CN109134754B

CN109134754B - Antibacterial micro-nano particles with adhesion function and preparation method and application thereof

Info

Publication number: CN109134754B
Application number: CN201810825106.3A
Authority: CN
Inventors: 朱韵; 邓舒丹; 董静; 顾衡
Original assignee: Chongqing Vocational Institute of Engineering
Current assignee: Chongqing Vocational Institute of Engineering
Priority date: 2018-07-25
Filing date: 2018-07-25
Publication date: 2020-06-05
Anticipated expiration: 2038-07-25
Also published as: CN109134754A

Abstract

The invention discloses a preparation method of an antibacterial micro-nano particle with an adhesion function. The prepared antibacterial particles have excellent antibacterial effect. The micro-nano particles are expected to be used as an antibacterial coating for improving the antibacterial property of biological materials, and the effects of effective adhesion and long-acting antibacterial are achieved. It has important significance for controlling bacterial infection and improving human life.

Description

Antibacterial micro-nano particles with adhesion function and preparation method and application thereof

Technical Field

The invention belongs to the field of antibacterial materials, and relates to an antibacterial micro-nano particle with an adhesion function, and a preparation method and application thereof.

Background

It is important to improve the antibacterial properties of the surface of the biomaterial because bacterial infection is often caused during the implantation of the biomaterial, which poses serious health risks to people. There are many strategies for improving the antibacterial properties of biological materials, and antibacterial strategies are generally constructed by studying the process of bacteria causing infection. Costerton et al found that bacteria first adhered to the surface of the material, then induced aggregation of the same species by secretion of signal molecules, and when the concentration of signal molecules increased to a certain level, bacterial cells were induced to secrete protein components constituting the extracellular matrix until a biofilm was finally formed. Subsequently, the biofilm gradually matures to form a cavity that releases planktonic bacteria, initiating a new process, forming an infection. The formation of the biofilm provides a protective layer for bacteria to maintain cell activity in the biofilm, but antibacterial substances outside the biofilm cannot contact with the biofilm, so that the resistance of the bacteria in the biofilm to antibacterial agents and the immune system of a human body is greatly enhanced, and the formation of the biofilm becomes the chief culprit of difficult cure of bacterial infection related to biological materials. Therefore, preventing the formation of bacterial biofilm on the surface of the biomaterial is the key to improve the antibacterial performance of the biomaterial, and the first step of biofilm formation is the adhesion of bacteria on the surface of the material, so that the prevention of the adhesion of bacteria on the surface of the material can effectively inhibit bacterial infection. The bacteria adhesion can be effectively inhibited by changing the hydrophilicity and the hydrophobicity of the body or the surface of the biological material, the main materials comprise polyethylene glycol, zwitterionic polymers and the like, and the bacteria adhesion is inhibited by forming a hydration layer on the surface of the material. Since the adhesion of a very small amount of bacteria can rapidly cause the formation of a biofilm, it is more common to graft an antibacterial group on the surface of a material or load an antibacterial substance into the material, which can rapidly kill a large amount of bacteria adhered to the surface and around the material, and prevent the occurrence of infection. A large number of researches show that the groups with positive charges can endow the material with good antibacterial property, such as spermidine quantum dots with amino-rich surfaces, amino-modified cellulose and the like. Commonly used antibacterial substances include inorganic silver, zinc dioxide, etc., organic synthetic quaternary ammonium salt, halamine, etc., and natural chitosan, antibacterial peptide, eugenol, etc. Wherein eugenol is a natural antibacterial agent extracted from plant flos Caryophylli, and has excellent antibacterial effect on gram-negative bacteria and gram-positive bacteria. The studies on eugenol are less than those of antibacterial agents having a long study history such as quaternary ammonium salts and chitosan, and therefore, it is necessary to conduct intensive studies.

Disclosure of Invention

In view of the above, the present invention provides a method for achieving the above object, and the method comprises the following steps:

the invention has the beneficial effects that: the method is characterized in that Dopamine Methacrylamide (DMA) is used as a monomer, Eugenol Methacrylate (EMA) is introduced as a second monomer to prepare a copolymerization micro-nano particle (P (DMA-co-EMA)) of the DMA and the EMA, the micro-nano particle has universal adhesion performance, and can be used as an antibacterial coating for improving the antibacterial property of a biological material, so that the effects of effective adhesion and long-acting antibacterial are achieved, and particularly, the antibacterial micro-nano particle has an excellent antibacterial effect on gram-negative bacteria. It has important significance for controlling bacterial infection and improving human life.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a scheme showing the synthesis scheme of P (DMA-co-EMA) copolymer.

FIG. 2 is an SEM image of P (DMA-co-EMA) at different EMA/DMA ratios.

FIG. 3 is a graph showing the adhesion effect of P (DMA-co-EMA).

FIG. 4 is a graph showing the antibacterial effect of P (DMA-co-EMA).

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

As shown in fig. 1, antibacterial micro-nano particles with an adhesion function are prepared by reacting monomeric dopamine methacrylamide and monomeric eugenol methacrylate in an ethanol/water mixed solution system under the initiation of an initiator azobisisobutyronitrile.

Example 1

1. Synthesis of Dopamine Methacrylamide (DMA)

10g of sodium borate and 4g of sodium carbonate are weighed and dissolved in 100mL of deionized water, and then the solution is added into a 250mL three-neck flask and stirred to be dissolved, so that the sodium borate and the sodium carbonate are supersaturated. Nitrogen bubbling for 20min removed the oxygen from the solution, and 5g dopamine hydrochloride was added and stirred continuously until dissolved. After 4.7mL of methacrylic anhydride was dissolved in 25mL of tetrahydrofuran, the solution was slowly added dropwise to a three-necked flask, and after the dropwise addition was completed, the pH was adjusted to 8 or more with 1M sodium hydroxide to protect the phenolic hydroxyl group of dopamine. The reaction was stirred at room temperature for 14h with aeration and the product was obtained as a white suspension. The post-treatment was as follows: and (3) filtering the reaction product by suction to remove redundant salt, adjusting the pH value to 2 by using 6M salt, extracting for 3 times by using 50mL ethyl acetate to obtain a yellow organic layer, drying by using anhydrous magnesium sulfate, filtering to obtain a clear organic layer, carrying out rotary evaporation to about 25mL, slowly dropwise adding 250mL n-hexane under stirring to obtain a gray suspension, and refrigerating and crystallizing in a refrigerator at 4 ℃. Filtering every other day, naturally drying to obtain gray powder, collecting the product, sealing, and refrigerating.

2. Synthesis of Eugenol Methacrylate (EMA)

6.56g of eugenol and 4.04g of triethylamine were weighed out and dissolved in 100mL of diethyl ether and introduced into a 250mL three-necked flask, and nitrogen was bubbled for 20min to remove oxygen. After 4.7g of methacryloyl chloride was dissolved in 25mL of diethyl ether in an ice bath, the solution was added dropwise slowly to the reaction flask. The reaction was stirred at room temperature with aeration for 48h and the product obtained was a yellow suspension. The post-treatment was as follows: and filtering the reaction liquid to remove triethylamine hydrochloride, washing the reaction liquid respectively by using a 5% sodium hydroxide solution and deionized water for 3 times, drying the reaction liquid by using anhydrous magnesium sulfate, and performing rotary evaporation to obtain a product, wherein the product is yellow liquid.

3. Synthesis of DMA and EMA copolymer P (DMA-co-EMA)

The total amount of fixed DMA and EMA is 0.1mol/L, and the solvent is a mixed solvent of ethanol and water with the volume ratio of 1/9 and the total volume is 10 mL. Weighing a proper amount of DMA and EMA in a 10mL polymerization tube, exhausting air and removing oxygen for 20min, and sealing. Weighing 0.0033g of AIBN, dissolving in 10mL of ethanol, introducing air for 20min, adding 1mL of initiator solution into a polymerization tube under the protection of nitrogen, continuously adding 9mL of deionized water which is introduced for 20min into the polymerization tube under the protection of nitrogen after reactants are completely dissolved, uniformly mixing, and standing for reaction for 8h at 70 ℃ in a dark place. The post-treatment was as follows: and precipitating the reaction product in a 50mL centrifuge tube containing 40mL deionized water, centrifuging for multiple times, removing a supernatant, and freeze-drying the precipitate for 24h to obtain a white powder as a final product. To explore the effect of EMA/DMA ratio on the response, a series of experiments were designed, with the specific experimental design shown in Table 1.

TABLE 1 amounts of monomers and initiators for different EMA/DMA ratios

As can be seen from fig. 2, as the second monomer EMA increases, the yield and particle size of the polymer particles gradually increase, and the morphology thereof significantly changes. After the addition of the second monomer EMA, the size of the copolymer P (DMA-co-EMA) was significantly smaller than that of the DMA homopolymer PDMA. This is because EMA is more hydrophobic than DMA, so that the copolymer of DMA and EMA has a lower solubility in the system than the DMA homopolymer, and a larger number of primary stable cores can be formed in a shorter time, and an increase in the number of primary stable cores inevitably leads to a decrease in the particle size of the final fine particles. FIG. 2 shows the EMA/DMA dose ratio n/n: SEM images of polymer particles at 3/7, 5/5, and 7/3. Obviously, the shape of the polymer particles is not greatly influenced when the EMA is added in a small amount, the spherical shapes in the systems of the graphs a-b are regular, and the particle size is slightly reduced along with the increase of the EMA. As EMA continues to increase, the regularity of the polymer particles is progressively affected (as shown in fig. 2 c), presumably because the hydrophobicity of the polymer particles increases, causing a large number of polymer particles to aggregate. Meanwhile, it was found experimentally that when EMA was further increased to an EMA/DMA ratio of 10/0(V/V), no polymer particles could be obtained and lumps were formed. In summary, when the monomer ratio EMA/DMA is 5/5(n/n), the polymer microspheres with regular morphology can be obtained.

Example 2

The total amount of fixed DMA and EMA is 0.15mol/L, and the solvent is a mixed solvent of ethanol and water with the volume ratio of 5/5 and the total volume is 10 mL. Weighing a proper amount of DMA and EMA in a 10mL polymerization tube, exhausting air and removing oxygen for 20min, and sealing. Weighing 0.00656g AIBN, dissolving in 10mL ethanol, introducing air for 20min, adding 1mL initiator solution into a polymerization tube under the protection of nitrogen, continuously adding 9mL deionized water which is introduced with air for 20min into the polymerization tube under the protection of nitrogen after reactants are completely dissolved, uniformly mixing, and standing and reacting for 10h at 80 ℃ in a dark place. The post-treatment was as follows: and precipitating the reaction product in a 50mL centrifuge tube containing 40mL deionized water, centrifuging for multiple times, removing a supernatant, and freeze-drying the precipitate for 24h to obtain a white powder as a final product.

Example 3

The total amount of fixed DMA and EMA is 0.08mol/L, and the solvent is 10mL of a mixed solvent with the volume ratio of ethanol to water being 1/10. Weighing a proper amount of DMA and EMA in a 10mL polymerization tube, exhausting air and removing oxygen for 20min, and sealing. Weighing 0.00495g AIBN, dissolving in 10mL ethanol, introducing air for 20min, adding 1mL initiator solution into a polymerization tube under the protection of nitrogen, continuously adding 9mL deionized water which is introduced with air for 20min into the polymerization tube under the protection of nitrogen after reactants are completely dissolved, uniformly mixing, and standing for reaction for 6h at 75 ℃ in a dark place. The post-treatment was as follows: and precipitating the reaction product in a 50mL centrifuge tube containing 40mL deionized water, centrifuging for multiple times, removing a supernatant, and freeze-drying the precipitate for 24h to obtain a white powder as a final product.

Example 4 adhesion Performance testing

Cutting the titanium sheet into a specification of 1.5cm multiplied by 1.5cm, performing ultrasonic treatment for 10min by using deionized water, absolute ethyl alcohol and acetone respectively to clean the surface of the titanium sheet, and drying the titanium sheet by using nitrogen for later use. 20mg of the polymer particles obtained in the R3 protocol in example 1 were weighed and dispersed in 10mL of ethanol, and the cleaned titanium sheets were respectively placed in each suspension and stirred at normal temperature for 24h, and the cleaned titanium sheets were used as blank control. And (3) respectively washing the titanium sheet with deionized water and ethanol, drying the titanium sheet with nitrogen, and observing the surface appearance of the titanium sheet through a scanning electron microscope to determine whether the polymer particles are adhered to the titanium sheet.

Fig. 3 shows SEM images of the surface of a pure titanium sheet and a surface of a titanium sheet to which P (DMA-co-EMA) was attached, respectively (microscopic in fig. b, macroscopic in fig. c). As can be seen, the surface of the pure titanium sheet is smooth and clean (as shown in FIG. 3 a). In contrast, the surface of the titanium sheet with P (DMA-co-EMA) adhered to the surface (as shown in FIG. 3 b), the polymer particles are clearly visible, indicating that the polymer particles successfully adhered to the surface of the titanium sheet and remained spherical.

Example 5 antimicrobial Property test

The antibacterial performance of P (DMA-co-EMA) is evaluated by measuring the Minimum Inhibitory Concentration (MIC), and gram-negative bacterium Escherichia coli (E.coli) is selected as an experimental strain. Experiment the experiment of the minimum inhibitory concentration was carried out using escherichia coli as the experimental species, and the experimental results are shown in fig. 4. As can be seen from FIG. 4, a-f respectively show the antibacterial test results of the concentrations of P (DMA-co-EMA) of 40, 20, 10, 5, 2.5 and 0mg/mL, and it can be seen from the graph that P (DMA-co-EMA) has a very good antibacterial effect on Escherichia coli compared with the control group f, and the antibacterial effect is enhanced with the increase of the concentration of P (DMA-co-EMA).

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A preparation method of antibacterial micro-nano particles with an adhesion function is characterized in that monomer dopamine methacrylamide and monomer eugenol methacrylate are reacted in an ethanol/water mixed solution system under the initiation of initiator azobisisobutyronitrile to prepare the antibacterial micro-nano particles with the adhesion function; the total concentration of the monomers is 0.1mol/L, the volume ratio of the ethanol to the water is 1:9, and the concentration of the initiator is 2% of the molar weight of the monomers; the molar ratio of the dopamine methacrylamide to the eugenol methacrylate is 1: 1.

2. The preparation method of the antibacterial micro-nano particles with the adhesion function according to claim 1, wherein the reaction is performed with oxygen removal before starting and is performed under inert gas protection and in a dark place.

3. The preparation method of the antibacterial micro-nano particles with the adhesion function according to claim 1, wherein the reaction condition is that the reaction is carried out at 70-80 ℃ for 6-10 h.

4. The preparation method of the antibacterial micro-nano particles with the adhesion function according to claim 1, wherein a product is precipitated in water after the reaction is finished, and the precipitate is freeze-dried.

5. The antibacterial micro-nano particles with the adhesion function prepared by the preparation method of the antibacterial micro-nano particles with the adhesion function according to any one of claims 1 to 4.

6. The use of the antibacterial micro-nano particle with adhesion function of claim 5 in resisting gram-negative bacteria.

7. The use according to claim 6, wherein the gram-negative bacterium is E.