Protein-based mineralization inhibitor with anti-fouling function

Abstract

The invention discloses a protein-based mineralization inhibitor with an anti-fouling function. The protein-based mineralization inhibitor is prepared from phosphorylated protein, a disulfide bond reducing agent and a pH regulator. According to the mineralization inhibitor, a flat protein-based coating can be obtained at an interface through methods of soaking, coating and the like; the deposition of hydroxyapatite and derivative biominerals thereof can be inhibited in a physiological environment, so that diseases such as osteoarthritis and dental calculus caused by abnormal mineralization are reduced. Besides, after a coating is formed by the mineralization inhibitor, the mineralization inhibitor has excellent anti-pollution capacity, impure protein and heteropolysaccharide in body fluid can be prevented from being adhered to the coating, and the phenomenon that the calcification inhibition capacity disappears due to pollutant adhesion is prevented. The mineralization inhibitor is non-toxic and non-irritant, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a protein-based mineralization inhibitor with anti-fouling function, which can prevent hydroxyapatite from mineralizing at the interface, thereby reducing the health hazards of abnormal mineralization. Background Art

[0002] Hydroxyapatite (HAP) is the primary inorganic mineral constituting human bones and teeth, and its physiological mineralization is crucial. However, abnormal HAP mineralization is a serious health problem. Effectively controlling this pathological nucleation and growth is of great clinical significance, as it directly contributes to a variety of diseases, such as vascular calcification, heart valve calcification, kidney stone formation, and heterotopic ossification. These lesions cause tissue sclerosis, loss of elasticity, luminal narrowing, and serve as nidus for inflammation and infection. Current clinical management strategies primarily include pharmacological treatment (such as phosphate binders and bisphosphonates) and physical removal (such as surgery and lithotripsy). However, these approaches generally have significant limitations: drug treatment has limited efficacy and is often associated with significant side effects (such as abnormal bone metabolism and nephrotoxicity); physical treatments are invasive and prone to recurrence. Some chemical interventions may also disrupt essential mineral homeostasis. Although research has thoroughly explored the complex influences of calcium-phosphate imbalance, the local microenvironment (pH, inflammation, matrix proteins), related diseases (chronic kidney disease, diabetes), and cellular phenotypic transformation on the pathological nucleation and crystal growth of HAP, the core molecular mechanisms remain incompletely elucidated, limiting the development of effective and specific intervention strategies. Surface engineering technologies offer potential solutions for medical devices (such as prosthetic valves and stents). However, such coatings face significant challenges in the dynamic and complex physiological environment: insufficient biostability and poor long-term durability. They also struggle to effectively resist the effects of persistent biofouling (such as protein adsorption and microbial biofilm formation) on the mineralization process, ultimately leading to coating failure and device calcification.

[0003] To address these issues, a new technology must be developed that simultaneously inhibits interfacial mineralization and provides good antifouling properties, preventing the loss of the interface mineralization inhibition effect due to the adhesion of pollutants. Therefore, designing functional nanocoatings that integrate both mineralization inhibition and antifouling properties is of great significance. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of traditional mineralization inhibitors being expensive, inefficient, and causing significant side effects on the human body, while also solving the problem of the disappearance of the mineralization inhibition effect due to coating contamination, and to provide a protein-based mineralization inhibitor with anti-fouling function.

[0005] To achieve the above-mentioned purpose, the protein-based mineralization inhibitor with anti-fouling function provided by the present invention is composed of the following raw materials in the following weight ratios:

[0006] Phosphorylated protein 1-20 parts

[0007] Disulfide bond reducing agent 1-10 parts

[0008] pH adjusting agent 2-20 parts

[0009] The above mineralization inhibitor is preferably composed of the following raw materials in the following proportions by weight:

[0010] Phosphorylated protein 4-10 parts

[0011] Disulfide bond reducing agent 2-6 parts

[0012] pH adjusting agent 6-10 parts

[0013] The above phosphorylated protein is a natural phosphorylated protein or is prepared from a non-phosphorylated protein by reaction with a phosphorylating agent selected from any one of sodium polyphosphate, sodium hexametaphosphate, sodium tripolyphosphate, sodium trimetaphosphate, adenosine triphosphate, phosphorylase kinase, phosphorus oxychloride, sodium pyrophosphate, etc.

[0014] Further, the protein in the above phosphorylated protein is selected from any one or more of lysozyme, bovine serum albumin, human serum albumin, lactoferrin, insulin, α-lactalbumin, fibrinogen, β-lactoglobulin, ribonuclease A, cytochrome c, α-amylase, horseradish peroxidase, pepsin, myoglobin, collagen, keratin, hemoglobin, DNA polymerase, casein, soybean protein isolate, pea protein isolate, zein, gliadin, oat protein, potato protein, hemp seed protein, walnut protein, rapeseed protein, hazelnut protein, quinoa protein, sunflower seed protein, pumpkin seed protein, black sesame seed protein, mushroom protein, red algae protein, conglutinin, barley protein, wheat protein, gliadin, kidney bean protein, catalase, transferrin, thyroglobulin, chymotrypsin, egg white albumin, glutelin, etc.

[0015] Further, the above disulfide bond reducing agent is selected from any one or more of tris(2-carboxyethyl)phosphine hydrochloride, glutathione, cysteine, dithiothreitol, dithiothreitol isomer, β-mercaptoethanol, mercaptopropionic acid, trishydroxypropylphosphine, urea, sodium ferrate, azobis isobutyramide hydrochloride, hexafluoroisopropanol, sodium bismuthate, hydrogen peroxide, trivalent cobalt salt, guanidine hydrochloride, trifluoroethanol, sodium borohydride, etc.

[0016] Further, the above pH adjusting agent is any one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, sodium citrate, etc.

[0017] The method for using the mineralization inhibitor of the present application is as follows: the mineralization inhibitor is added to deionized water, and various raw materials are fully dissolved by gently stirring to prepare a mineralization inhibitor solution of 1-400 mg / mL, and the pH of the mineralization inhibitor solution is controlled to be 3-7.5; then the mineralization inhibitor solution is sprayed on the surface of the substrate by a spray gun, or the substrate is directly immersed in the mineralization inhibitor solution for 10-15 min.

[0018] The above substrate refers to a stent material used for treatment of human diseases or recovery of surgery, such as an orthopedic bone plate, a vascular stent, a heart stent, and the like.

[0019] The method for using the mineralization inhibitor of the present application is as follows: the mineralization inhibitor is added to deionized water, and various raw materials are fully dissolved by gently stirring to prepare a mineralization inhibitor solution of 1-400 mg / mL, and the pH of the mineralization inhibitor solution is controlled to be 3-7.5; then the mineralization inhibitor solution is sprayed on the surface of the substrate by a spray gun, or the substrate is directly immersed in the mineralization inhibitor solution for 10-15 min.

[0020] The in-vitro experiment proves that the mineralization inhibitor solution can form a dense coating at the interface, and the coating can inhibit nucleation of salt ions at the interface in a salt ion environment. In addition, after the mineralization inhibitor solution forms the coating, it can effectively resist the adhesion of bacteria and proteins, prevent the formation of a contaminant film, and thus maintain the mineralization inhibition effect. The cell experiment proves that the mineralization inhibitor is non-toxic and harmless to cells, and can play a role in inhibiting cell mineralization.

[0021] The present application has the following beneficial effects:

[0022] 1. The mineralization inhibitor of the present application is simple to prepare and has good controllability. The main component is composed of proteins, which are non-toxic and non-irritating, have good biocompatibility, can be stably stored after large-scale preparation, and are convenient for subsequent use.

[0023] 2. The mineralization inhibitor of the present application is simple to use, and the coating effect can be achieved by simple spraying or immersion.

[0024] 3. Compared with the traditional mineralization inhibitor which has poor effect, serious waste, and obvious toxic and side effects, the mineralization inhibitor of the present application has good stability, excellent mineralization inhibition effect, and is non-toxic and non-polluting.

[0025] 4. The coating formed by the mineralization inhibitor of the present application has excellent anti-pollution ability, can prevent the adhesion of pollutants on the interface, and thus maintains the mineralization inhibition effect.

[0026] 5. The coating formed by the mineralization inhibitor of the present application can resist high temperature (100℃), resist chemical action of acid and alkali, and also resist physical action such as ultrasonic and adhesive tape tearing, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 This is the morphology of the film formed by the mineralization inhibitor of Example 1 on the interface.

[0028] Figure 2 These are scanning electron microscope photos of a titanium sheet with a film formed on the surface of the mineralization inhibitor of Example 1 and a blank titanium sheet immersed in a mineralization solution for one week.

[0029] Figure 3 This is an Alizarin Red staining effect diagram of the mineralization inhibitor of Example 1 inhibiting cell mineralization.

[0030] Figure 4 This is a scanning electron microscope photograph showing that the mineralization inhibitor of Example 1 inhibits mineralization in a cell experiment.

[0031] Figure 5 The figures are Alizarin red staining and scanning electron microscopy pictures showing that the mineralization inhibitor of Example 1 inhibits the mineralization of articular cartilage in an animal experiment.

[0032] Figure 6 This is a biological toxicity characterization of the mineralization inhibitor of Example 1 in inhibiting mineralization in animal experiments.

[0033] Specific implementation measures

[0034] The present invention is further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples.

[0035] Example 1

[0036] Mix 40 mg of phosphorylated lysozyme, 10 mg of tris(2-carboxyethyl)phosphine hydrochloride, and 60 mg of sodium bicarbonate to obtain a mineralization inhibitor.

[0037] Example 2

[0038] Mix 50 mg of phosphorylated lysozyme, 15 mg of tris(2-carboxyethyl)phosphine hydrochloride, and 70 mg of sodium carbonate to obtain a mineralization inhibitor.

[0039] Example 3

[0040] Mix 60 mg of phosphorylated lysozyme, 20 mg of tris(2-carboxyethyl)phosphine hydrochloride, and 80 mg of sodium carbonate to obtain a mineralization inhibitor.

[0041] Example 4

[0042] Mix 10 mg of phosphorylated lysozyme, 60 mg of tris(2-carboxyethyl)phosphine hydrochloride, and 20 mg of sodium bicarbonate to obtain a mineralization inhibitor.

[0043] Example 5

[0044] Mix 100 mg of phosphorylated lysozyme, 60 mg of tris(2-carboxyethyl)phosphine hydrochloride, and 100 mg of sodium bicarbonate to obtain a mineralization inhibitor.

[0045] Example 6

[0046] 150 mg of phosphorylated lysozyme, 100 mg of tris(2-carboxyethyl)phosphine hydrochloride, and 100 mg of sodium bicarbonate were mixed evenly to obtain a mineralization inhibitor.

[0047] Example 7

[0048] 200 mg of phosphorylated lysozyme, 100 mg of tris(2-carboxyethyl)phosphine hydrochloride, and 200 mg of sodium bicarbonate were mixed evenly to obtain a mineralization inhibitor.

[0049] The preparation method of the phosphorylated lysozyme in Examples 1 to 7 above is as follows: 200 mg of lysozyme and 1000 mg of sodium tripolyphosphate (STPP) are added to 10 mL of ultrapure water, stirred and mixed uniformly, and the pH of the mixed solution is adjusted to 8 using sodium hydroxide and hydrochloric acid; then, the pH-adjusted solution is placed in a constant temperature shaker at 50° C. for 8 hours. After the reaction, the solution is dialyzed in ultrapure water for 3 days and freeze-dried to obtain the phosphorylated lysozyme.

[0050] In the present invention, the phosphorylated lysozyme can also be prepared according to the above method, and polyphosphate can be used to phosphorylate the lysozyme, replacing the phosphorylated lysozyme in Examples 1 to 7.

[0051] The present invention can also use polyphosphoric acid to phosphorylate proteins such as bovine serum albumin, insulin, and α-lactalbumin according to the above-mentioned method for preparing phosphorylated lysozyme. The obtained phosphorylated bovine serum albumin, phosphorylated insulin, and phosphorylated α-lactalbumin can replace the phosphorylated lysozyme in Examples 1 to 7. This solution is within the scope of protection of the present invention.

[0052] To demonstrate the beneficial effects of the present invention, the inventors added 60 mg of the mineralization inhibitor prepared in Example 1 to 10 mL of deionized water and gently shook until the mineralization inhibitor dissolved, resulting in a 6 mg / mL mineralization inhibitor solution. Various performance tests were performed on this mineralization inhibitor solution, as follows:

[0053] Immerse the blank titanium sheet in the mineralization inhibitor solution and leave it at room temperature for 2 minutes before taking it out. Since the mineralization inhibitor will naturally adsorb on the interface to form a film, the morphology of the film can be characterized by atomic force microscopy. Figure 1 It shows that the film exists evenly on the surface of the titanium sheet.

[0054] The titanium sheet with the mineralization inhibitor forming a film on the surface and the blank titanium sheet were placed in the mineralization solution (2.5mMCa 2+, 1.5mM PO4 3- , pH = 7.4), and the ambient temperature was controlled at 37 ° C. The mineralization was continued for 1 week. The mineralized samples were taken out and the sample surface was observed by field emission scanning electron microscopy. Figure 2 It can be seen that the titanium sheet with a film formed on its surface remains clean and tidy after being immersed in the mineralizing solution for up to one week, while minerals appear on the surface of the blank titanium sheet.

[0055] Furthermore, the effect of mineralization inhibitors was evaluated at the cellular level. First, calcification of cells was induced by drugs. Then, calcified cells were treated with 6 mg / mL of mineralization inhibitor solution. The mineralization status of cells was observed by alizarin red staining. The redder the alizarin red staining, the more obvious the mineralization. Figure 3 It can be seen that after adding the mineralization inhibitor solution, the red color of the cells became significantly lighter than that of the calcified cells. Subsequently, the mineralization of the cells was observed by scanning electron microscopy. Figure 4 As can be seen from the figure, compared with untreated cells, the cell surface was covered with minerals after drug-induced cell mineralization, forming a large area of ​​minerals; while after the calcified cells were treated with mineralization inhibitors, the mineralization of the cell surface was significantly weakened, and the mineralized area was significantly reduced.

[0056] Finally, animal experiments demonstrated the mineralization-inhibiting effect of mineralization inhibitors. We established an osteoarthritis rat model for validation. By inducing apoptotic vesicles in chondrocytes within the rat joint cavity, the release of apoptotic vesicles increased the concentration of calcium and phosphate ions in the body fluids, leading to osteoarthritis. Subsequently, a 6 mg / mL solution of mineralization inhibitor was injected, and observation was performed at different times. The mineralization-inhibiting effect was evaluated by Alizarin red staining of joint sections and morphological photography. Figure 5 Alizarin red staining showed that the untreated articular cartilage was hardly stained red, while a large number of mineralized crystals appeared in the induced calcified cartilage. After the calcified cartilage was treated with a mineralization inhibitor, the number of minerals in the cartilage was significantly reduced, reaching the uninduced level. Scanning electron microscope photos show the morphological differences of the articular cartilage after mineralization. The morphology of the untreated articular cartilage is fibrous; the fibers in the induced calcified cartilage are wrapped and covered by inorganic mineralization; the morphology of the fibers is maintained after the calcified cartilage is treated with a mineralization inhibitor, indicating that the mineralization inhibitor of the present invention has an inhibitory effect on mineralization. As a mineralization inhibitor used in vivo, its biosafety is of paramount importance. Further data from animal organ heart, liver, spleen, lung, and kidney slices show that the mineralization inhibitor has non-toxic and harmless properties (such as Figure 6 ).

Claims

1. A protein-based mineralization inhibitor with antifouling function, characterized in that The inhibitor is composed of the following raw materials in the following weight proportions: 1-20 copies of phosphorylated protein 1-10 parts of disulfide bond reducing agent 2 to 20 parts of pH adjuster.

2. The protein-based mineralization inhibitor with antifouling function according to claim 1, characterized in that The inhibitor is composed of the following raw materials in the following weight proportions: 4-10 copies of phosphorylated protein 2-6 parts of disulfide bond reducing agent 6 to 10 parts of pH adjuster.

3. The protein-based mineralization inhibitor with antifouling function according to claim 1 or 2, characterized in that: The phosphorylated protein is a natural phosphorylated protein or is prepared by reacting a non-phosphorylated protein with a phosphorylation reagent, wherein the phosphorylation reagent is selected from any one of sodium polyphosphate, sodium hexametaphosphate, sodium tripolyphosphate, sodium trimetaphosphate, adenosine triphosphate, phosphorylation kinase, phosphorus oxychloride, and sodium pyrophosphate.

4. The protein-based mineralization inhibitor with antifouling function according to claim 1 or 2, characterized in that: The protein in the phosphorylated protein is selected from any one or more of lysozyme, bovine serum albumin, human serum albumin, lactoferrin, insulin, α-lactalbumin, fibrinogen, β-lactoglobulin, ribonuclease A, cytochrome c, α-amylase, horseradish peroxidase, pepsin, myoglobin, collagen, keratin, hemoglobin, DNA polymerase, casein, soy protein isolate, pea protein isolate, zein, gliadin, oat protein, potato protein, hemp kernel protein, walnut protein, rapeseed protein, hazelnut protein, quinoa protein, sunflower seed protein, pumpkin seed protein, black sesame protein, mushroom protein, red algae protein, legumin, barley protein, wheat protein, gluten, kidney bean protein, catalase, transferrin, thyroglobulin, chymosin, egg white albumin, and gluten.

5. The protein-based mineralization inhibitor with antifouling function according to claim 1 or 2, characterized in that: The disulfide bond reducing agent is selected from any one or more of tris(2-carboxyethyl)phosphine hydrochloride, glutathione, cysteine, dithiothreitol, dithiothreitol isomers, β-mercaptoethanol, mercaptopropionic acid, trihydroxypropylphosphine, urea, sodium ferrate, azobisisobutyramidine hydrochloride, hexafluoroisopropanol, sodium bismuthate, hydrogen peroxide, trivalent cobalt salt, guanidine hydrochloride, trifluoroethanol, and sodium borohydride.

6. The protein-based mineralization inhibitor with antifouling function according to claim 1 or 2, characterized in that: The pH regulator is any one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, and sodium citrate.

7. The protein-based mineralization inhibitor with antifouling function according to claim 1 or 2, characterized in that: The method for using the mineralization inhibitor is as follows: adding the mineralization inhibitor to deionized water, gently stirring to fully dissolve the various raw materials, preparing a 1-400 mg / mL mineralization inhibitor solution, and controlling the pH of the mineralization inhibitor solution to be 3-7.5; then spraying the mineralization inhibitor solution on the surface of the substrate with a spray gun, or directly immersing the substrate in the mineralization inhibitor solution for 10-15 minutes.

8. The protein-based mineralization inhibitor with anti-fouling function according to claim 7, characterized in that: The substrate refers to a stent material used for treating human diseases or recovering from surgery, including orthopedic bone plates, vascular stents, and cardiac stents.

9. The protein-based mineralization inhibitor with antifouling function according to claim 1 or 2, characterized in that: The method for using the mineralization inhibitor is as follows: adding the mineralization inhibitor to deionized water, gently stirring to fully dissolve the various raw materials, preparing a 1-10 mg / mL mineralization inhibitor solution, and controlling the pH of the mineralization inhibitor solution to be 7.0-7.5; then injecting the mineralization inhibitor solution into the lesion.

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