A mineralized and antibacterial "dual functional" nanoparticle and a preparation method and application thereof

Abstract

The application discloses a kind of mineralization and antibacterial " dual-functionality" nanoparticles and its preparation method and application, first according to amino acid sequence preparation trefoil factor 3 active peptide (Trefoil factor 3, TFF3), then preparation mineralization and antibacterial " dual-functionality" nanoparticles, preparation method is: stable agent, calcium salt and TFF3 additive are dissolved in solvent to obtain calcium-phosphorus mixed solution, amorphous calcium phosphate (Amorphous calcium phosphate, ACP) is synthesized by mixed precipitation method, after adjusting pH value, centrifugal forms nanoparticle, put into vacuum drying oven.The nanoparticle prepared in the application contains amorphous calcium phosphate and has mineralization potential, can provide stable calcium and phosphorus source, induce the mineralization of teeth, TFF3 and calcium ion chelation not only improve the mineralization performance of ACP, but also endow it with antibacterial function, which can be used for the prevention and treatment of dental white spots and early caries.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation technology, and relates to a method for preparing mineralization and antibacterial "dual-function" nanoparticles and their application. Background Technology

[0002] Dental caries, commonly known as cavities or tooth decay, is a chronic, progressive demineralization disease of the hard tissues of the teeth, primarily caused by bacteria and other factors. If left untreated, it can affect the pulp, leading to pulpitis, periapical periodontitis, and even tooth loosening, ultimately resulting in extraction. Dental caries has become a significant threat to human health. Biomimetic mineralization can regenerate hard tooth tissues, reversing early caries and achieving the goal of caries prevention and treatment. Simultaneously, bacteria in dental plaque biofilm are a prerequisite for caries development; effective bacterial inhibition can significantly reduce the caries rate. Biomimetic mineralization and antibacterial properties can minimally invasively treat demineralization of hard tooth tissues, allowing them to self-repair and halting caries progression. However, currently, there is a lack of "dual-function" anti-caries materials that combine biomimetic mineralization and antibacterial properties, and the mineralization mechanism remains unclear.

[0003] Most dental caries patients present with dentin involvement at the time of clinical diagnosis. Dentin biomimetic mineralization is essentially a form of collagen mineralization, promoting intramolecular mineralization of dentin collagen fibers and inducing inorganic remodeling, which is crucial for restoring tooth function. Streptococcus mutans is the main pathogen of dental caries. It can adhere to the tooth surface and form plaque biofilm by specifically binding to the glycoprotein 340 (gp340), a natural immune scavenger receptor in saliva, through its surface adhesin P1. Bacterial acid production leads to tooth demineralization. Currently commercially available mineralizing solutions or pastes (such as mouthwashes and dental protectants) contain liquid amorphous calcium phosphate (ACP) as their active ingredient. Liquid ACP is prone to phase transition and has a short interaction time with teeth, failing to provide a continuous and stable source of calcium and phosphate, thus failing to achieve the ideal biomimetic mineralization effect. Furthermore, existing biomimetic mineralization products rarely possess antibacterial properties and cannot effectively inhibit the adhesion of Streptococcus mutans, thereby disrupting plaque biofilm formation. Clinically, their mineralization and caries prevention effects are unsatisfactory. Metal cations such as Ag... 2+ Mg 2+ Zn 2+ 、Sr 2+ While some reports indicate that these products have antibacterial properties, issues such as biosafety, antibacterial durability, and damage to tooth tissue remain. Summary of the Invention

[0004] Based on this, the present invention provides a mineralizing and antibacterial dual-function nanoparticle, its preparation method, and its application. The main active ingredient of this mineralizing and antibacterial dual-function nanoparticle is amorphous calcium phosphate, with a particle size of 40-60 nm and a regular spherical shape. After doping with TFF3 (Trefoil factor 3), the TFF3-ACP nanoparticles possess antibacterial and mineralization-promoting properties. TFF3 has good biocompatibility and long-lasting antibacterial effects. Incorporating it into ACP to functionalize ACP and enhance its antibacterial and mineralizing properties may make it an ideal oral mineralizing and caries-preventing material with broad application prospects. The preparation method of this invention is simple and easy to implement, with low preparation cost, facilitating large-scale production and clinical application.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing mineralizing and antibacterial "dual-functional" nanoparticles, comprising the following steps:

[0007] 1) A calcium solution is obtained by adding a polyelectrolyte solution to a calcium salt solution. The polyelectrolyte reacts with Ca... 2+ The molar ratio is 1:10-1:20;

[0008] 2) Add TFF3 to the phosphate solution to obtain a phosphorus solution. The molar ratio of TFF3 to phosphorus is 1:10-1:20.

[0009] 3) Mix the calcium solution obtained in step 1) and the phosphorus solution obtained in step 2) in equal volumes. When mixing, the molar ratio of calcium in the calcium solution to TFF3 in the phosphorus solution is 1 to 10:1; the molar ratio of the sum of calcium and TFF3 to phosphorus is 10 to 20:6 to 10; adjust the pH value of the mixture and centrifuge to obtain mineralization and antibacterial "dual-function" nanoparticles.

[0010] According to a preferred embodiment of the present invention, in the calcium solution of step 1), the components are, by weight, 10-100 parts of polyelectrolyte, 50-500 parts of calcium salt, and 50-2000 parts of solvent.

[0011] According to a preferred embodiment of the present invention, in step 1), the polyelectrolyte is any one or a combination of polyelectrolytes such as polyaspartic acid, polyacrylic acid, polyglutamic acid, and polylysine;

[0012] The calcium salt is a soluble calcium salt selected from any one or more combinations of calcium chloride and / or its hydrate, calcium bicarbonate, calcium dihydrogen phosphate, calcium lactate and / or its hydrate, calcium acetate and / or its hydrate, and calcium citrate and / or its hydrate.

[0013] According to a preferred embodiment of the present invention, in the phosphorus solution of step 2), the components are as follows by weight: 50-500 parts of phosphate, 10-50 parts of TFF3, and 50-2000 parts of solvent.

[0014] According to a preferred embodiment of the present invention, in step 2), the phosphate is a soluble phosphate selected from any one or more combinations of sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, sodium phosphate, potassium phosphate, and ammonium phosphate.

[0015] According to a preferred embodiment of the present invention, in step 3), the pH value of the mixture is adjusted to 9.5 ± 0.5.

[0016] Secondly, the present invention provides a mineralization and antibacterial "dual-function" nanoparticle prepared by the above method. Preferably, the active ingredient of the mineralization and antibacterial "dual-function" nanoparticle is amorphous calcium phosphate, the particle size of which is 30-40 nm and the shape is spherical. The mineralization performance of the amorphous calcium phosphate is improved and it is endowed with antibacterial properties after being doped with TFF3.

[0017] Thirdly, the present invention provides the application of the above-mentioned mineralization and antibacterial "dual-function" nanoparticles in the preparation of drugs for the prevention and treatment of dental leukoplakia or early caries.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) This invention is the first to confirm that TFF3 has antibacterial activity against Streptococcus mutans (see Appendix Table 1). Furthermore, because TFF3 contains a large number of negatively charged amino acids, it can chelate calcium ions. Since calcium ion chelation may promote the mineralization of teeth and bones, we speculate that TFF3 may be a "dual-function" factor that promotes mineralization and has antibacterial properties. This invention confirms that ACP containing TFF3 has a "dual" effect of promoting mineralization and antibacterial activity (see Appendix Table 1). Figure 1-3 ).

[0020] (2) The main active ingredients of the formulation of the present invention are ACP and TFF3. TFF3 can improve the mineralization performance of traditional ACP and endow it with antibacterial properties. ACP solid nanoparticles are not prone to phase change, which is conducive to maintaining their mineralization potential and is beneficial to biomimetic mineralization.

[0021] (3) The preparation method of the tooth mineralization and antibacterial "dual-function" nanoparticles provided by the present invention is simple and easy to implement, does not require large-scale instruments and equipment, has high production efficiency and low cost, and is suitable for large-scale production.

[0022] (4) The artificially synthesized TFF3-amorphous calcium phosphate (ACP) nanoparticles of this invention possess both mineralization and antibacterial properties, and are expected to provide a new theoretical basis for the prevention and treatment of dental caries, with significant scientific and clinical value. They can be used for the prevention and treatment of oral diseases such as leukoplakia and early dental caries, and have huge market potential. Attached Figure Description

[0023] Figure 1 Transmission electron microscopy image of mineralized antibacterial "dual-function" nanoparticles;

[0024] Figure 2 Infrared characterization of mineralized antibacterial "dual-function" nanoparticles;

[0025] Figure 3 A diagram illustrating the type I collagen mineralization induced by "dual-function" nanoparticles for mineralization and antibacterial purposes. Detailed Implementation

[0026] The present invention will be further illustrated below with examples, including preferred embodiments. Any modifications and variations made within the principles of the present invention are within the scope of protection of the present invention.

[0027] Implementation Case 1

[0028] The preparation process of the "dual-functional" nanoparticles in this embodiment is as follows:

[0029] (1) Add PAsp (final PAsp concentration is 480 μg / mL) to 25 mL of 10 mM CaCl2 solution and mix evenly to prepare calcium solution;

[0030] (2) Prepare phosphorus solution by adding TFF3 (final concentration 100 μg / mL) to 25 mL of 6 mM NaH2PO4 solution;

[0031] (3) Mix the phosphorus solution and calcium solution for 10 min and adjust the pH value to 9.5±0.5. After mixing, the calcium-phosphorus molar ratio in the reaction solution is 10:6. After three centrifugations, TFF3-PAsp-ACP nanoparticles are formed and placed in a vacuum drying oven to obtain "dual-function" nanoparticles.

[0032] Implementation Case 2

[0033] The preparation process of the "dual-functional" nanoparticles in this embodiment is as follows:

[0034] (1) Add PAA (final PAA concentration of 500 μg / mL) to 25 mL of 10 mM CaCl2 solution and mix evenly to prepare calcium solution;

[0035] (2) Prepare phosphorus solution by adding 25 mL of 6 mM NaH2PO4 to TFF3 (final concentration 200 μg / mL);

[0036] (3) Mix the phosphorus solution and calcium solution for 10 min and adjust the pH value to 9.5±0.5. After mixing, the calcium-phosphorus molar ratio in the reaction solution is 10:6. After three centrifugations, TFF3-PAsp-ACP nanoparticles are formed and placed in a vacuum drying oven to obtain "dual-function" nanoparticles.

[0037] Implementation Case 3

[0038] The preparation process of the "dual-functional" nanoparticles in this embodiment is as follows:

[0039] (1) Add L-Glu (final L-Glu concentration is 500 μg / mL) to 25 mL of 10 mM CaCl2 solution and mix evenly to prepare calcium solution;

[0040] (2) Add TFF3 (final concentration of TFF3 is 150 μg / mL) to 25 mL of 6 mM NaH2PO4 solution to prepare phosphorus solution;

[0041] (3) Mix the phosphorus solution and calcium solution for 10 min and adjust the pH value to 9.5±0.5. After mixing, the calcium-phosphorus molar ratio in the reaction solution is 10:6. After centrifugation, TFF3-PAsp-ACP nanoparticles are formed and placed in a vacuum drying oven to obtain "dual-function" nanoparticles.

[0042] Implementation Case 4

[0043] The "dual-function" nanoparticles obtained in Case Study 1 were characterized using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The results are as follows: Figure 1 and Figure 2 As shown, from Figure 1 and Figure 2 As can be seen, the characterization results, infrared and TEM results of the "dual-function" nanoparticles of the present invention show that TFF3-PAsp-ACP is a spherical nanoparticle, and its infrared peak still has the characteristic peaks of amorphous calcium phosphate (580, 1050 cm-1), indicating that TFF3-PAsp-ACP nanoparticles are in an amorphous phase and have mineralization potential, which can be used for biomimetic mineralization research.

[0044] Implementation Case 5

[0045] The "bifunctional" nanoparticles obtained in Case 3 were used to conduct experiments related to anti-Streptococcus mutans. ① Culture of Streptococcus mutans: First, a Streptococcus mutans (S. mutans) bacterial suspension was prepared. Revived S. mutans were picked using an inoculation loop and placed in Brain Heart Infusion (BHI) liquid medium. The suspension was anaerobically cultured at 37°C for 24 hours. After at least two subcultures, the bacterial concentration was adjusted to 1×10⁶ CFU / mL using a spectrophotometer (600 nm). ② Antibacterial experiment: The effect of the "bifunctional" nanoparticles on S. mutans was analyzed using the MIC experiment. The "bifunctional" nanoparticles were used as the experimental group (TFF3-PAsp-ACP group), BHI liquid medium as the control group, and sterile deionized water as the blank control group to evaluate the antibacterial activity.

[0046] The results are shown in Table 1:

[0047] Table 1. Antibacterial activity of TFF3-PAsp-ACP against Streptococcus mutans.

[0048]

[0049] Note: The blank control group was a bacterial suspension with added sterile water.

[0050] Statistical analysis showed that the intergroup comparison P<0.01, indicating that the addition of TFF3 enhanced the antibacterial properties of PAsp-ACP.

[0051] The TFF3-PAsp-ACP group showed a significant difference compared with the blank control group, indicating that TFF3-PAsp-ACP has an anti-streptococcal effect.

[0052] Implementation Case 6

[0053] The "dual-functional" nanoparticles of this invention are used for recombining type I collagen. 10 μL of rat tail collagen solution was mixed with 1 ml of assembly solution (the assembly solution included glycine, KCl, and NaN3, wherein the concentration of glycine was 50 mM, the concentration of KCl was 200 mM, and the mass percentage of NaN3 was 0.1%). After standing at room temperature for 20 min, 3 μL of the liquid was dropped onto a nickel mesh and left overnight. After cross-linking with glutaraldehyde, the mixture was rinsed with deionized water, dried, and 8 mg of nanoparticles were placed in 240 μL of artificial saliva. The nickel mesh containing type I collagen was suspended on the surface. Samples were taken after 1, 4, and 7 days, and the collagen mineralization was observed by TEM. Figure 3 As shown: the control group is the left side, showing no obvious mineralization; the experimental group containing "dual-function" nanoparticles is... Figure 3 On the right side, it can be seen that the collagen is completely mineralized after 7 days.

[0054] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of a mineralizing and antibacterial "dual-function" nanoparticle in the preparation of drugs for the prevention and treatment of leukoplakia or early caries, characterized in that, The preparation method of the mineralization and antibacterial "dual-function" nanoparticles includes the following steps: 1) A calcium solution is obtained by adding a polyelectrolyte solution to a calcium salt solution. The polyelectrolyte reacts with Ca... 2+ The molar ratio is 1:10-1:20; in the calcium solution, the components by weight are: 10-100 parts of polyelectrolyte, 50-500 parts of calcium salt, and 50-2000 parts of solvent; the polyelectrolyte is polyaspartic acid, polyacrylic acid, or polyglutamic acid; the calcium salt is calcium chloride; 2) TFF3 is added to a phosphate solution to obtain a phosphorus solution, wherein the molar ratio of TFF3 to phosphorus is 1:10-1:20; the components in the phosphorus solution are, by weight: 50-500 parts of phosphate, 10-50 parts of TFF3, and 50-2000 parts of solvent; wherein the phosphate is sodium dihydrogen phosphate. 3) Mix the calcium solution obtained in step 1) and the phosphorus solution obtained in step 2) in equal volumes. When mixing, the molar ratio of calcium in the calcium solution to TFF3 in the phosphorus solution is 1~10:1; the molar ratio of the sum of calcium and TFF3 to phosphorus is 10~20:6~10; adjust the pH of the mixture to 9.5±0.5, and centrifuge to obtain mineralization and antibacterial "dual-function" nanoparticles.

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