Fluorescent probe based on lanthanide series metal organic framework, preparation method and application

Abstract

The invention relates to a fluorescent probe based on a lanthanide series metal organic framework as well as a preparation method and application thereof, and belongs to the technical field of nano materials. The fluorescent probe provided by the invention is obtained by covalent crosslinking of a lanthanide series metal organic framework, a neutrophil elastin binding peptide and a P-selectin binding peptide, and the surface of the lanthanide series metal organic framework is modified with tetracyclic antibiotics and arginine. The fluorescent probe prepared by the invention specifically targets activated platelets and neutrophils, does not influence the cell survival rate, does not cause red blood cell hemolysis, and can be applied to preparation of thrombosis disease diagnosis products. The fluorescent probe is used for loading urokinase, stable and efficient delivery of urokinase is achieved, aggregation of platelets is inhibited, blood clot contraction is slowed down, and the fluorescent probe has a good thrombolysis effect and has the potential of being used for preparing drugs for treating thrombosis diseases. The required materials are easy to obtain, the preparation method is simple, and a new method is provided for diagnosis and treatment of thrombosis 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 of cerebral blood supply disorder caused by cerebrovascular disease, leading to ischemia, hypoxia, necrosis of brain tissue and impaired neurological function. IS has the characteristics of "five highs" of high incidence, high recurrence rate, high disability rate, high mortality rate and high economic burden, and has become one of the major diseases threatening the health of Chinese people. Therefore, safe and efficient IS precise diagnosis and treatment plans are of great significance to reduce the recurrence rate, disability rate and mortality rate, and improve the prognosis of IS.

[0003] At present, the main diagnostic tests for IS include nuclear magnetic resonance imaging, X-ray computed tomography and angiography. These auxiliary tests indirectly judge thrombi through vascular blood flow conditions, cannot directly evaluate the size and nature of thrombi, and are difficult to track thrombi scattered in the brain. This indirect imaging test also has certain difficulties in distinguishing IS from other causes of lumen stenosis and occlusion (such as atherosclerotic plaques, etc.), which is not conducive to the early diagnosis of IS. Therefore, exploring high-sensitivity direct thrombus imaging technology is expected to achieve early diagnosis of IS. Metal-organic frameworks (MOFs) are a class of crystalline porous materials formed by combining organic ligands with metal nodes through coordination bonds. MOF has become one of the most promising drug delivery carriers due to its high specific surface area and porosity, diverse functions, and good biocompatibility. MOF usually uses host-guest interaction forces to adsorb and fix the target in the pores and surfaces. The MOF structure has a tight structural restriction effect on proteins, which improves the stability and biological activity of proteins. This fast and low-cost loading process brings new possibilities for the development and application of biomacromolecules. 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 makes it widely used in sensing, imaging, catalysis, etc. Therefore, multi-element Ln-MOFs fluorescent probes as drug carriers are expected to achieve stable loading of thrombolytic drugs while having high resolution, low background fluorescence imaging capability and antioxidant activity, ultimately achieving accurate 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] In order to solve the above technical problems, the present invention provides a fluorescent probe based on a lanthanide metal organic framework, a preparation method and an application thereof, wherein the surface of the lanthanide metal organic framework is modified with tetracyclic antibiotics and arginine, and the lanthanide metal organic framework is covalently cross-linked with a neutrophil elastin binding peptide and a P-selectin binding peptide, and the obtained fluorescent probe specifically targets activated platelets and neutrophils. The use of a fluorescent probe loaded with urokinase can achieve 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, 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 the clarity and accuracy of imaging. 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 (SEQID 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] The third object of the present invention is to provide a method for preparing the above 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 thiol 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 hemolysis of red blood cells, and can be used in the preparation of diagnostic products for thrombotic diseases;

[0032] (2) The fluorescent probe prepared by the present invention is used to load urokinase, with a loading rate of more than 80%, achieving stable and efficient delivery of urokinase, inhibiting platelet aggregation, slowing down blood clot contraction, having a good thrombolytic effect, and having 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, thus providing 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 according to 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 the 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 is a graph showing the release of urokinase uPA by NP-Ln-MOF under different pH conditions in Example 2 of the present invention;

[0042] Figure 8is a graph showing the biocompatibility test results of NP-Ln-MOF in Example 3 of the present invention;

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

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

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

[0046] Fig.12 is a diagram showing the contraction of a blood clot in Example 4 of the present invention;

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

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

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

[0050] Fig.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 is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0052] Example 1: Preparation of fluorescent probe

[0053] 1. Preparation method 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) transferring the precursor solution to a high-pressure reactor, reacting at 100° C. for 60 minutes, and washing 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 with 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 crosslinker DSPE-Hyd-PEG-Mal aqueous solution (1 mg / mL) 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 hours, 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 h, washed with deionized water, freeze-dried and stored in a 4 °C refrigerator to obtain the urokinase-loaded fluorescent probe NP-Ln-MOF@uPA, and 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 is revealed that probe loading and modification does 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 3 As shown, it is revealed that the probe can be excited by 405-460nm excitation light to emit Eu 3+ characteristic emission.

[0065] Zeta potential diagram of nanomaterials Figure 4 As shown, the change in potential reveals that the drug, cross-linker, and peptide are successfully bound to Ln-MOF.

[0066] TEM image and element distribution map of the fluorescent probe NP-Ln-MOF@uPA loaded with urokinase 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 shown in 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 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 the CCK-8 kit. The results are as follows: 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 a negative control and deionized water as a positive control. The absorbance of the samples at 544 nm was detected and the hemolysis rate was calculated. The results are shown in Fig. 9 As shown, 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, respectively. After the excess fluorescent probe NP-Ln-MOF was fully washed away, the cells were imaged using a laser scanning confocal microscope at a 63x objective lens and a 405nm excitation wavelength, and the signals in the range of 600-630nm were collected. The results are shown in Fig.10 and Fig.11 As shown, the fluorescent probe NP-Ln-MOF can specifically target activated platelets and neutrophils, but has weak interactions 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 the aggregation 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 respectively. 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. Fig.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 simple application of urokinase, while Ce / Eu-PTA-DOX without urokinase has no inhibitory effect on clot contraction. Fig.13 As shown, compared with the control group and Ce / Eu-PTA-DOX, the change in clot volume slowed down after the application of NP-Ln-MOF@uPA, and the clot still maintained about 60% of its volume after 50 minutes of action.

[0076] 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, platelets were washed with CGS buffer and then dispersed in MTB buffer. 9 / L of platelets were incubated with 200μg / mL fluorescent probe Ce / Eu-PTA-DOX without urokinase loading, fluorescent probe NP-Ln-MOF@uPA loaded with urokinase, and urokinase of equivalent concentration at 37°C for 30 minutes. After adding the aggregation inducer collagen (0.5μg / mL), the aggregation of platelets was detected by platelet aggregation instrument. The results are shown in Figure 2. Fig.14 and Fig.15 As shown, the platelet photoconductivity decreased significantly after treatment with the control group and Ce / Eu-PTA-DOX, indicating that the platelet aggregation was serious, while the platelets did not aggregate significantly after 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] The whole blood of wild-type mice was placed in a centrifuge tube and placed in a refrigerator at 4°C for 24 hours to form a blood clot. The blood clot was cut into a blood clot weighing about 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 of equivalent concentration at 37°C for 3 hours. The remaining blood clot was weighed again (W2), and the thrombolysis rate was calculated as (W1-W2) / W1×100%. The results are shown in Figure 2. Fig.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), trimesic acid (H3BTC) and pyromellitic 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 clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A fluorescent probe based on a lanthanide metal organic framework, characterized in that: 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 metal as metal ion, terephthalic acid as organic ligand, and is surface-modified with tetracyclic antibiotics and arginine.

2. The fluorescent probe according to claim 1, characterized in that: The lanthanide metals are cerium and europium.

3. The fluorescent probe according to claim 1, characterized in that: The tetracyclic antibiotic is doxycycline.

4. Use of the fluorescent probe according to any one of claims 1 to 3 in the preparation of diagnostic products for thrombotic diseases.

5. The method for preparing the fluorescent probe according to any one of claims 1 to 3, 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 tetracyclic antibiotics 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.

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

7. The preparation method according to claim 5, 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.

8. Use of the fluorescent probe according to any one of claims 1 to 3 in the preparation of drugs for treating thrombotic diseases.

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

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

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