Dopa-modified polyamidoamine dendrimers, methods of making and uses thereof

Abstract

The application provides a preparation method of dopamine (DA) modified polyamide-amine dendrimer, which comprises amino terminal dopamine carboxylation, synthesis of carboxylated dopamine with a protective group, synthesis of carboxylated dopamine with a protective group modified polyamide-amine dendrimer, and synthesis and application of dopamine modified polyamide-amine dendrimer. The application utilizes the firm adhesion performance of DA in a marine humid environment, the antibacterial performance and the osteogenic induction performance of PAMAM-NH2 cationic polymer, introduces DA into the surface of PAMAM-NH2 to synthesize a new high polymer material DA-PAMAM-NH2, detects the long-acting antibacterial and osteogenic induction performance of the system as a bifunctional coating on the surface of an implant, and evaluates the role of the system in improving the clinical durability of the implant.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high polymer material synthesis, and particularly relates to a preparation method and application of dopamine-modified polyamide-amine dendritic polymer composite material. BACKGROUND

[0002] With the improvement of medical level, implant materials have been widely used in dental and orthopedic surgery. Compared with traditional repair technology (fixed denture, removable denture), it has the advantages of not damaging adjacent teeth, strong load function, comfort and the like, and is accepted by the majority of patients. The key factor for successful repair of the implant is the formation of the interface bone between the implant and the bone. Titanium material has been widely used as an implant material for orthopedic and dental surgery due to its excellent biocompatibility and mechanical properties matching the bone tissue.

[0003] However, although strict surgical disinfection process and preventive systemic use of antibiotics, implant infection caused by bacteria is still an important factor leading to implant failure. Numerous studies have been made from different aspects to solve the problem of implant-related infection, and it is found that constructing an antibacterial coating on the surface of the implant to inhibit bacterial growth is a directional strategy to prevent implant infection. However, the current antibacterial coating generally has a too fast release speed, and it is difficult to maintain an effective antibacterial concentration locally, thereby being difficult to achieve the purpose of long-acting antibacterial. At the same time, due to the chemical inertness of the surface of the titanium implant, the combination with the surrounding bone tissue is poor, and it is difficult to ensure the stability of the implant in the early stage of implantation. The traditional surface modification method is rarely capable of simultaneously achieving the dual function, and secondly, these methods still need to be investigated to improve the clinical durability of the implant.

[0004] In summary, to improve the long-term success rate of the implant, on the one hand, the antibacterial performance of the implant needs to be improved to prevent the occurrence of infection around the implant, and on the other hand, the combination performance of the implant with the surrounding bone tissue needs to be improved to improve the long-term stability of the implant. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a dopamine-modified polyamide-amine dendritic polymer and a preparation method and application thereof in view of the deficiencies of the prior art. Inspired by the mussel foot protein, the present application optimizes and screens DA with specific adsorption of the implant from the mussel protein, and introduces the DA into PAMAM-NH2 to synthesize a new high polymer material DA-PAMAM-NH2. By using the specific adsorption performance of DA and the antibacterial performance and osteoinductive performance of PAMAM-NH2, a dual-function in-vivo implant coating material capable of long-acting antibacterial performance and promoting bone combination is constructed, thereby providing a new strategy and new idea for improving the long-term stability of the implant.

[0006] The technical scheme adopted by the present application is: a dopamine-modified polyamide-amine dendrimer, a structural formula of the dopamine-modified polyamide-amine dendrimer is:

[0007]

[0008] In the above structural formula, n is the number of grafted dopamines, n=4-5.

[0009] The present application also provides a method for preparing the dopamine-modified polyamide-amine dendrimer, and the method is:

[0010] S1, carboxylation of an amino-terminal dopamine:

[0011] Under the condition of room temperature and normal pressure, dopamine is dissolved in pyridine, then succinic anhydride and 4-dimethylaminopyridine (DMAP) are added, after stirring and dissolving, the reaction is carried out for 6 hours, then the reaction solution is poured into ice ether for precipitation for 2 hours, the product is collected by filtration, and vacuum drying is carried out, so as to obtain carboxylated dopamine; the structural formula of the carboxylated dopamine is

[0012]

[0013] S2, synthesis of carboxylated dopamine with a protecting group:

[0014] Under the condition of 0 DEG C, triethylamine, p-toluenesulfonyl chloride, DMAP and CH2Cl are added to the dopamine subjected to carboxylation treatment in S1, and the reaction is carried out for 6 hours under the condition of 0 DEG C, so as to obtain OTs (p-toluenesulfonyl) protected carboxylated dopamine, and the structural formula is

[0015]

[0016] S3, synthesis of carboxylated dopamine with a protecting group modified polyamide-amine dendrimer

[0017] Under the condition of room temperature and light shielding, the fifth-generation polyamide-amine dendrimer is dissolved in methanol, after stirring and dissolving, BOP (Benzotriazol-1-yloxy) and OTs protected carboxylated dopamine obtained in S2 are added, stirring and dissolving are carried out, and the reaction is carried out for 2 hours under the condition of room temperature, so as to obtain a reaction solution of carboxylated dopamine with a protecting group modified polyamide-amine dendrimer, and the structural formula of the carboxylated dopamine with a protecting group modified polyamide-amine dendrimer is

[0018]

[0019] S4, synthesis of dopamine-modified polyamide-amine dendrimer:

[0020] Under the protection of nitrogen, potassium tert-butoxide was added dropwise into the reaction solution obtained in S3, and then poured into acetone, sealed in the dark at room temperature, and precipitated overnight. The crude product was collected by filtration, then dissolved in dimethyl sulfoxide (DMSO) to fully dissolve, and transferred to a dialysis bag. Dialysis was performed for 24 h, the dialysate was collected, and freeze-drying was performed to obtain a dopamine-modified polyamide-amine dendrimer, with a structural formula of

[0021]

[0022] Preferably, the amount ratio of dopamine, pyridine, succinic anhydride and DMAP in S1 is 50 mg: 2 mL: 39.20 mg: 3.99 mg.

[0023] Preferably, the equivalent ratio of triethylamine and DMAP in S2 is 60:1.

[0024] Preferably, the amount ratio of the fifth generation polyamide-amine dendrimer, methanol, BOP and OTs-protected carboxylated dopamine in S3 is 50 mg: 2 mL: 1.54 mg: 2.66 mg; and the equivalent ratio of BOP in S3 and carboxylated dopamine in S2 is 1:5.

[0025] Preferably, the molecular weight cut-off of the dialysis bag in S4 is 8000-14000 DA.

[0026] Preferably, the preparation method of the fifth generation polyamide-amine dendrimer in S3 is as follows:

[0027] S301. Synthesis of G 0.5PAMAM-NH2

[0028] Under the conditions of ice bath and nitrogen, methyl acrylate was dissolved in methanol, then ethylenediamine was added, and the reaction was terminated after stirring at a rotation speed of 1000 r / min for 48 h in the dark. Then, the dissolved and partially reacted residues were removed by distillation under reduced pressure to obtain the reaction product G 0.5PAMAM-NH2. The molar ratio of methyl acrylate and methanol was 2.4:1.

[0029] S302. Synthesis of G 1.0PAMAM-NH2

[0030] Under the conditions of ice bath and nitrogen, G 0.5PAMAM-NH2 obtained in S301 was dissolved in methanol, then ethylenediamine was added to the obtained solution at room temperature, and the reaction was terminated after stirring at a rotation speed of 1000 r / min for 48 h in the dark. Then, the dissolved and partially reacted residues were removed by distillation under reduced pressure to obtain the reaction product G 1.0PAMAM-NH2. The molar ratio of G 0.5PAMAM-NH2 and ethylenediamine was 1:25.

[0031] S303, synthesis of G 5.0 PAMAM-NH2

[0032] G 1.0 PAMAM-NH2 obtained in S302 is added with ethylenediamine, and through continuous Michael addition reaction and amidation reaction, higher generation PAMAM-NH2 is prepared, until G 5.0 PAMAM-NH2, that is, the fifth generation of polyamidoamine dendrimer, is synthesized.

[0033] Polyamidoamine (PAMAM) is a kind of dendrimer, which is composed of a central core, an internal cavity, a rich branched structure and an easily modified surface functional group, and has the characteristics of controllable molecular weight, multifunctionality, good solubility and biocompatibility. Experiments have proved that PAMAM-NH2 can induce dentin remineralization as a nucleation template. At the same time, the fifth generation of PAMAM-NH2 has 128 amino groups on the surface, and the amino cation can produce electrostatic effect with the anion on the surface of bacteria, causing the bacterial membrane to be damaged and the cytoplasm to be leaked, and then causing the death of the bacteria, which is a spectrum antibacterial agent. Mussel is a kind of crustacean marine organism widely distributed in coastal and offshore areas. The byssus gland can secrete byssus, and the byssus disc formed by the byssus can fix the mussel on the surface of various solid substrates. The special adhesive protein secreted by the mussel contains a large amount of catechol compound 3,4-dihydroxy-l-phenylalanine (DOPA), which can firmly and quickly adhere to almost any substrate surface in a humid environment. There are many biomimetic synthetic polymers rich in dopamine units in nature, such as dopamine (DA), which can be regarded as a universal choice of anchoring functional molecules inspired by this natural adhesion mechanism.

[0034] The present application utilizes the strong adhesion performance of DA in a humid environment and the induction of hydroxyapatite deposition and antibacterial effect of PAMAM-NH2 cationic polymer, introduces DA into the surface of the fifth generation of PAMAM-NH2 to improve its adhesion performance, the purpose is to construct DA-PAMAM-NH2 bifunctional coating material, detect the ability of the system as a dual functional coating of the implant surface to improve bone integration remineralization and long-acting antibacterial, and evaluate the influence of the system on the durability of the implant.

[0035] The present application also provides the application of the dopamine-modified polyamidoamine dendrimer prepared by the above method, and the dopamine-modified polyamidoamine dendrimer is used for implant coating, can long-acting inhibit the occurrence of peri-implantitis, promote bone integration, and has potential value in improving the clinical durability of the implant.

[0036] Preferably, the dopamine-modified polyamide-amine dendrimer is used as a long-acting bifunctional coating system firmly adsorbed on the surface of titanium sheets, for improving the antibacterial property of implants and promoting the bone bonding around implants, improving the clinical durability of implants, and providing a new technology for effectively preventing and treating inflammation around implants and poor bone bonding.

[0037] Compared with the prior art, the present application has the following advantages:

[0038] 1. From the perspective of bionics, the catechol structure in the mussel molecule, which plays a key role in adhesion in a water environment, is introduced into polyamide-amine dendrimer substances, so that a coating system with dual functions can be obtained, in order to prevent and treat inflammation around implants and a series of problems of poor long-term durability of implants.

[0039] 2. The dopamine-modified polyamide-amine dendrimer has a firm adsorption effect on titanium sheets, can effectively resist the erosion of SBF, can still stably exist on the surface of titanium sheets even after being soaked in SBF for four weeks, realizes long-acting antibacterial property, can induce new bone deposition and promote bone bonding, and thus can be used as a bifunctional material for long-acting antibacterial property and promoting bone bonding on the surface of human implants.

[0040] 3. The raw material cost of the method is low, the preparation process is simple, the reaction purity is high, the method can be realized by using conventional raw materials and equipment, and industrialized production can be easily realized.

[0041] The present application will be further described in detail below in combination with the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a schematic diagram of the synthesis route of the dopamine-modified polyamide-amine dendrimer (DA-PAMAM-NH2) shown in the present application.

[0043] Figure 2 is the nuclear magnetic hydrogen spectrum of the dopamine-modified polyamide-amine dendrimer prepared in Example 1.

[0044] Figure 3 is the infrared spectrum of the dopamine-modified polyamide-amine dendrimer prepared in Example 1.

[0045] Figure 4 is the laser confocal microscope graph of the DA-PAMAM-NH2 labeled with fluorescein isothiocyanate in Example 2 after being adsorbed on the surface of titanium sheets, and after being washed with SBF after the DA-PAMAM-NH2 labeled with fluorescein isothiocyanate is adsorbed on the surface of titanium sheets (a is before washing, and b is after washing).

[0046] Figure 5 Figure 8 is the infrared spectrogram of the titanium sheet surface of the blank group (a), the titanium sheet surface after adsorption of DA-PAMAM-NH2(b), and the titanium sheet surface after adsorption of DA-PAMAM-NH2and SBF flushing (c) in Example 3.

[0047] Figure 6 Figure 11 is a bar chart of the bacterial colony count of the titanium sheet surface of each group in Example 4.

[0048] Figure 7 Figure 14 is a bar chart of the ratio of dead and live bacteria in the bacterial biofilm formed on the titanium sheet surface of each group in Example 5.

[0049] Figure 8 Figure 17 is a scanning electron microscope image, an atomic force microscope image, and an X-ray diffraction image of the amount of hydroxyapatite deposited on the titanium sheet surface of each group in Example 6.

[0050] Figure 9 Figure 20 is a bar chart of the cell adhesion density on the titanium sheet surface of each group after 1, 3, and 7 days of incubation in Example 7.

[0051] Figure 10 Figure 23 is a bar chart of the alkaline phosphatase expression on the titanium sheet surface of each group after 3 days and 7 days of incubation in Example 8.

[0052] Figure 11 Figure 26 is a CCK-8 chart of DA-PAMAM-NH2on mouse preosteoblast cells (MC3T3-E1) prepared in Example 9. DETAILED DESCRIPTION

[0053] Example 1

[0054] The dopa-modified polyamide-amine dendrimer of the present example has the following structural formula:

[0055]

[0056] In the above structural formula, n is the number of grafted dopamine, n = 4-5.

[0057] The present example also provides a method for preparing the above dopa-modified polyamide-amine dendrimer, the synthetic route is as shown in Figure 1 The method is as follows:

[0058] S1, carboxylation of the amino-terminal dopamine:

[0059] Carboxylated dopamine was prepared by dissolving 50 mg dopamine in 2 mL pyridine at room temperature and normal pressure, then adding 39.20 mg succinic anhydride and 3.99 mg 4-dimethylaminopyridine (DMAP), stirring and dissolving, and then reacting for 6 h. The reaction solution was then poured into 10 mL ice ether to precipitate for 2 h, the product was collected by filtration, and vacuum drying was performed to obtain the carboxylated dopamine; the structural formula of the carboxylated dopamine is

[0060]

[0061] S2, synthesis of dopamine with a protective group and carboxylation:

[0062] Carboxylated dopamine was prepared by dissolving 50 mg dopamine in 2 mL pyridine at room temperature and normal pressure, then adding 39.20 mg succinic anhydride and 3.99 mg 4-dimethylaminopyridine (DMAP), stirring and dissolving, and then reacting for 6 h. The reaction solution was then poured into 10 mL ice ether to precipitate for 2 h, the product was collected by filtration, and vacuum drying was performed to obtain the carboxylated dopamine; the structural formula of the carboxylated dopamine is

[0063]

[0064] S3, synthesis of polyamidoamine dendrimer modified with dopamine with a protective group and carboxylation:

[0065] Carboxylated dopamine was prepared by dissolving 50 mg dopamine in 2 mL pyridine at room temperature and normal pressure, then adding 39.20 mg succinic anhydride and 3.99 mg 4-dimethylaminopyridine (DMAP), stirring and dissolving, and then reacting for 6 h. The reaction solution was then poured into 10 mL ice ether to precipitate for 2 h, the product was collected by filtration, and vacuum drying was performed to obtain the carboxylated dopamine; the structural formula of the carboxylated dopamine is

[0066]

[0067] S4, synthesis of polyamidoamine dendrimer modified with dopamine with a protective group and carboxylation:

[0068] Under the protection of nitrogen, potassium tert-butoxide (6.0 eq) was added dropwise into the reaction solution obtained in S3, then poured into 10 mL of acetone, sealed in the dark at room temperature, precipitated overnight, the crude product was collected by filtration, then the crude product was dissolved in 1 mL of DMSO to fully dissolve, and transferred to a dialysis bag (molecular weight cut-off of 8000-14000 DA), dialyzed for 24 h, the dialysate was collected and freeze-dried to obtain dopamine-modified polyamide-amine dendrimer, denoted as DA-PAMAM-NH2, with the structural formula

[0069]

[0070] The preparation method of the fifth generation polyamide-amine dendrimer is as follows:

[0071] S301, synthesis of G 0.5PAMAM-NH2

[0072] Under the conditions of ice bath and nitrogen, methyl acrylate was dissolved in methanol, then ethylenediamine was added, the reaction was terminated after stirring at a speed of 1000 r / min for 48 h in the dark, then the dissolved and part of the reaction residues were removed by distillation under reduced pressure to obtain the reaction product G 0.5PAMAM-NH2; the molar ratio of methyl acrylate to methanol was 2.4:1;

[0073] S302, synthesis of G 1.0PAMAM-NH2

[0074] Under the conditions of ice bath and nitrogen, G 0.5PAMAM-NH2 obtained in S301 was dissolved in methanol, then ethylenediamine was added to the obtained solution at room temperature, the reaction was terminated after stirring at a speed of 1000 r / min for 48 h in the dark, then the dissolved and part of the reaction residues were removed by distillation under reduced pressure to obtain the reaction product G 1.0PAMAM-NH2; the molar ratio of G 0.5PAMAM-NH2 to ethylenediamine was 1:25;

[0075] S303, synthesis of G 5.0PAMAM-NH2

[0076] Ethylenediamine was added to G 1.0PAMAM-NH2 obtained in S302, and a higher generation of PAMAM-NH2 was prepared by continuous Michael addition reaction and amidation reaction, until G 5.0PAMAM-NH2 was synthesized, which was the fifth generation polyamide-amine dendrimer;

[0077] The related characterization of the dopamine-modified polyamide-amine dendrimer (DA-PAMAM-NH2) prepared in this example is as follows: Figure 2 (nuclear magnetic hydrogen spectrum) and Figure 3The characteristic peaks observed by referring to the literature and compared with the standard peaks are basically consistent, which indicates that the dopamine-modified fifth-generation polyamidoamine dendrimer is successfully synthesized.

[0078] Example 2

[0079] In this embodiment, the adsorption of the polyamidoamine dendrimer with polypeptide-modified end groups (DA-PAMAM-NH2) on the surface of a titanium sheet is determined. In this embodiment, the titanium sheet has a size of 10 mm x 1 mm.

[0080] The DA-PAMAM-NH2 prepared in Example 1 of the present application is labeled with fluorescein isothiocyanate (FITC). Specifically, the DA-PAMAM-NH2 is first mixed with an equal molar amount of FITC, and then stirred in the dark at room temperature for 24 h. After filtration, the obtained solution is dialyzed in a large beaker containing ultrapure water using a dialysis bag with a molecular weight cut-off of 8000-16000 daltons for 24 h. Subsequently, the obtained solution is frozen and dried to obtain the FITC-labeled DA-PAMAM-NH2. Then, 100 μL of the FITC-labeled DA-PAMAM-NH2 solution prepared by dissolving the DA-PAMAM-NH2 in deionized water at a concentration of 10 mg / mL is coated on the surface of a titanium sheet. After drying at room temperature, the surface of the titanium sheet is washed with SBF solution three times. The fluorescence distribution on the surface of the titanium sheet before and after washing is observed under an inverted confocal laser scanning microscope (CLSM), and the results are shown in Figure 4 The experimental results show that after washing with SBF solution, a large amount of DA-PAMAM-NH2 is still firmly adsorbed on the surface of the titanium sheet, which can resist the elution of the SBF solution, thereby indicating that the dopamine-modified polyamidoamine dendrimer synthesized in the present application has a firm adsorption property on the surface of the titanium sheet.

[0081] Example 3

[0082] In this embodiment, the adsorption of the polyamidoamine dendrimer with polypeptide-modified end groups (DA-PAMAM-NH2) on the surface of a titanium sheet is determined. In this embodiment, the titanium sheet has a size of 10 mm x 1 mm.

[0083] The DA-PAMAM-NH2 of the present application is dissolved in deionized water to prepare a solution with a concentration of 10 mg / mL. 100 μL of the solution is coated on the surface of a titanium sheet. After drying at room temperature, the surface of the titanium sheet is washed with SBF solution three times. The adsorption of the DA-PAMAM-NH2 on the surface of the titanium sheet before and after washing is further observed by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), and the results are shown in Figure 5As shown, the characteristic peaks of DA-PAMAM-NH2 still exist even after 1.5X SBF washing, which indicates that it has strong adsorption capacity to the titanium surface, which is sufficient to resist the erosion of SBF, which provides a strong guarantee for the subsequent application of DA-AMAM-NH2 in the moist and complex environment of the oral cavity. The experimental results are shown in the following table.

[0084] Example 4

[0085] In this embodiment, the instant antibacterial performance of the dopamine-modified polyamide-amine dendrimer (DA-PAMAM-NH2) with terminal groups as described in the present application as a coating adsorbed on the surface of titanium pieces was quantitatively determined, and whether it still has antibacterial performance after being immersed in 1.5X SBF solution for 4 weeks was detected. In this embodiment, the titanium pieces are 10 mm x 1 mm in size.

[0086] A solution of DA-PAMAM-NH2 prepared in Example 1 was prepared with deionized water to a concentration of 10 mg / mL, and titanium pieces with a size of 10 mm x 1 mm were randomly divided into a control group, an experimental group 1, and an experimental group 2 (3 titanium pieces as repeats in each group).

[0087] (1) The preparation of the coating was as follows:

[0088] At room temperature, 100 μL of deionized water was dropped onto the surface of the titanium pieces in the control group using a pipette, and they were naturally air-dried; 10 mg / mL of PAMAM-NH2 aqueous solution prepared in advance was dropped onto the surface of the titanium pieces in experimental group 1, and they were naturally air-dried, and then the unadsorbed substances on the surface of the titanium pieces were washed with 1.5X SBF; similarly, 10 mg / mL of DA-PAMAM-NH2 aqueous solution was dropped onto the surface of the titanium pieces in experimental group 2 at room temperature, and they were dried in air, then the unadsorbed substances on the surface were washed away with 1.5X SBF, then the titanium pieces in experimental group 2 were immersed in 1.5X SBF, stored at 37°C, and the liquid was replaced every 2 days. After 4 weeks of immersion, they were taken out, washed again with 1.5X SBF, naturally air-dried at room temperature, and then used.

[0089] (2) Culture of bacterial biofilm and detection of the inhibition of bacterial biofilm by each experimental group:

[0090] In the present application, S. aureus and E. coli are selected as the characteristic bacteria, and BHI medium is used for culture. Each group of titanium samples is placed in a 24-well plate, and each group of titanium samples has three parallel samples. 1.8 mL of BHI medium is added to each well, and 200 μL of S. aureus or E. coli bacterial solution is inoculated, respectively. After mixing the bacterial suspension with a pipette gun, it is incubated in a 37°C constant temperature incubator overnight to culture bacterial biofilm on the surface of the titanium sheet. Then, the titanium sheet with biofilm is transferred to an EP tube containing 2 mL of PBS in a clean bench, and the bacterial biofilm formed on the surface of the titanium sheet is peeled off by ultrasonic oscillation for 30 min. The bacterial biofilm is then plated on agar plates by the method of continuous dilution, and the number of colonies is counted (CFU) by selecting appropriate colonies. The results are shown in Table 1. Figure 6 As shown in Table 1, after 4 weeks of 1.5X SBF immersion and flushing, the survival rate of bacteria on the surface of the titanium sheet coated with DA-PAMAM-NH2 is not statistically different from that before immersion, and the bacteria are mainly dead, still maintaining good antibacterial performance. This shows that DA-PAMAM-NH2 can be firmly adsorbed on the surface of the titanium sheet, and can exert its long-acting antibacterial effect.

[0091] Example 5

[0092] In this example, the immediate antibacterial performance of the dopamine-modified polyamide-amine dendrimer (DA-PAMAM-NH2) as a coating adsorbed on the surface of the titanium sheet is quantitatively determined, and whether it still has antibacterial performance after being immersed in 1.5X SBF solution for 4 weeks is detected. In this example, the titanium sheet has a size of 10 mm x 1 mm.

[0093] (1) The preparation of the coating is as follows:

[0094] At room temperature, 100 μL of deionized water is dropped onto the titanium sheet of the control group with a pipette gun, and it is naturally air-dried. 100 μL of 10 mg / mL DA-PAMAM-NH2 aqueous solution is dropped onto the surface of the titanium sheet of the experimental group 1, and it is naturally air-dried in the air. Then, the titanium sheet is washed with 1.5X SBF to remove the unadsorbed substances on the surface. Similarly, 100 μL of 10 mg / mL DA-PAMAM-NH2 aqueous solution is dropped onto the surface of the titanium sheet of the experimental group 2 at room temperature, and it is naturally air-dried. Then, the unadsorbed substances on the surface are washed away with 1.5X SBF. The titanium sheet of the experimental group 2 is then immersed in the buffer 1.5X SBF, and it is stored at 37°C. The liquid is replaced every 2 days. After 4 weeks of immersion, the titanium sheet is taken out, washed with 1.5X SBF again, naturally air-dried at room temperature, and then reserved.

[0095] (2) Culture of bacterial biofilm and detection of the inhibition of bacterial biofilm by each experimental group

[0096] In the present application, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are selected as the detection bacteria, and BHI medium is used for culture. The treated titanium sheets of each experimental group are placed in a 24-well plate, 1.8 mL of BHI medium is added to each well, and 200 μL of S. aureus and E. coli bacterial liquid is inoculated respectively. After mixing the bacterial suspension with a pipette gun, it is incubated in a 37°C constant temperature incubator overnight to culture bacterial biofilm on the surface of the titanium sheet. Then, the planktonic bacteria not forming biofilm on the surface of the titanium sheet are washed off with PBS in a clean bench, and the surface is dried for several minutes at room temperature. Then, under the condition of avoiding light, according to the reagent instruction, dye 1 and dye 2 are mixed uniformly at a ratio of 1:1, and then 200 μL of the staining agent is added dropwise on the surface of each sample with a pipette gun to ensure that the entire titanium sheet surface is coated with the staining agent. After standing for 25 min, the excess dye on the surface of the sample is washed off with PBS buffer, and then dried at room temperature. Subsequently, it is transferred to a confocal special small dish, and the distribution of live and dead bacteria on the surface of the sample is observed by CLSM.

[0097] Figure 7 The results show that DA-PAMAM-NH2 has an antibacterial effect on bacteria by live / dead bacterial staining. In the control group treated with pure water, the biofilm on the surface of the titanium sheet is mainly composed of live bacteria (S. aureus and E. coli); in contrast, the biofilm on the surface of Ti6Al4V treated with PAMAM-NH2 or DA-PAMAM-NH2 is mainly composed of dead bacteria. This is because PAMAM-NH2 or DA-PAMAM-NH2 has a large number of amino positive charges on the amino terminal, which produces electrostatic interaction with the negative charge on the surface of the bacteria, leading to cell membrane damage, cytoplasm leakage, and bacterial death. After soaking in 1.5X SBF for 4 weeks, the surface of Ti6Al4V treated with PAMAM-NH2 is mainly composed of live bacteria; in contrast, the surface of Ti6Al4V treated with DA-PAMAM-NH2 is mainly composed of dead bacteria. And there is no significant difference in the ratio of dead bacteria to live bacteria in the DA-PAMAM-NH2 group before and after SBF incubation.

[0098] The experimental results of Example 4 and Example 5 both show that the polyamide-amine dendrimer with dopamine-modified end groups described in the present application can be used as a long-acting antibacterial coating material for the surface of a dental implant.

[0099] Example 6

[0100] In this embodiment, the adhesion of MC3T3-E1 cells on the surface of each group of titanium sheets treated with the coating is detected, and the morphology and adhesion density of the cells on different surfaces are recorded after incubation for 1, 3 and 7 days.

[0101] (1) The preparation of the coating is as follows:

[0102] Similar to the coating preparation method in the preceding Example 6, except that the titanium sheet substrate was subjected to high-temperature autoclave sterilization and ultraviolet irradiation sterilization treatment, and each reagent used in the experiment was subjected to bacterial filtration treatment.

[0103] (2) Cell culture and staining

[0104] The mouse preosteoblast cells (MC3T3-E1) were obtained from the laboratory of the School of Stomatology, Lanzhou University. They were cultured in an α-MEM medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. The cells were incubated in an incubator (37°C) with 5% CO2 and 95% relative humidity.

[0105] After the MC3T3-E1 cells reached 80% confluence, they were seeded on the surface of each substrate at a density of 1.0 x 10 4 After 1, 3, and 7 days of incubation, the samples were taken out, washed twice with PBS buffer, and then fixed with 4% paraformaldehyde fixing solution. After 20 min, the cells were discarded, and then treated with 0.5% Triton X-100 permeabilization for 3 min. Finally, the cytoskeleton and nucleus were stained with phalloidin and DAPI, respectively. After the staining was completed, the excess dye was washed away with PBS, and then the samples were dried and transferred to a small dish for CLSM observation of cell adhesion. As shown in Figure 8 more osteoblasts adhered to the surface of the titanium substrate coated with DA-PAMAM-NH2 after 3 and 7 days of culture, compared with the other two groups. This experimental result shows that the titanium sheet coated with DA-PAMAM-NH2 creates a more favorable environment for osteogenesis, and osteoblasts play an important role in the synthesis, secretion, and mineralization of bone matrix.

[0106] Example 7

[0107] In this example, the bone differentiation ability of MC3T3-E1 cells on different substrate surfaces was evaluated by detecting the activity of the early osteogenesis-related protein alkaline phosphatase (ALP). The coating preparation and cell culture methods are described in Example 7.

[0108] After the MC3T3-E1 cells reached 80% confluence, 1.0 x 10 4The surface of each substrate was seeded with a density of 1 x 104cells / well and incubated in an incubator. After 1 day of culture in ordinary a-MEM medium, the medium was replaced with osteogenic induction medium. The formula of the osteogenic induction medium was as follows: 250 mL of a-MEM medium was added with 10% FBS, 0.1 μmol / L of dexamethasone, 50 μmol / L of ascorbic acid and 10 μmol / L of β-glycerophosphate sodium. The medium was replaced every two days. The ALP expression level was detected after 3 days and 7 days. The specific method was as follows: the original osteogenic induction medium was discarded, the cells were washed with PBS for 3 times, 100 μL of Ripa lysis solution was used to lyse the cells for 30 min to obtain cell lysate, the total protein content in the cell lysate was detected by using a micro-BCA protein detection kit against a standard curve, and the corresponding ALP activity was detected by using an ALP activity detection kit.

[0109] The osteogenic potential mediated by MC3T3-E1 cells was evaluated by assessing the ALP expression level, as shown in Table 1. Figure 9 As shown in Table 1, the ALP activity of the MC3T3-E1 cells grown on the surface of the titanium sheet coated with DA-PAMAM-NH2 was significantly higher than that of the other two groups at all time periods. Based on the above results, it is assumed that the DA-PAMAM-NH2 coating promotes the proliferation and osteogenic differentiation of MC3T3-E1 cells by providing a more favorable microenvironment, thereby enhancing the early stability of the implant.

[0110] Example 8

[0111] In this example, the osteogenic induction ability of the dopamine-modified polyamide-amine dendrimer (DA-PAMAM-NH2) described in the present application as a coating adsorbed on the surface of a titanium sheet was determined. In this example, the titanium sheet had a size of 10 mm x 1 mm.

[0112] (1) The preparation of the coating was performed as follows:

[0113] At room temperature, 100 μL of deionized water was dropped on the titanium sheet of the control group by using a pipette, and the titanium sheet was naturally air-dried; 100 μL of an aqueous solution of PAMAM-NH2 at a concentration of 10 mg / mL was dropped on the surface of the titanium sheet of experimental group 1, and the titanium sheet was naturally air-dried; 100 μL of an aqueous solution of PAMAM-NH2 at a concentration of 10 mg / mL was dropped on the surface of the titanium sheet of experimental group 2, and the titanium sheet was naturally air-dried, and was ready for use.

[0114] (2) Preparation of 1.5X simulated body fluid (1.5X SBF): 0.1M NaCl, 4.2mM NaHCO3, 3mM KCl, 1mM K2PO4.3H2O, 1.5mM MgCl2.6H2O, 2.6mM CaCl2 and 0.5mM Na2SO4, 1.5M Tris-HCl to adjust pH to 7.4, stored in plastic bottles, 4℃ refrigerator storage. The treated titanium pieces of each experimental group were placed in a 24-well plate, three in each group, and 2mL of prepared SBF was added, and mineralized in an 80r, 37℃ constant temperature incubator for 7 days, and the mineralization solution was changed every day. After 7 days, the titanium pieces were taken out at the predetermined time point, the samples were washed with deionized water for 3 times, and then naturally dried at room temperature. The surface morphology of the substrate was observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), and the phase and orientation of the newly formed hydroxyapatite (HA) crystals on the substrate surface were detected by X-ray diffraction (XRD) instrument. The experimental results are shown in Figure 10 As shown in Table 1, the SEM results show that the original Ti6Al4V substrate has limited ability to induce calcium and phosphate deposition, which leads to the formation of only a few small crystals on the surface of the titanium piece, and the PAMAM-NH2 coated Ti6Al4V surface also shows only sparse mineral crystal deposition; in contrast, a large number of mineral crystals are found on the surface of the DA-PAMAM-NH2 coated Ti6Al4V, and elemental analysis results confirm that the Ca / P ratio of these mineral crystals is close to the HA Ca / P ratio in normal bone tissue. AFM calculation and three-dimensional reconstruction results of the surface roughness of the samples show that the surface roughness of the DA-PAMAM-NH2 coated titanium piece is higher than that of the other two groups. The XRD instrument detection results after 7 days of mineralization show that obvious diffraction peaks are found at 2θ = 26° and 32° on the surface of the DA-PAMAM-NH2 coated titanium, which are characteristic peaks of HA, further proving that more HA crystals are deposited on the titanium surface.

[0115] Example 9

[0116] In this embodiment, the biological safety of dopamine-modified polyamide-amine dendrimers (DA-PAMAM-NH2) as coating materials for mouse preosteoblast cells (MC3T3-E1) was studied.

[0117] The cytotoxicity of DA-PAMAM-NH2 was determined by cell counting kit-8 (CCK-8). After the MC3T3-E1 cells grew and fused to 80%, the cells were diluted to 1.0×10 4The density of 1 cell / hole is inoculated in a 96-well plate and incubated for 24 hours. Then 100 μL fresh culture medium containing different concentrations of DA-PAMAM-NH2 is used to replace the original culture medium. After overnight incubation, 10 μL CCK-8 solution is added to each well, and the cells are incubated in a cell culture medium at 37 DEG C for 4 hours. Then the absorbance at 570 nm is measured by an enzyme marker.

[0118] Cell viability (%) = ([A] test - [A] blank) / ([A] control - [A] blank) x 100%,

[0119] Wherein [A] test represents the absorbance of the well containing DA-PAMAM-NH2 solution, cell culture medium and cells; [A] blank represents the absorbance of the well containing only the medium; and [A] control represents the absorbance of the well containing cells and medium.

[0120] The parallel absorbance of 6 wells is calculated respectively. The cell viability detection diagram of MC3T3-E1 cells is as follows Figure 11 As shown in the figure, when the concentration of DA-PAMAM-NH2 is 0.625 mg / mL to 20 mg / mL, the relative survival rate of cells decreases from 142% at 0.625 mg / mL to about 50% at 20 mg / mL, and the cell survival rate decreases significantly with the gradient increase of the concentration of DA-PAMAM-NH2, especially when the concentration of DA-PAMAM-NH2 is 20 mg / mL, the cell activity is greatly affected, and when the concentration of DA-PAMAM-NH2 is 10 mg / mL, the cell activity remains at about 90%,

[0121] The analysis and synthesis of DA inspired by the mussel protein have the ability to firmly adsorb on the surface of the implant, and the DA-PAMAM-NH2 high molecular compound is synthesized by introducing DA to the surface of PAMAM-NH2 by chemical synthesis. The introduction of DA enables DA-PAMAM-NH2 to firmly and durably adhere to the surface of the implant, and can effectively resist the flushing of SBF, which is a key factor to ensure the long-term stability of the implant; In the in-vitro environment, the DA-PAMAM-NH2 coating can maintain good antibacterial performance for a long time, and can induce the production of hydroxyapatite crystals, which is beneficial to the adhesion of osteoblasts and promotes bone bonding; At the same time, the coating can still maintain good long-acting antibacterial performance and osteogenic induction effect in the complex environment of the animal body. Therefore, the application of DA-PAMAM-NH2 dual functional coating will provide a new technology for effectively preventing and treating inflammation around the implant and poor bone bonding.

[0122] The present application mainly aims at the research of dopamine modified polyamide-amine dendrimer as an implant coating in reducing the occurrence of peri-implantitis and promoting the bone bonding around the implant, thereby desirably improving the clinical durability of the implant. The summary is as follows:

[0123] (1) From the mussel protein inspired DA with strong adsorption capacity, it is successfully introduced into the surface of PAMAM-NH2, and a new dendritic polymer material DA-PAMAM-NH2 with good biocompatibility is synthesized.

[0124] (2) DA-PAMAM-NH2 can be firmly adsorbed on the surface of titanium sheet, and still has strong adsorption capacity under the environment of human simulated body fluid immersion and flushing, which is the key to the long-acting of the bifunctional coating.

[0125] (3) DA-PAMAM-NH2 coating has excellent immediate and long-acting antibacterial performance in vitro, which is due to its strong adsorption capacity on the surface of titanium sheet; at the same time, DA-PAMAM-NH2 coating is more easy to induce the production of new bone on the surface of titanium sheet.

[0126] (4) The titanium implant loaded with DA-PAMAM-NH2 bifunctional coating has long-acting antibacterial performance and dual function of inducing osteogenesis in the model of rats with concurrent infection and bone defect, significantly reduces the incidence of postoperative infection of implant, promotes the bone integration around the implant, has good clinical application potential, and provides a new technology in the direction of improving the clinical life of implant.

[0127] The raw material cost of the method is low, the preparation process is simple, the reaction purity is high, the method can be realized by using conventional raw materials and equipment, and the industrialized production is easy to realize.

[0128] The above is only the preferred embodiment of the present application, and does not limit the present application. Any simple modification, change and equivalent change of the above embodiment according to the technical essence of the present application still belongs to the protection scope of the technical scheme of the present application.

Claims

1. A dopamine-modified polyamidoamine dendrimer, characterized in that, The structural formula of the dopamine-modified polyamide-amine dendrimer is: ； In the above structural formula, n is the number of grafted dopamine, n = 4-5.

2. A method of preparing the dopamine-modified poly(amido-amine) dendrimers of claim 1, characterized in that, The method is: S1, carboxylation of dopamine at the amino end: Under the condition of room temperature and normal pressure, dopamine is dissolved in pyridine, then succinic anhydride and 4-dimethylaminopyridine are added, after stirring and dissolving, the reaction is carried out for 6 hours, then the reaction solution is poured into ice ether for precipitation for 2 hours, the product is collected by filtration, and vacuum drying is carried out, to obtain carboxylated dopamine; the structural formula of the carboxylated dopamine is: ； S2, synthesis of carboxylated dopamine with a protecting group: Under the condition of 0℃, triethylamine, p-toluenesulfonyl chloride, DMAP and CH2Cl are added to the carboxylated dopamine obtained in S1, and the reaction is carried out for 6 hours under the condition of 0℃, to obtain carboxylated dopamine with a p-toluenesulfonyl protecting group, and the structural formula is: ； S3, synthesis of dopamine-modified polyamide-amine dendrimer with a protecting group Under the condition of room temperature and light shielding, the fifth generation polyamide-amine dendrimer is dissolved in methanol, then K2CO3 is added, and the reaction is carried out for 2 hours under the condition of room temperature, to obtain a reaction solution of dopamine-modified polyamide-amine dendrimer with a protecting group, and the structural formula of the dopamine-modified polyamide-amine dendrimer with a protecting group is: ； S4, synthesis of dopamine-modified polyamide-amine dendrimer: Under the protection of nitrogen, potassium tert-butoxide is added dropwise into the reaction solution obtained in S3, then poured into acetone, sealed under the condition of room temperature and light shielding, and precipitated overnight, the crude product is collected by filtration, then dissolved in dimethyl sulfoxide, transferred into a dialysis bag, dialyzed for 24 hours, the dialysate is collected, and freeze-drying is carried out, to obtain dopamine-modified polyamide-amine dendrimer, and the structural formula is: 。 3. The method of claim 2, wherein, The dosage ratio of dopamine, pyridine, succinic anhydride and DMAP in S1 is 50 mg:2 mL:39.20 mg:3.99 mg.

4. The method of claim 2, wherein, The equivalent ratio of triethylamine and DMAP in S2 is 60:

1.

5. The method of claim 2, wherein, The dosage ratio of the fifth generation polyamide-amine dendrimer, methanol, BOP and OTs-protected carboxylated dopamine in S3 is 50 mg:2 mL:1.54 mg:2.66 mg, and the equivalent ratio of BOP in S3 and carboxylated dopamine in S2 is 1:

5.

6. The method of claim 2, wherein, The molecular weight cut-off of the dialysis bag in S4 is 8000-14000 DA.

7. The method of claim 2, wherein, The preparation method of the fifth generation polyamide-amine dendrimer in S3 is: S301, synthesis of G 0.5 PAMAM-NH2 The methyl acrylate is dissolved in methanol under ice bath and nitrogen condition, then ethylenediamine is added, the reaction is terminated after stirring at 1000r / min for 48h under dark condition, then the dissolved and part of reaction residues are removed by distillation under reduced pressure to obtain the reaction product G 0.5 PAMAM-NH2; the molar ratio of the methyl acrylate and methanol is 2.4:1; S302, synthesis of G 1.0 PAMAM-NH2 The G 0.5 PAMAM-NH2 obtained in S301 is dissolved in methanol under ice bath and nitrogen condition, then ethylenediamine is added to the obtained solution under normal temperature, the reaction is terminated after stirring at 1000r / min for 48h under dark condition, then the dissolved and part of reaction residues are removed by distillation under reduced pressure to obtain the reaction product G 1.0 PAMAM-NH2; the molar ratio of the G 0.5 PAMAM-NH2 and ethylenediamine is 1:25; S303, synthesis of G 5.0 PAMAM-NH2 The G 1.0 PAMAM-NH2 obtained in S302 is added with ethylenediamine, and a higher generation of PAMAM-NH2 is prepared through continuous Michael addition reaction and amidation reaction until G 5.0 PAMAM-NH2, i.e. the fifth generation of polyamidoamine dendrimer, is synthesized.

8. Use of the method according to any one of claims 2 to 7 for the preparation of dopamine-modified poly(amidoamine) dendrimers, characterized in that, The dopamine-modified polyamidoamine dendrimer is used for implant coating to inhibit the occurrence of implant peri-implantitis, promote bone integration and improve long-term stability of the implant.

9. Use according to claim 8, characterized in that, The dopamine-modified polyamidoamine dendrimer is used as a long-acting bifunctional coating system firmly adsorbed on the surface of titanium sheet to improve the antibacterial property of the implant, promote peri-implant bone integration and improve the clinical durability of the implant.

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