Dynamic covalent colloid material as well as preparation method and application thereof

Abstract

The invention discloses a dynamic covalent colloid material as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The invention provides a novel method for constructing dynamic covalent colloidal glue (DCCGs) based on a reaction induced self-assembly (RISA) strategy, and the prepared DCCGs are used for combined treatment of oral periodontitis and tooth demineralization. Experimental results show that the prepared DCCGs can be efficiently adhered and have outstanding functions in the aspects of scavenging free radicals and pro-inflammatory related molecules, inhibiting bacteria colonization, promoting tooth remineralization and the like. The prepared DCCGs can meet the requirements of oral cavity anti-inflammation and mineralization promotion synergistic treatment, a novel multifunctional integrated material is provided for treatment of periodontal diseases, and a foundation is laid for a multi-disease co-treatment strategy.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and in particular to a dynamic covalent colloidal material, its preparation method, and its application. Background Technology

[0002] Currently, a dynamic covalent drug delivery nanonetwork preparation technology based on reaction-induced self-assembly has been developed, which is often used in the treatment of bacterial infectious diseases and inflammatory diseases. It realizes the responsive release of active components or drugs in acidic / high reactive oxygen environments and has targeted delivery characteristics.

[0003] Periodontal disease is a group of diseases characterized by inflammation and infection of some or all of the tooth-supporting structures (gingiva, cementum, periodontal ligament, alveolar bone, and other peri-dental tissues). Periodontitis is the main form of periodontal disease, often accompanied by tooth demineralization. Current dynamic covalent nanonetwork drug delivery systems are micellar suspensions, unable to exert interfacial regulation effects and thus difficult to apply directly to the treatment of periodontal disease. Furthermore, no existing technology has been found to provide combined treatment for periodontal inflammation and tooth demineralization.

[0004] Therefore, there is an urgent need to design a novel dynamic covalent gel structure with interface regulation function, which can synergistically promote tooth remineralization while regulating local inflammatory response. Summary of the Invention

[0005] The purpose of this invention is to provide a dynamic covalent colloidal material, its preparation method, and its application to solve the problems existing in the prior art. The DCCGs prepared by this invention can meet the needs of synergistic treatment of oral inflammation and mineralization, providing a novel multifunctional integrated material for the treatment of periodontal diseases and laying the foundation for a multi-disease co-treatment strategy.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing a dynamic covalent colloidal material, comprising the following steps:

[0008] The following mother liquors were prepared: polyamide-amine dendritic polymer mother liquor, epigallocatechin gallate mother liquor, and 2-formylphenylboronic acid mother liquor.

[0009] The epigallocatechin gallate mother liquor, the 2-formylphenylboronic acid mother liquor, and ultrapure water are mixed and stirred. The polyamide-amine dendritic polymer mother liquor is then added dropwise to carry out a self-assembly reaction until a white colloidal gel appears, thus obtaining the dynamic covalent colloidal gel material.

[0010] Furthermore, the solvent for the polyamide-amine dendritic polymer mother liquor and the epigallocatechin gallate ester mother liquor is ultrapure water;

[0011] The solvent for the 2-formylphenylboronic acid mother liquor is dimethyl sulfoxide.

[0012] Furthermore, in the reaction system of the self-assembly reaction, the molar ratio of the polyamide-amine dendritic polymer, the 2-formylphenylboronic acid, and the epigallocatechin gallate is 2:10:5;

[0013] The molar amount of the polyamide-amine dendritic polymer is calculated based on the amino groups in the polyamide-amine dendritic polymer.

[0014] The molar amount of 2-formylphenylboronic acid is based on the ortho-dihydroxy structural unit in 2-formylphenylboronic acid;

[0015] The molar amount of the epigallocatechin gallate is based on the catechol structural units in the epigallocatechin gallate.

[0016] Furthermore, the stirring speed is 2500 rpm; the temperature of the self-assembly reaction is 25°C.

[0017] The present invention also provides a dynamic covalent colloidal adhesive material obtained according to the above preparation method.

[0018] The present invention also provides the application of the above-mentioned dynamic covalent colloidal material in the preparation of drugs for treating periodontal diseases.

[0019] Furthermore, the periodontal disease includes periodontitis and tooth demineralization.

[0020] Furthermore, the periodontal disease is periodontitis combined with tooth demineralization.

[0021] The present invention also provides a medicament for treating periodontitis and / or tooth demineralization, with the above-mentioned dynamic covalent colloidal adhesive material as the main active ingredient.

[0022] Furthermore, it also includes pharmaceutically acceptable excipients.

[0023] The present invention discloses the following technical effects:

[0024] This invention provides a novel method for constructing dynamic covalent gels (DCCGs) based on a reaction-induced self-assembly (RISA) strategy, and applies the prepared DCCGs to the combined treatment of periodontal inflammation and tooth demineralization. Experimental results show that the DCCGs prepared by this invention exhibit efficient adhesion and outstanding functions in scavenging free radicals and pro-inflammatory molecules, inhibiting bacterial colonization, and promoting tooth remineralization. The DCCGs prepared by this invention can meet the needs of synergistic anti-inflammatory and promineralization treatment in oral cavity, providing a novel multifunctional integrative material for the treatment of periodontal diseases and laying the foundation for multi-disease co-treatment strategies. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A schematic diagram of the synthetic route for preparing PFE CGs;

[0027] Figure 2 The images show the particle size and representative TEM images at different time points during the preparation of PFE CGs.

[0028] Figure 3 FT-IR spectra of PAMAM, EGCG, FPBA, and PFE;

[0029] Figure 4 Figure showing the experimental results of the effect of pH and H2O2 on PFE particle size;

[0030] Figure 5 Statistical graph showing the scavenging rates of ABTS+ radicals (a) and DPPH radicals (b) by different concentrations of PFE CGs;

[0031] Figure 6 The chart shows the adsorption capacity of different materials for CpG and LPS; where a represents the CpG residual rate and b represents the LPS residual rate.

[0032] Figure 7 A graph showing the quantitative statistical results of the adhesion force of different materials on the surface of dental grinding discs;

[0033] Figure 8 Representative images of rat maxillary teeth reconstructed using Micro-CT three-dimensional reconstruction after treatment with different materials, and quantitative statistical graphs of gray values;

[0034] Figure 9Representative images of oral plaque staining in rats after treatment with different materials and quantitative plaque staining index; Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Epigallocatechin gallate (EGCG), with the molecular formula C0.05 22 H 18 O 11 The CAS number is 989-51-5.

[0041] 2-Formylphenylboronic acid (2-FPBA), with the molecular formula C7H7BO3 and CAS number 40138-16-7.

[0042] Polyamide-amine dendritic polymer (PAMAM) is a nanomaterial with a three-dimensional dendritic structure, which is a colorless to pale yellow transparent liquid at room temperature; the PAMAM of this invention is an ethylenediamine core, generation 3.0; purchased from Maclean; CAS: 153891-46-4.

[0043] Example 1: Method for constructing DCCGs

[0044] In this embodiment, EGCG was selected as a polyphenol compound to construct DCCGs, and the constructed DCCGs were named PFECGs.

[0045] 10 mg PAMAM and 10 mg EGCG were dissolved in 1 mL of ultrapure water to prepare 10 mg / mL PAMAM stock solution and EGCG stock solution, respectively; 10 mg 2-FPBA was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare 10 mg / mL 2-FPBA stock solution. The preparation temperature was 25℃, and the solution was subjected to vortexing and ultrasonic vibration for 5 min.

[0046] Then, 725 μL of ultrapure water was taken, and 106 μL of EGCG stock solution and 70 μL of 2-FPBA stock solution were added. With magnetic stirring at 2500 rpm, 100 μL of PAMAM stock solution was added dropwise at a rate of 10 μL every 10 seconds. The reaction was carried out at 25°C for 5 min. The initial suspension stage was completed when the solution turned into transparent milky blue micelles, yielding PFE. The reaction time was extended to 30 min at 25°C until a white colloidal gel appeared, indicating the reaction was complete, yielding PFE CGs.

[0047] The synthetic route of the PFE CGs of the present invention is as follows: Figure 1 As shown.

[0048] Example 2 Material Characterization of DCCGs

[0049] The PFE CGs prepared in Example 1 were subjected to structural characterization studies.

[0050] 1. Particle size and material morphology

[0051] The detection steps are as follows: PFE / PFE CGs at different time points during the preparation process were diluted 10 times with ultrapure water, and the particle size was obtained using a nanoparticle size analyzer. The morphology of the material was then photographed using a transmission electron microscope (TEM).

[0052] Figure 2 The images show the particle size of PFE / PFE CGs at different time points during the preparation process and the corresponding TEM images. The results show that PFE / PFE CGs gradually assemble from the colloidal suspension into a three-dimensional network colloidal structure with increasing time.

[0053] 2. Chemical bond detection

[0054] The detection steps are as follows: After centrifuging PFE CGs at 5000 rpm, the precipitate was collected and freeze-dried. The freeze-dried PFE powder and PAMAM, EGCG, and 2-FPBA raw materials were subjected to Fourier transform infrared spectroscopy (FT-IR) testing.

[0055] Figure 3 The FT-IR plot of PFE shows that PAMAM is at 3271 cm⁻¹. -1 The characteristic peak values ​​of NH vibration, 2-FPBA, and EGCG at 3345 cm⁻¹ -1 Nearby OH vibration, 1673 cm -1 2-FPBA C=O vibration at 1362 cm -1 The characteristic peaks of BO vibration at 1615 cm⁻¹ all disappeared. In contrast, PFE at 1615 cm⁻¹... -1 (C=N), 1000 cm -1 (BOC) and 760 cm -1 Three new characteristic peaks were observed at (B←N), indicating the formation of imine bonds, borate ester bonds, and B←N coordination bonds in the PFE.

[0056] Example 3: Verification of the effect of DCCGs

[0057] The therapeutic efficacy of the PFE CGs prepared in Example 1 was verified.

[0058] 1. Response Release

[0059] The particle size variation of PFE was detected under different pH conditions and with or without hydrogen peroxide (H2O2).

[0060] The detection steps are as follows: PFE is added to phosphate buffer (10 mM) at pH 7.4 or 6.5, and then co-incubated with 100 μM H2O2 at 37°C. The particle size change under different conditions is detected using a nanoparticle size analyzer.

[0061] The results are as follows Figure 4 As shown, the presence of acidity and high ROS increases the particle size of PFE, proving that dynamic covalent bonds break.

[0062] 2. Free radical scavenging

[0063] The results of the determination of the ability of PFE CGs to scavenge ABTS+ and DPPH free radicals.

[0064] The detection steps are as follows: Mix equal volumes of different concentrations of PFE CGs with ABTS+ working solution, incubate in the dark, and then measure the absorbance at 734 nm using a microplate reader. Mix equal volumes of different concentrations of PFE CGs with DPPH working solution, incubate in the dark, and then measure the absorbance at 517 nm using a microplate reader.

[0065] The results are as follows Figure 5 As shown, PFE CGs possess concentration-dependent broad-spectrum free radical scavenging capabilities.

[0066] 3. Adsorption of inflammation-related molecules

[0067] The adsorption of oligonucleotides (CpG) and lipopolysaccharides (LPS) by different materials was detected.

[0068] The detection steps are as follows: Different materials were incubated with FITC-labeled CpG / LPS in the dark for 1 h, and then centrifuged at 10,000 rpm for 15 min to separate the complex bound to the materials from the free FITC-CpG / LPS. The fluorescence intensity of the supernatant was measured using an ELISA reader, which represents the unbound CpG / LPS.

[0069] The results are as follows Figure 6 As shown, PFE CGs exhibited a superior ability to adsorb inflammation-related molecules compared to other control groups.

[0070] 4. Highly efficient adhesion

[0071] The adhesion force distribution of different materials on the surface of dental grinding discs was detected.

[0072] The testing steps are as follows: different materials are dropped onto the surface of clinically obtained human extracted tooth sections, and the adhesion of the materials is examined using atomic force microscopy (AFM).

[0073] The results are as follows Figure 7 As shown, PFE CGs exhibit significantly better adhesion strength on the surface of dental radiographs than EGCG and the Mix group.

[0074] 5. Promotes remineralization

[0075] Micro-CT three-dimensional reconstruction results of rat teeth after treatment with different materials were detected.

[0076] The detection steps are as follows: The demineralized rat maxillary first molar was treated with the material for 14 consecutive days. After sacrifice, the rat maxillary teeth were photographed with Micro-CT.

[0077] The results are as follows Figure 8 As shown, the demineralized areas of the maxillary teeth of rats treated with PFE CGs appear whiter, indicating a higher degree of mineralization.

[0078] 6. Inhibits dental plaque biofilm

[0079] The detection steps are as follows: Rat teeth were ligated and bacteria were administered to establish an animal model. Different materials were used to treat the rats continuously for 14 days. Plaque indicator was used to stain the rat maxillary teeth.

[0080] Figure 9 Images of dental plaque indicator staining after treatment with different materials were obtained. The results showed that the plaque content in the oral cavity of rats was significantly reduced after PFE CGs treatment.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a dynamic covalent colloidal material, characterized in that, Includes the following steps: The following mother liquors were prepared: polyamide-amine dendritic polymer mother liquor, epigallocatechin gallate mother liquor, and 2-formylphenylboronic acid mother liquor. The epigallocatechin gallate mother liquor, the 2-formylphenylboronic acid mother liquor, and ultrapure water are mixed and stirred. The polyamide-amine dendritic polymer mother liquor is then added dropwise to carry out a self-assembly reaction until a white colloidal gel appears, thus obtaining the dynamic covalent colloidal gel material.

2. The preparation method according to claim 1, characterized in that, The solvent for the polyamide-amine dendritic polymer mother liquor and the epigallocatechin gallate mother liquor is ultrapure water; The solvent for the 2-formylphenylboronic acid mother liquor is dimethyl sulfoxide.

3. The preparation method according to claim 1, characterized in that, In the reaction system of the self-assembly reaction, the molar ratio of the polyamide-amine dendritic polymer, the 2-formylphenylboronic acid and the epigallocatechin gallate is 2:10:5; The molar amount of the polyamide-amine dendritic polymer is calculated based on the amino groups in the polyamide-amine dendritic polymer. The molar amount of 2-formylphenylboronic acid is based on the ortho-dihydroxy structural unit in 2-formylphenylboronic acid; The molar amount of the epigallocatechin gallate is based on the catechol structural units in the epigallocatechin gallate.

4. The preparation method according to claim 1, characterized in that, The stirring speed is 2500 rpm; the temperature of the self-assembly reaction is 25°C.

5. A dynamic covalent colloidal adhesive material obtained by the preparation method according to any one of claims 1-4.

6. The use of the dynamic covalent colloidal material as described in claim 5 in the preparation of a medicament for treating periodontal disease.

7. The application as described in claim 6, characterized in that, The periodontal diseases mentioned include periodontitis and tooth demineralization.

8. The application as described in claim 6, characterized in that, The periodontal disease mentioned is periodontitis combined with tooth demineralization.

9. A medicament for treating periodontitis and / or tooth demineralization, characterized in that, The dynamic covalent colloidal adhesive material described in claim 5 is the main effective component.

10. The medicament as claimed in claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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