CN115873233A - Multifunctional polypeptide polymer and preparation method and application thereof - Google Patents
Multifunctional polypeptide polymer and preparation method and application thereof Download PDFInfo
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- CN115873233A CN115873233A CN202211578379.5A CN202211578379A CN115873233A CN 115873233 A CN115873233 A CN 115873233A CN 202211578379 A CN202211578379 A CN 202211578379A CN 115873233 A CN115873233 A CN 115873233A
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A multifunctional polypeptide polymer and a preparation method and application thereof, wherein the multifunctional polypeptide polymer comprises the following raw materials: polyethylene glycol, and a targeting antimicrobial peptide capable of completely reacting the polyethylene glycol; the tail end of the targeted antibacterial peptide is provided with a sulfydryl, the polyethylene glycol is polyethylene glycol with a methoxy end cap and a maleimide end group, and the sulfydryl at the tail end of the targeted antibacterial peptide and maleimide of the polyethylene glycol are subjected to Michael addition reaction to synthesize the polypeptide polymer. The targeting antibacterial peptide adopted by the invention has weaker self-assembly antibacterial performance, forms a polypeptide polymer after grafting polyethylene glycol, mainly plays a role in resisting pollution under a neutral condition, and reduces the adhesion of nonspecific molecules such as bacteria, protein and the like to maintain the health of teeth; once the pH value of the oral cavity is changed, arginine and tryptophan in the antibacterial peptide molecules show an acidic self-enhancing antibacterial function along with the change of the pH value, and can effectively kill harmful flora by cooperating with the osmotic function of polyethylene glycol.
Description
Technical Field
The invention relates to the field of oral cavity high molecular materials, in particular to a multifunctional polypeptide polymer and a preparation method and application thereof.
Background
In a healthy oral environment, beneficial flora and harmful flora are in a dynamic equilibrium state, and once the oral flora is unbalanced, the pH value of the oral cavity is changed to cause bacteria-related oral diseases. In the oral environment, oral mucosa, tongue surface, dental restorations and tooth surfaces, especially near the gingival margin, are present in large amounts of microorganisms and proteins, which are easily cleared from their surfaces due to rapid epithelial tissue turnover metabolism (three times daily). In contrast, in the absence of metabolic renewal of teeth, dentures, and implants, microbial masses, saliva, and food residues, i.e., dental plaque, adhered to the surface of teeth are difficult to remove, and biofilm is easily formed particularly in areas lacking a self-cleaning function, which may cause dental diseases such as caries, periodontitis, and implant implantation failure. To prevent the development of periodontal diseases such as caries and periodontitis, it is an effective method to cut the formation of oral biofilms.
However, on one hand, due to the dilution and flow of a large amount of oral saliva and the existence of enzyme, most antibacterial agents are inactivated due to the retention in the oral cavity and short acting time, and cannot achieve the ideal antibacterial effect; on the other hand, the effect of killing harmful flora only and not affecting the activity of beneficial flora is achieved without any antibacterial agent; therefore, the design of a material which has a targeting effect and is adaptive to antibiosis along with the change of the oral environment has great research significance.
Disclosure of Invention
Based on the above, the invention provides a multifunctional polypeptide polymer, and a preparation method and application thereof, so as to solve the technical problem that the existing antibacterial agent applied to oral cavity tooth care is greatly influenced by the oral environment, so that the antibacterial effect is not ideal.
In order to achieve the above object, the present invention provides a multifunctional polypeptide polymer, which comprises the following raw materials: polyethylene glycol, and a targeting antimicrobial peptide capable of completely reacting the polyethylene glycol; the tail end of the targeted antibacterial peptide is provided with a sulfydryl, the polyethylene glycol is methoxy-terminated and maleimide terminal group modified polyethylene glycol, and the sulfydryl at the tail end of the targeted antibacterial peptide reacts with maleimide of the polyethylene glycol to synthesize a polypeptide polymer.
As a further preferable technical scheme, the amino acid sequence of the targeted antibacterial peptide is DDDEEKRWRWC, and the molecular weight is 1880.01g/mol.
In a further preferred embodiment of the present invention, the molecular weight of the polyethylene glycol is 2000 to 5000g/mol.
In a further preferred embodiment of the present invention, the molar ratio of the targeting antibacterial peptide to the polyethylene glycol is 1.5 to 3.
According to another aspect of the present invention, there is provided a method for preparing the above multifunctional polypeptide polymer, comprising the steps of:
s1, introducing nitrogen into a phosphate buffer solution with the pH = 7.2-7.4, and deoxidizing;
s2, respectively dissolving polyethylene glycol and the targeted antibacterial peptide by using the deoxidized phosphate buffer solution to obtain a polyethylene glycol solution and a targeted antibacterial peptide solution;
and S3, slowly dripping the targeted antibacterial peptide solution into a polyethylene glycol solution for addition reaction, dialyzing and freeze-drying to obtain the polypeptide polymer.
In a more preferred embodiment of the present invention, in step S1, the pH of the phosphate buffer =7.2, and the nitrogen gas is supplied for 30min.
As a further preferred embodiment of the present invention, in step S3, the conditions of the addition reaction are: and reacting for 12 hours at room temperature under an inert atmosphere.
According to another aspect of the present invention, the present application also provides a use of the above multifunctional polypeptide polymer for preparing a mouthwash, or spraying the polypeptide polymer onto a tooth surface to form a polypeptide polymer-modified tooth surface.
The multifunctional polypeptide polymer and the preparation method and the application thereof can achieve the following technical scheme
Has the advantages that:
1) The multifunctional polypeptide polymer with targeting, oral environment self-adaptive antibacterial, antifouling and the like is synthesized by a simple and mild method, and is applied to dental care to reduce the adhesion definite value of non-specific molecules such as proteins and bacteria on the tooth surface and inhibit the formation of dental plaque, so that bacterial infectious periodontal disease is prevented;
2) The targeting antibacterial peptide adopted by the invention has weaker self-assembly antibacterial performance, forms a polypeptide polymer after being grafted with polyethylene glycol, mainly plays an anti-fouling effect under a neutral condition, and reduces the adhesion of non-specific molecules such as bacteria, protein and the like to maintain the health of teeth; once the pH value of the oral cavity is changed, arginine and tryptophan in the antibacterial peptide molecules show an acidic self-enhanced antibacterial function along with the change of the pH value, and can effectively kill harmful flora in cooperation with the penetration function of polyethylene glycol; deprotonation is generated along with the increase of the pH value of the oral cavity after the harmful flora is removed, so that the damage of the antibacterial property of the oral cavity to the beneficial flora is reduced, and the balance of the whole oral flora is maintained;
3) The polypeptide polymer of the present invention can be bound to the component Ca of teeth 2+ The polypeptide polymer is expected to be prepared into mouthwash or sprayed on the surface of teeth to form the polypeptide polymer modified tooth surface.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a schematic diagram of the synthesis of the polypeptide polymer DRW-PEG in example 1.
FIG. 2 is a schematic diagram of the synthesis of the polypeptide polymer DC-PEG in example 2.
FIG. 3 is a nuclear magnetic hydrogen spectrum of the polypeptide polymer in example 1, 2.
FIG. 4 is a time-of-flight mass spectrum of the polypeptide polymer of example 1, 2.
FIG. 5 shows the antimicrobial properties of various polypeptide polymers and polypeptides of example 3.
FIG. 6 is a graph of the antimicrobial properties of the polypeptide polymer of example 4 at various pH values.
FIG. 7 is a graph of saturation adsorption concentration and targeted fluorescence for the polypeptide polymer of example 5.
FIG. 8 is the anti-fouling performance of the example 6 polypeptide polymer.
FIG. 9 is the in vitro cytotoxicity of the polypeptide polymer of example 7 on HOK cells.
The objects, features and advantages of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are all conventional methods unless otherwise specified.
The invention provides a multifunctional polypeptide polymer, which comprises the following raw materials: polyethylene glycol, and a targeting antimicrobial peptide capable of completely reacting the polyethylene glycol; the tail end of the targeted antibacterial peptide is provided with a sulfydryl, the polyethylene glycol is methoxy-terminated and maleimide terminal group modified polyethylene glycol, the sulfydryl at the tail end of the targeted antibacterial peptide and maleimide of the polyethylene glycol are subjected to Michael addition reaction to synthesize a polypeptide polymer, wherein excessive targeted antibacterial peptide ensures complete reaction of the polyethylene glycol. Preferably, the molar ratio of the targeting antibacterial peptide to the polyethylene glycol is 1.5-3.
Preferably, the amino acid sequence number of the targeted antibacterial peptide is DDDEEKRWRWRWC, and the molecular weight is 1880.01g/mol; the molecular weight of the polyethylene glycol is 2000-5000 g/mol. Wherein, the larger the molecular weight of the polyethylene glycol is, the longer the molecular chain is, which leads to the reduction of the solubility, and when the molecular weight of the polyethylene glycol is 2000g/mol, which is grafted to the surface of the targeted antibacterial peptide, the antifouling property is the best.
Example 1
The preparation method of the multifunctional polypeptide polymer (denoted as DRW-PEG) of this example has the following synthetic steps, as shown in FIG. 1:
ventilating PBS under liquid for 30min to remove oxygen in the solution; 0.05mmol polyethylene glycol PEG (Mn =2000g/mol,100 mg) was dissolved in 3ml deoxygenated PBS (in a 10ml reaction tube, aerated for 15min under nitrogen protection); then, 0.075mmol of DRW (Mn =1880.01g/mol,141 mg) was dissolved in 2ml of PBS, and then added dropwise to the reaction tube to react at room temperature for 12 hours, and then dialyzed with a dialysis bag (MWCO = 2500), and after one day, the sample was lyophilized to obtain the final product DRW-PEG, which was stored in a refrigerator at-20 ℃.
Wherein, DRW is targeted antibacterial peptide, the sequence is DDDEEKRWRWRWC, and the molecular weight is 1880.01g/mol.
Comparative example 1
This comparative example provides a comparative experiment to example 1, and provides a control for the preparation of a polypeptide polymer (designated DC-PEG) starting from a targeting peptide (DC), the synthetic route of which is shown in figure 2 and is as follows:
ventilating PBS liquid for 30min to remove oxygen in the solution; 0.05mmol polyethylene glycol PEG (Mn =2000g/mol,100 mg) was dissolved in 3ml deoxygenated PBS (in a 10ml reaction tube, aerated for 15min under nitrogen protection); then, 0.075mmol of DDDEEKC (DC) (Mn =852.8g/mol,64 mg) was dissolved in 2ml of PBS, and then added dropwise to the reaction tube to react at room temperature for 12 hours, followed by dialysis with a dialysis bag (MWCO = 2500) for one day, after which the sample was lyophilized to obtain the final product DC-PEG, which was stored in a refrigerator at-20 ℃.
Wherein, DC is a targeting peptide without antibacterial function, the sequence of the targeting peptide is DDDEEKC, and the molecular weight is 852.8g/mol.
Fig. 3 and 4 are nuclear magnetic and mass spectra of example 1 and comparative example 1.
To further demonstrate the beneficial technical effects of the multifunctional polypeptide polymer of the present invention (denoted as DRW-PEG), the following tests were performed:
test 1
In this test, the above synthesized DC-PEG and DRW-PEG were compared.
Firstly, in order to detect different polypeptide polymers and antibacterial activity of the polypeptide, various polypeptides and polypeptide polymer coatings are constructed on the surface of the dental film. All dental pieces were dipped into a solution containing S.mutans (10) 6 CFU/mL) for 12h, washing the bacterial suspension with PBS 3 times, staining with LIVE/DEAD backlight kit reagent for 15min at room temperature in the dark, washing gently 3 times, and observing with laser confocal microscope.
According to the experimental result, a large amount of live bacteria (green) are adhered to the surface of the blank dental film; DC, although increasing the hydrophilicity of the coating to some extent, still has a large amount of live bacteria adhering to its surface; the DRW group has a large amount of positive charges on the surface, so a large amount of dead bacteria (in red) are adhered to the surface of the DRW group, and the accumulation of the dead bacteria can also shield the antibacterial efficiency of the coating to lose the function of the DRW group along with the prolonging of time; the DC-PEG and DRW-PEG groups make it difficult for bacteria to adhere to their surfaces because PEG rapidly forms a hydrated layer by interacting with water, and there is only a very small amount of bacteria adhering to their surfaces on both surfaces; in contrast, the surface of DC-PEG is adhered with green living bacteria, more bacteria are adhered to the surface of DC-PEG possibly along with the prolonging of time to form a biological film, while the surface of DRW-PEG is mostly killed dead bacteria, and the DRW-PEG cannot develop into the biological film.
FIG. 5 shows the antimicrobial properties of various polypeptide polymers and polypeptides.
Test 2
In the test, an experimental detection method for the antibacterial performance of the polypeptide polymer DRW-PEG under different pH (7.4, 5.5, 4) values is provided, which comprises the following steps:
in order to test the antibacterial activity of the polypeptide polymer at different pH values, 10 is included 6 Solutions of streptococcus mutans (PBS, 0.05M) at different pH values (pH =7.4,5.5, 4) at CFU/mL were incubated with different concentrations of polypeptide polymer (0.125, 0.25, 0.5, 1,2 mM) at 37 ℃ for 4,12h and plated to test for antibacterial performance.
With the change of pH, tryptophan and arginine contained in DRW-PEG molecules can be (de) protonated, so that the dry powder has a self-adaptive antibacterial function, the damage of materials to surrounding mucosal tissues is reduced, and the balance and diversity of oral flora are maintained.
Fig. 6 shows the antibacterial performance of the polypeptide antibacterial polymer at different pH.
Test 3
In the test, the saturated adsorption concentration detection of the polypeptide polymer and the synthesis experiment method of the FITC marked polypeptide polymer under different pH (7.4, 5.5 and 4) values are provided, and the steps are as follows:
(1) Detection of saturation adsorption concentration of polypeptide Polymer at different pH (7.4, 5.5, 4) values
In order to quantitatively detect the saturated adsorption amount of the polypeptide polymer per unit of Hydroxyapatite (HA), 50mg of HA powder was added to PBS solutions (pH =7.4,5.5, 4) of various concentrations of 0.25-7mg/ml, the mixture was stirred at 37 ℃ for 24 hours, centrifuged for 3min (10000 rpm/min), the supernatant was extracted, and then the absorbance at 210nm was measured using a microplate reader, and a standard curve was drawn to calculate the apparent adsorption amount. Dropping the water solution with various saturated adsorption concentrations on the surface of HA or dental film (taken from human or animal teeth), incubating for 4h at 37 ℃, washing for 3 times by using ultrapure water, drying by using nitrogen, and preparing the functional coating for subsequent test characterization.
(2) FITC-labeled polypeptide polymer
First, 5mg/ml PEG, polypeptide and polypeptide polymer solutions were prepared with 0.1M carbonate buffer (pH = 9); FITC was dissolved in DMSO (1 mg/ml), the FITC solution was slowly added dropwise to the stirring polypeptide solution (FITC: polypeptide =1, medium) with a syringe, reacted at room temperature for 24h and dialyzed for 2-3 days until colorless, lyophilized and stored at 4 ℃ in the dark for use. Preparing various solutions of 1mg/mL FITC marker, placing a dental film (taken from teeth of human or animals) which is polished smooth by sand paper into a 24-hole plate, adding 1mL of the solution, standing for 1h, washing with ultrapure water for 3 times, and performing laser confocal test at the wavelength of 488 nm.
According to the experimental result, PEG is adsorbed on HA only in a trace amount due to lack of the target binding capacity with HA, while DRW-PEG can be combined with Ca 2+ The adsorption capacity is higher due to electrostatic interaction, and the adsorption capacity is increased along with the reduction of pH, because the polypeptide contains abundant carboxyl groups, the increase of the pH value leads the deprotonation of the polypeptide, the negative potential of the HA surface is increased along with the increase of the pH value, and the enhanced electrostatic repulsion is generated between the polypeptide and the HA so as to block the adsorption process.
FIG. 7 is a graph of saturation adsorption concentration and targeted fluorescence of polypeptide polymers.
In the test, an antifouling performance detection experimental method for adsorbing a polypeptide polymer on the surface of a dental film is provided, which comprises the following steps:
dental films with dental surfaces modified with the polypeptide polymer of the present invention were prepared according to test 3.
The prepared dental plaque with the polypeptide polymer coating was immersed in PBS fresh PBS for 1h at room temperature and then immersed in 1mL of BSA and lysozyme (Ly) solution (2 mg/mL), incubated at 37 ℃ for 2h, and then the dental plaque was washed away from non-adhered proteins by gently rinsing with PBS 3 times. Then adding 300 mu L SDS solution, carrying out ultrasonic treatment for 20min to remove the adhered bacteria, and finally detecting the protein content by using a BCA kit; to more visually indicate the anti-protein adhesion ability of the surface, 2mg/mL FITC-labeled BSA and Ly were incubated with the dental plaque under the same conditions for 2h, and after 3 gentle washes with PBS, the amount of protein adhesion on the surface was observed with CLSM.
According to the experimental results, the PEG-containing coatings all had excellent anti-protein adhesion properties, except that the amount of lysozyme adhered to the surface of the blank tooth was greater due to the negative charge on the tooth surface compared to the negatively charged BSA.
FIG. 8 is a graph of the antifouling performance of a polypeptide polymer coating.
Test 5
In this embodiment, an experimental method for in vitro cytotoxicity of a polypeptide polymer coating is provided, which includes the following steps:
cytotoxicity of the coating was tested using Oral Keratinocytes (HOK) 2X 10 5 After culturing in a carbon dioxide incubator for 24h, the sterilized dental film and the cells are incubated for 1d and 3d, and then the OD value at 450nm is detected by a microplate reader, and the Cell activity (Cell viability) is calculated, the calculation formula is as follows:
according to the experimental result, after the HOK cells and the polypeptide polymer coating are co-cultured for 24h and 72h, the cell survival rate is over 85 percent, and the difference with the blank control group is not significant, so that the polypeptide polymer coating is proved to have good biocompatibility and can be applied to oral antibiosis, antifouling and prevention of formation of related dental diseases.
Fig. 9 is a graph of the cytotoxicity of the polypeptide polymer coating.
The invention also provides application of the multifunctional polypeptide polymer, wherein the polypeptide polymer is applied to preparing mouthwash or is sprayed on the surface of teeth to form the polypeptide polymer modified tooth surface.
When the polypeptide polymer is dissolved in a solvent (water) and used as a mouthwash, the polypeptide polymer is used as an antibacterial agent and is targeted to the tooth surface for antibacterial and antifouling when the mouthwash is used for gargling.
The polypeptide polymer coating can be formed on the tooth surface by spraying the polypeptide polymer on the tooth surface, and the using method comprises the following steps:
grinding the tooth surfaces of the teeth by using abrasive paper with different meshes; and (3) dropwise adding a solution of the polypeptide polymer with a certain concentration on the surface of the tooth surface, cleaning for three times after adsorption, and blow-drying by using nitrogen to obtain the polypeptide polymer modified tooth surface.
Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely examples and that many variations or modifications may be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims.
Claims (8)
1. A multifunctional polypeptide polymer is characterized by comprising the following raw materials: polyethylene glycol, and a targeting antimicrobial peptide capable of completely reacting the polyethylene glycol; the tail end of the targeted antibacterial peptide is provided with a sulfydryl, the polyethylene glycol is methoxy-terminated and maleimide terminal group modified polyethylene glycol, and the sulfydryl at the tail end of the targeted antibacterial peptide and maleimide of the polyethylene glycol are subjected to Michael addition reaction to synthesize the polypeptide polymer.
2. The multifunctional polypeptide polymer of claim 1 wherein the amino acid sequence of the targeting antibacterial peptide is dddeekrrwrwrwrwrwc with a molecular weight of 1880.01g/mol.
3. The multifunctional polypeptide polymer of claim 1 wherein said polyethylene glycol has a molecular weight of 2000 to 5000g/mol.
4. The multifunctional polypeptide polymer of claim 1 wherein the molar ratio of the targeting antimicrobial peptide to the polyethylene glycol is 1.5 to 3.
5. A method for preparing a multifunctional polypeptide polymer according to any one of claims 1 to 4 comprising the steps of:
s1, introducing nitrogen into a phosphate buffer solution with the pH = 7.2-7.4, and deoxidizing;
s2, respectively dissolving polyethylene glycol and the targeted antibacterial peptide by using the deoxidized phosphate buffer solution to obtain a polyethylene glycol solution and a targeted antibacterial peptide solution;
and S3, slowly dripping the targeted antibacterial peptide solution into a polyethylene glycol solution for addition reaction, dialyzing and freeze-drying to obtain the polypeptide polymer.
6. The method for preparing the multifunctional polypeptide polymer of claim 5, wherein the pH of the phosphate buffer solution is =7.2 and the nitrogen gas is introduced for 30min in step S1.
7. The method for preparing the multifunctional polypeptide polymer of claim 5, wherein in step S3, the addition reaction conditions are as follows: and reacting for 12 hours at room temperature under an inert atmosphere.
8. The use of the multifunctional polypeptide polymer of claim 1 wherein said polypeptide polymer is used in the preparation of a mouthwash or sprayed onto a tooth surface to form a polypeptide polymer modified tooth surface.
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| CN202211578379.5A CN115873233B (en) | 2022-12-09 | Multifunctional polypeptide polymer and preparation method and application thereof |
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| CN202211578379.5A CN115873233B (en) | 2022-12-09 | Multifunctional polypeptide polymer and preparation method and application thereof |
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| CN117100621A (en) * | 2023-10-24 | 2023-11-24 | 山东一飞环保材料科技有限公司 | Antibacterial nanofiber dry mask and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117100621A (en) * | 2023-10-24 | 2023-11-24 | 山东一飞环保材料科技有限公司 | Antibacterial nanofiber dry mask and preparation method thereof |
| CN117100621B (en) * | 2023-10-24 | 2024-01-09 | 山东一飞环保材料科技有限公司 | Antibacterial nanofiber dry mask and preparation method thereof |
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