CN113214479B

CN113214479B - Mineralized anticarious material and its prepn

Info

Publication number: CN113214479B
Application number: CN202110501625.6A
Authority: CN
Inventors: 李继遥; 陶思颖; 梁坤能; 杨佼佼; 高源�
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2022-03-18
Anticipated expiration: 2041-05-08
Also published as: CN113214479A

Abstract

The present disclosure provides a mineralized anticarious material and a preparation method thereof. The mineralized anticarious material comprises polyamide-amine, wherein a part of functional groups on the surface of the polyamide-amine are grafted with polypeptide fragments capable of specifically binding collagen molecules. According to the present disclosure, a novel mineralized anticarious material that simultaneously stabilizes the collagen structure and induces biomimetic remineralization of collagen in the MMPs environment can be obtained.

Description

Mineralized anticarious material and its prepn

Technical Field

The disclosure relates to the technical field of materials, in particular to a mineralized anticarious material for oral teeth and a preparation method thereof.

Background

Dental caries is a common disease and frequently encountered disease of human oral cavity, leads to morphological defect and dysfunction of tooth, and seriously harms human oral cavity and general health. The effective prevention and treatment of caries is of great significance. Dentin is a highly mineralized hard dental tissue, mainly composed of inorganic minerals, organic matrix and water. Whereas in organic matrices, type I collagen fibers account for about 90%, the remaining 10% being predominantly non-collagenous proteins (NCPs). When caries progresses to the dentinal layer, demineralization of dentin and exposure of type I collagen fibers are caused. The traditional treatment technology mainly removes carious tissues through surgical instruments, fills cavities with repair materials such as resin and the like, and has certain invasiveness to healthy tooth tissues. With the proposition and popularization of the minimally invasive dental concept, the biomimetic remineralization technology is utilized to promote the remineralization of demineralized dentin collagen, reverse the caries process and recover the dentin structure, form and function, thus becoming a new direction for preventing and treating caries.

Disclosure of Invention

In view of the deficiencies in the prior art, it is an object of the present invention to address one or more of the problems in the prior art as set forth above. For example, one of the objectives of the present invention is to provide a mineralized anticaries material capable of protecting collagen from MMPs, maintaining the structural integrity and stability of collagen in the microenvironment of the oral cavity, and a preparation method thereof

One aspect of the present disclosure provides a mineralized anticaries material comprising polyamidoamine, wherein a part of functional groups on the surface of the polyamidoamine is grafted with a polypeptide fragment capable of specifically binding to a collagen molecule.

According to embodiments of the present disclosure, at most half of the functional groups of the polyamidoamine surface can be grafted with the polypeptide fragments, the polypeptide fragments can be symmetrically grafted to the functional groups of the polyamidoamine surface, 4, 16 or 64 of the polypeptide fragments can be grafted to the functional groups of the polyamidoamine surface, each of the polypeptide fragments is grafted to one functional group.

According to embodiments of the present disclosure, the functional group of the polyamide-amine surface may be one or more of amino, carboxyl, hydroxyl, phosphate, and alendronate.

According to embodiments of the present disclosure, the polypeptide fragments may include one or more of DSESSEEDR and/or SEENRDSDSQDSSR sequences in dentin matrix protein-1, NGVFKYRPRYFLYKHAYFYPPLKRFPVQ sequences in bone sialoprotein, and CQDSETRTFY sequences in fibronectin.

According to embodiments of the present disclosure, the hydrophobic cavity inside the polyamidoamine may be loaded with matrix metalloproteinase inhibitors including one or more of chlorhexidine, quaternary ammonium salts, galanin, and tetracyclines.

According to another aspect of the present disclosure, there is provided a method of making a mineralized anticaries material as described above, the method comprising: synthesizing a polypeptide fragment capable of specifically binding to a collagen molecule; synthesizing polyamide-amine and grafting the polypeptide fragment on a part of functional groups on the surface of the polyamide-amine; and loading a matrix metalloproteinase inhibitor into a hydrophobic cavity inside the polyamidoamine.

According to the embodiment of the disclosure, a michael addition method can be adopted to synthesize polyamide-amine with 128 amino functional groups on the surface, a part of amino functional groups on the surface of the polyamide-amine is acylated with acryloyl chloride symmetrically through click reaction, and then the rest amino groups on the surface of the polyamide-amine are subjected to carboxyl modification by succinic anhydride; connecting cysteine to the tail end of the polypeptide fragment, and grafting the polypeptide fragment to an amino functional group on the surface of polyamidoamine after acylation treatment by utilizing a mercapto-Michael addition reaction to obtain the polyamidoamine with the polypeptide fragment grafted on the surface; mixing the polyamide-amine with the polypeptide fragments grafted on the surface with a matrix metalloproteinase inhibitor by adopting any one of an organic solvent method, a water-soluble method and a co-soluble method, and preparing the polyamide-amine with the polypeptide fragments grafted on the surface and the matrix metalloproteinase inhibitor loaded in a hydrophobic cavity inside through ultrasonic and oscillation modes.

According to an embodiment of the present disclosure, the method may further include: detecting the ability of the polyamidoamine with the polypeptide segment grafted on the surface to crosslink collagen, and screening and optimizing the polyamidoamine with the polypeptide segment grafted on the surface by analyzing the result of the detection step, wherein the detection step specifically comprises the following steps: observing the binding sites of the polyamidoamine with the polypeptide segments grafted on the surface and the collagen fibers, and detecting the binding force of the polyamidoamine with the polypeptide segments grafted on the surface and collagen molecules; determination of different pH and Ca²⁺The effect of the polyamidoamine with the polypeptide segment grafted on the surface under the concentration on the collagen fibril generation time and the binding force among collagen molecules; observing the change of the secondary structure of the collagen before and after crosslinking; and detecting the change of the mechanical property and the enzymolysis resistance of the collagen hydrogel before and after crosslinking.

According to an embodiment of the present disclosure, the method may further include: detecting the polyamidoamine with the polypeptide fragment grafted on the surface and the matrix metalloproteinase inhibitor loaded in the internal hydrophobic cavity, and screening and optimizing the polyamidoamine with the polypeptide fragment grafted on the surface and the matrix metalloproteinase inhibitor loaded in the internal hydrophobic cavity by analyzing the result of the detection step, wherein the detection step specifically comprises the following steps: detecting the physical and biological properties of the system; qualitatively detecting the loading condition of the matrix metalloproteinase inhibitor, and quantitatively detecting the loading amount of the matrix metalloproteinase inhibitor; detecting the slow release efficiency of the matrix metalloproteinase inhibitor under different buffer systems and different pH conditions; the ability of the polyamidoamines, surface grafted with polypeptide fragments and loaded with matrix metalloproteinase inhibitor in the interior hydrophobic cavity, to inhibit matrix metalloproteinase activity was evaluated.

Compared with the prior art, the method can obtain the novel mineralized anticarious material which synchronously stabilizes the collagen structure and induces the biomimetic remineralization of the collagen in the MMPs environment.

Drawings

The accompanying drawings, which are included to provide a further understanding of the inventive concepts, are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the inventive concepts and together with the description serve to explain the principles of the inventive concepts.

FIG. 1 shows a schematic view of a mineralized anticaries material according to an exemplary embodiment of the present disclosure.

FIG. 2 shows the NMR of SEENRDSDSQDSSRC-PAMAM (SP) grafted with 4 polypeptide fragments.

FIG. 3 shows a circular dichroism spectrum of SEENRDSDSQDSSRC-PAMAM (SP) grafted with 4 polypeptide fragments.

FIG. 4 shows a High Performance Liquid Chromatography (HPLC) profile of SEENRDSDSQDSSRC-PAMAM grafted with 4 polypeptide fragments.

FIG. 5 shows the hydroxyproline assay results of SEENRDSDSQDSSRC-PAMAM grafted with 4 polypeptide fragments. SP on the graph represents SEENRDSDSQDSSRC-PAMAM.

Detailed Description

The applicant successfully induces the regeneration of mineral crystals in and among collagen fibers of type I collagen and demineralized dentin by taking dendrimer Polyamidoamine (PAMAM) as a mineralization template. Realizes the bionic remineralization of collagen in an in vitro environment. However, the clinical application of biomimetic remineralization needs to further consider the influence caused by the complex environment in the human body. Among them, the destruction of collagen structural integrity by Matrix Metalloproteinases (MMPs) present in large amounts in the oral cavity is not favorable for the clinical development of biomimetic remineralization. Under normal physiological conditions, MMPs are in an inactivated state. However, in pathological conditions such as caries, MMPs are activated by repeated low pH stimulation. In addition, intake of acidic foods, beverages, dentinal adhesion, etc. may activate MMPs. Activated MMPs are capable of enzymatically hydrolyzing exposed collagen fibers, disrupting the structural integrity of the collagen, and even causing disintegration of the collagen fibers. In the natural structure of dentin, the organic collagen matrix is a basic scaffold for inorganic mineral growth, and plays the roles of mechanical support, nucleation template and the like, and once the structure is damaged, the induction of biomimetic remineralization can also lose the structural basis. The complete and stable structure of the collagen matrix is a precondition for effectively inducing the biomimetic remineralization.

In the artificial saliva environment in the presence of collagenase, if the collagen structure is destroyed by enzymolysis, the remineralization repair effect of demineralized dentin is significantly reduced. Therefore, how to protect collagen from being invaded by MMPs and maintain the complete and stable structure of the collagen in the microenvironment of the oral cavity becomes a key problem to be solved urgently in clinical application of the biomimetic remineralization technology. Unfortunately, existing biomimetic remineralization studies do not take into account the MMPs environment in the oral cavity, and little attention has been paid to the preservation of the structural integrity of the mineralization-induced procollagen.

Based on the problems faced by the research of biomimetic remineralization of collagen, the novel mineralized caries-preventing material which can synchronously stabilize the collagen structure and induce biomimetic remineralization of collagen in the MMPs environment is obtained, and has great significance for promoting the clinical application of the biomimetic remineralization technology.

In order to solve the key problems that the collagen scaffold structure in the microenvironment of the oral cavity of a human body is easy to be damaged by enzymolysis and is not beneficial to biomimetic remineralization induction, the applicant proposes that on the basis of the study on the biomimetic remineralization of the PAMAM, the PAMAM is subjected to double modification of an external functional group and an internal cavity, and under the premise of keeping the biomimetic remineralization capability of the PAMAM, the functions of crosslinking collagen fibers and inhibiting MMPs are given to the PAMAM, so that the effective induction on the biomimetic remineralization of the collagen in the environment containing the MMPs is realized.

Mineralized anticaries materials according to exemplary embodiments of the present disclosure include Polyamidoamines (PAMAMs) having grafted onto functional groups on the surface of the polyamidoamines polypeptide fragments that can specifically bind to collagen molecules.

The PAMAM dendrimer is composed of core molecules, internal cavities, branched structures and surface functional groups, and has the characteristics of monodispersity, clear structure, excellent biocompatibility and the like. Since PAMAM is similar in molecular size and structure to many biological proteins and has a protein-like self-assembly effect, it can mimic the function of natural proteins, PAMAM is reputed as an "artificial protein". In addition, the PAMAM is a good drug carrier, a hydrophobic cavity in the PAMAM can be used as a storage base of insoluble small-molecule drugs, and the PAMAM can be combined with the drugs through electrostatic interaction to realize loading and slow release of the drugs, improve the solubility and stability of the drugs and prolong the action time of the drugs.

Previous studies by the applicant found that PAMAM (PAMAM-NH) with amino as a surface functional group₂) Can stabilize and induce ACP to deposit in and among collagen fibers; the carboxyl modified PAMAM (PAMAM-COOH) can be self-assembled into a multi-stage structure of nanospheres, ball chains and micro-strips in artificial saliva, so that the regeneration of minerals in collagen fibers is promoted; the hydroxyl modified PAMAM (PAMAM-OH) can enter deep layer of dentinal tubule to induce mineral crystal to close dentinal tubule; phosphate group modified PAMAM (PAMAM-PO)₃H₂) A remineralizing layer having a certain thickness can be induced on the surface of the demineralized dentin.

First, applicants simulated the organic nucleation and mineralization modulation functions of NCPs using PAMAM, achieving biomimetic remineralization of demineralized dentin and type I collagen in an in vitro environment (figure 1). Like other mineralized materials, PAMAM does not have the ability to stabilize collagen structure and does not achieve the protection of collagen structure and the induction of biomimetic remineralization in MMPs environments. Therefore, how to endow the PAMAM with the collagen protection function on the premise of not influencing the biomimetic remineralization capability of the PAMAM becomes an important scientific problem.

The easily modifiable nature of PAMAM macromolecules offers the possibility of solving the above scientific problems. On one hand, the PAMAM surface functional group can be correspondingly modified according to different requirements. For example, the remineralization rate is regulated by carboxyl, hydroxyl, phosphate group modification; the adsorption capacity of tooth enamel is enhanced through the modification of the alendronate; grafting organic polypeptide fragments on the surface functional groups to obtain the functions of the polypeptides. On the other hand, the PAMAM molecule internal cavity can be used for loading and slowly releasing the drug, so that the multifunctional composition of the PAMAM and the loaded drug is realized. For example, the use of PAMAM to load and release the antibacterial agent triclosan inhibits the growth of cariogenic bacteria while promoting remineralization of demineralized enamel. Therefore, the application designs a novel mineralized anticarious material which is based on PAMAM molecules and can simultaneously stabilize a collagen structure and induce biomimetic remineralization on a molecular level by modifying the external functional groups and the internal cavities of the PAMAM. Thus, the search for collagen protective materials that can be used to modify PAMAM is a breakthrough to solve the aforementioned scientific problems.

During the natural development of the collagen matrix, collagen molecules undergo a cross-linking reaction to form collagen fibrils, which further aggregate to form thicker and longer collagen fibers. The phenomenon of mutual cross-linking of collagen molecules greatly enhances the structural stability of the collagen matrix. Therefore, a conventional method for stabilizing the collagen structure is to promote crosslinking of collagen fibers using a protein crosslinking agent. For example, the mechanical properties and the enzymolysis resistance of demineralized dentin are effectively enhanced by pretreating demineralized dentin collagen with epigallocatechin gallate (EGCG). The procyanidine is used for crosslinking the dentin collagen fibers, so that the structural stability of the dentin collagen is obviously improved. Other more studied protein crosslinking agents or methods include glutaraldehyde, polycarboxylic acids, and ultraviolet irradiation. These protein cross-linkers are all capable of stabilizing the collagen structure to some extent, but unfortunately they are not suitable for modifying PAMAM.

In addition to the well-known mineralization induction and regulation functions, dentin Non-collagenous proteins (NCPs) are also Non-covalent cross-linking agents (Non-covalent cross-linkers) of collagen fibers, and can be enriched around the collagen fibers through the firm combination with collagen molecules to play a role of a bridge, so that the collagen molecules are connected together to promote the cross-linking in the collagen molecules and among the molecules. It has been found that once the NCPs on the collagen fibers are removed by proteases, the structural integrity and stability of the collagen fiber web is compromised. The strong binding force of NCPs to collagen molecules is the basis for their non-covalent crosslinking, and mainly benefits from the amino acid sequences in NCPs that specifically bind collagen. For example, two amino acid sequences DSESSEEDR and SEENRDSDSQDSSR at the C-terminal end of dentin matrix protein-1 (DMP-1) are considered as the main sequences of DMP-1 binding collagen molecules. The structure and conformation of amino acids in the 19 th-46 th sequence of Bone Sialoprotein (BSP) are related to its specific adsorption to collagen. Fibronectin (FN) binds tightly to collagen molecules through the CQDSETRTFY sequence in its structure. The polypeptide fragments which specifically adsorb collagen also show non-covalent cross-linking effect similar to that of complete NCPs after being combined with collagen fibers, and are particularly characterized in that the self-assembly of collagen molecules is accelerated, the generation time of collagen fibrils is shortened, the diameters of the collagen fibers are increased, the folding and the spiral in secondary structures of the collagen are increased, and the like. According to the application, polypeptide sequences which are specifically combined with collagen are modified on the surface of PAMAM with a protein-like structure, and the advantages of the adsorption of the specific collagen of the former and the high-density branching sites of the latter are combined, so that the polypeptide modified PAMAM (Peptide-PAMAM) which can be synchronously and specifically combined with a plurality of collagen molecules and can promote collagen crosslinking is obtained.

Wherein more than half of the functional groups on the surface of the PAMAM are reserved so as to preserve the capability of attracting calcium and phosphorus ions. At most half of the functional groups on the surface of the PAMAM are grafted with polypeptide fragments, which are symmetrically grafted to the functional groups on the surface of the polyamidoamine.

For example, in the examples, 4, 16 or 64 of the above-described polypeptide fragments are grafted onto functional groups on the polyamide surface, each polypeptide fragment being grafted onto a functional group.

In addition, since MMPs are an important cause of collagen structure destruction, according to embodiments of the present disclosure, the use of an MMPs inhibitor reduces the activity of MMPs, reduces degradation of collagen fibers by MMPs, and stabilizes the collagen structure. Inhibitors of MMPs may include Chlorhexidine (CHX), quaternary ammonium salts, tetracyclines, galardin (galardin), and the like. Among them, garradin (galardin) has been studied more recently as a specific MMPs inhibitor capable of selectively inhibiting MMP-2, -3, -8 and-9. galardin has strong MMPs inhibition ability, and can effectively inhibit MMPs at lower concentrations. In the dental field, the galardin is added into the treatment fluid of the adhesive, so that the degradation of collagen fibers at the bonding interface is effectively reduced. However, galardin is poorly soluble in water and not suitable for clinical use. In addition, the effect of galardin in continuously inhibiting MMPs is not yet ideal because of weak binding force between galardin and MMPs. The PAMAM adopted by the application is an excellent carrier of insoluble small molecular drugs, and can improve the solubility of the drugs and prolong the action time of the drugs through loading and slow release of the drugs. Therefore, by loading and slowly releasing galardin by utilizing the hydrophobic cavity in the PAMAM molecule, the activity of MMPs can be continuously inhibited, thereby reducing the degradation of the MMPs to collagen fibers.

That is, in the examples, the Peptide-PAMAM-galardin system was constructed by symmetrically grafting multiple Peptide fragments that specifically bind collagen in non-collagenous proteins on the PAMAM surface and loading galardin using the hydrophobic cavity inside PAMAM. In the MMPs environment, the system can specifically combine a plurality of collagen molecules through peptide, promote collagen crosslinking, release galardin slowly, continuously inhibit the activity of the MMPs, and adsorb calcium and phosphorus to deposit on a collagen scaffold by utilizing the carboxyl groups on the surface of PAMAM to induce the bionic remineralization of the collagen. The present disclosure is not so limited and matrix metalloproteinase inhibitors may include one or more of chlorhexidine, quaternary ammonium salts, galanin, and tetracyclines. Polypeptide (peptide) fragments may include one or more of DSESSEEDR and/or SEENRDSDSQDSSR sequences in DMP-1, NGVFKYRPRYFLYKHAYFYPPLKRFPVQ sequences in bone sialoprotein, and CQDSETRTFY sequences in fibronectin. The functional groups of the PAMAM surface are modified via one or more of amino, carboxyl, hydroxyl, phosphate and alendronate groups.

FIG. 1 shows a schematic view of a mineralized anticaries material according to an exemplary embodiment of the present disclosure. As shown in figure 1, a Peptide-PAMAM-galardin system is firmly combined with collagen molecules through a polypeptide sequence for specifically adsorbing collagen, and is enriched around the collagen; secondly, the function of a bridge is exerted, a plurality of collagen molecules are connected, and collagen crosslinking is promoted; ③ sustained release of galardin, continuous inhibition of MMPs activity; and fourthly, the carboxyl groups on the surface attract ACP to deposit in the collagen scaffold, so that the bionic remineralization of the collagen fibers is realized.

According to another aspect of the present disclosure, there is provided a method of making a mineralized anticaries material as described above, comprising:

s1, synthesizing a polypeptide fragment capable of specifically binding to the collagen molecule.

First, polypeptide sequences (amino acid sequences) that specifically bind to collagen in NCPs are determined and synthesized, and the structural stability and collagen-binding ability of the polypeptide fragments are examined, and optimized polypeptide fragments are screened. Polypeptide fragments may include one or more of DSESSEEDR and/or SEENRDSDSQDSSR sequences in DMP-1, NGVFKYRPRYFLYKHAYFYPPLKRFPVQ sequences in Bone Sialoprotein (BSP) protein, and CQDSETRTFY sequences in Fibronectin (FN). The polypeptide fragments are synthesized in a solid phase, and a polypeptide sequence with stable secondary structure and strong binding capacity with collagen molecules is screened out through an in vitro collagen adsorption experiment.

S2, synthesizing dendritic polyamide-amine (PAMAM-COOH) with terminal carboxyl, and grafting the polypeptide fragment synthesized in the step S1 on part of functional groups on the surface of the polyamide-amine.

Specifically, synthesizing PAMAM-COOH, reserving more than half of carboxyl groups on the surface of the PAMAM, and preserving the capability of the PAMAM to attract calcium and phosphorus ions; then, a plurality of polypeptide fragments which are specifically combined with collagen are symmetrically grafted on the surface of the PAMAM to synthesize the Peptide-PAMAM, so that the Peptide-PAMAM can be synchronously and specifically combined with a plurality of collagen molecules, and the collagen crosslinking is promoted. The grafting number of the polypeptide fragments can be 4, 16 or 64, and the Peptide-PAMAM (polyamide amine with the polypeptide fragments grafted on the surface) is synthesized, the structure of the polypeptide-PAMAM is characterized in detail, and the physical properties and the biocompatibility of the polypeptide-PAMAM are tested.

For example, in the examples, the synthesis of polyamidoamines (PAMAM-NH) bearing 128 amino functional groups on the surface is carried out by Michael addition or classical diffusion₂) Acylating part of amino functional group symmetrical acryloyl chloride on the surface of polyamide-amine by click reaction, and then carrying out carboxyl modification on the rest amino groups on the surface of polyamide-amine by succinic anhydride; cysteine (C) is attached to the end of the polypeptide fragment. The synthesis process is shown in the following synthesis scheme (1):

then, the polypeptide fragment is grafted to the amino functional group of the polyamide-amine surface after acylation by using Thiol-Michael addition reaction (thio-Michael reaction), thereby obtaining the polyamide-amine with the polypeptide fragment grafted on the surface. As shown in the following synthetic scheme (2), SEENRDSDSQDSSRC-PAMAM grafted with 4 polypeptide fragments was synthesized.

The ability of SEENRDSDSQDSSRC-PAMAM to stabilize the collagen structure was preliminarily verified by the hydroxyproline assay, fig. 2 shows NMR of SEENRDSDSQDSSRC-PAMAM (sp) grafted with 4 polypeptide fragments, and fig. 3 shows the circular dichroism spectrum of SEENRDSDSQDSSRC-PAMAM (sp) grafted with 4 polypeptide fragments. FIG. 4 shows a High Performance Liquid Chromatography (HPLC) profile of SEENRDSDSQDSSRC-PAMAM grafted with 4 polypeptide fragments. FIG. 5 shows the hydroxyproline assay results of SEENRDSDSQDSSRC-PAMAM grafted with 4 polypeptide fragments. Wherein the different letters a and b represent that the difference is statistically different (P < 0.05).

As can be seen in FIG. 5, the degradation of collagen by MMPs produced significantly less hydroxyproline than the control group after SP-treated collagen fibers.

Preferably, the ability of the polyamidoamine crosslinked collagen fibers surface-grafted with polypeptide fragments can also be tested. The detection step may specifically include:

a. and observing the binding sites of the polyamidoamine with the polypeptide segments grafted on the surface and the collagen fibers, and detecting the binding force of the polyamidoamine with the polypeptide segments grafted on the surface and collagen molecules. Specifically, the Peptide-PAMAM is respectively subjected to fluorescent staining and zinc phosphate quantum dot marking, then is co-cultured with a collagen I fiber solution, and the binding sites of the Peptide-PAMAM and collagen fibers are observed by using a laser confocal microscope (CLSM) and a Transmission Electron Microscope (TEM); detecting the amount of collagen I adsorbed by Peptide-PAMAM by QCM-D; binding of Peptide-PAMAM to collagen molecules was determined using AFM and SFA.

b. Determination of different pH and Ca²⁺The effect of the polyamidoamine with polypeptide segments grafted on the surface at concentration on the collagen fibril formation time and the binding force between collagen molecules. Specifically, formulations containing different Ca²⁺Adjusting the pH of a collagen fiber I solution with a concentration (0.1mM/1mM/10mM) to 4, 7, 10, and co-culturing Peptide-PAMAM with the collagen solution; detecting the turbidity change of the collagen solution by using a spectrophotometer, measuring the retention period and the growth period of the collagen fibrils, and calculating the generation time of the collagen fibrils; AFM and SFA were used to detect the binding force between collagen molecules.

c. Observing the change of the secondary structure of the collagen before and after crosslinking; and detecting the change of the mechanical property and the enzymolysis resistance of the collagen hydrogel before and after crosslinking. Specifically, single-layer recombinant type I collagen fibers are treated by Peptide-PAMAM solutions with different concentrations, and the change of the diameter of collagen before and after crosslinking is observed by AFM and TEM; detecting changes in secondary structure of collagen before and after crosslinking by circular dichroism spectroscopy (CD); detecting the change of storage modulus and loss modulus of the collagen hydrogel before and after crosslinking by using a rheometer, and evaluating the capability of the Peptide-PAMAM in enhancing the mechanical property of the collagen; and (3) soaking the monolayer recombinant collagen hydrogel before and after crosslinking in MMPs solution, and measuring the concentration of hydroxyproline in the solution by using a hydroxyproline detection method after 5 minutes, 6 hours and 3 days respectively to evaluate the capability of the Peptide-PAMAM in enhancing the enzymolysis resistance of the collagen.

And (3) screening and optimizing the Peptide-PAMAM by comprehensively analyzing the results of the detection steps.

S3, loading the matrix metalloproteinase inhibitor into the hydrophobic cavity in the polyamide-amine.

Mixing the Peptide-PAMAM with a matrix metalloproteinase inhibitor (e.g. galardin) by any one of an organic solvent method, a water-soluble method and a co-soluble method, and preparing polyamide-amine (Peptide-PAMAM-galardin) with polypeptide fragments grafted on the surface and matrix metalloproteinase inhibitor loaded in an internal hydrophobic cavity by means of ultrasound and vibration.

The method may further comprise: and detecting the polyamide-amine of which the surface is grafted with the polypeptide fragment and the inner hydrophobic cavity is loaded with the matrix metalloproteinase inhibitor, and screening and optimizing the polyamide-amine of which the surface is grafted with the polypeptide fragment and the inner hydrophobic cavity is loaded with the matrix metalloproteinase inhibitor by analyzing the result of the detection step. Wherein, the detecting step may specifically include: detecting the physical and biological properties of the system; qualitatively detecting the loading condition of the matrix metalloproteinase inhibitor, and quantitatively detecting the loading amount of the matrix metalloproteinase inhibitor; detecting the slow release efficiency of the galardin under different buffer systems and different pH conditions; the ability of polyamidoamines, surface grafted with polypeptide fragments and loaded with matrix metalloproteinase inhibitor in the interior hydrophobic cavity, to inhibit matrix metalloproteinase activity was evaluated.

For example, the system is characterized using time-of-flight mass spectrometry, HPLC, NMR, FTIR, etc.; the toxicity of the system to cells such as oral mucosa, periodontal and dental pulp is detected through a cytotoxicity experiment; qualitatively detecting the loading condition of the galardin by using NMR and FTIR; the amount of galardin loading was quantified using HPLC.

Preparing artificial saliva containing different calcium and phosphorus ion concentrations, adjusting the pH value to be 4, 7 and 10, placing a Peptide-PAMAM-galardin system in a dialysis bag, quantitatively detecting the galardin concentration at a plurality of continuous time points by using HPLC, drawing a drug release curve, and evaluating the sustained release effect of the drug.

Preparing an MMPs activating solution containing calcium and zinc ions, adding a Peptide-PAMAM-galardin system into the solution, and detecting the activity of the MMPs at a plurality of continuous time points by using an MMPs activity determination kit; comprehensively analyzing the experimental results, and screening and optimizing the Peptide-PAMAM-galardin system.

3) Preparing artificial saliva containing different calcium and phosphorus ion concentrations, adjusting the pH value to be 4, 7 and 10, placing a Peptide-PAMAM-galardin system in a dialysis bag, quantitatively detecting the galardin concentration at a plurality of continuous time points by using HPLC, drawing a drug release curve, and evaluating the sustained release effect of the drug.

In addition, research on inducing type I collagen biomimetic remineralization by a Peptide-PAMAM-galardin system in an environment containing MMPs can be carried out, and the research specifically comprises the following steps:

(1) preparing artificial saliva containing matrix metalloproteinase, and stabilizing calcium and phosphorus ions in the artificial saliva by polyacrylic acid; preparing a single-layer recombinant I-type collagen model on a Transmission Electron Microscope (TEM) nickel screen, treating the TEM nickel screen by polyamide-amine with a polypeptide fragment grafted on the surface and a matrix metalloproteinase inhibitor loaded in an internal hydrophobic cavity, soaking the TEM nickel screen into prepared artificial saliva, and standing for 1, 3, 7, 14 and 28 days; determining the concentration of hydroxyproline in the artificial saliva by hydroxyproline detection; the diameter and structure of the collagen fibers were observed by Atomic Force Microscopy (AFM) and TEM, and the secondary structure of the collagen fibers was detected by circular dichroism spectroscopy (CD).

(2) In the same step (1), after soaking artificial saliva containing MMPs in a TEM nickel screen for 1, 3, 7, 14 and 28 days, observing the number, size, structure, morphology and arrangement mode of the regenerated mineral crystals by using a scanning electron microscope and a TEM; phase analysis of the regenerated crystals using Energy Dispersive Spectroscopy (EDS) and Selected Area Electron Diffraction (SAED); detecting the storage modulus and the loss modulus of the collagen fibers by using a rheometer; the results of the above experiments were analyzed in combination to evaluate the ability of polyamidoamines (e.g., Peptide-PAMAM-galardin) with polypeptide fragments grafted on the surface and matrix metalloproteinase inhibitors loaded in the interior hydrophobic cavity to protect collagen structures and induce the biomimetic remineralization of type I collagen in an environment containing MMPs.

In light of the present disclosure, it has been discovered through research that the ability of NCPs to stabilize collagen structures is primarily due to their non-covalent crosslinking of collagen molecules, which is closely related to the polypeptide sequences that specifically bind collagen. Therefore, the applicant grafts the polypeptide fragments specifically binding to the collagen on the surface of the PAMAM with a protein-like structure symmetrically, and by combining the advantages of the adsorption of the specific collagen of the former and the high-density branching site of the latter, the Peptide-PAMAM which can synchronously and specifically bind a plurality of collagen molecules and promote the crosslinking of collagen fibers is obtained, and secondly, the PAMAM is an excellent drug carrier, and the drug-carrying object of the PAMAM is mainly a small slightly soluble molecule; galardin, which has a well-defined inhibitory effect on MMPs, is precisely this class of drugs. Thus, loading and sustained release of galardin using PAMAM can continue to inhibit the activity of MMPs.

The invention innovatively provides a method for modifying a polypeptide sequence specifically combined with collagen in NCPs to the surface of PAMAM with a protein-like structure, combines the advantages of the adsorption of the specific collagen and the high-density branching site of the PAMAM, endows the PAMAM with a function of crosslinking collagen molecules on the premise of keeping the bionic remineralization capability of the PAMAM, and is an innovation of thought in the field of bionic remineralization research.

The design and construction of the Peptide-PAMAM-galardin system with the integrated functions of adsorbing and crosslinking collagen fibers, inhibiting the activity of MMPs and inducing the deposition of calcium and phosphorus ions in a collagen scaffold fundamentally solve the key problem that the structure of the collagen scaffold is easy to be damaged by enzymolysis in the clinical application of the existing bionic remineralization material, and is a technical and material innovation in the field of minimally invasive treatment of caries.

The method is an innovation of an experimental method for mineralization induction, wherein MMPs are added into traditional in-vitro artificial saliva to simulate the MMPs environment of a human oral cavity, and an attempt is made to induce the biomimetic remineralization of collagen in the environment.

Although certain exemplary embodiments and implementations have been described herein, other embodiments and modifications will be apparent from the description. The inventive concept is therefore not limited to the exemplary embodiments, but is to be defined by the appended claims along with their full scope of equivalents.

Claims

1. A mineralized anticaries material, which comprises polyamidoamine, wherein a part of functional groups on the surface of the polyamidoamine is grafted with a polypeptide fragment which is specifically bound with collagen molecules in non-collagen, and the polypeptide fragment comprises one or more of DSESSEEDR and/or SEENRDSDSQDSSR sequences in dentin matrix protein-1, NGVFKYRPRYFLYKHAYFYPPLKRFPVQ sequences in bone sialoprotein, and CQDSETRTFY sequences in fibrous fibronectin.

2. The mineralized anticaries material according to claim 1, wherein at most half of the functional groups on the polyamidoamine surface are grafted with the polypeptide segments, the polypeptide segments are symmetrically grafted to the functional groups on the polyamidoamine surface, 4, 16 or 64 polypeptide segments are grafted to the functional groups on the polyamidoamine surface, and each of the polypeptide segments is grafted to one functional group.

3. The mineralized anticaries material according to claim 1, wherein the functional groups on the polyamidoamine surface are one or more of amino, carboxyl, hydroxyl, and phosphate groups.

4. The mineralized anticaries material according to claim 3, wherein the phosphate group comprises an alendronate group.

5. The mineralized anticaries material according to claim 1, wherein the hydrophobic cavities inside the polyamidoamine are loaded with matrix metalloproteinase inhibitors comprising one or more of chlorhexidine, quaternary ammonium salts, galadine, and tetracyclines.

6. A method of preparing the mineralized anticaries material according to any one of claims 1 to 5, comprising:

synthesizing a polypeptide fragment that specifically binds to a collagen molecule in a non-collagen protein, the polypeptide fragment comprising one or more of an DSESSEEDR and/or SEENRDSDSQDSSR sequence in dentin matrix protein-1, a NGVFKYRPRYFLYKHAYFYPPLKRFPVQ sequence in bone sialoprotein, and a CQDSETRTFY sequence in fibronectin;

synthesizing polyamide-amine and grafting the polypeptide segment on a part of functional groups on the surface of the polyamide-amine; and

loading a matrix metalloproteinase inhibitor into a hydrophobic cavity inside the polyamidoamine.

7. The method for preparing the mineralized anticaries material according to claim 6, wherein the polyamide-amine with 128 amino functional groups on the surface is synthesized by Michael addition, part of the amino functional groups on the surface of the polyamide-amine is acylated with acryloyl chloride symmetrically by click reaction, and then the rest of the amino groups on the surface of the polyamide-amine are subjected to carboxyl modification by succinic anhydride;

connecting cysteine to the tail end of the polypeptide fragment, and grafting the polypeptide fragment to an amino functional group on the surface of polyamide-amine after acylation treatment by utilizing a mercapto-Michael addition reaction to obtain the polyamide-amine with the polypeptide fragment grafted on the surface;

mixing the polyamide-amine with the polypeptide fragments grafted on the surface with a matrix metalloproteinase inhibitor by adopting any one of an organic solvent method, a water-soluble method and a co-soluble method, and preparing the polyamide-amine with the polypeptide fragments grafted on the surface and the matrix metalloproteinase inhibitor loaded in a hydrophobic cavity inside through ultrasonic and oscillation modes.

8. The method for preparing a mineralized anticaries material according to claim 7, further comprising: detecting the ability of the polyamidoamine cross-linked collagen fibers with the polypeptide segments grafted on the surface, and screening and optimizing the polyamidoamine with the polypeptide segments grafted on the surface by analyzing the result of the detection step, wherein the detection step specifically comprises the following steps:

observing the binding sites of the polyamidoamine with the polypeptide segments grafted on the surface and the collagen fibers, and detecting the binding force of the polyamidoamine with the polypeptide segments grafted on the surface and collagen molecules;

determination of different pH and Ca²⁺The influence of the polyamidoamine with the polypeptide segment grafted on the surface under the concentration on the collagen fibril generation time and the binding force among collagen molecules;

observing the change of the secondary structure of the collagen before and after crosslinking;

and detecting the change of the mechanical property and the enzymolysis resistance of the collagen hydrogel before and after crosslinking.

9. The method for preparing a mineralized anticaries material according to claim 7, further comprising: detecting the polyamide-amine with the surface grafted with the polypeptide fragment and the internal hydrophobic cavity loaded with the matrix metalloproteinase inhibitor, and screening and optimizing the polyamide-amine with the surface grafted with the polypeptide fragment and the internal hydrophobic cavity loaded with the matrix metalloproteinase inhibitor by analyzing the result of the detection step, wherein the detection step specifically comprises the following steps:

detecting the physical and biological properties of the matrix metalloproteinase inhibitor loaded in the hydrophobic cavity of which the surface is grafted with the polypeptide segment;

qualitatively detecting the loading condition of the matrix metalloproteinase inhibitor, and quantitatively detecting the loading amount of the matrix metalloproteinase inhibitor;

detecting the slow release efficiency of the matrix metalloproteinase inhibitor under different buffer systems and different pH conditions;

the ability of the polyamidoamines, surface grafted with polypeptide fragments and loaded with matrix metalloproteinase inhibitor in the interior hydrophobic cavity, to inhibit matrix metalloproteinase activity was evaluated.