CN107711828A - Silver/cation copolymer modified Nano diamond compound particle and preparation method thereof - Google Patents

Abstract

The invention discloses a silver/cation copolymer modified nano-diamond composite particle and a preparation method thereof. The composite particle is obtained by modifying the copolymer to alkylated nano-diamond, and then loading nano-silver on its surface. Wherein, the copolymer adopts the product after copolymerization of 4-vinylpyridine and hydroxyethyl methacrylate. The obtained antibacterial agent has good inhibitory and killing effects on Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus, and the corresponding minimum inhibitory concentrations are 50 μg/mL and 25 μg/mL respectively, and the inhibition zone Obviously, the 90min sterilization rate of Escherichia coli reaches 100%; the antibacterial agent prepared by the present invention can be used as an additive to join in some medical composite materials, and is used in the fields such as orthopedics, bone joint and/or internal prosthesis, especially for In situations where there is a high risk of infection, while enhancing its mechanical properties, it is endowed with excellent antibacterial properties, prolonging the service life of medical devices and reducing the risk of infection for patients.

Description

Silver/cationic copolymer modified nano-diamond composite particles and preparation method thereof

technical field

The invention belongs to the field of antibacterial agent synthesis and relates to a medical composite material, in particular to a silver/cation copolymer modified nano-diamond composite particle and a preparation method thereof.

Background technique

How to effectively prevent and treat post-operative infection has always attracted attention. The traditional method of treating bacterial infection is through injection or oral antibiotics. However, with the emergence of drug-resistant bacteria, the cure rate of traditional antibiotic therapy is decreasing year by year. In recent years, nano-antibacterial agents have been extensively studied by researchers as a new way to treat drug-resistant bacterial infections. Jang et al. (non-patent literature) prepared silica nanoparticles modified with small molecule quaternary ammonium salts, which greatly improved the antibacterial efficiency of quaternary ammonium salts, and their antibacterial properties gradually increased with the decrease of the particle size of silica. big.

As a new type of carbon-based material, nanodiamond (ND) has been widely used in polishing, superhard coating, imaging and drug delivery in the past few decades. Because of its good stability, excellent biocompatibility and extremely small particle size, it can be used as a good carrier for antibacterial agents. CN105253882A patent document uses polytetravinylpyridinium salt cationic homopolymer to carry out simple functional modification on ND, which not only solves the serious agglomeration problem of ND, but also endows ND with excellent antibacterial properties, opening up a new era for the application of ND in the field of antibacterial a new path. CN104987052A patent document mixes nano-diamonds and antibacterial agents into ceramics to obtain ceramics with enhanced mechanical strength and antibacterial properties. CN105802046A patent document uses the addition of nano-diamond and other raw materials to enhance the wear resistance of the rubber plate, and adds N-(trichloromethylsulfur)phthalimide, 1,2-benzisothiazoline- 3-ketone, so that the rubber has the function of antibacterial and antifungal. As an important class of inorganic nano-antibacterial agents, silver nanoparticles have broad antibacterial spectrum, high bactericidal efficiency, low toxicity to mammalian cells and are not easy to cause microbial drug resistance. CN1241662A patent document attaches ultrafine silver (Ag) with a particle size of 1 to 100 nanometers between the fibers of the fabric or on the fibers. The surface layer of the ultrafine silver is silver oxide, and the core is metallic silver. Sterilization is achieved by releasing Ag ⁺ effect. The Ag- _TiO2 coating invented by CN102497892A patent document is used in numerous existing medical tools and implants.

Existing research uses the simple physical mixing of nano-diamonds and antibacterial agents as nano-antibacterial materials. Among them, the nano-diamonds mixed directly without treatment have limited their ability to strengthen and toughen due to their large particle size and wide particle size distribution. ability. On the other hand, Ag is mostly directly deposited on porous materials such as fabrics and dressings, so the Ag nanoparticles obtained in this way inevitably have the problem of being easy to fall off, and the antibacterial ability of nano-silver antibacterial agents against Gram-positive bacteria is relatively insufficient.

Contents of the invention

The object of the present invention is to provide a high-efficiency composite antibacterial agent and its preparation method, which can be used in the biomedical industry to enhance the service life of medical devices and endow them with antibacterial properties.

The technical solution that realizes the object of the present invention is:

The silver/cationic copolymer modified nano-diamond composite particle of the present invention is obtained by modifying the copolymer to alkylated nano-diamond, and then loading nano-silver on its surface.

Further, the copolymer is a product obtained by copolymerization of 4-vinylpyridine and hydroxyethyl methacrylate.

Further, the alkylated nano-diamond is obtained by reacting the nano-diamond with bromopropyltrimethoxysilane in an aqueous alcohol solution.

Further, the modification is obtained by reacting the copolymer with alkylated nano-diamonds in ethanol, and using excess hexyl bromide to fully quaternize the copolymer.

Further, the loading amount of nano-silver is controlled at 50-60%.

The preparation method of above-mentioned composite particle, comprises the following steps:

Step 1, preparation of copolymer (PVP-co-HEMA): azobisisobutyronitrile (AIBN) as a thermal initiator, mixing 4-vinylpyridine and hydroxyethyl methacrylate, heating and stirring, purification and vacuum drying ;

Step 2, nanodiamonds react with bromopropyltrimethoxysilane in an aqueous alcohol solution, and the product (BrND) is centrifuged, washed, and dried;

Step 3, react the product obtained in step 2 with the copolymer described in step 1 in ethanol, and add excess bromohexane to fully quaternize the copolymer, and the product (QND) is centrifuged, washed, and dried;

Step 4, disperse the product obtained in step 3 in distilled water, mix with AgNO ₃ aqueous solution and boil, add reducing agent sodium citrate, boil together, centrifuge, wash and vacuum dry.

In a preferred embodiment, in step 1, the molar ratio of 4-vinylpyridine to hydroxyethyl methacrylate is 100:(1~100), the reaction temperature is 60° C., and the reaction time is 24 hours.

In a preferred embodiment, in step 2, ethanol in the alcohol aqueous solution, water volume ratio 9: 1, under the condition of a small amount of water, silane coupling agent can be hydrolyzed; The mass ratio of nano-diamond and alcohol aqueous solution ( 1~5 : 100), too large or too small mass fraction of nanodiamond in alcohol aqueous solution is not conducive to its full reaction with silane coupling agent.

In a preferred embodiment, in step 3, the product BrND obtained in step 2 and the copolymer PVP-co-HEMA described in step 1 are in a mass ratio of 100:(50~300), the reaction temperature is 50~80 ^o C, and the time is 10 ~24h.

In a preferred embodiment, in step 4, the mass ratio of nano-diamond, silver nitrate and sodium citrate in step 2 is 1:(1-1.5):(1-2).

The nano-diamond provided by the invention is used in composite materials, biomedical fields, especially in situations with high risk of infection.

Compared with the prior art, the present invention has the remarkable advantages of:

1. The present invention combines the polymer antibacterial agent of the cationic copolymer on the ND carrier, which can not only effectively avoid the loss of the polymer and the pollution to the environment, but also can be reused.

2. The small size and large specific surface area of cationic copolymer-modified nano-diamonds can provide more contact targets with bacteria, which can greatly improve the antibacterial ability of polymer antibacterial agents and increase the antibacterial efficiency.

3. The polymer acts as the active center to generate silver nanoparticles in situ, and through the coordination with the silver nanoparticles, the diameter of the prepared silver nanoparticles is relatively stable.

4. The antibacterial agent obtained by the present invention is a contact-type and release-type composite antibacterial agent, and has a fast and excellent antibacterial effect, and the 90-min sterilization rate for Escherichia coli reaches 100%.

5. Nano-diamond has a hard texture and good wear resistance. Adding it to resin and other materials may greatly improve the mechanical properties of the material.

6. The antibacterial agent prepared by the present invention can be added as an additive to some medical composite materials for fields such as orthopedics, bone joints and/or endoprostheses, especially for situations with a high risk of infection. The mechanical properties endow it with excellent antibacterial properties, which prolongs the service life of medical devices and reduces the risk of infection for patients.

Description of drawings

Figure 1 is the infrared spectra of (a) raw material ND, (b) QND, (c) QND@Ag in Example 1 of the present invention.

Fig. 2 is the XRD patterns of raw materials (a) ND, (b) QND, (c) QND@Ag in Example 1 of the present invention.

Fig. 3 is an enlarged transmission electron microscope image of (a) raw material ND, (b) QND, (c, d) QND @Ag in Example 1 of the present invention.

Fig. 4 is the hydrated particle size distribution diagram of raw materials ND, BrND, QND, QND@Ag in Example 1 of the present invention.

Figure 5 is the results of the inhibition zone experiment in Example 1 of the present invention (a) E. coli , (b) S. aureus : (1) blank control, (2) raw material ND, (3) QND, (4) QND @Ag.

Figure 6 is the antibacterial kinetics curve of Example 1 of the present invention.

detailed description

The preparation method of silver/cationic copolymer modified nano-diamond composite particle comprises the following steps:

a. Preparation of PVP-co-HEMA: Azobisisobutyronitrile (AIBN) was used as a thermal initiator, 4-vinylpyridine and hydroxyethyl methacrylate were mixed, heated and stirred, and the resulting mixture was precipitated in hexane three times and dried in vacuo.

b. ND was reacted with bromopropyltrimethoxysilane in aqueous alcohol solution, and the product (BrND) was centrifuged, washed and dried.

c.BrND reacts with PVP-co-HEMA in ethanol, and add excess hexyl bromide to fully quaternize PVP-co-HEMA, the product (QND) is centrifuged, washed, dried, and quaternized by adding excess hexyl bromide The degree is improved, the dispersibility of ND and the antibacterial performance of cationic polymer are improved.

d. Disperse the QND prepared above in distilled water, mix with AgNO ₃ aqueous solution and boil, add sodium citrate, boil together, centrifuge, wash, and vacuum dry, and the organic macromolecule is coordinated with silver nanoparticles to make The diameter of the prepared silver nanoparticles is relatively uniform and stable, and the particle diameter of the obtained silver nanoparticles is 20-50nm.

Example 1

Step a: Azobisisobutyronitrile AIBN is used as a thermal initiator to synthesize a copolymer by free radical polymerization. The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30. The mixture was stirred and heated in an oil bath at 60°C for 24 hours. Finally, the reaction mixture was precipitated three times in hexane and dried in a vacuum oven to obtain a pale yellow product.

Step b: Weigh 100mg of raw material ND and add it to 20mL alcohol aqueous solution (V _ethanol : V _water = 9:1), and then add silane coupling agent (bromopropyltrimethoxysilane) to it according to 10% of ND mass (10mg) , magnetically stirred at 50°C for 24h, the product was centrifuged, washed three times with deionized water, and dried in a vacuum oven at 60°C.

Step c: Dissolve 50mg BrND in 20ml ethanol solution, add 50mg copolymer PVP-co-HEMA, react at 70°C for 24h, then add excess bromide hexane (0.12g) to make it fully quaternized, centrifuge the product, and use Washed three times with ethanol, dried in vacuum at 50℃ for 12h to obtain quaternized modified nanodiamond (QND).

Step d: Disperse 50 mg of the QND prepared above in 20 mL of distilled water, mix with 100 mL of AgNO ₃ (60 mg) aqueous solution and boil, add 68 mg of sodium citrate (mass ratio, nanodiamond: silver nitrate: sodium citrate = 1:1.2:1.4), keep the solution boiling for 1 h. After centrifugation, washing with deionized water three times, vacuum drying at 60 °C for 24 h, the QND@Ag was obtained.

The obtained QND@Ag was characterized by infrared, XRD, TEM, and DLS, and compared with the raw materials ND, QND, etc.

It can be seen from Figure 1 that compared with ND original powder, the number of peaks in the infrared spectra of QND and QND@Ag has increased, of which 2880-2970 cm ^-1 is the stretching vibration peak of -CH ₃ and -CH ₂ , and 1601 cm ^-1 and 1569 cm ^-1 are the stretching vibration peaks of C=N, 1515 and 1466 cm ^-1 are the stretching vibration peaks of C=C, 1714 cm ^-1 is the stretching vibration peak of C=O, and 1045 cm ^-1 corresponds to the stretching vibration peak of COC Symmetrical stretching vibration peaks. In addition, since the peak of C=N ⁺ appeared at 1640 cm ^-1 , it indicated that the cationic polymer was successfully introduced into the nanodiamond surface. In addition, the comparison of QND @Ag and QND infrared spectra shows that: due to the formation of coordination bonds between Ag NPs and the hydroxyl and pyridine groups of the ND surface polymer, its infrared spectra are at 3400 cm ^-1 , 1601 cm ^-1 and 1569 cm The peaks at ^-1 are all significantly weakened, which proves that there is an interaction between the nano-Ag and the polymer grafted on the ND surface, and this interaction can stabilize the Ag NPs supported on the ND surface.

In Figure 2, 43 ^o and 75 ^o are the characteristic diffraction peaks of nano-diamond, corresponding to the (1 1 1) and (2 20) cubic crystal planes of nano-diamond, respectively. The crystal form of nano-diamond has no obvious effect after grafting polymer, but the intensity of diffraction peak of nano-diamond is weakened due to the wrapping of organic matter, which proves that nano-diamond has been successfully modified from another aspect. Due to the strong tendency of silver to crystallize, its peak covers the diffraction peak of nano-diamond. In the ^XRD diffraction pattern of ^{QND @ Ag} , silver (1 1 1 ), (2 0 0), (2 2 0), and (3 1 1) four crystal plane characteristic diffraction peaks, thus proving that silver nanoparticles were successfully loaded on the ND surface.

It can be seen from Figure 3 that the agglomerated particle size of the raw material ND reaches the micron level, and after the cationic copolymer is functionalized on its surface, the particle size is reduced to 10-100nm, and the deagglomeration effect is very obvious. This is because the functionalized ND surface has a large number of positive charges, the repulsive force between particles is greatly increased, which plays a role in deagglomeration, and this repulsive force hinders the re-agglomeration of nanoparticles. Figures (c) and (d) show that QND has successfully loaded Ag nanoparticles, which is consistent with the results of XRD. The particle size of the loaded Ag nanoparticles is relatively uniform, about 10-100 nm, and the composite nanoparticles after silver loading are still Maintain good particle dispersion performance. The hydrated particle size distribution diagram in Figure 4 is consistent with the TEM observation results, and the modified nano-diamond has a stable dispersion.

In terms of antibacterial properties, the composite antibacterial agent has obvious inhibition zones against both Escherichia coli and Staphylococcus aureus, as shown in Figure 5. The antibacterial kinetics test shows that QND@Ag can kill 100% of Escherichia coli within 90 minutes, as shown in Figure 6. In addition, the antibacterial activity of the compound antibacterial agent against Escherichia coli and Staphylococcus aureus with a colony concentration of 10 ⁶ CFU/mL was tested, and the minimum inhibitory concentration (MIC) was 50 μg/mL and 25 μg/mL respectively, showing good antibacterial activity. performance.

Embodiments 2~4 According to the experimental method of embodiment 1, different monomer molar ratios of 4-vinylpyridine and hydroxyethyl methacrylate, different silver nitrate concentrations, and different sodium citrate amounts are given to obtain different composites. antibacterial material.

The antibacterial agent prepared by the present invention can be added as an additive to some medical composite materials, and is used in the fields of orthopedics, bone joint and/or internal prosthesis, especially for situations with high risk of infection, in order to enhance its mechanical properties At the same time, it endows it with excellent antibacterial properties, prolongs the service life of medical devices, and reduces the risk of infection for patients.

Example 2

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 95/5.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.2: 1.4, keep the solution boiling for 1 h.

Example 3

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 90/10.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.2: 1.4, keep the solution boiling for 1 h.

Example 4

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 50/50.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.2: 1.4, keep the solution boiling for 1 h.

Example 5

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30.

Step d: mass ratio, nano-diamond: silver nitrate: sodium citrate=1:1:1, keep the solution boiling for 1 h.

Example 6

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate=1:1:2, keep the solution boiling for 1 h.

Example 7

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.5: 1, keep the solution boiling for 1 h.

Example 8

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.5: 2, keep the solution boiling for 1 h.

Example 9

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.2: 1.4, keep the solution boiling for 0.5 h.

Example 10

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30.

Step d: mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.2: 1.4, keep the solution boiling for 1.5 h.

Example 11

Step a: The molar ratio of 4-vinylpyridine monomer to hydroxyethyl methacrylate is 70/30.

Step d: Mass ratio, nano-diamond: silver nitrate: sodium citrate = 1: 1.2: 1.4, keep the solution boiling for 2 h.

After testing, Table 1 lists the minimum inhibitory concentration (MIC) and the size of the inhibition zone of the composite antibacterial materials given in Examples 1 to 4 respectively for Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) .

Claims (10)

1. Silver/cationic copolymer modified nano-diamond composite particle, it is characterized in that, by copolymer, the nano-diamond of alkylation is modified, then nano-silver is supported on its surface and obtains, and wherein, copolymer adopts 4- The product of copolymerization of vinylpyridine and hydroxyethyl methacrylate. 2. The composite particle according to claim 1, wherein the alkylated nano-diamond is obtained by reacting the nano-diamond with bromopropyltrimethoxysilane in an aqueous alcohol solution. 3. Composite particle as claimed in claim 1, is characterized in that, described modification is the nano-diamond reaction of copolymer and alkylation in ethanol, and adopts excessive hexyl bromide to make copolymer fully quaternized after getting. 4. The composite particle according to claim 1, wherein the loading of nano-silver is controlled at 50-60%. 5. The preparation method of the composite particle as described in any one of claims 1-4, is characterized in that, comprises the following steps: Step 1, preparation of the copolymer: azobisisobutyronitrile is used as a thermal initiator, 4-vinylpyridine and hydroxyethyl methacrylate are mixed, heated and stirred, purified, and vacuum-dried; Step 2, reacting nano-diamonds with bromopropyltrimethoxysilane in an aqueous alcohol solution, centrifuging the product, washing, and drying; In step 3, the product obtained in step 2 is reacted with the copolymer described in step 1 in ethanol, and excess bromohexane is added to fully quaternize the copolymer, and the product is centrifuged, washed, and dried; Step 4, disperse the product obtained in step 3 in distilled water, mix with AgNO ₃ aqueous solution and boil, add reducing agent sodium citrate, boil together, centrifuge, wash and vacuum dry. 6. The preparation method according to claim 5, wherein in step 1, the molar ratio of 4-vinylpyridine to hydroxyethyl methacrylate is 100:1~100, the reaction temperature is 60°C, and the reaction time is 24h. 7. the preparation method as claimed in claim 5, is characterized in that, in step 2, the ethanol in the alcohol aqueous solution, water volume ratio 9:1; The mass ratio 1～5:100 of nano-diamond and alcohol aqueous solution. 8. The preparation method according to claim 5, wherein in step 3, the product obtained in step 2 and the copolymer described in step 1 are reacted in ethanol at a mass ratio of 100:50~300, and the reaction temperature is 50~80 ^° C C, time 10~24h. 9. The preparation method according to claim 5, characterized in that, in step 4, the mass ratio of nano-diamond, silver nitrate, and sodium citrate described in step 2 is 1:1-1.5:1-2. 10. The antibacterial agent prepared by the composite particle as described in any one of claims 1-4.