CN116496492B - Polyamide-amine dendritic polymer double-modified by phytic acid and polypeptide, and preparation method and application thereof - Google Patents
Polyamide-amine dendritic polymer double-modified by phytic acid and polypeptide, and preparation method and application thereof Download PDFInfo
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- CN116496492B CN116496492B CN202310352967.5A CN202310352967A CN116496492B CN 116496492 B CN116496492 B CN 116496492B CN 202310352967 A CN202310352967 A CN 202310352967A CN 116496492 B CN116496492 B CN 116496492B
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- 239000000412 dendrimer Substances 0.000 title claims abstract description 72
- 229920000736 dendritic polymer Polymers 0.000 title claims abstract description 72
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- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 title claims abstract description 64
- 239000000467 phytic acid Substances 0.000 title claims abstract description 64
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- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Polyamides (AREA)
- Peptides Or Proteins (AREA)
- Dental Preparations (AREA)
Abstract
The invention discloses a polyamide-amine dendritic polymer double-modified by phytic acid and polypeptide, and a preparation method and application thereof. The method utilizes the easily modified functional group of the polyamide-amine dendritic Polymer (PAMAM) to carry out double modification on the PAMAM, grafts phytic acid on the surface of the PAMAM, utilizes the strong chelation of the phytic acid to enhance the chelating ability of the PAMAM to dentin calcium ions, grafts polypeptide, endows the PAMAM with antibacterial effect and reduces the occurrence of clinical relay caries. The molecular weight of the obtained polymer can reach 152kDa, the requirement of the molecular weight of more than 40kDa is met, the polymer has higher solubility, can effectively and rapidly induce selective external demineralization of collagen fibers, and can synchronously realize antibiosis. The dentin bonding pretreatment agent has high solubility, can effectively and rapidly induce selective external demineralization of collagen fibers and has high efficiency and antibacterial effect, and the pretreatment agent is applied to a dentin acid etching-washing bonding system, so that the purposes of external demineralization and synchronous antibacterial effect of the fibers are achieved, and the service life of the resin restoration can be prolonged.
Description
Technical Field
The invention belongs to the technical field of tooth restoration, and particularly relates to a polyamide-amine dendritic polymer, and a preparation method and application thereof.
Background
The long-term durability of the resin-dentin adhesive interface tends to be inadequate, mainly due to mixed layer degradation and the occurrence of secondary caries. The existing dentin bonding system is based on an acid etching technology to simultaneously lose the hydroxyapatite inside and outside the collagen fiber, so that excessive demineralization is caused, and resin is difficult to infiltrate into the fiber, so that enzymolysis of naked collagen and resin hydrolysis are caused. In addition, along with popularization of the minimally invasive dental concept, the traditional mechanical means are required to treat caries and store the rest of dental tissues to the maximum extent, so that bacterial residues are inevitably remained on the surfaces of the dentin of the cavities and in the dentin tubules, and secondary caries is caused between the teeth and the resin restoration by the embedded bacteria and metabolites thereof. Therefore, improving the long-term durability of the resin-dentin adhesive interface is an important problem to be solved in oral clinic.
In recent years, selective extrafibrous demineralization techniques have been focused on the treatment of dentin with substances having a molecular weight of > 40kDa, which is based on the fact that type I collagen fibers have molecular size exclusion properties (i.e., molecules having a relative molecular mass of less than 6kDa can freely penetrate into the interior of the collagen fibers, molecules between 6 and 40kDa can be partially entered, while molecules having a molecular mass of greater than 40kDa are completely excluded from entering the interior of the fibers). The technology well reserves mineral crystals in the fibers, avoids the formation of a weak area of an adhesive interface, effectively avoids the damage of the structural integrity of the interface caused by the acid etching technology, and plays a key role in prolonging the service life of the resin restoration. However, the existing material realizes selective external demineralization of fibers to a certain extent, but has weak effect of chelating calcium ions and long action time, has poor effect on dentin demineralization, has insufficient resin bonding strength, and has the defects of poor solubility, insolubility and the like, thereby greatly limiting the potential clinical application of the material.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a polyamide-amine dendritic polymer (IP 6-PAMAM-GL 13K) with double modification of phytic acid and polypeptide, and a preparation method and application thereof, so as to obtain a dentin bonding pretreatment agent which can effectively and rapidly induce selective external demineralization of collagen fibers, has high solubility and high antibacterial effect, and can be applied to a dentin acid etching-washing bonding system, thereby realizing external demineralization and synchronous antibacterial effect, improving bonding strength and prolonging the service life of a resin restoration.
Polyamide-amine (PAMAM) is a dendritic macromolecule with molecular weight controllability and with easily modified surface functional groups. The inventor of the present invention has found through earlier studies on a polyamide-amine type dendritic polymer (G5-PAMAM-NH 2) with a fifth-generation end group as an amino group that PAMAM can effectively chelate calcium ions in a solution, but the chelating ability on calcium ions on the dentin surface is still weak, so that dentin demineralization and adhesion effects are still not ideal. The invention utilizes the functional group which is easy to modify PAMAM to carry out double modification on PAMAM, grafts phytic acid (IP 6) on the surface of the PAMAM, and utilizes the strong chelation of the phytic acid to enhance the chelation capability of PAMAM on dentin calcium ions, thereby improving the bonding strength; meanwhile, polypeptide (GL 13K) is grafted, so that the PAMAM has an antibacterial effect, and the occurrence of clinical secondary caries is reduced. The molecular weight of the obtained polymer (IP 6-PAMAM-GL 13K) can reach 153kDa, the requirement of the molecular weight of more than 40kDa is met, the selective external demineralization of collagen fibers can be induced effectively and rapidly, the bonding strength is improved, and the polymer has higher antibacterial property and solubility.
The invention provides a phytic acid and polypeptide double-modified polyamide-amine dendritic polymer, which has the structural formula
The following are provided:
wherein n is the number of grafted polypeptides, n=53-63, m is the number of grafted phytic acid, and m=65-75.
Preferably, n=53, m=75.
The preparation method of the polyamide-amine dendritic polymer with double modification of phytic acid and polypeptide provided by the invention comprises the following steps:
(1) Synthesis of a Phytic acid-modified Polyamide-amine dendrimer with protecting groups (IP 6-PAMAM)
Dissolving a polyamide-amine dendritic polymer (G5-PAMAM-NH 2) with a fifth generation end group of a protecting group as an amino group in a solvent to prepare a first solution; dissolving phytic acid (IP 6) with a structural formula shown in a formula (I) in a mixed solution of N, N Dimethylformamide (DMF), triethylamine and methylmorpholine, slowly adding condensing agents Dicyclohexylcarbodiimide (DCC) and 1-Hydroxybenzotriazole (HOBT), and uniformly mixing to obtain a second solution; the mass ratio of phytic acid in the second solution to the amino-terminated polyamide-amine dendritic polymer in the first solution is 3:144;
Slowly raising the temperature of the first solution to 26-28 ℃, slowly dripping the second solution into the first solution under stirring, continuing to react for 1-1.5 h after all the second solution is dripped, gradually sampling and detecting in the reaction process, controlling the number of the reaction groups of PAMAM, lowering the temperature to 0 ℃ after the reaction is finished, slowly stopping stirring, standing, concentrating the reaction solution to remove the solvent, filtering, dissolving the obtained solid by ethyl acetate, adding petroleum ether for crystallization, and drying to obtain dry powder; dissolving the obtained dry powder in dimethyl sulfoxide (DMSO) to obtain a phytic acid modified polyamide-amine dendritic polymer solution with a protecting group;
(2) Synthesis of polyamide-amine dendritic polymer with double modification of phytic acid and polypeptide with protecting group
Dissolving a polypeptide (GL 13K, only exposing an N-terminal amino group) with a structural formula shown in a formula (II) with a protecting group in N, N-Dimethylformamide (DMF), adding a mixed solution of Dicyclohexylcarbodiimide (DCC) and N-Hydroxysuccinimide (HOSU), stirring or vibrating to dissolve the polypeptide, and filtering to obtain a third solution; wherein the polypeptide: the mass ratio of the polyamide-amine dendritic polymer with the end group being amino is 7:144;
Slowly adding the third solution into the phytic acid modified polyamide-amine dendritic polymer solution with the protecting group obtained in the step (2), and reacting for 18-26 h at 0-10 ℃; after the reaction is finished, removing the reaction solution, drying to obtain dry powder, dissolving the obtained dry powder in trifluoroacetic acid aqueous solution, and reacting for 3.5-4.5 hours at room temperature under a sealing condition to obtain a polyamide-amine type dendritic polymer solution with double modification of phytic acid and polypeptide and a protecting group;
(3) Deprotection groups
And (3) adding diethyl ether into the solution obtained in the step (2) to obtain a crystal, filtering and drying to obtain the polyamide-amine dendritic polymer double-modified by phytic acid and polypeptide.
Further, the polyamide-amine type dendritic polymer having an amino group as a terminal group is prepared by the following method:
① Synthesis of 0.5 generation PAMAM-NH 2
Uniformly mixing ethylenediamine and ethanol in an ice water bath in a nitrogen atmosphere, dropwise adding methyl acrylate at room temperature (25 ℃), and continuing to react for 24 hours after the dropwise addition, wherein ethylenediamine: the molar ratio of methyl acrylate is 1:8; after the reaction, the solvent and excessive reactants are removed by reduced pressure distillation, and a pale yellow transparent liquid G0.5-PAMAM-NH 2 is obtained.
② Synthesis of generation 1 PAMAM
Uniformly mixing G0.5-PAMAM and methanol in an ice water bath in a nitrogen atmosphere, dropwise adding ethylenediamine at 25 ℃, and continuing to react for 24 hours after the dropwise adding, wherein G0.5-PAMAM: the molar ratio of ethylenediamine is 1:24, removing the solvent and the excessive ethylenediamine after the reaction is finished to obtain a light yellow viscous liquid which is G1-PAMAM-NH 2.
Then, using the synthesized G1.0-PAMAM-NH 2 as a raw material, increasing the dosage of methyl acrylate, and synthesizing G1.5-PAMAM-NH 2 according to the method of the step ①, wherein G1.0-PAMAM-NH 2: the molar ratio of methyl acrylate is 1:16; and then using the synthesized G1.5-PAMAM-NH 2 as a raw material, increasing the consumption of ethylenediamine, and synthesizing G2.0-PAMAM-NH 2 according to the method of the step ②, wherein G1.5-PAMAM-NH 2: the molar ratio of ethylenediamine is 1:48.
According to the method, the feeding of methyl acrylate and ethylenediamine is increased by multiple steps, and Michael addition reaction and amidation reaction are alternately repeated until G5-PAMAM-NH 2 is obtained.
The reaction principle is that ethylenediamine is taken as an initial core, and the catalyst is prepared by repeatedly carrying out Michael addition reaction on methyl acrylate and then carrying out amidation reaction on the methyl acrylate and ethylenediamine.
Further, the polyamide-amine type dendritic polymer having the protecting group as the end group of the fifth generation in the step (1) is prepared by the following method:
taking a fifth generation polyamide-amine dendritic polymer with an end group as an amino group and chloroformic acid-9-fluorenylmethyl methyl ester (Fmoc-C1) as raw materials, and reacting for 2-4 hours at room temperature under the action of dichloromethane, sodium bicarbonate and water to obtain Fmoc (9-fluorenylmethoxycarbonyl) -protected G5-PAMAM.
Further, in the step (1), samples are taken at 15 minutes intervals, and the number of remaining Fmoc (9-fluorenylmethoxycarbonyl) groups is measured by Fmoc (Fmoc) group assay to determine the number of unreacted amino groups on the PAMAM, thereby determining the number of amino groups reacted on the PAMAM (i.e., the number of grafting of phytic acid) to control the number of grafted phytic acid to 65 to 75.
Further, in the step (1), the polyamide-amine dendritic polymer with the end group of amino is dissolved in a mixed solvent of anhydrous dimethyl sulfoxide and N, N-dimethylformamide at the temperature of-12 ℃.
Further, in the preparation process of the second solution in the step (1), the solute: solvent: the mass ratio of the condensing agent is 3:6:2, and the condensing agent is dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, wherein the mass ratio of dicyclohexylcarbodiimide to 1-hydroxybenzotriazole is 7:5.
Further, the crystallization operation in the step (1) is to add petroleum ether into the concentrated reaction liquid for crystallization, and filter to obtain crystals; after dissolving the crystal by ethyl acetate, adding petroleum ether again for crystallization, and dissolving the obtained crystal in dimethyl sulfoxide (DMSO) solution to obtain the phytic acid modified polyamide-amine dendritic polymer solution (IP 6-PAMAM) with the protecting group.
Further, in the step (2), a mixed solution of Dicyclohexylcarbodiimide (DCC) and N-Hydroxysuccinimide (HOSU) is added, wherein Dicyclohexylcarbodiimide (DCC): N-Hydroxysuccinimide (HOSU): the mass ratio of N, N dimethylformamide is 16:14:8.
Further, the amount of each solvent used in the above preparation method is limited by the complete dissolution of the corresponding solute.
The invention also provides application of the polyamide-amine dendritic polymer double-modified by the phytic acid and the polypeptide in the field of dentin bonding, in particular application in a dentin acid etching-rinsing bonding system. Preferably, the application is as a dentin bonding pretreatment.
The polyamide-amine dendritic polymer with the dual modification of the phytic acid and the polypeptide has the advantages that the polyamide-amine dendritic polymer has a surface functional group which is easy to modify, the graft phytic acid improves the defect that PAMAM chelated dentin calcium ions are weak, the polypeptide has stronger antibacterial property, stable physicochemical property, better biocompatibility, difficult enzymolysis and extremely low cytotoxicity. The polypeptide modifies 53 amino groups of the polyamide-amine dendritic polymer, so that the polyamide-amine dendritic polymer is endowed with better antibacterial property.
The invention researches the bonding performance and bonding strength of the dentin bonding interface, the MMPs activity of the bonding interface mixed layer, the antibacterial performance and biosafety of the bonding interface. The results of the micro-stretching bonding experiments show that the phytic acid and polypeptide modified polyamide-amine dendritic polymer has good solubility and strong-efficiency and rapid induction of selective external demineralization of collagen fibers, and can obtain good bonding strength in both dry bonding and wet bonding modes. The results of the scanning electron microscope showed that less demineralized matrix appeared on the dentin surface treated with IP6-PAMAM-GL13K, and that peri-and inter-tubular minerals were present. The collagen fiber net is not collapsed under the two modes of HMDS drying and air drying, the existence of a pore structure is observed, the formed bonding interface is better in sealing performance, the demineralized collagen can resist the shrinkage and deformation of the fiber itself under the drying condition, and the three-dimensional bracket structure of the collagen fiber net is maintained, so that a space for the adhesive monomer to enter is still reserved between the demineralized collagen fibers under the air drying state. Therefore, the IP6-PAMAM-GL13K prepared by the invention is particularly suitable for dentin dry adhesion technology with low technical sensitivity and more friendly clinic, and can well improve the stability of dentin adhesion interface.
The atomic force microscope results are consistent with the micro-stretching bonding experiments and scanning electron microscope experiments. The experimental results jointly verify that after the dentin pretreated by the IP6-PAMAM-GL13K is subjected to dry bonding, sufficient adhesive permeation occurs in the mixed layer, so that the demineralized collagen fibers and the resin adhesive monomer are matched, the relatively durable bonding is realized, the influence factors of external water molecules on the bonding interface are eliminated, and the integrity of the interface is maintained. The problem of defects of the bonding interface structure constructed by the traditional acid etching technology is solved greatly. The in-situ zymogram experiment result shows that the IP6-PAMAM-GL13K serving as a pretreatment agent has a good inhibition effect on dentin endogenous MMP. The bacterial dead-dyeing experiment result shows that the IP6-PAMAM-GL13K serving as the pretreatment agent has good inhibition effect on bacterial biomembrane. The cytotoxicity experiment result shows that after the IP6-PAMAM-GL13K acts on the human dental pulp cells and the fibroblasts, the cell survival rate is higher than 80%, and the biosafety is good.
Compared with the prior art, the invention has the following beneficial effects:
1. The relative molecular weight of the IP6-PAMAM-GL13K prepared by the invention is more than 40kDa, and the agent is used as a dentin bonding pretreatment agent, so that the dentin bonding technology of selective external demineralization of collagen fibers is realized, the defect of bonding interface structure caused by simultaneous loss of internal and external apatite of the collagen fibers due to the acid etching technology in the traditional dentin bonding system is overcome, the key effect is exerted on prolonging the clinical service life of a resin prosthesis, and the method is a new idea in the field of dentin bonding.
2. The polyamide-amine dendritic polymer with double modification of phytic acid and polypeptide is a novel strong-effect rapid induction selective external demineralization of collagen fibers, has an antibacterial function and is a functional integrated dentin bonding pretreatment agent, and the problem of insufficient resin bonding durability caused by secondary caries can be effectively avoided.
3. The method provided by the invention has the advantages that the preparation process is simple, the preparation can be realized by adopting conventional raw materials and equipment, and the industrial production is easy to realize.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of a polyamide-amine dendrimer double modified with phytic acid and polypeptides according to the present invention;
FIG. 2 is a nuclear magnetic resonance spectrum of a protective group-containing phytic acid-modified polyamide-amine type dendrimer prepared in example 1;
FIG. 3 is an infrared spectrum of a phytic acid modified polyamide-amine dendrimer with protecting groups prepared in example 1;
FIG. 4 is a nuclear magnetic resonance spectrum of a polyamide-amine dendrimer double modified with a protecting group and a polypeptide prepared in example 1;
FIG. 5 is an infrared spectrum of a polyamide-amine dendrimer double modified with a protecting group and a polypeptide prepared in example 1;
FIG. 6 is a schematic diagram of the structure of a polyamide-amine dendrimer double modified with phytic acid and polypeptides prepared in example 1;
FIG. 7 is a high performance liquid chromatogram of a polyamide-amine dendrimer double modified with a protecting group-containing phytic acid and a polypeptide prepared in example 2;
FIG. 8A is a graph showing the micro-stretching adhesion of the treatment dentin with IP6-PAMAM-GL13K at different concentrations and for different times prepared in example 2, and the adhesion strength and adhesion performance after wet adhesion were studied. Concluding that: IP6-PAMAM-GL13K of 60s at 50mg/mL compared with phosphoric acid gives a similar adhesive strength;
FIG. 8B is a micro-stretch bonding chart of example 2 further testing 50mg/mL 60s of IP6-PAMAM-GL13K for bond strength and bond performance in a dry-wet bond mode;
FIG. 9 is a scanning electron microscope image obtained by treating dentin collagen matrix morphology with IP6-PAMAM-GL13K prepared in example 3;
FIG. 10 is an atomic force microscope image obtained by treating dentin collagen matrix morphology with IP6-PAMAM-GL13K prepared in example 4;
FIG. 11 is a bar graph of atomic force microscope roughness obtained by treating dentin collagen matrix morphology with IP6-PAMAM-GL13K prepared in example 4;
FIG. 12 is an in situ zymogram image obtained from the resin-dentin interface pretreated with IP6-PAMAM-GL13K prepared in example 5;
FIG. 13 is positional in situ zymogram data obtained from the resin-dentin interface pretreated with IP6-PAMAM-GL13K prepared in example 5;
FIG. 14 is a confocal microscope image of the treatment of dentin surfaces with IP6-PAMAM-GL13K prepared in example 6 to form bacterial biofilm;
FIG. 15 is a bar graph showing the proportion of dead bacteria in the formation of bacterial biofilm on the surface of dentin sheet by IP6-PAMAM-GL13K prepared in example 6;
FIG. 16 is a CCK-8 diagram of the IP6-PAMAM-GL13K prepared in example 7 versus L929 fibroblasts and human dental pulp cells.
Detailed Description
The polyamide-amine dendritic polymer with the end groups of phytic acid and polypeptide double modification and the preparation method and application thereof are further described below by the examples.
In example 1 below, the phytic acid and the polypeptides used in steps (2) and (3) were purchased from Shanghai Biotechnology Co., ltd, and their structural formulas are shown as formula (I) (phytic acid) and formula (II) (polypeptide),
Example 1
In this embodiment, the synthetic route of the 5-generation polyamide-amine dendritic polymer with the end group being double modified by phytic acid and polypeptide is shown in fig. 1, and the specific preparation method is as follows:
(1) Synthesis of phytic acid modified polyamide-amine dendritic polymer with protecting group
150Mg of a polyamide-amine dendritic polymer (G5-PAMAM-NH 2) with a protecting group end group of the fifth generation is taken and dissolved in 1500 mL of N, N-Dimethylformamide (DMF) and 600mL of dimethyl sulfoxide (DMSO) at a temperature of minus 12 ℃ to prepare a first solution with a concentration of 0.5-1.5G/mL.
330Mg of phytic acid (IP 6) was dissolved in a mixed solution of 330mg of N, N Dimethylformamide (DMF), 82.5mg of triethylamine and 247.5mg of methylmorpholine, and 70mg of Dicyclohexylcarbodiimide (DCC) and 50mg of 1-Hydroxybenzotriazole (HOBT) were slowly added thereto and mixed uniformly to obtain a second solution. . Slowly raising the temperature of the first solution to 27 ℃, slowly dropwise adding the second solution into the first solution under stirring, sampling every 15 minutes after the reaction is completed after the dropwise adding, detecting the residual Fmoc (9-fluorenylmethoxycarbonyl) group number by adopting an Fmoc (Fmoc) group assay method to determine the unreacted amino number on PAMAM, thereby determining the amino number (namely the grafting number of phytic acid) reacted on the PAMAM, and controlling the grafting phytic acid number to be 65-75; after the reaction is finished, the temperature is reduced to 0 ℃, stirring is slowly stopped, and the mixture is stood for 3 hours; concentrating the reaction solution to remove the solvent, adding petroleum ether for crystallization, and filtering to obtain crystals; dissolving the crystal by ethyl acetate, adding petroleum ether again for crystallization, leaching, and drying to obtain 270mg; 270mg of dry powder is added into dimethyl sulfoxide (DMSO) solution, and the solution is slowly dissolved to obtain phytic acid modified polyamide-amine dendritic polymer solution (IP 6-PAMAM) with protective groups.
(2) Synthesis of polyamide-amine dendritic polymer with double modification of phytic acid and polypeptide with protecting group
700Mg of polypeptide (GL 13K, only N-terminal amino group is exposed) with a protecting group is dissolved in N, N-Dimethylformamide (DMF), meanwhile, a mixed solution of 2 times of N, N-Dimethylformamide (DCC) and (HOSU) is added for slow dissolution, and a constant THZ-103B constant temperature culture shaking table is used for shaking for 17 hours in the Shanghai, and then a third solution is obtained by filtration.
Slowly adding the third solution into the phytic acid modified polyamide-amine dendritic polymer solution with the protecting group prepared in the step (2), keeping the temperature within 0-10 ℃ and reacting for 24 hours; after the reaction is finished, 550mg of dry powder is obtained after low-pressure distillation and drying; dissolving the dry powder in trifluoroacetic acid solution, and reacting for 4 hours at room temperature under a sealing condition to obtain the polyamide-amine dendritic polymer solution with double modification of phytic acid and polypeptide.
(4) Deprotection groups
And (3) adding diethyl ether into the solution in the step (3) to obtain crystals (removing the protective groups of the residual polypeptide), filtering and drying the crystalline substances to remove liquid, and thus obtaining the polyamide-amine dendritic polymer with double modification of phytic acid and polypeptide.
The synthesis process of the selected 5 th generation amino group-terminated polyamide-amine dendritic polymer is as follows:
Firstly, 0.15mol of ethylenediamine and 1.0mol of ethanol are weighed and stirred to be uniformly mixed. 0.9mol of methyl acrylate was added dropwise over 2 hours at 25℃and the reaction was continued for 24 hours. Excess reactant was distilled off under reduced pressure to give a clear liquid of yolk color G0.5-PAMAM-NH 2. Then 0.05mol G0.5-PAMAM and 2.0mol methanol are weighed and stirred to be evenly mixed. 1.2mol of ethylenediamine is added dropwise at 25 ℃ for 24 hours. The obtained yolk-colored viscous liquid is-G1.0 PAMAM-NH 2.
Then, using the synthesized G1.0-PAMAM-NH 2 as a raw material, increasing the dosage of methyl acrylate, and synthesizing G1.5-PAMAM-NH 2 according to the method of the step ①, wherein G1.0-PAMAM-NH 2: the molar ratio of methyl acrylate is 1:16; and then using the synthesized G1.5-PAMAM-NH 2 as a raw material, increasing the consumption of ethylenediamine, and synthesizing G2.0-PAMAM-NH 2 according to the method of the step ②, wherein G1.5-PAMAM-NH 2: the molar ratio of ethylenediamine is 1:48.
According to the method, the feeding of methyl acrylate and ethylenediamine is increased by multiple steps, and Michael addition reaction and amidation reaction are alternately repeated until G5-PAMAM-NH 2 is obtained.
The polyamide-amine dendritic polymer with the protecting group and the fifth generation end group being amino is prepared by the following method:
taking a fifth generation polyamide-amine dendritic polymer with an end group as an amino group and chloroformic acid-9-fluorenylmethyl methyl ester (Fmoc-C1) as raw materials, and reacting for 2-4 hours at room temperature under the action of dichloromethane, sodium bicarbonate and water to obtain Fmoc (9-fluorenylmethoxycarbonyl) -protected G5-PAMAM.
The nuclear magnetic hydrogen spectrum of the polyamide-amine dendritic polymer (IP 6-PAMAM-GL 13K) modified by the end group prepared in the embodiment is shown in FIG. 4, and FIG. 5 is an infrared spectrum of the polyamide-amine dendritic polymer (IP 6-PAMAM-GL 13K) modified by the end group with the protecting group prepared in the embodiment 1; FIGS. 4 and 5 demonstrate successful synthesis of IP6-PAMAM-GL 13K. The structural formula is shown in figure 6.
Example 2
In this example, a micro-stretching adhesion test was performed to determine the conditions of investigation of dentin adhesion strength and adhesion performance by using a polyamide-amine dendrimer (IP 6-PAMAM) with double modification of phytic acid and polypeptide as the end groups as a pretreatment agent for dentin adhesion. In this example, the IP6-PAMAM-GL13K was immediately dissolved in deionized water at room temperature under standard atmospheric pressure, and the solubility was high, the purity was > 95%, and the experimental requirements were met, as shown in FIG. 7.
The IP6-PAMAM-GL13K prepared in example 1 was placed on an electronic balance, and different masses of IP6-PAMAM-GL13K were weighed and dissolved in deionized water to prepare 100. Mu.l of each of 25 and 50mg/mL solutions. Freshly extracted, intact human third molars were obtained at the university of lan oral hospital. All teeth were stored at 4℃and 0.9% NaCl and used within 1 month after removal. The isolated tooth is fixed on a white nylon block by using memory wax, the extracted tooth root is cooled by a low-speed cutting saw at the position of 2-3mm of the tooth enamel juncture, the tooth enamel is removed perpendicular to the longitudinal axis of each tooth, and the smooth coronal dentin is exposed. The exposed crown dentin surface was polished with 600 mesh silicon carbide wet paper for 60s to form a standard smear layer. Tooth specimens were divided into 6 groups (10 teeth per group) according to enamel conditioning mechanism, respectively, 37% phosphoric acid (H 3PO4) treatment for 15s, 1% IP6 (phytic acid) treatment for 60s, IP6-PAMAM-GL13K 25mg mL -1 and 50mg mL -1 treatment for 30 or 60s. And (3) respectively coating Single Bond 2 full acid etching adhesives according to manufacturer application instructions, curing by a light curing lamp for 20s, gradually stacking 3M Z350 composite resin on the surface of the adhesive, and irradiating the adhesive for 20s by the light curing lamp at 1mm increment each time to finally form the composite resin with the thickness of 4 mm. The strips of each tooth were stored in deionized water at 37 ℃ for 24 hours, cut into 0.9mm x 7mm beams, with the resin-enamel interface of each beam located in the middle of the bar. Each beam is connected to a test fixture by cyanoacrylate glue, an instrument is started, the pulling speed is set to be 1mm/min until the samples are broken, a universal tensile testing machine is used for carrying out tensile stress failure, and the obtained maximum force is divided by the cross section area of an adhesive interface, namely the micro-tensile strength value (Mpa) of each sample is used for ensuring that each tensile break is in an interface breaking mode. Each specimen was performed in 6 replicates. Data analysis was performed in statistical units of teeth (n=10 teeth).
Based on the above results, the optimal concentration and time of IP6-PAMAM-GL13K were selected and the adhesive strength was measured on 10 enamel specimens by wet and dry adhesive methods, respectively, and dentin blocks were treated with H 3PO4 s (wet/dry adhesive), 1% IP6 s (wet/dry) and 50mg/mL IP6-PAMAM-GL13K 60s (wet/dry), respectively. And (3) respectively coating Single Bond2 full acid etching adhesives according to manufacturer application instructions, curing by a light curing lamp for 20s, gradually stacking 3M Z350 composite resin on the surface of the adhesive, and irradiating the adhesive for 20s by the light curing lamp at 1mm increment each time to finally form the composite resin with the thickness of 4 mm. The strips of each tooth were stored in deionized water at 37 ℃ for 24 hours, cut into 0.9mm x 7mm beams, with the resin-enamel interface of each beam located in the middle of the bar. Each beam is connected to a test fixture by cyanoacrylate glue, an instrument is started, the pulling speed is set to be 1mm/min until the samples are broken, a universal tensile testing machine is used for carrying out tensile stress failure, and the obtained maximum force is divided by the cross section area of an adhesive interface, namely the micro-tensile strength value (Mpa) of each sample is used for ensuring that each tensile break is an interface breaking mode. As shown in fig. 8A, the experimental results indicate that: only 50mg/ml 60s of IP6-PAMAM-GL13K gave a similar adhesive strength as the control group phosphoric acid. As shown in fig. 8B, the experimental results indicate that: the bonding strength of 50mg/ml and 60s of IP6-PAMAM-GL13K in a dry-wet bonding mode has no statistical difference, can achieve better bonding strength, and has the capability of selective ore removal.
Example 3
This example measures the investigation of the microscopic morphology of the demineralized dentin collagen matrix using the polyamide-amine dendrimer (IP 6-PAMAM-GL 13K) with the end groups of phytic acid and polypeptide double modification as treatment agent described in example 1.
Freshly extracted, intact human third molars were obtained at the university of lan oral hospital. All teeth were stored at 4℃and 0.9% NaCl and used within 1 month after removal. The isolated tooth is fixed on a white nylon block by using memory wax, the extracted tooth root is cooled by a low-speed cutting saw at the position of 2-3mm of the tooth enamel juncture, the tooth enamel is removed perpendicular to the longitudinal axis of each tooth, and the smooth coronal dentin is exposed. Grinding the exposed surface of the dentin of the middle crown with 600-mesh sand paper for 1min, performing ultrasonic treatment for 15min under an ultrasonic cleaner, and selecting 4 dentin sheets with good states for standby. The samples were separated into an HDMS drying group and an air drying group, wherein 2 samples were treated with IP6-PAMAM-GL13K and phosphoric acid, and then were fixed in 2.5wt% glutaraldehyde solution for 4 hours, and were dehydrated in ascending ethanol series (50%, 70%,85% and 90%), each time for 30 minutes. The samples were then immersed in (HMDS) -ethanol solutions (70%, 80%,95% and 100%) containing different volume fractions, and after removal, the surface residual liquid was blotted with filter paper and placed in disposable plastic petri dishes. The remaining half of the sample was dehydrated and then dried in a glass dryer for 24 hours. Placing the dried sample on a sample holder, placing the sample in an ion sputtering instrument after cementing conductive adhesive, spraying metal, taking out the sample, observing the sample by using a field emission scanning electron microscope, wherein the low-power image scale is 5 mu m, and the high-power image scale is 1 mu m. As shown in fig. 9, the experimental results show that after the dentin is treated by IP6-PAMAM-GL13K, the adhesive can better permeate into the gaps outside the fibers, the sealing performance of the formed bonding interface is better, the demineralized collagen can resist the shrinkage and deformation of the fibers under the drying condition, the three-dimensional bracket structure of the collagen fiber net is maintained, and the space for the adhesive monomer to enter is still reserved between the demineralized collagen fibers under the air drying. Based on the above results, it can be derived that the IP6-PAMAM-GL13K is more suitable for dentin dry adhesion technology with low technical sensitivity and more clinically friendly, and has the ability to improve the stability of dentin adhesion interface.
Example 4
In this example, the study of dentin surface roughness was performed by determining the pretreatment agent, which is polyamide-amine dendritic polymerization (IP 6-PAMAM-GL 13K) in which the terminal group described in example 1 was double-modified with phytic acid and a polypeptide.
Dentin sheets 1 mm thick (collection of isolated teeth incorporating standard and dentin preparation method and micro-stretching adhesion experiment) were prepared, sequentially ground with 600 mesh, 1200 mesh, 2000 mesh, 4000 mesh, 7000 mesh and 10000 mesh sandpaper for 1min, and then finely polished with w1 and w0.5 diamond grinding paste. The prepared dentin sheet is randomly treated with IP6-PAMAM-GL13K and phosphoric acid, cleaned by ultrapure water for 10s, and the filter paper is used for sucking the residual liquid on the surface and is placed in a vacuum drying oven for drying. And under normal temperature and standard atmospheric pressure conditions, using an atomic force microscope to randomly and fixedly measure the surfaces of the two groups of dentin. The obtained images were 3D synthesized using Nano Scope analysis1.9 software to analyze the surface roughness of the two sets of samples. The results are shown in fig. 10 and 11, and the experimental results show that the dentin surface roughness of the treatment with IP6-PAMAM-GL13K is significantly higher than that of the phosphoric acid group. The result is consistent with the result of the micro-stretching bonding experiment and the scanning electron microscope experiment, the dentin dry bonding processed by the IP6-PAMAM-GL13K is verified, and the mixed layer is fully permeated by the adhesive, so that the demineralized dentin is matched with the resin monomer, the relatively durable bonding can be realized, and the problem of the defect of the bonding interface structure constructed under the traditional acid etching technology is solved.
Example 5
In this example, the condition of investigation of MMP activity in dentin was examined using polyamide-amine dendrimer (IP 6-PAMAM-GL 13K) with end groups of phytic acid and polypeptide double modification as a pretreatment agent as described in example 1.
Dentin sheets 0.5mm thick (collection and inclusion standard of isolated teeth and dentin preparation method and micro-stretching adhesion experiment) were prepared and then ultrasonically cleaned with deionized water for 5min. Mixing rhodamine dye powder with 1 drop of Single Bond 2 adhesive, and diluting with deionized water for 10 times to form uniform solution for later use. 1mg/ml of fluorescein-labeled gelatin solution was prepared by dropping 1ml of deionized water into a reagent tube containing 1ml of DQ-gelatin, and the gelatin solution was prepared by: fading-resistant agent: the buffer solution is diluted in a ratio of 1:1:8 for later use.
Four designated adjustment protocols, 37% H 3PO4 for 15 seconds, IP6 for 60 seconds, 50mg/ml IP6-PAMAM-GL13K for 60 seconds were used to apply dentin strips, followed by deionized water rinse for 15 seconds, and gently blow-dry. The surface was coated with rhodamine-added adhesive for 10s, photo-cured for 10s, and a 1mm thick layer of flowable resin composite was deposited on the adhered dentin and lightly cured for 20s. After 24h storage in deionized water, the bonded specimens were cut vertically into 1mm thick sections, the resin-dentin interface was exposed, fixed on glass slides, and polished to a thickness of about 50 μm.
In situ enzymatic analysis was performed using the EnzChek TM gelatinase/collagenase assay kit to determine MMP active sites in the hybridization layer. 50 μl of the prepared fluorescent gelatin solution was added dropwise to the polished resin-dentin surface, lightly covered with glass flakes, and air was not allowed to enter. Finally, the sample is placed in a dark environment in a 37 ℃ incubator for 48 hours. Green fluorescence (λexcitation/λemission=494/521 nm) generated after gelatin hydrolysis and red fluorescence (λexcitation/λemission=553/627 nm) generated by the adhesive were imaged using a two-photon Confocal Laser Scanning Microscope (CLSM). Sections with a thickness of 85 μm were obtained from different focal planes for each bonded specimen. These images were stacked and processed with ZEN 2010 software (Carl Zeiss). Fluorescein intensity was quantitatively analyzed using Image J software. As shown in FIGS. 12 and 13, the experimental results showed that almost no green fluorescence was observed in the mixed layer of the IP6-PAMAM-GL13K group, and that the IP6 group could see a green fluorescence similar to that of the phosphoric acid group. The relative percentage of green fluorescence region was shown after contacting with highly quenched fluorescein-conjugated gelatin substrate in each mixed layer, and 37% phosphoric acid > IP6-PAMAM-GL13K, indicating that IP6-PAMAM-GL13K has inhibitory effect on dentinal endogenous MMP.
Example 6
In this example, the antibacterial property of the adhesion interface was examined by measuring the double-modified polyamide-amine dendritic polymerization (IP 6-PAMAM-GL 13K) with phytic acid and polypeptide as terminal groups as the treating agent in example 1. In this example, the concentration of the IP6-PAMAM-GL13K is 50mg/ml and the action time is 60s.
Streptococcus mutans (ATCC 700610), actinomyces naeslundii (ATCC 12104), enterococcus faecalis (ATCC 29212) were selected as the test strain for detecting the antibacterial properties of IP6-PAMAM-GL 13K. Respectively picking up the liquid of three strains by using a disposable inoculating loop, culturing for 18h by using a BHI solid culture medium containing 0.2% glucose and under the aerobic environment (5% CO 2) at 37 ℃ for the next day, picking up single bacterial colony by using a sterile inoculating loop, inoculating the bacterial colony into 10mL of BHI culture medium under the aerobic environment at 37 ℃ and placing the bacterial colony on a constant temperature bacterial horizontal shaking table (200 rpm) for culturing, when the bacterial liquid is proliferated to the logarithmic proliferation phase, the concentration of the bacterial liquid is diluted by a BHI liquid culture medium, the OD value is 0.5 (the bacterial concentration is about 1.0X10 6 CFU/mL) measured at the wavelength of 600nm of an enzyme-labeled instrument, and the bacterial liquid is used as an experimental inoculation bacterial liquid for standby, and when a biological film is cultured, 1% (w/v) sucrose is additionally added into the BHI liquid culture medium. The dentin sheet with the thickness of 1mm is prepared by a micro-stretching bonding experiment, phosphoric acid etching the dentin sheet for 15s, washing with deionized water for 1min, ultrasonic cleaning with an ultrasonic cleaner for 10min, and sterilizing with a conventional high-pressure steam sterilizing pot for later use. The sterilized dentin sheet was placed in 24-well plates, each well plate was inoculated with 1mL of diluted fresh bacterial liquid (chain, line and faecal intestine), and the inoculated 24-well plates were placed in an incubator for overnight culture for 24 hours. The L-7012 bacteria activity kit is adopted to label the dead living bacteria in the biological film, deionized water (negative control), IP6-PAMAM (positive control) and IP6-PAMAM-GL13K are respectively used for acting dentin, and PBS is used for cleaning dental films to remove unbound dye. The treated dead live-stained image was observed at CLSM 20 x, dead bacteria were stained red, live bacteria were stained green (live bacteria: λ Excitation /λ Emission of =480/500 nm, dead bacteria: λ Excitation /λ Emission of =590/635 nm). The qualitative fluorescence results are shown in FIG. 14, and the quantitative results of three groups of dead/live bacteria plotted on the staining chart of dentin blocks are shown in FIG. 15. The experimental results show that: the surface of the biomembrane treated by 50 mg/mlIP-PAMAM-GL 13K is covered with a large amount of dead bacteria, and the dental film treated by the deionized water group and the IP6-PAMAM group has a large amount of green fluorescence, which shows that the dental film is a living bacteria, so that 50mg/ml of the IP6-PAMAM-GL13K has a good inhibition effect on bacterial biomembrane.
Example 7
In this example, the biosafety of Human Dental Pulp Cells (HDPCs) and mouse fibroblasts L929 was examined when the polyamide-amine dendrimer (IP 6-PAMAM-GL 13K) with the end groups modified by both phytic acid and polypeptide as described in example 1 was used as a pretreatment agent.
Healthy teeth that were required to be pulled out for orthodontic treatment were collected for 10-20 years of age with written informed consent from the donor. The dental pulp is hidden from the dental tissue which is ground by the high-speed turbine handpiece under the drill until the position of the pulp cavity is seen, and then the dental pulp is stored in a DMEM culture solution. The crowns were separated with forceps on an ultra clean bench, the exposed dental pulp tissue was extracted by pulling out the intramedullary nail, the ophthalmic scissors were cut into 1mm x 1mm size pieces, dispersed in 3mg/ml type I collagenase for 1.5h, centrifuged at 800rpm/min for 3min, and the supernatant was discarded. Then adding 3ml of FBS complete culture medium, transferring to a T25 culture flask, placing in a cell incubator for culturing, standing for cell adhesion, taking care that the culture flask cannot be shaken vigorously 3-5 days after primary cells are extracted, and preventing adhesion tissues from separating.
The test was performed using third to fifth generation Human Dental Pulp Cells (HDPCs) and L929 fibroblasts, and when the cells grew to 80% confluence, old medium PBS was discarded and washed 3 times, 500. Mu.L trypsin was added to digest the cells, 2 volumes of trypsin was added to stop digestion by complete culture, and the supernatant was centrifuged off. A volume of complete medium was added to resuspend, and cells were counted under a microscope and diluted to 1X 10 5 cells/ml. A blank group, i.e. a group without inoculated cells, is set, and only sterile medium is added. The control group was a 0 dosing group, and only cells were inoculated. 100 microliters of the diluted cell fluid in each well is inoculated into a 96-well plate for culturing for 24 hours, and the original culture medium is replaced by PBS, IP6, 50mg/mL IP6-PAMAM-GL13K or 50mg/mL IP6-PAMAM-GL13K according to 1/100 or 1/1000 dilution, and the culture is carried out for 24 hours. The PBS solution was steam sterilized and the IP6 and IP6-PAMAM-GL13K solutions were filter sterilized through a cellulose acetate filter having a pore size of 0.22. Mu.m. Finally, 10. Mu.l of CCK-8 kit was added to each well according to the manufacturer's instructions, incubated for 4 hours at 37℃under 100% relative humidity in the dark, and absorbance at 570nm was measured using an enzyme-labeled instrument. Cell viability (%) = ([ a test- [ a ] blank)/([ a control- [ a ] blank) ×100%). For each sample, the average absorbance of 6 wells run in parallel was calculated. The results are shown in FIG. 16, and the experimental results show that the cell survival rate of the human dental pulp cells and the mouse fibroblasts is higher than 80%, so that the IP6-PAMAM-GL13K has lower cytotoxicity under the working concentration and good biological safety.
Claims (10)
1. The polyamide-amine dendritic polymer with double modification of phytic acid and polypeptide is characterized by having the following structural formula:
wherein n is the number of grafted polypeptides, n=53-63, m is the number of grafted phytic acid, and m=65-75.
2. The method for preparing a polyamide-amine dendrimer double modified by phytic acid and polypeptide according to claim 1, comprising the following steps:
(1) Synthesis of protective group-containing phytic acid modified polyamide-amine dendritic polymer
Dissolving a polyamide-amine dendritic polymer with a protecting group and a fifth generation end group of amino into a solvent to prepare a first solution; dissolving phytic acid with a structural formula shown in a formula (I) in a mixed solution of N, N dimethylformamide, triethylamine and methylmorpholine, slowly adding condensing agent dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, and uniformly mixing to obtain a second solution; the mass ratio of phytic acid in the second solution to the amino-terminated polyamide-amine dendritic polymer in the first solution is 3:144;
Slowly raising the temperature of the first solution to 26-28 ℃, slowly dripping the second solution into the first solution under stirring, continuing to react for 1-1.5 h after all the second solution is dripped, gradually sampling and detecting in the reaction process, controlling the number of the reaction groups of PAMAM, lowering the temperature to 0 ℃ after the reaction is finished, slowly stopping stirring, standing, concentrating the reaction solution to remove the solvent, filtering, dissolving the obtained solid by ethyl acetate, adding petroleum ether for crystallization, and drying to obtain dry powder; dissolving the obtained dry powder in dimethyl sulfoxide to obtain a phytic acid modified polyamide-amine dendritic polymer solution with a protecting group;
(2) Synthesis of polyamide-amine dendritic polymer with double modification of phytic acid and polypeptide with protecting group
Dissolving the polypeptide with the full protective group and the structural formula shown as the formula (II) in N, N dimethylformamide, adding a mixed solution of dicyclohexylcarbodiimide and N-Hydroxysuccinimide (HOSU), stirring or vibrating to dissolve the polypeptide, and filtering to obtain a third solution; wherein the polypeptide: the mass ratio of the polyamide-amine dendritic polymer with the end group being amino is 7:144;
Slowly adding the third solution into the phytic acid modified polyamide-amine dendritic polymer solution with the protecting group obtained in the step (2), and reacting for 18-26 h at 0-10 ℃; after the reaction is finished, removing the reaction solution, drying to obtain dry powder, dissolving the obtained dry powder in trifluoroacetic acid aqueous solution, and reacting for 3.5-4.5 hours at room temperature under a sealing condition to obtain a polyamide-amine type dendritic polymer solution with double modification of phytic acid and polypeptide and a protecting group;
(3) Deprotection groups
And (3) adding diethyl ether into the solution obtained in the step (2) to obtain a crystal, filtering and drying to obtain the polyamide-amine dendritic polymer double-modified by phytic acid and polypeptide.
3. The method according to claim 2, characterized in that the polyamide-amine dendrimer whose end groups are amino groups is prepared by the following method:
① Synthesis of 0.5 generation PAMAM-NH 2
Uniformly mixing ethylenediamine and ethanol in an ice water bath in a nitrogen atmosphere, dropwise adding methyl acrylate at room temperature (25 ℃), and continuing to react for 24 hours after the dropwise addition, wherein ethylenediamine: the molar ratio of methyl acrylate is 1:8; after the reaction is finished, the solvent and excessive reactants are removed by reduced pressure distillation, and a pale yellow transparent liquid G0.5-PAMAM-NH 2 is obtained;
② Synthesis of generation 1 PAMAM
Uniformly mixing G0.5-PAMAM and methanol in an ice water bath in a nitrogen atmosphere, dropwise adding ethylenediamine at 25 ℃, and continuing to react for 24 hours after the dropwise adding, wherein G0.5-PAMAM: the molar ratio of ethylenediamine is 1:24, removing the solvent and excess ethylenediamine after the reaction is finished to obtain a light yellow viscous liquid which is G1-PAMAM-NH 2;
Then, using the synthesized G1.0-PAMAM-NH 2 as a raw material, increasing the dosage of methyl acrylate, and synthesizing G1.5-PAMAM-NH 2 according to the method of the step ①, wherein G1.0-PAMAM-NH 2: the molar ratio of methyl acrylate is 1:16; and then using the synthesized G1.5-PAMAM-NH 2 as a raw material, increasing the consumption of ethylenediamine, and synthesizing G2.0-PAMAM-NH 2 according to the method of the step ②, wherein G1.5-PAMAM-NH 2: the molar ratio of the ethylenediamine is 1:48;
According to the method, the feeding of methyl acrylate and ethylenediamine is increased by multiple steps, and Michael addition reaction and amidation reaction are alternately repeated until G5-PAMAM-NH 2 is obtained.
4. The method according to claim 2, wherein the fifth-generation amino-terminated polyamide-amine dendrimer with protecting groups in step (1) is prepared by the following method:
Taking a fifth-generation polyamide-amine dendritic polymer with an end group as an amino group and chloroformic acid-9-fluorenylmethyl ester as raw materials, and reacting for 2-4 hours at room temperature under the action of dichloromethane, sodium bicarbonate and water to obtain Fmoc-protected G5-PAMAM.
5. The method according to claim 2, wherein the step (1) is performed by sampling every 15 minutes, and detecting the remaining number of Fmoc groups by Fmoc group assay to control the amount of grafted phytic acid to 65 to 75.
6. The method according to claim 2, wherein in the step (1), the polyamide-amine type dendritic polymer having an amino group as a terminal group is dissolved in a mixed solvent of anhydrous dimethyl sulfoxide and N, N-dimethylformamide at-12 ℃.
7. The method of claim 2, wherein during the second solution of step (1), the solute: solvent: the mass ratio of the condensing agent is 3:6:2, and the condensing agent is dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, wherein the mass ratio of dicyclohexylcarbodiimide to 1-hydroxybenzotriazole is 7:5.
8. The method according to claim 2, wherein the crystallization operation in the step (1) is to add petroleum ether into the concentrated reaction solution for crystallization, and filter the crystals; after dissolving the crystal by ethyl acetate, adding petroleum ether again for crystallization, and dissolving the obtained crystal in dimethyl sulfoxide solution to obtain the phytic acid modified polyamide-amine dendritic polymer solution with the protecting group.
9. The method according to claim 2, wherein in the step (2), a mixed solution of dicyclohexylcarbodiimide and N-hydroxysuccinimide is added in an amount 2 times the amount of N, N-dimethylformamide.
10. Use of the polyamide-amine dendrimer double modified with phytic acid and polypeptide according to claim 1 in the field of dentin adhesion.
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