Catechol and metal ion-based nano-microsphere as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, and particularly discloses catechol and metal ion-based nano-microspheres as well as a preparation method and application thereof. The catechol and metal ion-based nano-microspheres are prepared from catechol-based small molecules, metal ions, ammonia water, an organic solvent and water; the catechol-based small molecules comprise catechol, gallic acid or dopamine, and the concentration of the catechol-based small molecules is 5-50 mM. The metal ions comprise Mg < 2 + >, Zn < 2 + >, Ca < 2 + > or Cu < 2 + >, and the concentration is 2.5 mM to 25mM. The content of the ammonia water is 5vol%-10vol%. The preparation method comprises the following steps: dissolving catechol-based micromolecular monomers and metal ions in a mixed solution of an organic solvent and water, and continuously stirring for reaction to obtain the nano-microspheres. The prepared nano-microspheres have excellent cell compatibility, can be used for inflammation treatment and tissue regeneration and repair, and can be used for preparing anti-inflammatory drugs and drugs for promoting tissue repair and regeneration in the biomedical field.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a catechol and metal ion-based nanosphere and its preparation method and application. Background Art

[0002] Catechol-based small molecule monomers, such as catechol, gallic acid, dopamine, etc., are widely used in the biomedical field due to their high-efficiency antioxidant and free radical scavenging functions. Some metal ions, such as Mg 2+ , Zn 2+ , Ca 2+ and Cu 2+ etc., exhibit excellent biological activities, antibacterial, anti-inflammatory and other properties. In addition, these metal ions can also form metal-phenolic networks by coordinating with polyphenols.

[0003] Currently, commonly used anti-inflammatory drugs in clinics (such as non-steroidal anti-inflammatory drugs NSAIDs, glucocorticoids, biological agents, etc.) have the following problems: non-specific inhibition of inflammation: NSAIDs (such as ibuprofen, aspirin) reduce prostaglandin synthesis by inhibiting COX enzymes, but may cause gastrointestinal damage, cardiovascular risks and renal dysfunction. Glucocorticoids (such as dexamethasone) broadly inhibit the immune response, and long-term use leads to increased osteoporosis, metabolic disorders and infection risks. Low bioavailability: Some antibody-based anti-inflammatory drugs (such as TNF-α inhibitors) need to be administered by injection, with poor patient compliance and high prices. Unable to target delivery: Traditional drugs are distributed throughout the body and are difficult to precisely act on the inflammatory site, resulting in insufficient efficacy or side effects. Unable to synergistically promote tissue repair: Existing drugs mainly inhibit the inflammatory response, but lack the function of promoting tissue regeneration, delaying the healing process. The limitations of current tissue repair drugs (such as growth factors, stem cell therapy, biomaterials, etc.) include: poor stability of growth factors: exogenous growth factors (such as EGF, FGF) are easily degraded by proteases, have a short half-life, and need to be administered frequently. High risk of stem cell therapy: Stem cell transplantation has immune rejection, tumorigenicity and ethical controversies, and the preparation cost is high. Single function of biomaterials: Some scaffold materials (such as collagen, hydroxyapatite) only provide mechanical support and lack the ability of active anti-inflammatory or signal regulation. Inflammatory microenvironment interferes with repair: Chronic inflammation (such as diabetic wounds, arthritis) inhibits the regeneration process, and existing repair drugs are difficult to regulate inflammation and regeneration simultaneously. Based on the above defects, a catechol and metal ion-based nanosphere and its preparation method and application are proposed. Summary of the Invention

[0004] The object of the present invention is to address the deficiencies of the prior art and provide a catechol and metal ion-based nanosphere and its preparation method and application to solve the problems raised in the above background art.

[0005] To achieve the above object, the present invention provides the following technical solution: A catechol and metal ion-based nanosphere, which is composed of a catechol-based small molecule, a metal ion, ammonia water, an organic solvent, and water.

[0006] As a preferred technical solution of the present invention, the catechol-based small molecule includes catechol, gallic acid, or dopamine, and the concentration is 5 mM - 50 mM.

[0007] As a preferred technical solution of the present invention, the metal ion includes Mg 2+ 、Zn 2+ 、Ca 2+ or Cu 2+ , and the concentration is 2.5 mM - 25 mM.

[0008] As a preferred technical solution of the present invention, the content of the ammonia water is 5 vol% - 10 vol%.

[0009] As a preferred technical solution of the present invention, the organic solvent is methanol, ethanol, and isopropanol.

[0010] A preparation method of the catechol and metal ion-based nanosphere as described above, the specific steps are as follows:

[0011] S1. Prepare a mixed solution of an organic solvent and water;

[0012] S2. Ultrasonically dissolve the catechol-based small molecule monomer and the metal ion solution in the mixed solution;

[0013] S3. Add ammonia water and ultrasonically mix evenly;

[0014] S4. Prepare the catechol and metal ion-based nanosphere through continuous stirring reaction.

[0015] As a preferred technical solution of the present invention, the water content in the mixed solution of the organic solvent and water is 20 vol% - 80 vol%.

[0016] As a preferred technical solution of the present invention, the stirring reaction time is 1 h - 24 h, and the reaction condition is room temperature.

[0017] An application of the catechol and metal ion-based nanosphere as described above in the preparation of anti-inflammatory drugs.

[0018] An application of the catechol and metal ion-based nanosphere as described above in the preparation of drugs for promoting tissue repair and regeneration.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. The preparation steps of the catechol and metal ion-based nanospheres disclosed in the present invention are simple, and the raw materials are inexpensive;

[0021] 2. The present invention provides new ideas and technical support for the preparation research and application of polymer-metal-based nanomaterials;

[0022] 3. The nanospheres are formed by catechol-based small molecule monomers with high antioxidant efficiency and various active metal ions through forces such as coordination bonds, and have excellent cell compatibility, and can be used in biomedical fields such as inflammation treatment and tissue regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Scanning electron microscope photograph of the catechol / Mg 2+ nanospheres of the present invention;

[0024] Figure 2 Scanning electron microscope photograph of the gallic acid / Mg 2+ nanospheres of the present invention;

[0025] Figure 3 Photograph of the GelMA hydrogel loaded with gallic acid / Mg 2+ nanospheres after photocrosslinking of the present invention;

[0026] Figure 4 FTIR test results of the gallic acid / Mg 2+ nanospheres of the present invention;

[0027] Figure 5 Cytotoxicity detection results of the dopamine / Zn 2+ nanospheres of the present invention;

[0028] Figure 6 Live-dead staining photograph of the gallic acid / Zn 2+ nanospheres of the present invention co-cultured with rBMSCs for 24 h;

[0029] Figure 7 Anti-inflammatory characteristics of the gallic acid / Mg 2+ nanospheres of the present invention, *P<0.05;

[0030] Figure 8 Osteogenic differentiation performance of the gallic acid / Ca 2+ nanospheres of the present invention for promoting stem cells. DETAILED DESCRIPTION OF THE INVENTION

[0031] The following describes in detail the preferred embodiments of the present invention with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0032] Example 1: A method for preparing catechol and metal ion-based nanospheres

[0033] Prepare an ethanol solution containing 50 vol% water. Ultrasonically dissolve 10 mM catechol and 3.5 mM MgSO4 in this solution. Add 7 vol% ammonia water, stir and react for 12 h, and centrifuge to collect the reaction product. After vacuum drying, observe the morphology of the catechol / Mg 2+ nanospheres by scanning electron microscopy. The results are as Figure 1 shown. It can be seen that the particle size of the nanospheres is less than 100 nm.

[0034] Example 2: A method for preparing catechol and metal ion-based nanospheres

[0035] Prepare an ethanol solution containing 30 vol% water. Ultrasonically dissolve 20 mM gallic acid and 7 mM MgCl2 in this solution. Add 10 vol% ammonia water, stir and react for 8 h, and centrifuge to collect the reaction product. Observe the morphology of the gallic acid / Mg 2+ nanospheres by scanning electron microscopy. The results are as Figure 2 shown. The particle size of the nanospheres is about 100 nm. Load the nanoparticles (1 mg / mL) into the GelMA hydrogel. It can be seen that ( Figure 3 ), the introduction of the nanospheres does not affect the photocrosslinking performance of the GelMA hydrogel; the nanospheres are uniformly and stably dispersed in the hydrogel. Characterize the nanoparticles by FTIR. The results are as Figure 4 shown. It can be seen that the intensities of the carboxyl peak (1680 - 1700 cm -1 ) and hydroxyl peak (~3500 cm -1 ) in gallic acid are significantly reduced. It is speculated that these functional groups form coordination bonds with Mg 2+ .

[0036] Example 3: A method for preparing catechol and metal ion-based nanospheres

[0037] Prepare a methanol solution containing 20 vol% water. Ultrasonically dissolve 35 mM dopamine and 10 mM Zn(NO3)2 in this solution. Add 5 vol% ammonia water, stir and react for 24 h, and centrifuge to collect the reaction product. After co-culturing the nanoparticles (0.1 mg / mL) with rat bone marrow mesenchymal stem cells (rBMSCs) for 1 and 3 days, detect their cytotoxicity by the CCK8 kit. As Figure 5 shown, it can be seen that the relative proliferation degree of the cells is 98.7 - 113.3%, indicating that the nanospheres have excellent cell compatibility and the cytotoxicity is at level 0 - 1.

[0038] Example 4: A method for preparing catechol and metal ion-based nanospheres

[0039] Prepare an isopropanol solution containing 60 vol% water, ultrasonically dissolve 15 mM gallic acid and 8 mM ZnCl₂ in this solution, add 6 vol% ammonia water, stir and react for 10 h, and centrifuge to collect the reaction product. After co-culturing the nanoparticles (0.05 mg / mL) with rBMSCs for 24 h, perform live / dead staining on the cells, and observe the adhesion and spreading state of the cells through a fluorescence microscope. The results are as Figure 6 shown. It can be seen that the live cells show a spindle shape, the cell spreading state is good, and almost no dead cells are observed, indicating that the nano-microspheres exhibit excellent cell compatibility.

[0040] Example 5: Application of a catechol and metal ion-based nano-microsphere in the preparation of an anti-inflammatory drug

[0041] Catechol and metal ions form a dynamic cross-linked network through coordination to form nano-microspheres. The advantages of this microsphere are as follows: (1) pH / ROS responsiveness: controllable drug release at the inflammatory site (acidic or high reactive oxygen environment); (2) Synergistic antioxidant and anti-inflammatory effects: catechol scavenges free radicals, and metal ions regulate the inflammatory signaling pathway; (3) Targeted delivery: The surface of the microsphere can be modified with targeting molecules (such as hyaluronic acid) to enrich in the inflammatory tissue.

[0042] Prepare an ethanol solution containing 70 vol% water, ultrasonically dissolve 8 mM catechol and 3 mM Mg(NO₃)₂ in this solution, add 8 vol% ammonia water, stir and react for 8 h, and centrifuge to collect the reaction product. Co-culture the prepared nano-microspheres (0.1 mg / mL) with lipopolysaccharide (100 ng / mL, 1 h)-stimulated mouse macrophages RAW264.7 for 24 h, and analyze the expression of inflammatory genes in the macrophages ( Figure 7 ), and it can be seen that the nano-microspheres have anti-inflammatory properties and inhibit the expression of inflammatory-related genes TNF-α, IL-6, and IL-1β in lipopolysaccharide-stimulated macrophages.

[0043] Example 6: Application of a catechol and metal ion-based nano-microsphere in the preparation of a drug for promoting tissue repair and regeneration

[0044] Catechol and metal ions form a three-dimensional cross-linked network through dynamic coordination, constituting nanospheres with the following characteristics: (1) Self-assembly ability: Catechol and metal ions form a stable network with a coordination ratio of 3:1 or 2:1, endowing the microspheres with good mechanical strength and controllable degradability. (2) pH / ROS responsiveness: Drugs or bioactive factors can be controllably released in damaged or inflamed tissues (acidic, high ROS microenvironment). (3) Synergistic effect of antioxidant and repair promotion: Catechol scavenges free radicals and reduces oxidative stress; metal ions promote cell migration and collagen deposition. (4) Biomimetic adhesion: The adhesion property of catechol (similar to mussel protein) enables the microspheres to closely adhere to the wound surface or tissue defect, enhancing the retention time.

[0045] Prepare an ethanol solution containing 60 vol% water, ultrasonically dissolve 16 mM gallic acid and 8 mM CaCl2 in this solution, add 5 vol% ammonia water, stir and react for 10 h, and centrifuge to collect the reaction product. Co-culture the prepared nanospheres (0.2 mg / mL) with rBMSCs for 21 days, and change the medium every two days. Subsequently, add alizarin red S staining solution for staining, and observe the expression of intracellular calcium nodules through an optical microscope. From Figure 8 It can be seen that the number of calcium nodules in the control group is small, and almost no deposition of calcium nodules can be observed. In the nanoparticle intervention group, obvious deposition of calcium nodules can be observed, and the number is large and the color is deep. The results show that the nanospheres can promote the osteogenic differentiation of stem cells.

[0046] The above embodiments only express the implementation modes of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A catechol and metal ion-based nano microsphere, characterized in that: It consists of a catechol-based small molecule, metal ions, ammonia water, an organic solvent and water.

2. The catechol and metal ion-based nanospheres according to claim 1, characterized in that: The catechol-based small molecule includes catechol, gallic acid or dopamine, and the concentration is 5 mM - 50 mM.

3. The catechol and metal ion-based nanospheres according to claim 2, wherein: The metal ions include Mg 2+ , Zn 2+ , Ca 2+ or Cu 2+ , with a concentration of 2.5 mM - 25 mM.

4. The catechol and metal ion-based nano-microspheres according to claim 3, wherein: The content of the ammonia water is 5 vol% - 10 vol%.

5. The catechol and metal ion-based nano-microspheres according to claim 4, characterized in that: The organic solvent is methanol, ethanol or isopropanol.

6. A method for preparing catechol and metal ion-based nanospheres according to any one of claims 1-5, characterized in that: The specific steps are as follows: S1. Prepare a mixed solution of an organic solvent and water; S2. Ultrasonically dissolve the catechol-based small molecule monomer and the metal ion solution in the mixed solution; S3. Add ammonia water and ultrasonically mix evenly; S4. Through continuous stirring reaction, prepare catechol and metal ion-based nano microspheres.

7. The preparation method according to claim 6, characterized in that: The content of water in the mixed solution of the organic solvent and water is 20 vol% - 80 vol%.

8. The preparation method according to claim 6, characterized in that: The time of the stirring reaction is 1 h - 24 h, and the reaction condition is room temperature.

9. Use of the catechol and metal ion-based nano microspheres according to any one of claims 1 - 5 in the preparation of an anti-inflammatory drug.

10. Use of the catechol and metal ion-based nano microspheres according to any one of claims 1 - 5 in the preparation of a drug for promoting tissue repair and regeneration.

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