After purification by column chromatography using dichloromethane as the elution solvent, a white solid product MB-ES was obtained.
[0007] The synthesized esterase-specific activated nanoaggregate probe MB-ES can be used to release methylene blue signaling molecules in response to esterases in vitro, and the probe can also be used in the early non-invasive diagnosis of acute kidney injury.
[0008] Particle size characterization of the MB-ES nanoparticle-associated probe: MB-ES probes were dissolved in dimethyl sulfoxide (DMSO) to prepare a 2 mM stock solution. 10 μL of this stock solution was added to 1 mL of deionized water, vortexed, and the hydrodynamic diameter was measured using a Malvern zetasizer. The results showed that MB-ES can self-assemble into nanoaggregates with a hydrodynamic diameter of approximately 644.9 nm in aqueous media. Optical response performance test of MB-ES to esterase: Optical response tests were performed in phosphate-buffered saline (PBS, 10 mM, pH 7.4). 10 μL of MB-ES stock solution was mixed with an appropriate amount of esterase in a 2 mL centrifuge tube, and PBS was added to a total volume of 2 mL. After vortexing, the mixture was incubated at 37℃. After the reaction, the mixture was transferred to a 3 mL quartz cuvette, and its UV-Vis absorption and fluorescence spectra were measured. The results showed that after reacting with esterase, the absorbance of MB-ES in the 500–710 nm band was significantly enhanced, and a distinct characteristic absorption peak appeared at 665 nm. Simultaneously, its fluorescence intensity at 696 nm increased by approximately 137 times compared to before the reaction, indicating that this probe has a sensitive optical response to esterase. High-performance liquid chromatography (HPLC) validation of methylene blue release from MB-ES activated by esterase: HPLC was used to validate the release of methylene blue from MB-ES after activation by esterase. The chromatographic conditions were as follows: mobile phase A was deionized water, and mobile phase B was acetonitrile; the gradient elution program was: 0–5 min, 50% A phase, 50% B phase; 5–25 min, 10% A phase, 90% B phase; 25–30 min, 50% A phase, 50% B phase; injection volume 20 μL. The results showed that the retention times of MB-ES and methylene blue were 27.6 min and 3.1 min, respectively. After esterase treatment, the chromatographic peak at 27.6 min attributed to MB-ES gradually weakened, while a new chromatographic peak appeared at 2.8 min. This peak's retention time was essentially consistent with that of free methylene blue, confirming that MB-ES was consumed by the esterase and effectively released methylene blue. Application of MB-ES in the early non-invasive diagnosis of acute kidney injury: Healthy ICR mice were used to establish an acute kidney injury model by intraperitoneal injection of cisplatin (20 mg / kg), while the control group was injected with an equal volume of PBS. At 6 h, 12 h, 24 h, 48 h, and 72 h after cisplatin induction, mice in each group were orally administered MB-ES (200 μM, 300 μL). Two h after administration, urine samples were collected from each group, and fluorescence imaging analysis was performed using a small animal optical imaging system. The results showed that the fluorescence intensity of urine in the cisplatin-induced model group was significantly lower than that in the healthy control group at the same time points, indicating that MB-ES can effectively distinguish between AKI model mice and healthy mice, and has the potential for early non-invasive diagnosis of acute kidney injury.
[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention establishes for the first time a novel non-invasive diagnostic method for acute kidney injury based on oral-urine analysis, completely eliminating the need for puncture procedures and achieving truly non-invasive detection; 2. The diagnostic sensitivity of this method is significantly better than the clinical gold standard (serological diagnosis). It can detect abnormalities within 6 hours after the onset of kidney injury, which is 42 hours earlier than the traditional method, and has extremely high potential for translational application. Attached Figure Description
[0010] Figure 1 MB-ES characterization by proton NMR spectroscopy Figure 2 MB-ES C NMR characterization Figure 3 MB-ES mass spectrometry characterization Figure 4 Hydrodynamic particle size distribution of MB-ES in aqueous media Figure 5 UV-Vis spectra of MB-ES and esterase response Figure 6 Fluorescence spectra of MB-ES and esterase response Figure 7 High-performance liquid chromatography analysis of methylene blue released after MB-ES activation by esterase. Figure 8 Comparison of urinary fluorescence intensity between healthy control group and acute kidney injury model group mice after oral administration of MB-ES. Detailed Implementation
[0011] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Example 1
[0012] Preparation and characterization of MB-ES: Reaction formula: .
[0013] Preparation steps: (1) Synthesis of compound 1: Methylene blue (1 mmol, 384 mg), anhydrous sodium carbonate (4 mmol, 424 mg), 6 mL of distilled water and 9 mL of dichloromethane were mixed in a two-necked flask and stirred at 40 °C under nitrogen protection. Subsequently, an aqueous solution of sodium dithionite (4 mmol, 696 mg) was injected and the mixture was stirred for 30 minutes to obtain compound 1.
[0014] (2) Synthesis of Compound 2: The reaction solution of Compound 1 was cooled to room temperature and placed in an ice-water bath. Under N2 protection, a dichloromethane solution containing triphosgene (0.6 mmol, 113 mg) was added dropwise. The reaction was stirred at room temperature for 2 hours to obtain a yellowish-brown solution. After the reaction was complete, the solution was extracted with saturated sodium chloride solution and ethyl acetate. The organic phase was collected and the solvent was removed by vacuum evaporation. Compound 2 was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluent.
[0015] (3) Synthesis of compound 3: p-hydroxybenzyl alcohol (5 mmol, 620.6 mg) was dissolved in 15 mL of dichloromethane and stirred in an ice-water bath. After 5 minutes, triethylamine (15 mmol, 2 mL) was added, and stirring was continued for 30 minutes. Then, benzoyl chloride (5 mmol, 700 mg) dissolved in 10 mL of dichloromethane was added stepwise, and the mixture was stirred overnight. The organic phase was separated, washed with saturated sodium chloride solution (3 × 50 mL), and the aqueous phase was extracted with dichloromethane (3 × 50 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The compound 3 was purified by column chromatography with ethyl acetate / petroleum ether (1 / 3) as the eluent to give a white solid compound 3.
[0016] (4) Synthesis of MB-ES: Compound 2 (347 mg, 1 mmol), compound 3 (228 mg, 1 mmol), 4-dimethylaminopyridine (122 mg, 1 mmol), and anhydrous sodium carbonate (318 mg, 3 mmol) were dissolved in 20 mL of anhydrous dichloromethane. The reaction mixture was stirred at room temperature for 3 hours, and the reaction progress was monitored by thin-layer chromatography. After purification by column chromatography using dichloromethane as the elution solvent, a white solid product MB-ES (135 mg, 25%) was obtained. 1 H NMR (400 MHz, DMSO- d 6) δ8.15 – 8.12 (m, 2H), 7.74 (t, J = 7.2 Hz, 1H), 7.60 (t,J = 7.5 Hz, 3H), 7.51(d, J = 7.8 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.37 – 7.31 (m, 2H), 7.30 –7.26 (m, 2H), 6.67 (d, J = 14.4 Hz, 3H), 5.21 (s, 2H), 2.88 (s, 12H). 13 C NMR(101 MHz, DMSO) δ 165.01, 154.88, 154.07, 151.12, 150.72, 149.11, 134.65,134.50, 133.72, 132.55, 130.26, 130.10, 129.42, 129.26, 128.08, 127.49,122.50, 122.37, 111.37, 110.25, 67.04, 40.68. MS: m / z Calcd 539.1879, Found[M+H] + 540.200, [M+Na] + 562.250, [M+K] + 578.150. (See) Figure 1 , Figure 2 , Figure 3 ).
[0017] Example 2 Particle size characterization of the MB-ES nanoparticle-aggregated probe: The probe MB-ES was dissolved in dimethyl sulfoxide (DMSO) to prepare a 2 mM stock solution. 10 μL of this stock solution was added to 1 mL of deionized water, vortexed, and the hydrodynamic diameter was measured using a Malvern Zetasizer. The results showed that MB-ES could self-assemble into nanoaggregates with a hydrodynamic diameter of approximately 644.9 nm in aqueous media (see...). Figure 4 ). Example 3
[0018] MB-ES optical response performance test to esterases: Optical response tests were performed in phosphate-buffered saline (PBS, 10 mM, pH 7.4). 10 μL of MB-ES stock solution was mixed with an appropriate amount of esterase in a 2 mL centrifuge tube, and PBS was added to a total volume of 2 mL. After vortexing, the mixture was incubated at 37 °C. After the reaction, the mixture was transferred to a 3 mL quartz cuvette, and its UV-Vis absorption and fluorescence spectra were measured. The results showed that after reacting with the esterase, the absorbance of MB-ES in the 500–710 nm band was significantly enhanced, and a distinct characteristic absorption peak appeared at 665 nm; simultaneously, its fluorescence intensity at 696 nm increased by approximately 137 times compared to before the reaction, indicating that the probe has a sensitive optical response to the esterase (see [link to relevant documentation]). Figure 5 , Figure 6 ).
[0019] Example 4 High-performance liquid chromatography validation of esterase activation of MB-ES to release methylene blue: High-performance liquid chromatography (HPLC) was used to verify the release of methylene blue from MB-ES after activation by esterase. The chromatographic conditions were as follows: mobile phase A was deionized water, and mobile phase B was acetonitrile; the gradient elution program was: 0–5 min, 50% A phase, 50% B phase; 5–25 min, 10% A phase, 90% B phase; 25–30 min, 50% A phase, 50% B phase; injection volume 20 μL. The results showed that the retention times of MB-ES and methylene blue were 27.6 min and 3.1 min, respectively. After esterase treatment, the chromatographic peak at 27.6 min attributed to MB-ES gradually weakened, while a new chromatographic peak appeared at 2.8 min, whose retention time was basically consistent with that of free methylene blue, confirming that MB-ES was consumed by esterase and effectively released methylene blue (see [link to HPLC]). Figure 7 ).
[0020] Example 5 Application of MB-ES in the early non-invasive diagnosis of acute kidney injury: Healthy ICR mice were selected and an acute kidney injury (AKI) model was established by intraperitoneal injection of cisplatin (20 mg / kg), while the control group received an equal volume of PBS. Mice in each group were orally administered MB-ES (200 μM, 300 μL) by gavage at 6 h, 12 h, 24 h, 48 h, and 72 h after cisplatin induction. Two h after administration, urine samples were collected from each group, and fluorescence imaging analysis was performed using a small animal optical imaging system. The results showed that the fluorescence intensity of urine in the cisplatin-induced model group was significantly lower than that in the healthy control group at the same time points, indicating that MB-ES can effectively distinguish AKI model mice from healthy mice and has the potential for early non-invasive diagnosis of acute kidney injury (see [link to relevant documentation]). Figure 8 ).
Claims
1. A methylene blue-based esterase-activated nanoaggregating probe, MB-ES, characterized in that, The structural formula is: 。 2. The method for synthesizing the methylene blue-based esterase-activated nanoaggregate probe MB-ES as described in claim 1, characterized in that, The reaction formula is: 。 3. The method for synthesizing the methylene blue-based esterase-activated nanoaggregate probe MB-ES as described in claim 2, characterized in that, Includes the following steps: (1) Mix 1 mmol methylene blue, 4 mmol anhydrous sodium carbonate, distilled water and dichloromethane in a two-necked flask and stir under nitrogen protection at 40°C; then, inject 4 mmol of sodium dithionite aqueous solution and stir for 30 minutes to obtain compound 1; (2) The reaction solution of compound 1 was cooled to room temperature and placed in an ice-water bath. Under nitrogen protection, 0.6 mmol of triphosgene in dichloromethane solution was added dropwise. The reaction was stirred at room temperature for 2 hours to obtain a yellow-brown solution. After the reaction was completed, the solution was extracted with saturated sodium chloride solution and ethyl acetate. The organic phase was collected and the solvent was removed by vacuum evaporation. Compound 2 was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluent. (3) Dissolve 5 mmol of p-hydroxybenzyl alcohol in dichloromethane and stir in an ice-water bath; after 5 minutes, add 15 mmol of triethylamine and continue stirring for 30 minutes; then add 5 mmol of benzoyl chloride dissolved in dichloromethane in portions and stir overnight. Separate the organic phase, wash three times with 50 mL of saturated sodium chloride solution, and then extract the aqueous phase three times with 50 mL of dichloromethane. Combine the organic phases and dry them on anhydrous sodium sulfate. Purify by column chromatography with ethyl acetate / petroleum ether = 1 / 3 as the eluent to obtain a white solid compound 3; (4) 1 mmol of compound 2, 1 mmol of compound 3, 1 mmol of 4-dimethylaminopyridine and 3 mmol of anhydrous sodium carbonate were dissolved in anhydrous dichloromethane; the reaction mixture was stirred at room temperature for 3 hours and the reaction progress was monitored by thin-layer chromatography; after purification by column chromatography using dichloromethane as the elution solvent, a white solid product MB-ES was obtained.
4. The application of the nano-aggregated probe MB-ES as described in claim 1 in the preparation of esterase detection reagents or detection kits.
5. The use of the nano-aggregated probe MB-ES as described in claim 1 in the preparation of a detection reagent or kit for the early non-invasive diagnosis of acute kidney injury.