Preparation method and application of metal carrier loaded with deaminotyrosine

Abstract

The invention discloses a preparation method and application of a metal carrier loaded with deamination tyrosine, the preparation method comprises the following steps: ZrOCl28H2O, TCPP and benzoic acid with a specific ratio and concentration are synthesized into Zr-MOFs with a specific particle size and structure, then the Zr-MOFs react with deamination tyrosine with a certain concentration, and DAT-Zr-MOF is prepared through a two-step method. The prepared DAT (at) Zr-MOF loaded deamination tyrosine is high in content and good in stability, can effectively solve the problem that deamination tyrosine is subjected to enzymolysis and oxidation, and can be used for treating wounds and promoting tissue repair through external application.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically relates to a method for preparing a metal carrier loaded with deaminotyrosine and its application. Background Art

[0002] Deaminotyrosine (also known as 4-hydroxyphenylpropionic acid, Desaminotyrosine, DAT) is a metabolite derived from gut microbiota. Research has shown that DAT has functions such as antioxidant, anti-inflammatory, and regulation of lipid metabolism, and exhibits good application prospects in reducing inflammatory responses, controlling infections, anti-aging, antioxidant therapy, and protecting neurons. However, due to the fact that DAT is easily enzymatically hydrolyzed or oxidized and inactivated in the physiological environment, its free molecules have a fast metabolism in the body and it is difficult to reach an effective concentration.

[0003] Metal-organic frameworks (MOFs) are porous materials formed by the self-assembly of metal ions / clusters and organic ligands, with characteristics such as high specific surface area, adjustable pore size, and surface functionalization, and are widely used in fields such as drug delivery, catalysis, and gas adsorption. The structural flexibility of MOFs allows the regulation of properties by designing ligands and metal nodes. Among them, Zr 4+ -based metal-organic frameworks (Zr-MOFs) are a class of porous materials with zirconium ions (Zr 4+ ) as metal nodes and organic ligands as linking units. Due to their ultra-high stability, structural diversity, and tunable functionality, they have become a research hotspot in the field of MOFs. The high bond energy of the Zr-O bond in Zr-MOFs (about 800 kJ / mol) endows the material with excellent chemical stability and is not easily decomposed in body fluids (pH 4 - 8), blood, and high-temperature environments, avoiding premature drug leakage. Compared with liposomes or polymer micelles, Zr-MOFs have strong resistance to the degradation of biological enzymes such as proteases and nucleases and are suitable for long-term in vivo circulation. The specific surface area of Zr-MOFs can reach 3000 m² / g, and the microporous / mesoporous structure (0.5 - 3 nm) can physically adsorb or chemically bond drug molecules, with a drug loading capacity as high as 30% - 50% (weight ratio). At the same time, Zr 4+ is a biologically inert metal, and its in vivo metabolites (such as ZrO2) are non-toxic and can be slowly excreted through the kidneys, with relatively high safety for long-term use.

[0004] In order to overcome the problems of easy enzymatic hydrolysis and oxidation of deaminotyrosine in our laboratory, Zr-MOFs were combined with deaminotyrosine to explore a metal carrier capable of loading deaminotyrosine. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing a metal carrier loaded with deaminotyrosine and its application to solve the problems mentioned in the background art.

[0006] The technical solution adopted by the present invention to achieve the above purpose is as follows: A method for preparing a metal carrier loaded with deaminotyrosine, comprising the following steps: S1. Mix the ZrOCl2•8H2O solution, TCPP solution and benzoic acid solution with each other, and reflux and heat at 90 °C for 5 h. After the reaction is completed, the product Zr-MOFs is obtained; S2. Mix deaminotyrosine with the Zr-MOFs obtained in step S1, and reflux and heat at 90 °C for 5 h. After the reaction is completed, dialyze 3-4 times with deionized water to obtain the product DAT@Zr-MOF.

[0007] Preferably, the mass concentration ratio of ZrOCl2•8H2O, TCPP and benzoic acid is 6:1-1.5:28.

[0008] Preferably, the mass concentration of ZrOCl2•8H2O is not less than 15 mg / mL.

[0009] Preferably, the mass concentration of deaminotyrosine is not less than 10 mg / mL.

[0010] The preparation method of the metal carrier loaded with deaminotyrosine according to the present invention first synthesizes Zr-MOFs with a specific particle size and structure from ZrOCl2•8H2O, TCPP and benzoic acid in specific proportions and concentrations, and then reacts with deaminotyrosine at a certain concentration. The DAT@Zr-MOF is prepared by a one-step method. The prepared DAT@Zr-MOF has a high loading of deaminotyrosine and good stability, and can effectively overcome the problems of deaminotyrosine being enzymatically hydrolyzed and oxidized.

[0011] Preferably, the present invention also provides the application of the above-prepared metal carrier of deaminotyrosine in the preparation of drugs for external application to treat wounds and promote tissue repair.

[0012] The present invention has the following beneficial effects: 1. The metal carrier loaded with deaminotyrosine prepared by the present invention is spherical nanoparticles, the loaded deaminotyrosine is evenly distributed, has good stability, and the preparation method is simple and the conditions are mild; 2. The metal carrier loaded with deaminotyrosine prepared by the present invention has low cytotoxicity, is not easily decomposed in body fluids, blood and high-temperature environments, and will not cause premature leakage of drugs; 3. The carrier prepared by the present invention can be used to prepare an externally applied drug loaded with deaminotyrosine, enabling it to treat wounds and rapidly promote tissue repair; BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a flow chart for preparing a metal framework loaded with deaminotyrosine; Figure 2 is a transmission electron micrograph of the metal framework loaded with deaminotyrosine prepared (scale bar: 200 nm); Figure 3 shows the promotion of wound healing in mice by the metal framework loaded with deaminotyrosine prepared by the present invention. The upper figure is the HE tissue staining of the skin wound of the mouse; the lower figure is the Masson tissue staining of the skin wound of the mouse. DETAILED DESCRIPTION OF THE INVENTION

[0014] To further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. EXAMPLE

[0015] Prepare a metal framework M1 loaded with deaminotyrosine, including the following steps (as Figure 1 ) S1. Mix the ZrOCl2•8H2O solution (2.5 mL, 15 mg / mL, dissolved in DMF), the TCPP solution (5 mL, 2.5 mg / mL, dissolved in DMF), and benzoic acid (5 mL, 70 mg / mL, dissolved in DMF) with each other. The above reaction solution is refluxed and heated at 90 °C for 5 h. After the reaction is completed, the product Zr-MOFs is obtained.

[0016] S2. Mix the above reaction product with the deaminotyrosine solution (DAT, 3.75 mL, 10 mg / mL, dissolved in DMF) and reflux and heat at 90 °C for 5 h. After the reaction is completed, dialyze 3 - 4 times with deionized water to obtain the product M1. EXAMPLE

[0017] Prepare a metal framework M2 loaded with deaminotyrosine, including the following steps: S1. Mix the ZrOCl2•8H2O solution (2.5 mL, 15 mg / mL, dissolved in DMF), the TCPP solution (5 mL, 2.5 mg / mL, dissolved in DMF), and benzoic acid (5 mL, 70 mg / mL, dissolved in DMF) with each other. The above reaction solution is refluxed and heated at 90 °C for 5 h. After the reaction is completed, the product Zr-MOFs is obtained.

[0018] S2. Mix the above reaction product with a deaminotyrosine solution (DAT, 3.75 mL, 12 mg / mL, dissolved in DMF) and reflux at 90 °C for 5 h. After the reaction is complete, dialyze 3 - 4 times with deionized water to obtain product M2. Example

[0019] Prepare a metal framework M3 loaded with deaminotyrosine, including the following steps: S1. Mix a ZrOCl₂•8H₂O solution (2.5 mL, 15 mg / mL, dissolved in DMF), a TCPP solution (5 mL, 3.75 mg / mL, dissolved in DMF), and benzoic acid (5 mL, 70 mg / mL, dissolved in DMF) with each other. The above reaction solution is refluxed and heated at 90 °C for 5 h. After the reaction is complete, obtain product Zr-MOFs.

[0020] S2. Mix the above reaction product with a deaminotyrosine solution (DAT, 3.75 mL, 12 mg / mL, dissolved in DMF) and reflux at 90 °C for 5 h. After the reaction is complete, dialyze 3 - 4 times with deionized water to obtain product M3. Example

[0021] Characterize the particle size distribution of M1 - M3 prepared in Examples 1 - 3 by transmission electron microscopy (TEM), ultraviolet - visible absorption spectroscopy, infrared spectroscopy, and dynamic light scattering technology. The results show that the prepared M1 - M3 are all uniformly distributed spherical nanoparticles. Among them, the particle size of the prepared product M2 is the smallest, which is 143.8 nm, as Figure 2 shown. Example

[0022] Prepare the M2 product prepared in Example 2 into an external application for treating wounds and test its ability to promote tissue repair.

[0023] S1. Mix M2 with poloxamer 407 (1 mL, 20%, dissolved in DAT@Zr - MOF aqueous solution) and dissolve in a shaker at 4 °C for 8 hours to obtain product DAT@Zr - MOF hydrogel.

[0024] S2. Take 6 - 8 - week - old C57 mice, anesthetize them with 1% pentobarbital at a dose of 50 mg / kg, depilate the back skin and disinfect it with iodine tincture, and then create a circular skin wound with a diameter of 9 mm using ophthalmic scissors.

[0025] S3. After disinfecting again with iodine tincture, add 80 μL of DAT@Zr - MOF hydrogel. After the hydrogel solidifies, wrap the wound with a gauze.

[0026] S4. After 14 days, the back skin of the mice was fixed in 4% paraformaldehyde, and the wound healing of the mouse skin was examined by paraffin embedding, sectioning, HE staining, and Masson staining.

[0027] S5. It can be seen from Figure 3 that the metal-organic framework DAT@Zr-MOF hydrogel loaded with deaminotyrosine can effectively promote the healing of the wounds on the surface of the mouse skin.

Claims

1. A method for preparing a metal carrier loaded with deaminotyrosine, characterized in that, It includes the following steps: S1. Mix the ZrOCl₂•8H₂O solution, TCPP solution and benzoic acid solution with each other, and then reflux and heat at 90 °C to obtain Zr-MOFs; S2. Mix deaminotyrosine with Zr-MOFs, reflux and heat at 90 °C. After the reaction is completed, dialyze 3-4 times with deionized water to obtain a metal carrier loaded with deaminotyrosine.

2. The method for preparing a metal carrier loaded with deaminotyrosine according to claim 1, characterized in that, The mass concentration ratio of ZrOCl₂•8H₂O, TCPP and benzoic acid is 6:1-1.5:

28.

3. The method for preparing a metal carrier loaded with deaminotyrosine according to claim 1, characterized in that, The mass concentration of ZrOCl₂•8H₂O is not less than 15 mg / mL.

4. The method for preparing a metal carrier loaded with deaminotyrosine according to claim 1, characterized in that, The mass concentration of deaminotyrosine is not less than 10 mg / mL.

5. Use of the metal carrier of deaminotyrosine prepared as described in claims 1-4 in the preparation of a drug for external application in treating wounds and promoting tissue repair.

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