Flavone-metal coordination nano-structure Zn-Fisetin as well as preparation method and application thereof

Abstract

The invention discloses a flavone-metal coordination nano-structure Zn-Fisetin, the Zn-Fisetin is a spatial mesh material formed by coordination of fisetin molecules and zinc ions, the central metal ions of the Zn-Fisetin are the zinc ions, the organic ligand is fisetin, and the nano-structure is formed by a dissolution heat method through coordination. The invention also discloses a preparation method and application thereof. The flavone-metal coordination nanostructure synthesized by the invention has the advantages of better stability, controllable size and pore diameter, low toxicity and good biocompatibility, changes the characteristic that fisetin is insoluble in water, and improves the bioavailability of fisetin. The fisetin has a good anti-cell aging capability and an immunoregulation effect of zinc ions, and can be applied to removal of atherosclerotic lipid plaques of senile frequent diseases. The structure has strong superiority in removing senescent cells, inhibiting the atherosclerosis process and relieving plaque load, and an effective way is provided for the design of multifunctional biological materials based on natural active products.

Description

Technical Field

[0001] The invention relates to a flavonoid-metal coordination nanostructure Zn-Fisetin and a preparation method and application thereof, belonging to the technical field of biomedical materials. Background Art

[0002] Metal-coordinated nanostructures are spatial network structures formed by the coordination of metal ions and organic ligands. They have adjustable physical and chemical properties, are easy to modify on the surface, and have high biocompatibility. By selecting different metal / ligand combinations, a series of metal-coordinated nanostructures with diverse functions can be developed to meet the needs of personalized diagnosis and treatment; surface modification can further improve pharmacokinetic behavior and pharmacokinetic properties to maximize drug efficacy; in addition, if drugs are directly coordinated with metals as ligands, the drug delivery amount and delivery efficiency can be greatly increased.

[0003] Fisetin is a flavonoid compound with multiple biomedical functions. It has shown significant anti-cell aging activity in basic research and clinical trials, and has development prospects for the treatment of geriatric diseases (such as atherosclerosis). However, Fisetin has poor water solubility and low bioavailability in vivo. How to better increase the bioavailability of Fisetin and improve its pharmacokinetics is a biomedical problem that needs to be solved urgently. Using metal coordination technology, Fisetin can be used as an organic molecular bridge, and by coordination with metal zinc ions, a nanoscale coordination material can be formed, which can significantly improve the water solubility of Fisetin and combine it with the biological function of zinc ions (such as immunomodulation) for application in the treatment of geriatric diseases. Flavonoid-metal coordination nanostructure Zn-Fisetin uses natural product active ingredients as organic ligands and is synthesized with zinc ions as nodes. It can be used to have good anti-cell aging effects and has better application prospects in the treatment of geriatric diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a flavonoid-metal coordination nanostructure Zn-Fisetin.

[0005] The technical solution adopted by the present invention is:

[0006] A flavonoid-metal coordination nanostructure Zn-Fisetin is a spatial network material formed by coordination of fisetin molecules and zinc ions, wherein the central metal ion is zinc ion and the organic ligand is fisetin, and the nanostructure is formed by coordination through the method of heat of solution.

[0007] The molecular formula of fisetin is C 15 H 10 O 6 , the chemical structure is shown in formula (I):

[0008]

[0009] Preferably, in the Zn-Fisetin, the mass ratio of zinc ions is 18% to 20%.

[0010] Preferably, in the Zn-Fisetin, the zinc ion is Zn 2+ .

[0011] Preferably, the size of the Zn-Fisetin is 100 - 120 nm, with a uniform morphology and uniform dispersion.

[0012] Preferably, Zr is also incorporated into the Zn-Fisetin 89 Zr, 89 The mass content of Zr in Zn-Fisetin is between 0.01% and 0.1%.

[0013] Preferably, the surface of the Zn-Fisetin is also modified with Py-PAA-PEG-Mal molecules.

[0014] Preferably, the Zn-Fisetin is also conjugated with functional molecules through Py-PAA-PEG-Mal molecules, and the functional molecules are one or a combination of several of polypeptides, small proteins, antibodies, nanobodies, nucleic acids, medical isotopes, aptamers.

[0015] The present invention also discloses a preparation method of the above-mentioned flavonoid-metal coordination nanostructure Zn-Fisetin, and its steps include:

[0016] (1) Dissolve fisetin and zinc acetate in the organic solvent DMA respectively;

[0017] (2) Mix the two evenly, control the mass ratio of the added fisetin to zinc acetate to be 2:1, heat and react for a period of time, after washing the reaction product, centrifuge and collect to obtain Zn-Fisetin.

[0018] Step (2) is specifically to mix the fisetin solution and zinc acetate solution, and then add a small amount of absolute ethanol, ultrasonicate for 10 min, heat and react at 120 - 140 °C for 14 h, the product is centrifuged and collected at 13000 rpm for 15 min, and washed three times with DMA, absolute ethanol, and pure water in sequence, and the obtained Zn-Fisetin is resuspended and dispersed in pure water.

[0019] Preferably, zirconium oxalate-89 is also added in step (1) to obtain Zn-Fisetin that can be used for PET imaging.

[0020] The present invention also discloses the application of the flavonoid-metal coordination nanostructure Zn-Fisetin as described above in the preparation of anti-cell aging drugs, drugs for clearing senescent cells or drugs for alleviating atherosclerosis.

[0021] The present invention also discloses a drug delivery system, comprising the above-mentioned flavonoid-metal coordination nanostructure Zn-Fisetin, and at least one active substance, wherein the active substance is an imaging agent or a drug; the imaging agent is one or a combination of several of a radionuclide, a radionuclide label or a molecular imaging agent, and the drug is one or a combination of several of a chemical drug, a biological drug, a photothermal therapy or a photodynamic therapy drug capable of clearing senescent cells. The Zn-Fisetin is coupled with a functional molecule through a Py-PAA-PEG-Mal molecule, and the functional molecule is one or a combination of several of a polypeptide, a small protein, an antibody, a nanobody, a nucleic acid, a medical isotope, an aptamer, etc.

[0022] Beneficial effects: The flavonoid-metal coordination nanostructure synthesized by the present invention has good stability, controllable size and pore size, low toxicity and good biocompatibility. The synthesized Zn-Fisetin has good dispersibility and stability in aqueous solution, changes the characteristic that fisetin is insoluble in water, improves the bioavailability of fisetin, and can perform plaque PET imaging. With the good anti-aging activity of fisetin and the immunomodulatory effect of zinc ions, it can be applied to the treatment of senile diseases. This coordination nanomaterial has strong superiority in the treatment of atherosclerosis and the identification of vulnerable plaques, providing an effective way for the design of multi-functional biomaterials based on natural active products.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) By virtue of the stable coordination of fisetin and zinc ions, the present invention obtains a coordination nanomaterial with stable properties by means of a solvothermal method. This design method is simple and easy to operate and can be synthesized efficiently on a large scale.

[0025] (2) The coordination nanomaterial synthesized by the present invention has good senescence targeting effect, can be used as a targeted drug delivery system or a visualization drug carrier, is safe and reliable, and has a very broad application prospect in the identification and clearance of atherosclerotic plaques.

[0026] (3) Other organic ligands can be added or connected to the structure of the coordination nanomaterial of the present invention to introduce new diagnostic and imaging characteristics. In addition, the coordination structure of the present invention also involves the surface modification of the material with organic molecules, such as polyethylene glycol, other polymer molecules, proteins or polypeptide derivatives. The Zn-Fisetin of the present invention can be surface-modified with pyrene-polyacrylic acid-polyethylene glycol (Py-PAA-PEG-Mal) and coupled with functional polypeptides.

[0027] (4) The nanostructure or its derivatives according to the present invention are conjugated or mixed with a preparation capable of clearing senescent cells to form a targeted drug delivery system. The preparations that can be conjugated include: small molecules, polypeptides, nucleic acids (DNA and RNA), proteins (including antibody-like proteins), medical isotopes, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a material morphology diagram of Zn-Fisetin of the present invention measured by a scanning electron microscope (SEM).

[0029] Figure 2 It is the hydrated particle size of Zn-Fisetin of the present invention measured by dynamic light scattering (DLS).

[0030] Figure 3 It is a composition content diagram of Zn-Fisetin of the present invention measured by thermogravimetric analysis (TGA). In the figure, the abscissa is temperature, the left ordinate is mass, and the right ordinate is the rate of mass reduction. The red line represents the curve of mass change with temperature, and the blue line represents the rate curve of mass change with temperature.

[0031] Figure 4 It is the X-ray photoelectron spectroscopy (XPS) measured for Zn-Fisetin of the present invention.

[0032] Figure 5 It is a dissolution comparison diagram of Zn-Fisetin and Fisetin.

[0033] Figure 6 It is a fluorescence image of Zn-Fisetin of the present invention being taken up by senescent mouse macrophages Raw264.7.

[0034] Figure 7 It is a β-galactosidase staining image of Zn-Fisetin of the present invention inhibiting senescent phenotypes in mouse macrophages Raw264.7 and bone marrow-derived macrophages BMDM.

[0035] Figure 8 It is an effect diagram of Zn-Fisetin of the present invention clearing senescent cells in mouse macrophages Raw264.7.

[0036] Figure 9 It is a section staining image of Zn-Fisetin of the present invention clearing senescent cells in plaques in atherosclerotic model mice.

[0037] Figure 10 It is an effect diagram of Zn-Fisetin of the present invention alleviating plaque burden in atherosclerotic model mice.

[0038] Figure 11 This is the structural diagram of fisetin. Specific implementation methods

[0040] The present invention will be further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0041] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. Additionally, although this text may provide examples containing specific values of parameters, it should be understood that the parameters do not necessarily exactly equal the corresponding values, but may approximate the corresponding values within an acceptable error tolerance or design constraint.

[0042] The substances or instruments used in the following embodiments, if not specifically stated, can be obtained from conventional commercial channels.

[0043] Example 1 Solvothermal synthesis of Zn-Fisetin

[0044] Dissolve 20 mg of fisetin (structural formula as Figure 10 shown) and 10 mg of zinc acetate in 1 mL of DMA respectively. Then mix the fisetin solution and the zinc acetate solution, add 0.5 mL of absolute ethanol, sonicate for 10 min, react at 120 °C for 14 h. The product is collected by centrifugation at 13000 rpm for 15 min and washed three times with DMA, absolute ethanol, and pure water in sequence. The obtained Zn-Fisetin is resuspended and dispersed in pure water. The product Zn-Fisetin is formed by the coordination of fisetin molecules and zinc ions to form a nanostructure.

[0045] The hydrodynamic radius of Zn-Fisetin can be measured by a particle size analyzer ( Figure 2 ), or its size and morphology can be determined by SEM ( Figure 1 ), the content composition of its organic components can be determined by TGA ( Figure 3 ), and its ionic valence state can be determined by XPS ( Figure 4 ). It can be seen from Figure 4 that the valence state of zinc ions in this nanostructure is divalent.

[0046] Example 2 Polyethylene glycol coating of Zn-Fisetin

[0047] 20 mg of the Py-PAA-PEG-Mal aqueous solution (10 mL) was added dropwise to 10 mg of the Zn-Fisetin solution (10 mL), and ultrasonic treatment was performed in an ice bath for 2 hours to coat PEG on the material surface. Subsequently, the material coated with PEG was collected by centrifugation and washed three times with deionized water to obtain Zn-Fisetin modified with maleimide groups. Zn-Fisetin was resuspended in pure water, and it had excellent dispersibility. From Figure 5 It can be seen that the water solubility of Zn-Fisetin is significantly better than that of Fisetin.

[0048] Example 3 Incorporation of Zirconium-89 to Synthesize Radioactive Zn-Fisetin

[0049] Take 70 - 700 MBq of zirconium-89 oxalate solution with a volume of 10 - 100 μL, add 200 μL of 0.1 M hydrochloric acid solution, heat it in an oven at 150 - 200 °C. After the solvent has evaporated, add 1 mL of DMA solution to the bottle. After ultrasonic treatment, transfer it to the reaction system of 20 mg of fisetin and 10 mg of zinc acetate in Example 1, and react at a high temperature of 120 °C for 14 h. The product was collected by centrifugation at 13000 rpm for 15 min and washed three times with DMA, absolute ethanol, and pure water in sequence. The obtained Zn-Fisetin was resuspended and dispersed in pure water.

[0050] Example 4 Detection of the Uptake of Zn-Fisetin by Senescent Cells

[0051] The characteristic fluorescence absorption of Zn-Fisetin was detected by a laser confocal scanning microscope (LSCM) to explore the endocytosis of Zn-Fisetin by senescent cells. First, oxidized low-density lipoprotein (oxLDL) was used to induce the senescence of mouse macrophages Raw264.7. Ordinary and senescent Raw264.7 were inoculated at a concentration of 1×10 5 cells / dish and incubated overnight in an incubator at 37 °C. Subsequently, Zn-Fisetin (20 μg / mL) was added to the cells and incubated for 4 h. After the incubation, the cells were fixed with 4% paraformaldehyde for 10 min, washed 3 times with PBS, stained with DAPI for 10 min, and after dropping anti-fluorescence quenching mounting medium, CLSM was used for observation ( Figure 6 ). Figure 6 Ctrl (Control) in [[ ]] refers to the result of the non-treatment control group, representing the cell group without adding Zn-Fis or any other special treatment. The results show that Zn-Fis can be effectively taken up by senescent macrophages.

[0052] Example 5 Detection of Zn-Fisetin Inhibiting Cell Senescence

[0053] Bone marrow-derived macrophages (BMDMs) were extracted from the leg bones of C57 mice and cultured for 7 days before being used in subsequent experiments. In this experiment, oxLDL (50 μg / mL) was used as a cell senescence inducer. Raw264.7 and BMDMs in the logarithmic growth phase were seeded in 12-well plates and cultured for 24 h. The next day, the cells were treated with different drug combinations (Ctrl, oxLDL, oxLDL+Zn-Fisetin) and incubated for another 24 h. After incubation, the cells were stained with a β-galactosidase staining kit ( Figure 7 ). The results showed that Zn-Fis could inhibit the cell senescence phenotype induced by oxLDL.

[0054] Example 6 Detection of the in vitro clearance of senescent cells by Zn-Fisetin

[0055] Raw264.7 in the logarithmic growth phase was seeded in 12-well plates and cultured overnight. The next day, the cells were treated with different drugs (Ctrl, Zn-Fisetin, oxLDL, oxLDL+Zn-Fisetin) and incubated for another 24 h. After incubation, the cells were stained with a Calcein AM-PI cell viability / cytotoxicity assay kit and observed using an inverted fluorescence microscope. Dead cells were labeled with red fluorescence and live cells were labeled with green fluorescence ( Figure 8 ). The results showed that Zn-Fis could induce apoptosis of senescent cells with less impact on normal cells.

[0056] Example 7 Detection of the clearance of senescent cells and alleviation of plaque burden by Zn-Fisetin in atherosclerotic mice

[0057] Eight-week-old male ApoE knockout mice were purchased and fed a high-fat diet (HFD) for 12 weeks to establish a mouse model of atherosclerosis. Mice in the treatment group were treated with Zn-Fisetin (8 mg / kg) by tail vein injection starting from the fifth week of HFD. The dosing frequency was twice a week for eight consecutive weeks. The body weight of the mice was monitored during the treatment period. After the treatment, the mice were sacrificed and the heart, liver, spleen, lungs, kidneys, and aortic valves were collected. The aortic roots were sectioned and stained with β-galactosidase to detect senescent cells ( Figure 9 ), and stained with Oil Red O, Masson, and H&E to examine the plaque burden ( Figure 10 ). Young represents young mice that were not treated with a high-fat diet (HFD) and were used as a control group. The results of β-galactosidase staining showed that compared with the model group, Zn-Fis could clear senescent cells in the plaques and reduce the senescence phenotype. Staining of aortic root sections showed that Zn-Fis reduced the size of the plaque area, restored collagen production, and effectively alleviated the plaque burden.

[0058] The above embodiments are only used to illustrate the technical concept and features of the present invention. The purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A flavonoid-metal coordination nanostructure Zn-Fisetin, characterized in that: The Zn-Fisetin is a nanometer space network material formed by the coordination of fisetin molecules and zinc ions, wherein the central metal ion is zinc ion and the organic ligand is fisetin.

2. The flavonoid-metal coordination nanostructure Zn-Fisetin according to claim 1, characterized in that: In the Zn-Fisetin, the weight ratio of zinc ions is 18% to 20%.

3. The flavonoid-metal coordination nanostructure Zn-Fisetin according to claim 2, characterized in that: In the Zn-Fisetin, the zinc ion is Zn 2+ .

4. The flavonoid-metal coordination nanostructure Zn-Fisetin according to any one of claims 1 to 3, characterized in that: The size of the Zn-Fisetin is 100-120 nm.

5. The flavonoid-metal coordination nanostructure Zn-Fisetin according to claim 4, characterized in that: The Zn-Fisetin is also incorporated 89 Zr, 89 The mass content of Zr in Zn-Fisetin is between 0.01% and 0.1%.

6. The flavonoid-metal coordination nanostructure Zn-Fisetin according to claim 4, characterized in that: The surface of Zn-Fisetin is also modified with Py-PAA-PEG-Mal molecules, and functional molecules are coupled through Py-PAA-PEG-Mal molecules. The functional molecules are one or a combination of polypeptides, small proteins, antibodies, nucleic acids, medical isotopes, and aptamers.

7. The method for preparing the flavonoid-metal coordination nanostructure Zn-Fisetin according to any one of claims 1 to 6, characterized in that: The steps include: (1) Fisetin and zinc acetate were dissolved in an organic solvent DMA at a mass ratio of 2:1; (2) Mix the two ingredients evenly, heat and react for a period of time, wash the reaction product, and collect it by centrifugation to obtain Zn-Fisetin.

8. The preparation method according to claim 7, characterized in that: In step (2), the reaction temperature is 120-140° C. and the reaction time is 14 hours.

9. Use of the flavonoid-metal coordination nanostructure Zn-Fisetin according to any one of claims 1 to 6 in the preparation of anti-cell aging drugs, drugs for removing senescent cells or drugs for alleviating atherosclerosis.

10. A drug delivery system, characterized in that: The invention comprises the flavonoid-metal coordination nanostructure Zn-Fisetin according to any one of claims 1 to 6, and at least one active substance, wherein the active substance is an imaging agent or a drug; the imaging agent is one or a combination of radionuclides, radionuclide markers or molecular imaging agents, and the drug is one or a combination of chemical drugs, biological drugs, photothermal therapy drugs or photodynamic therapy drugs that can eliminate senescent cells.

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