A fluorescent probe based on lanthanide metal-organic framework, preparation method and application

Abstract

The present invention relates to a fluorescent probe based on a lanthanide metal organic framework, a preparation method and an application, and belongs to the field of nanomaterial technology. The fluorescent probe provided by the present invention is obtained by covalently cross-linking a lanthanide metal organic framework with a neutrophil elastin binding peptide and a P-selectin binding peptide, and the surface of the lanthanide metal organic framework is modified with a tetracyclic antibiotic and arginine. The fluorescent probe prepared by the present invention specifically targets activated platelets and neutrophils, does not affect cell survival rate, does not cause hemolysis of red blood cells, and can be applied to the preparation of diagnostic products for thrombotic diseases. The use of a fluorescent probe loaded with urokinase achieves stable and efficient delivery of urokinase, inhibits platelet aggregation, slows down blood clot contraction, has a good thrombolytic effect, and has the potential to be used in the preparation of drugs for the treatment of thrombotic diseases. The materials required for the present invention are easy to obtain, the preparation method is simple, and a new method is provided for the diagnosis and treatment of thrombotic diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a fluorescent probe based on a lanthanide metal organic framework, a preparation method and an application thereof. Background Art

[0002] Ischemic stroke (IS), also known as cerebral infarction, refers to a syndrome characterized by impaired blood supply to the brain due to cerebrovascular disease, leading to ischemia, hypoxia, necrosis, and impaired neurological function. IS is characterized by high morbidity, recurrence, disability, mortality, and economic burden, making it a major health threat to the Chinese public. Therefore, safe and effective IS diagnosis and treatment options are crucial for reducing recurrence, disability, and mortality rates, and improving the prognosis of IS.

[0003] Currently, diagnostic tests for IS primarily include magnetic resonance imaging (MRI), X-ray computed tomography (CT), and angiography. These auxiliary tests indirectly assess thrombi by monitoring vascular blood flow, but cannot directly assess thrombus size and properties, nor can they easily track scattered thrombi within the brain. This indirect imaging approach also has difficulties distinguishing IS from other causes of luminal stenosis and occlusion, such as atherosclerotic plaques, hindering early diagnosis of IS. Therefore, the development of highly sensitive direct thrombus imaging techniques holds promise for early diagnosis of IS. Metal-organic frameworks (MOFs) are a class of crystalline porous materials composed of organic ligands bound to metal nodes via coordination bonds. MOFs are one of the most promising drug delivery vehicles due to their high surface area and porosity, diverse functionality, and excellent biocompatibility. MOFs typically utilize host-guest interactions to adsorb and immobilize targets within their pores and surfaces. The MOF structure provides tight structural constraints on proteins, enhancing their stability and bioactivity. This rapid and low-cost loading process brings new possibilities for the development and application of biomacromolecules. Lanthanide metal-organic frameworks (Ln-MOFs) are Ln-based 3+ MOFs constructed for metal nodes. Ln-MOFs not only have the loading capacity of MOFs, but also retain the Ln 3+ The inherent advantages (such as Eu 3+ 、Tb 3+ The fluorescence lifetime is long, the monochromaticity is good, the Stokes shift is large, and Ce 4+ 、Ce 3+The redox activity of Ln-MOFs has led to their widespread application in sensing, imaging, catalysis, and other fields. Therefore, multi-element Ln-MOFs fluorescent probes as drug carriers are expected to achieve stable loading of thrombolytic drugs while simultaneously exhibiting high-resolution, low-background fluorescence imaging capabilities and antioxidant activity, ultimately enabling precise diagnosis and synergistic treatment of IS.

[0004] Therefore, it is necessary to develop Ln-MOF-based fluorescent probes for the diagnosis and treatment of IS, which is expected to provide new ideas and methods for the optimization of IS nanodiagnosis and treatment systems. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a lanthanide metal-organic framework-based fluorescent probe, preparation method, and application. The surface of the lanthanide metal-organic framework is modified with a tetracyclic antibiotic and arginine. The lanthanide metal-organic framework is covalently cross-linked with a neutrophil elastin-binding peptide and a P-selectin-binding peptide. The resulting fluorescent probe specifically targets activated platelets and neutrophils. Urokinase is loaded onto the fluorescent probe, enabling efficient and stable delivery of urokinase.

[0006] The first object of the present invention is to provide a fluorescent probe based on a lanthanide metal organic framework, wherein the fluorescent probe is obtained by covalently cross-linking a lanthanide metal organic framework with a neutrophil elastin binding peptide and a P-selectin binding peptide;

[0007] The lanthanide metal organic framework uses lanthanide metal as metal ion and terephthalic acid as organic ligand, and is surface-modified with tetracyclic antibiotics and arginine.

[0008] Furthermore, tetracyclic antibiotics change the outer electron configuration and energy level structure of lanthanide metal ions.

[0009] Furthermore, tetracyclic antibiotics and arginine sensitized lanthanide metal ions to achieve red fluorescence emission.

[0010] Furthermore, the lanthanide metals are cerium and europium.

[0011] Europium ions have a large Stokes shift, which can reduce fluorescence background interference and improve imaging clarity and accuracy. Cerium ions have good redox properties and chemical stability, which can give fluorescent probes antioxidant activity.

[0012] Furthermore, the tetracyclic antibiotic is doxycycline.

[0013] Furthermore, the amino acid sequence of the neutrophil elastin-binding peptide is CGEAIPMSIPPEVK (SEQ ID NO. 1).

[0014] Furthermore, the amino acid sequence of the P-selectin binding peptide is CDAEWVDVS (SEQ ID NO. 2).

[0015] The second object of the present invention is to provide the use of the above fluorescent probe in the preparation of diagnostic products for thrombotic diseases.

[0016] A third object of the present invention is to provide a method for preparing the fluorescent probe, comprising the following steps:

[0017] Step S1, dissolving an organic ligand in a first solvent, dissolving a metal salt in a second solvent, mixing the first solvent and the second solvent, heating, and drying to obtain a lanthanide metal-organic framework;

[0018] Step S2, modifying the lanthanide metal-organic framework using tetracyclic antibiotics and arginine;

[0019] Step S3: adding a cross-linking agent, a neutrophil elastase binding peptide, and a P-selectin binding peptide to the modified lanthanide metal organic framework to obtain the fluorescent probe.

[0020] Furthermore, in step S1, the first solvent is N,N-dimethylformamide, and the second solvent is deionized water.

[0021] Furthermore, in step S1, the temperature is heated to 100-150° C. for 1-2 hours.

[0022] Furthermore, the cross-linking agent is phospholipid-hydrazone bond-polyethylene glycol-maleimide.

[0023] Furthermore, the neutrophil elastase binding peptide and the P-selectin binding peptide form covalent bonds with the maleimide functional groups in the phospholipid-hydrazone bond-polyethylene glycol-maleimide coated on the surface of the lanthanide metal organic framework through sulfhydryl groups, thereby being connected to the surface of the lanthanide metal organic framework.

[0024] Furthermore, the usage ratio of the tetracyclic antibiotic, arginine and lanthanide metal organic framework is (50-800) mmol: (50-300) mmol: (1-2) mg.

[0025] Furthermore, in step S3, the covalent cross-linking temperature is 25° C., and the covalent cross-linking time is 2 hours.

[0026] The fourth object of the present invention is to provide the use of the above fluorescent probe in the preparation of drugs for treating thrombotic diseases.

[0027] Furthermore, the fluorescent probe is loaded with urokinase.

[0028] Furthermore, the loading amount of urokinase is 3000-7500U.

[0029] Preferably, the loading amount of urokinase is 3000U.

[0030] Beneficial effects of the present invention:

[0031] (1) The fluorescent probe prepared by the present invention specifically targets activated platelets and neutrophils, has high luminescence intensity and luminescence efficiency, does not affect cell survival rate, and does not cause red blood cell hemolysis, and can be used in the preparation of diagnostic products for thrombotic diseases;

[0032] (2) The fluorescent probe prepared by the present invention is loaded with urokinase, with a loading rate exceeding 80%, achieving stable and efficient delivery of urokinase, inhibiting platelet aggregation, slowing blood clot contraction, and having a good thrombolytic effect. It has the potential to be used in the preparation of drugs for the treatment of thrombotic diseases;

[0033] (3) The materials required for the present invention are easily available and the preparation method is simple, which provides a new method for the diagnosis and treatment of thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0035] Figure 1 It is a schematic diagram of the working process of the present invention;

[0036] Figure 2 is the PXRD pattern of NP-Ln-MOF@uPA in Example 2 of the present invention;

[0037] Figure 3 is a fluorescence spectrum of NP-Ln-MOF@uPA in Example 2 of the present invention;

[0038] Figure 4 is the zeta potential diagram of the nanomaterial in Example 2 of the present invention;

[0039] Figure 5 TEM image and element distribution diagram of NP-Ln-MOF@uPA in Example 2 of the present invention;

[0040] Figure 6 This is a graph showing the loading capacity of NP-Ln-MOF for urokinase uPA in Example 2 of the present invention;

[0041] Figure 7 This is a graph showing the release of urokinase uPA from NP-Ln-MOF under different pH conditions in Example 2 of the present invention;

[0042] Figure 81 is a graph showing the biocompatibility test results of NP-Ln-MOF in Example 3 of the present invention;

[0043] Figure 9 This is a graph showing the results of a hemolysis experiment on NP-Ln-MOF in Example 3 of the present invention;

[0044] Figure 10 This is a fluorescence confocal image of NP-Ln-MOF targeting and activating platelets in Example 3 of the present invention;

[0045] Figure 11 This is a fluorescence confocal image of NP-Ln-MOF targeted activation of neutrophils in Example 3 of the present invention;

[0046] Figure 12 This is a diagram showing the shrinkage of blood clots in Example 4 of the present invention;

[0047] Figure 13 This is a statistical graph of blood clot shrinkage volume in Example 4 of the present invention;

[0048] Figure 14 This is a diagram of platelet aggregation in Example 4 of the present invention;

[0049] Figure 15 This is a statistical graph of platelet aggregation rate in Example 4 of the present invention;

[0050] Figure 16 This is a graph showing the dissolution rate of blood clots by NP-Ln-MOF@uPA in Example 5 of the present invention. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0052] Example 1: Preparation of fluorescent probe

[0053] 1. Preparation of Ln-MOF:

[0054] (1) Dissolve 145 mg of cerium ammonium nitrate and 111 mg of europium nitrate hexahydrate in 1 mL of deionized water;

[0055] (2) Dissolve 88.5 mg of terephthalic acid (PTA) in 3 mL of N,N-dimethylformamide;

[0056] (3) The precursor solution was transferred to a high-pressure reactor, reacted at 100°C for 60 minutes, and washed with deionized water to obtain Ce / Eu-PTA;

[0057] (4) Ce / Eu-PTA was dispersed in 1 mL of deionized water, 100 μL of the solution was added to 100 μL of doxycycline aqueous solution and 200 μL of arginine aqueous solution, stirred for 1 hour, washed with deionized water, and freeze-dried to obtain Ce / Eu-PTA-DOX.

[0058] 2. Preparation of fluorescent probes:

[0059] (1) 1 mL of a 1 mg / mL aqueous solution of the crosslinker DSPE-Hyd-PEG-Mal was added to 2 mg of Ln-MOF, stirred for 4 hours, and then washed with deionized water.

[0060] (2) 500 μL of neutrophil elastase binding peptide (N) and P-selectin binding peptide aqueous solution (P) (1 mg / mL) were added to Ln-MOF, stirred for 2 h, washed with deionized water, and freeze-dried to obtain the fluorescent probe NP-Ln-MOF.

[0061] Example 2: Fluorescent probe loaded with thrombolytic drug urokinase

[0062] 2 mg of fluorescent probe NP-Ln-MOF and urokinase uPA were added to 1 mL of HEPES buffer and stirred for 2 hours. The mixture was washed with deionized water, freeze-dried, and stored in a refrigerator at 4°C to obtain the urokinase-loaded fluorescent probe NP-Ln-MOF@uPA. Various nanomaterials were characterized.

[0063] The X-ray diffraction pattern PXRD results of the fluorescent probe NP-Ln-MOF@uPA loaded with urokinase are as follows: Figure 2 As shown, it was revealed that probe loading and modification did not change the crystal structure of Ce / Eu-PTA.

[0064] The fluorescence spectrum of the fluorescent probe NP-Ln-MOF@uPA loaded with urokinase is shown in Figure 2. Figure 3 As shown, it is revealed that the probe can be excited to emit Eu by 405-460nm excitation light. 3+ characteristic emission.

[0065] Zeta potential diagram of nanomaterials Figure 4 As shown, the potential changes revealed that the drug, cross-linker, and peptide were successfully bound to the Ln-MOF.

[0066] The electron microscope TEM image and element distribution map of the fluorescent probe NP-Ln-MOF@uPA loaded with urokinase are shown in Figure 2. Figure 5 As shown, it is revealed that the composition of the probe is Ce / Eu-PTA containing cerium and europium elements, doxycycline and arginine containing nitrogen elements, and a binding peptide containing sulfur elements.

[0067] The loading capacity of fluorescent probe NP-Ln-MOF for urokinase is as follows Figure 6 As shown, the loading rates of the fluorescent probe for 7500U, 6000U and 3000U urokinase were 75.2%, 83.2% and 94.8%, respectively.

[0068] When the urokinase loading was 6000U, the release of urokinase was detected using a protein detection kit under different pH conditions. The results are as follows: Figure 7 As shown, under the condition of pH = 6.8, the release rate of urokinase in 3 hours reached 60%, while under the condition of pH = 7.5, the release rate of urokinase in 3 hours was only around 40%.

[0069] Example 3: Fluorescence imaging of neutrophils and platelets using the fluorescent probe NP-Ln-MOF

[0070] Different concentrations of fluorescent probe NP-Ln-MOF were dispersed in serum-free culture medium and incubated with bEnd.3 cells for 12 hours. The cell survival rate was determined using CCK-8 kit. The results are shown in Figure 2. Figure 8 As shown, the survival rate of cells treated with fluorescent probes at concentrations of 50-250 μg / mL remained above 80%, demonstrating the biocompatibility of the fluorescent probe NP-Ln-MOF.

[0071] Different concentrations of fluorescent probe NP-Ln-MOF were incubated with red blood cells for 3 hours, with normal saline as negative control and deionized water as positive control. The absorbance of the samples at 544 nm was detected and the hemolysis rate was calculated. The results are shown in Figure 2. Figure 9 As shown in the figure, the hemolysis rate of red blood cells caused by the fluorescent probe NP-Ln-MOF at a concentration of 50-300 μg / mL was less than 20%.

[0072] Thrombin was used to activate platelets, and phorbol ester PMA was used to activate neutrophils. The fluorescent probe was dispersed in serum-free culture medium (200 μg / mL) and incubated with activated platelets and neutrophils for 1 hour. After fully washing away the excess fluorescent probe NP-Ln-MOF, the cells were imaged using a laser scanning confocal microscope at a 63x objective lens and a 405nm excitation wavelength. Signals within the range of 600-630nm were collected. The results are shown in Figure 2. Figure 10 and Figure 11 As shown, the fluorescent probe NP-Ln-MOF can specifically target activated platelets and neutrophils, but has weak interaction with resting platelets and neutrophils.

[0073] The above experimental results reveal that the fluorescent probe NP-Ln-MOF has the potential to be used in the diagnosis of thrombotic diseases.

[0074] Example 4: Effects of fluorescent probes on blood clot retraction and platelet aggregation

[0075] The whole blood of wild-type mice was added to an equal volume of physiological saline and centrifuged at 1100 rpm for 11 minutes. The upper layer was aspirated, a small amount of red blood cells were added, and 400 μL was added to a collection tube. The fluorescent probe Ce / Eu-PTA-DOX without urokinase loading, the fluorescent probe NP-Ln-MOF@uPA loaded with urokinase, and urokinase uPA with the same concentration were added. The shrinkage of the blood clot was observed by taking pictures, and the changes in the blood clot volume were recorded using image J software. Figure 12 As shown in the figure, the anti-clot contraction effect of NP-Ln-MOF@uPA loaded with urokinase is not much different from that of the simple application of urokinase, while Ce / Eu-PTA-DOX without urokinase has no inhibitory effect on clot contraction. Figure 13 As shown in the figure, compared with the control group and Ce / Eu-PTA-DOX, the change of blood clot volume slowed down after the application of NP-Ln-MOF@uPA, and the blood clot still maintained about 60% of its volume after 50 minutes of action.

[0076] The whole blood of wild-type mice was added with an equal volume of physiological saline and centrifuged at 1100 rpm for 11 min. The upper layer was aspirated and the platelets were washed with CGS buffer and then dispersed in MTB buffer. 9 / L of platelets were incubated with 200μg / mL of fluorescent probe Ce / Eu-PTA-DOX without urokinase loading, fluorescent probe NP-Ln-MOF@uPA loaded with urokinase, and urokinase at the same concentration at 37℃ for 30 minutes. After adding the aggregation inducer collagen (0.5μg / mL), the platelet aggregation was detected using a platelet aggregometer. The results are shown in Figure 2. Figure 14 and Figure 15 As shown in the figure, the platelet photoconductivity decreased significantly after treatment with the control group and Ce / Eu-PTA-DOX, indicating that the platelet aggregation was serious. However, the platelets did not aggregate significantly after treatment with urokinase and NP-Ln-MOF@uPA, and the effect of NP-Ln-MOF@uPA was better than that of urokinase alone.

[0077] Example 5: Thrombolytic effect of urokinase delivered by fluorescent probe

[0078] Whole blood from wild-type mice was placed in a centrifuge tube and allowed to stand in a refrigerator at 4°C for 24 hours to form a blood clot. The blood clot was cut into approximately 40 mg (W1) and incubated with the fluorescent probe Ce / Eu-PTA-DOX without urokinase loading, the fluorescent probe NP-Ln-MOF@uPA loaded with urokinase, and urokinase at equivalent concentrations at 37°C for 3 hours. The remaining clot was weighed again (W2), and the thrombolysis rate was calculated as (W1-W2) / W1×100%. The results are shown in Figure 2. Figure 16As shown, the thrombolytic effect of the fluorescent probe loaded with urokinase is basically the same as that of urokinase with equivalent concentration, which is better than the fluorescent probe not loaded with urokinase and can ensure the thrombolytic effect of urokinase.

[0079] Comparative Example

[0080] Ln-MOF was prepared using the method of Example 1, except that europium nitrate was used as the metal ion, and terephthalic acid (PTA), pyromellitic acid (H3BTC) and pyromellitic tetracarboxylic acid (H4BTEC) were used as the organic ligands. Eu-PTA, Eu-BTC and Eu-BTEC were synthesized and loaded with urokinase uPA at a concentration of 1 mg / mL, with loading rates of 58.4%, 22.6% and 17.4%, respectively.

[0081] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A fluorescent probe based on a lanthanide metal-organic framework, characterized by: The fluorescent probe is obtained by covalently cross-linking a lanthanide metal organic framework with a neutrophil elastin binding peptide and a P-selectin binding peptide; The lanthanide metal organic framework uses lanthanide metals as metal ions, terephthalic acid as organic ligands, and is surface-modified with tetracyclic antibiotics and arginine; The lanthanide metals are cerium and europium.

2. The fluorescent probe according to claim 1, wherein: The tetracyclic antibiotic is doxycycline.

3. Use of the fluorescent probe according to claim 1 or 2 in the preparation of diagnostic products for thrombotic diseases.

4. The method for preparing the fluorescent probe according to claim 1 or 2, characterized in that: The following steps are involved: Step S1, dissolving an organic ligand in a first solvent, dissolving a metal salt in a second solvent, mixing the first solvent and the second solvent, heating, and drying to obtain a lanthanide metal-organic framework; Step S2, modifying the lanthanide metal-organic framework using a tetracyclic antibiotic and arginine; Step S3: adding a cross-linking agent, a neutrophil elastase binding peptide, and a P-selectin binding peptide to the modified lanthanide metal organic framework to obtain the fluorescent probe.

5. The preparation method according to claim 4, characterized in that: The cross-linking agent is phospholipid-hydrazone bond-polyethylene glycol-maleimide.

6. The preparation method according to claim 4, characterized in that: The usage ratio of the tetracyclic antibiotic, arginine and lanthanide metal organic framework is (50-800) mmol: (50-300) mmol: (1-2) mg.

7. Use of the fluorescent probe according to claim 1 or 2 in the preparation of drugs for treating thrombotic diseases.

8. The use according to claim 7, characterized in that: The fluorescent probe is loaded with urokinase.

9. The use according to claim 8, characterized in that: The loading amount of urokinase is 3000-7500 U.

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