Copper ion responsive near-infrared two-region fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared two-region fluorescent probe with copper ion responsiveness and a preparation method and application thereof, and belongs to the technical field of fluorescent probes, 2ClOH-OBDP dye and 2-picolinic acid are subjected to an esterification reaction to obtain the near-infrared two-region fluorescent probe with the copper ion responsiveness, the preparation method is simple, the operation is easy, and the cost is low. Nanoparticles formed by the copper ion responsive near-infrared two-region fluorescent probe can realize high-sensitivity detection of copper ions, can perform in-vivo imaging on an MPTP-induced Parkinson's disease model, and have important significance in development of early diagnosis markers of Parkinson's disease.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a copper ion-responsive near-infrared II fluorescent probe, its preparation method, and its application. Background Technology

[0002] Parkinson's disease, a common neurodegenerative disorder, is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. Recent studies have shown that an imbalance in brain metal ion homeostasis, particularly the abnormal accumulation of copper ions, plays a crucial role in the pathogenesis of this disease.

[0003] Under physiological conditions, copper ions are essential cofactors for many important enzymes, such as superoxide dismutase and cytochrome C oxidase. However, in the progression of Parkinson's disease, copper ions are overexpressed in specific brain regions, such as the substantia nigra, leading to severe homeostasis. This excess of copper ions can catalyze the production of reactive oxygen species through the Fenton reaction, triggering oxidative stress, resulting in mitochondrial dysfunction, abnormal aggregation of α-synuclein and the formation of toxic oligomers, ultimately accelerating neuronal apoptosis.

[0004] Therefore, achieving precise and dynamic monitoring of copper ion levels in the brains of Parkinson's disease models is crucial for in-depth exploration of its specific mechanisms of action in the disease's development. Traditional detection methods often struggle to achieve in-situ, real-time, and highly sensitive detection. Against this backdrop, developing highly selective and sensitive fluorescence detection technologies can intuitively visualize the spatiotemporal distribution and concentration fluctuations of copper ions. This not only provides a key technical tool for elucidating copper ion-mediated neurotoxic pathways but also offers new strategies and insights for evaluating the efficacy of potential drugs and developing early diagnostic biomarkers for Parkinson's disease. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a copper ion-responsive near-infrared II fluorescent probe, its preparation method, and its application. A copper ion-responsive near-infrared II fluorescent probe was obtained by esterification of a dye with 2-pyridinecarboxylic acid. This preparation method is simple. The nanoparticles formed by the copper ion-responsive near-infrared II fluorescent probe can achieve high-sensitivity detection of copper ions and can perform in vivo imaging of MPTP-induced Parkinson's disease models. This is of great significance for the development of early diagnostic biomarkers for Parkinson's disease.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention provides a copper ion-responsive near-infrared II fluorescent probe, the structural formula of which is:

[0008] .

[0009] This invention also provides a method for preparing the copper ion-responsive near-infrared II fluorescent probe, the method comprising the following steps: ... Mixed with 2-pyridinecarboxylic acid, a condensing agent and a catalyst are added, and under an inert atmosphere, 0 o At C, a solvent was added, and the reaction was carried out at room temperature for 6-12 h. After post-processing, the copper ion-responsive near-infrared fluorescent probe Cu-OBDP was obtained.

[0010] The The structural formula is: .

[0011] The The molar ratio of 2-pyridinecarboxylic acid, condensing agent, and catalyst is 1:2.5~4:2.5~4:0.25~0.4.

[0012] The condensing agent is N,N'-dicyclohexylcarbodiimide.

[0013] The catalyst is 4-dimethylaminopyridine.

[0014] The solvent is anhydrous dichloromethane; the inert atmosphere is nitrogen.

[0015] The present invention also provides a copper ion-responsive near-infrared II fluorescent nanoprobe, which is prepared by the following method: dissolving the copper ion-responsive near-infrared II fluorescent probe of the present invention in an organic solvent miscible with water, and then adding it to water containing a surfactant under high-speed stirring.

[0016] Furthermore, the water-miscible organic solvent is any one or more of N,N-dimethylformamide, dioxane, dimethyl sulfoxide, and tetrahydrofuran;

[0017] The water containing the surfactant is water containing DSPE-PEG.

[0018] The present invention also provides the application of the copper ion-responsive near-infrared II fluorescent nanoprobe in copper ion detection or in the preparation of MPTP-induced fluorescent imaging drugs for Parkinson's disease models.

[0019] This invention will... A copper ion-responsive near-infrared II fluorescent probe (Cu-OBDP) was obtained by esterification of dye with 2-pyridinecarboxylic acid. Further preparation of Cu-OBDP nanoparticles revealed that, in the presence of free copper ions at pH 7.4, the absorption peak at 785 nm continuously increased, and the fluorescence emission peak at 905 nm was enhanced by 30 times. Using a 1000 nm long-pass filter, Cu... 2+ The detection limit can reach 3.4 nM, and the probe has excellent selectivity. It also has very good in vivo imaging performance in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced Parkinson's disease model.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The method for preparing the copper ion-responsive near-infrared II fluorescent probe provided by this invention is simple. After further preparation into nanoparticles, it exhibits superior performance for Cu ions. 2+ It has high responsiveness and can achieve Cu 2+ The high-sensitivity detection enables in vivo imaging of MPTP-induced Parkinson's disease models, which is of great significance for the development of early diagnostic biomarkers for Parkinson's disease. Attached Figure Description

[0022] Figure 1 This is a synthetic route diagram of Cu-OBDP in Example 1;

[0023] Figure 2 The 1H NMR spectrum of Cu-OBDP in Example 1;

[0024] Figure 3 The carbon NMR spectrum of Cu-OBDP in Example 1;

[0025] Figure 4 This is a high-resolution mass spectrum of Cu-OBDP from Example 1;

[0026] Figure 5 (a) TEM image and (b) dynamic light scattering pattern of Cu-OBDP nanoparticles in Example 2;

[0027] Figure 6 Cu-OBDP nanoparticles and Cu 2+ A graph showing the changes in the ultraviolet-visible-near-infrared absorption spectrum during the reaction process;

[0028] Figure 7 Cu-OBDP nanoparticles and Cu 2+ A diagram illustrating the reaction mechanism;

[0029] Figure 8Cu-OBDP nanoparticles and Cu 2+ Changes in fluorescence spectra before and after the reaction;

[0030] Figure 9 Experimental diagrams showing the selectivity of Cu-OBDP nanoparticles for different substances;

[0031] Figure 10 (a) Cu-OBDP nanoparticles with different concentrations of Cu 2+ (a) Changes in camera brightness after the reaction; (b) Linear relationship between camera brightness and copper ion concentration;

[0032] Figure 11 The images show the near-infrared II fluorescence imaging results for each experimental group in Application Example 2.

[0033] Figure 12 The graph shows the change in fluorescence intensity of brain regions in each experimental group over time in Example 2. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the embodiments.

[0035] Example 1

[0036] A copper ion-responsive near-infrared II fluorescent probe, Cu-OBDP, has the following structural formula: .

[0037] The preparation method of the copper ion-responsive near-infrared II fluorescent probe Cu-OBDP includes the following steps:

[0038] Will 30 mg (0.0413 mmol, 1.0 equivalent), 2-pyridinecarboxylic acid (20.3 mg, 0.1652 mmol, 4.0 equivalent), DCC (34 mg, 0.1652 mmol, 4.0 equivalent), and DMAP (2 mg, 0.01652 mmol, 0.4 equivalent) were placed in a reaction flask equipped with a magnetic stir bar. 5 mL of anhydrous dichloromethane was added at 0°C under nitrogen protection, followed by stirring overnight at room temperature. After the reaction was complete, the solid was removed by filtration, washed several times with saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After concentrating the solvent under reduced pressure, the residue was further purified by silica gel column chromatography using dichloromethane and petroleum ether in a 1:1 volume ratio as the eluent to obtain Cu-OBDP (22 mg, yield: 57%).

[0039] That 1H NMR (400 MHz, CDCl3): δ 8.93 (ddd, J = 4.8, 1.8, 0.9 Hz, 2H), 8.38 (dt, J = 7.8, 1.1 Hz, 2H), 8.19 – 8.02 (m, 8H), 7.98 (td, J = 7.8, 1.8Hz, 2H), 7.63 (ddd, J = 7.7, 4.7, 1.2 Hz, 2H), 7.07 – 6.98 (m, 4H), 6.95 (d,J = 1.2 Hz, 2H), 3.92 (s, 6H). like Figure 2 As shown.

[0040] 13 C NMR (101 MHz, CDCl3): δ 161.50, 161.33, 156.79, 155.70, 150.52, 146.13, 145.73, 144.79, 137.35, 135.06, 131.18, 129.72, 128.14, 127.90, 126.40, 124.84, 117.35, 114.41, 55.52. (e.g.) Figure 3 As shown.

[0041] MALDI-TOF-MS (m / z): calcd. for [M+Na] + , 958.0752, found, 958.443. (For example) Figure 4 As shown.

[0042] Example 2

[0043] A method for preparing a copper ion-responsive near-infrared II fluorescent nanoprobe (Cu-OBDP nanoparticles) includes the following steps:

[0044] 1) The Cu-OBDP prepared in Example 1 was dispersed in DMSO to prepare a 2.0 mM Cu-OBDP solution;

[0045] 2) Take 2 mg of DSPE-PEG 2k Dissolved in 10 mL of deionized water, 100 μL of the above Cu-OBDP solution was quickly added under high-speed stirring. Then, the solution was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 2000 Da to remove the organic solvent. The water was changed every 4 hours to obtain Cu-OBDP nanoparticle solution.

[0046] Its TEM such as Figure 5As shown in Figure a, a distinct spherical structure can be observed in the figure.

[0047] Its dynamic light scattering pattern is as follows Figure 5 As shown in Figure b, the Cu-OBDP nanoparticles prepared by this method have a particle size of 94 nm and a particle size distribution of 0.12.

[0048] The 20 μM Cu-OBDP nanoparticle solution prepared in this example was mixed with PBS buffer solution at pH 7.4 at a volume ratio of 1:1, and then Cu was added to a final concentration of 40 μM. 2+ Track the changes in UV-Vis-NIR absorption spectra at different reaction times, such as Figure 6 As shown in the figure, with the extension of reaction time, the absorption peak of the system at 550-705 nm gradually decreases, while the main peak at 785 nm gradually increases. This is because the Cu-OBDP probe generates [a specific substance] in the presence of copper ions. The mechanism diagram of this reaction process is as follows: Figure 7 As shown.

[0049] The 20 μM Cu-OBDP nanoparticle solution prepared in this example was mixed with PBS buffer solution at pH 7.4 at a volume ratio of 1:1, and then 20 μM Cu was added. 2+ After 8 hours of reaction, fluorescence detection was performed at an excitation wavelength of 808 nm. The detection results are as follows: Figure 8 As shown in the figure, the fluorescence of the system at 905 nm increased by nearly 30 times before and after the reaction, indicating that copper ions can effectively promote the conversion of Cu-OBDP to... This enhances the fluorescence.

[0050] The 20 μM Cu-OBDP nanoparticle solution prepared in this example was mixed with a PBS buffer solution at pH 7.4 at a ratio of 1:1, and then different metal ions or redox substances, including ClO-, H2O2, and O2, were added respectively. - Na + K + Mg 2+ Ca 2+ Zn 2+ Fe 2+ Fe 3+ Cu +All concentrations were 100 μM, and the mixture was incubated for 24 h. A system containing only the Cu-OBDP probe was used as a blank control. Fluorescence was detected in each system at an excitation wavelength of 808 nm. The ratio of the fluorescence intensity of each system at 905 nm to that of the blank system at 905 nm was plotted on the ordinate. The results are shown below. Figure 9 As shown in the figure, the fluorescence of these systems is almost unchanged compared to the blank system, while the fluorescence of the system with added Cu... 2+ The fluorescence change in the reaction system was very obvious, indicating that the Cu-OBDP nanoprobe was effective against Cu. 2+ It has excellent selectivity.

[0051] Application Example 1

[0052] Application of Cu-OBDP nanoparticles in in vitro copper ion detection

[0053] The 20 μM Cu-OBDP nanoparticle solution prepared in Example 2 was mixed with a PBS buffer solution at pH 7.4 at a ratio of 1:1. Then, Cu was added to the mixture at final concentrations of 0, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.7 μM, 0.8 μM, and 0.9 μM, respectively. 2+ After 8 hours of reaction, images were taken using a near-infrared II camera with a 1000 nm long-pass filter. The results are as follows. Figure 10 As shown in the figure, within this concentration range, the near-infrared II region brightness exhibits a good linear relationship with the copper ion concentration, and its detection limit can reach 3.4 nM. This demonstrates that the Cu-OBDP nanoparticle solution can achieve effective detection of Cu ions. 2+ High-sensitivity detection.

[0054] Application Example 2

[0055] Application of Cu-OBDP nanoparticles in highly sensitive in vivo imaging of copper ions in the brain in an MPTP-induced Parkinson's model

[0056] The Parkinson's disease model was rapidly induced by a single intraperitoneal injection of MPTP at a concentration of 30 mg / kg. The patients were divided into three groups:

[0057] Experimental group 1, i.e. control group: without MPTP induction, Cu-OBDP nanoprobe (concentration of 2mM, injection of 100 μL) was injected into the tail vein. Near-infrared fluorescence imaging of the brain was performed at 10 min, 30 min, 60 min and 90 min respectively, using 808 nm excitation and 1300 nm filter.

[0058] Experimental group 2: The experimental group received a single intraperitoneal injection of 30 mg / kg MPTP. 24 h later, Cu-OBDP nanoprobe (concentration of 2 mM, 100 μL) was injected into the tail vein. Near-infrared fluorescence imaging of the brain was performed at 10 min, 30 min, 60 min, and 90 min, respectively, using 808 nm excitation and a 1300 nm filter.

[0059] Experimental group 3, using Cu produced by L-dopamine consumption 2+ Following a single intraperitoneal injection of 30 mg / kg MPTP, L-dopamine was simultaneously injected intraperitoneally at 30 mg / kg to clear copper ions generated in the brain. Twenty-four hours later, Cu-OBDP nanoprobes (2 mM, 100 μL) were injected via tail vein. Near-infrared fluorescence imaging of the brain was performed at 10 min, 30 min, 60 min, and 90 min, using 808 nm excitation and a 1300 nm filter.

[0060] Its imaging results are as follows Figure 11 As shown, in the PBS control group (experimental group 1), the intensity of cerebral vascular fluorescence did not significantly increase over time; while in the MPTP-induced experimental group (experimental group 2), the fluorescence intensity gradually increased; and in mice induced simultaneously with MPTP / L-dopamine (experimental group 3), the change in cerebral vascular fluorescence intensity was relatively small. Figure 12 The quantitative results show that after 90 min, the fluorescence intensity of the mouse brain induced by MPTP was about 5 times that of the control group, indicating that Cu-OBDP nanoparticles can achieve high-sensitivity detection of Parkinson's disease.

[0061] The above detailed description of a copper ion-responsive near-infrared II fluorescent probe, its preparation method, and its application, with reference to the embodiments, is illustrative rather than limiting. Several embodiments may be listed within the defined scope. Therefore, variations and modifications that do not depart from the overall concept of the present invention should be within the protection scope of the present invention.

Claims

1. A copper ion-responsive near-infrared II fluorescent probe, characterized in that, The structural formula of the copper ion-responsive near-infrared II fluorescent probe is: 。 2. The method for preparing a copper ion-responsive near-infrared II fluorescent probe as described in claim 1, characterized in that, The preparation method includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or a missing section. Mixed with 2-pyridinecarboxylic acid, a condensing agent and a catalyst are added, and under an inert atmosphere, 0 o At C, a solvent was added, and the reaction was carried out at room temperature for 6-12 h. After post-processing, the copper ion-responsive near-infrared fluorescent probe Cu-OBDP was obtained. The The structural formula is: .

3. The preparation method according to claim 2, characterized in that, The The molar ratio of 2-pyridinecarboxylic acid, condensing agent, and catalyst is 1:2.5~4:2.5~4:0.25~0.

4.

4. The preparation method according to claim 2 or 3, characterized in that, The condensing agent is N,N'-dicyclohexylcarbodiimide.

5. The preparation method according to claim 2 or 3, characterized in that, The catalyst is 4-dimethylaminopyridine.

6. The preparation method according to claim 2 or 3, characterized in that, The solvent is anhydrous dichloromethane; the inert atmosphere is nitrogen.

7. A copper ion-responsive near-infrared II fluorescent nanoprobe, characterized in that, The copper ion-responsive near-infrared II fluorescent nanoprobe is prepared by the following method: the copper ion-activated near-infrared fluorescent probe of claim 1 is dissolved in an organic solvent miscible with water, and then added to water containing a surfactant under high-speed stirring.

8. The copper ion-responsive near-infrared II fluorescent nanoprobe according to claim 7, characterized in that, The water-miscible organic solvent is any one or more of N,N-dimethylformamide, dioxane, dimethyl sulfoxide, and tetrahydrofuran.

9. The copper ion-responsive near-infrared II fluorescent nanoprobe according to claim 7, characterized in that, The water containing the surfactant is water containing DSPE-PEG.

10. The application of the copper ion-activated near-infrared fluorescent probe as described in claim 9 in copper ion detection or in the preparation of MPTP-induced fluorescent imaging drugs for Parkinson's model.

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