CN115414386A - Preparation method of bioactive glass nanocomposite with catalytic antibacterial performance - Google Patents

Preparation method of bioactive glass nanocomposite with catalytic antibacterial performance Download PDF

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CN115414386A
CN115414386A CN202211122821.3A CN202211122821A CN115414386A CN 115414386 A CN115414386 A CN 115414386A CN 202211122821 A CN202211122821 A CN 202211122821A CN 115414386 A CN115414386 A CN 115414386A
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bioactive glass
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CN115414386B (en
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薛语萌
徐子琪
尚利
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Northwestern Polytechnical University
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Abstract

The invention belongs to the technical field of preparation of nano biological materials, and discloses a preparation method of a bioactive glass nano composite material with catalytic antibacterial property, which comprises the following steps: preparing bioactive glass nanoparticles by a microemulsion method; the gold nanoclusters are subjected to positive electrification and then react with the bioactive glass nanoparticles under stirring. The invention firstly utilizes the positive-electrochemical gold nanoclusters to modify the bioactive glass nanomaterial to endow the bioactive glass nanomaterial with high-efficiency enzyme catalysis property and antibacterial property. The preparation method of the bioactive glass nano composite material is simple and easy to implement, the reaction condition is mild, complex instruments and equipment are not needed, and the prepared bioactive glass nano composite material not only has excellent biocompatibility, catalase-like catalysis and antibacterial activity, but also has the functions of fluorescence imaging, cell behavior regulation and control and the like, and has great application potential in the biomedical fields of tissue regeneration and the like.

Description

Preparation method of bioactive glass nanocomposite with catalytic antibacterial performance
Technical Field
The invention belongs to the technical field of preparation of nano biological materials, and relates to a preparation method of a bioactive glass nano composite material with catalytic antibacterial performance.
Background
The bioactive glass is made of SiO 2 、CaO、P 2 O 5 The typical silicate material with the same composition can play an important role in various stages of wound healing, and various commercial products based on bioactive glass are used for wound healing at present. However, the antibacterial performance of the existing novel dressing based on bioactive glass is not very obvious and the function is single, so that the application requirement of a biological system in a complex environment is difficult to meet. Therefore, there is a need for further functionalization and modification of bioactive glass to enhance its efficacy in the treatment of complex wounds.
In order to improve the performance of bioactive glass in the treatment of infectious wounds, its antibacterial performance is generally achieved mainly by two ways: (1) In the process of preparing the bioactive glass, metal ions with antibacterial ability are directly doped into a glass network, and the aim of killing bacteria is achieved along with continuous dissolution of the ions in the later period. Common dopant ion packetsAg drawing + 、Cu 2 + 、Zn 2+ And the like, the metal ions can kill bacteria through various mechanisms such as destroying the bacterial membrane structure, inhibiting protein synthesis, damaging DNA and RNA and generating Reactive Oxygen Species (ROS). (2) The antibiotic and other medicines are loaded in the bioactive glass to improve the antibacterial activity of the bioactive glass. The bioactive glass nano material with the mesoporous structure and the Si-OH rich on the surface thereof can further realize the gradual release of the medicine in a physiological environment through the physical adsorption, the hydrogen bond action and the van der Waals force and the medicine action, thereby playing the role of sterilization.
Although the ion-doped bioactive glass has certain antibacterial activity, the ion release process is uncontrollable and the potential toxicity is large, which greatly limits the further application and clinical transformation of the ion-doped bioactive glass in the treatment of infectious wounds. The bioactive glass is an ideal choice for loading the antibacterial drugs, and has obvious burst release when the antibiotics are released, so that the long-acting loading and slow release of the antibiotics cannot be realized, and the safe and long-acting antibacterial effect is difficult to realize. In addition, because the antibiotics are widely used for a long time, the bacteria easily generate drug resistance, and the treatment effect of the drugs is obviously reduced. Therefore, a novel bioactive glass nanomaterial having high biosafety and excellent antibacterial properties has not yet been developed.
Disclosure of Invention
Aiming at the defects of an antibacterial strategy of a bioactive glass nano material in the prior art, the invention aims to provide a preparation method of the bioactive glass nano composite material with catalytic antibacterial performance, the preparation method endows gold nanoclusters (BSA-AuNCs) with excellent enzyme-like catalytic antibacterial activity by performing amination modification on the gold nanoclusters, and meanwhile, the BGN is efficiently functionalized by virtue of rich positive charges on the surface of the cBSA-AuNCs, so that the BGN can show obviously enhanced antibacterial activity at a lower material concentration; the preparation method has mild reaction conditions, does not need complex instruments and equipment, and is easy to operate. The bioactive glass nano composite material synthesized by the method also has excellent functions of fluorescence imaging, promotion of fibroblast proliferation and the like, and has wider application scenes compared with the traditional bioactive glass material.
In order to achieve the purpose, the technical scheme adopted by the application is as follows:
a preparation method of a bioactive glass nano composite material with catalytic antibacterial performance comprises the following steps:
1) Preparing bioactive glass nanoparticles by a microemulsion method;
2) And reacting the bioactive glass nano particles with the positive-electrochemical gold nanoclusters in a reaction solvent to obtain the bioactive glass nano composite material with catalytic antibacterial property.
Further, the bioactive glass nano-particles comprise silicon element and calcium element in a molar ratio of 4:1-4:2.
Further, the gold nanoclusters for positive electrification are prepared by the following processes: and performing amination modification reaction on the gold nanocluster, ethylenediamine hydrochloride and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to obtain the positive electrochemical gold nanocluster.
Further, the dosage ratio of the gold nanoclusters to the ethylenediamine hydrochloride is 10mmol:1-5mol.
Further, the dosage ratio of the gold nanocluster to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 10mmol:4-20mg.
Further, the time for the amination modification reaction is 0.5 to 6 hours.
Further, the mass ratio of the bioactive glass nanoparticles to the electropositive gold nanoclusters is 1:1-3:1.
Further, the reaction solvent in the step 2) is ultrapure water, and the reaction time is 12-48 hours.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the positive-electrochemical gold nanoclusters are utilized to modify the bioactive glass nanoparticles, so that the bioactive glass nanocomposite BGN-AuNCs has excellent catalase-like antibacterial activity, and the concentration threshold of the antibacterial material of the bioactive glass can be obviously reduced. In a trace amount of H 2 O 2 In the presence of 75 mug/mL bioactive glass nano-compositeThe composite material has excellent antibacterial performance, and the traditional antibacterial bioactive glass depending on ion doping and antibiotic loading can show ideal antibacterial effect at the concentration level of milligram per milliliter. The preparation method of the bioactive glass nanocomposite material is simple and easy to implement, the reaction conditions are mild, complex instruments and equipment are not needed, and the prepared bioactive glass nanocomposite material has excellent biocompatibility, catalase-like catalysis and antibacterial activity, also has the functions of fluorescence imaging, cell behavior regulation and the like, and has great application potential in the biomedical fields of tissue regeneration and the like.
Furthermore, the invention utilizes gold nanoclusters (BSA-AuNCs) protected by bovine serum albumin to modify the bioactive glass for the first time, and the bovine serum albumin is subjected to amination modification, so that the surface of the bioactive glass has rich positive charges, and the electropositive modification technology can effectively enhance the binding force between the BSA-AuNCs and BGN with negative charges on the surface, and can also enhance the catalase-like catalytic ability of the BSA-AuNCs in a physiological environment and improve the bacterial killing effect, so that the bioactive glass can generate an excellent sterilization effect (100% can kill escherichia coli and staphylococcus aureus) at an extremely low concentration (2.5 mu g/mL).
Drawings
FIG. 1 is a fluorescence spectrum of a bioactive glass nanocomposite of the present invention, wherein samples BA1, BA2, BA3 and BA4 are BGN-AuNCs composite materials prepared when the mass ratio of BGN to cBSA-AuNCs is 3;
FIG. 2 is a Zeta potential diagram of BA1, BA2, BA3 and BA4 of the bioactive glass nanocomposite of the present invention;
FIG. 3 is a transmission electron micrograph and elemental distribution of a bioactive glass nanocomposite BA4 of the present invention, wherein (a) is a transmission electron micrograph of a sample BA4, (b) is an energy spectrum scanning region of the sample BA4, and (c), (d), (e), (f) and (g) are distribution maps of Si, O, ca, au and S elements, respectively;
FIG. 4 is a catalase-like catalytic effect study of the bioactive glass nanocomposite BA4 of the present invention;
FIG. 5 is a graph showing the antibacterial effect of the bioactive glass nanocomposite BA4 of the present invention, wherein (a) is the survival rate of Escherichia coli and (b) is the survival rate of Staphylococcus aureus.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the technical scheme of the invention is clearly and completely described below by combining the specific embodiment of the invention and the attached drawings.
The invention utilizes the positive-electrochemical gold nanoclusters to modify the bioactive glass to prepare the bioactive glass nanocomposite with catalytic antibacterial performance. And selecting a gold nanocluster protected by bovine serum albumin with good biocompatibility, and carrying out amination modification on the gold nanocluster. Although bovine serum albumin Bai Jincu has excellent biocompatibility, it has poor antibacterial effect and poor catalase-like activity under neutral and weakly alkaline conditions. Through amination modification, on one hand, the enzyme-like catalytic antibacterial activity of bovine serum albumin gold clusters can be improved, and on the other hand, because the bioactive glass nano material is electronegative in a physiological environment, the binding force of the bovine serum albumin Bai Jincu and bioactive nano particles can be obviously enhanced through a positive electrochemical modification process, so that the high-efficiency functionalization of bioactive glass is realized, and the bioactive glass nano composite material with the catalytic antibacterial performance is prepared.
The invention relates to a preparation method of a bioactive glass nano composite material with catalytic antibacterial performance, which comprises the following steps:
1) Preparation of bioactive glass: the preparation method comprises the steps of taking cetyl pyridinium bromide as a surfactant and a template agent, fully hydrolyzing the cetyl pyridinium bromide and urea in water, adding cyclohexane and n-butyl alcohol, violently stirring to obtain uniform emulsion, gradually adding tetraethyl orthosilicate and calcium nitrate tetrahydrate to react to obtain milky bioactive glass sol-gel, washing, drying and calcining to obtain Bioactive Glass Nanoparticles (BGN).
Wherein the mass ratio of urea to bromohexadecyl pyridine is 0.6; the volume ratio of cyclohexane to water is 1:1; the volume ratio of the cyclohexane to the n-butanol is 30, and the molar ratio of the tetraethyl orthosilicate to the calcium nitrate tetrahydrate is 4:1-4:2; the reaction temperature is 65-75 ℃ and the reaction time is 8-16 hours.
2) Preparing positive charged gold nanoclusters: preparing gold nanoclusters (BSA-AuNCs) by taking bovine serum albumin as a template agent and a reducing agent, performing amination modification reaction for 0.5-6 hours with ethylenediamine hydrochloride and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) after ultrafiltration purification, and performing ultrafiltration purification on a reaction final solution to obtain the positive-electrochemical gold nanoclusters (cBSA-AuNCs) which are stored at the temperature of 4 ℃.
The concentration of BSA-AuNCs was 10mmol/L and the volume was 5mL.
The concentration of the ethylenediamine hydrochloride is 1-5mol/L, the volume is 5mL, and the ethylenediamine hydrochloride is slowly added into the BSA-AuNCs under the condition of vigorous stirring.
EDC is used in an amount of 20-100mg.
3) Preparation of bioactive glass nanocomposite: reacting the prepared BGN with cBSA-AuNCs under vigorous stirring for 12-48 hours, centrifuging at 8000rpm for 15 minutes, washing and precipitating for 3 times to remove the cBSA-AuNCs which are not effectively combined, and obtaining the bioactive glass nano composite material (BGN-AuNCs) with catalytic antibacterial property.
Wherein, 15mg of BGN should be dispersed in 500 μ L deionized water solution for 5 minutes of ultrasonic treatment, and then cBSA-AuNCs should be added.
The mass ratio of BGN to cBSA-AuNCs is 1:1-3:1, preferably 3:1,2:1,1.5 and 1:1.
Example 1
1) Preparation of bioactive glass: 0.6g of urea and 1g of bromocetylpyridine were taken and dissolved thoroughly in 30mL of deionized water for 20 minutes, then 30mL of cyclohexane and 1.1mL of n-butanol were added slowly and stirred vigorously for 30 minutes to form a homogeneous emulsion. 2.7mL of TEO (tetraethyl orthosilicate) were then added slowly dropwise, hydrolyzed for 30 minutes, then warmed to 70 ℃ and allowed to react for 8 hours with vigorous stirring. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed with acetone, ethanol, and water 3 times each. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparing positive charged gold nanoclusters: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (5 mol/L,5 mL) was added to purified BSA-AuNCs (10 mmol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 100mg of EDC was added and the reaction was continued with stirring at 1000rpm for 2 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared sample cBSA-AuNCs was stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 3:1. After 15mg of BGN was dispersed in a small volume of deionized water by sonication thoroughly, it was slowly added to 3.3mL of cBSA-AuNCs (1.5 mg/mL), the reaction was stirred vigorously at room temperature for 48 hours, the final reaction solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
Example 2
1) Preparing BGN: 0.6g of urea and 1g of bromohexadecylpyridine were taken to be sufficiently dissolved in 30mL of deionized water for 20 minutes, and then 30mL of cyclohexane and 1.1mL of n-butanol were slowly added and vigorously stirred for 30 minutes to form a uniform emulsion. 2.7mL of TEOS was then added slowly dropwise, hydrolyzed for 30 minutes and then warmed to 70 ℃ and allowed to react for 8 hours with continued vigorous stirring. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed with acetone, ethanol, and water 3 times each. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparation of cBSA-AuNCs: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (5 mol/L,5 mL) was added to purified BSA-AuNCs (10 mmol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 100mg of EDC was added and the reaction was continued with stirring at 1000rpm for 2 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared sample cBSA-AuNCs was stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 2:1. 15mg of BGN was dispersed in a small volume of deionized water by sonication thoroughly and slowly added to 5mL of cBSA-AuNCs (1.5 mg/mL), the reaction was stirred vigorously at room temperature for 48 hours, the reaction final solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
Example 3
1) Preparing BGN: 0.6g of urea and 1g of bromohexadecylpyridine were taken to be sufficiently dissolved in 30mL of deionized water for 20 minutes, and then 30mL of cyclohexane and 1.1mL of n-butanol were slowly added and vigorously stirred for 30 minutes to form a uniform emulsion. 2.7mL of TEOS was then added slowly dropwise, hydrolyzed for 30 minutes and then warmed to 70 ℃ and allowed to react for 8 hours with continued vigorous stirring. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed with acetone, ethanol, and water 3 times each. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparation of cBSA-AuNCs: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (5 mol/L,5 mL) was added to purified BSA-AuNCs (10 m mol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 100mg of EDC was added and the reaction was continued with stirring at 1000rpm for 2 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared sample cBSA-AuNCs was stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 1.5. 15mg of BGN was dispersed in a small volume of deionized water by sonication thoroughly and slowly added to 6.7mL of cBSA-AuNCs (1.5 mg/mL), the reaction was stirred vigorously at room temperature for 48 hours, the reaction final solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
Example 4
1) Preparing BGN: 0.6g of urea and 1g of bromohexadecylpyridine were taken to be sufficiently dissolved in 30mL of deionized water for 20 minutes, and then 30mL of cyclohexane and 1.1mL of n-butanol were slowly added and vigorously stirred for 30 minutes to form a uniform emulsion. 2.7mL of TEOS was then added slowly dropwise, hydrolyzed for 30 minutes and then warmed to 70 ℃ and the reaction continued with vigorous stirring for 8 hours. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed with acetone, ethanol, and water 3 times each. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparation of cBSA-AuNCs: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (5 mol/L,5 mL) was added to purified BSA-AuNCs (10 mmol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 100mg of EDC was added and the reaction was continued with stirring at 1000rpm for 2 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared samples cBSA-AuNCs were stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 1:1. 15mg of BGN was dispersed in a small volume of deionized water by sonication thoroughly and slowly added to 10mL of cBSA-AuNCs (1.5 mg/mL), the reaction was stirred vigorously at room temperature for 48 hours, the reaction final solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
Example 5
1) Preparing BGN: 0.6g of urea and 1g of bromohexadecylpyridine were taken to be sufficiently dissolved in 30mL of deionized water for 20 minutes, and then 30mL of cyclohexane and 1.1mL of n-butanol were slowly added and vigorously stirred for 30 minutes to form a uniform emulsion. 2.7mL of TEOS was then added slowly dropwise, hydrolyzed for 30 minutes and then warmed to 65 ℃ and allowed to react for 16 hours with continued vigorous stirring. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed with acetone, ethanol, and water 3 times each. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparation of cBSA-AuNCs: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (1 mol/L,5 mL) was added to purified BSA-AuNCs (10 mmol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 20mg of EDC was added and the reaction was continued with stirring at 1000rpm for 6 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared sample cBSA-AuNCs was stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 1:1. 15mg of BGN was dispersed in a small volume of deionized water by sonication thoroughly and slowly added to 10mL of cBSA-AuNCs (1.5 mg/mL), the reaction was stirred vigorously at room temperature for 48 hours, the reaction final solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
Example 6
1) Preparing BGN: 0.6g of urea and 1g of bromohexadecylpyridine were taken to be sufficiently dissolved in 30mL of deionized water for 20 minutes, and then 30mL of cyclohexane and 1.1mL of n-butanol were slowly added and vigorously stirred for 30 minutes to form a uniform emulsion. 2.7mL of TEOS was then added slowly dropwise, hydrolyzed for 30 minutes and then warmed to 75 deg.C, and the reaction was continued for 8 hours with vigorous stirring. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed with acetone, ethanol, and water 3 times each. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparation of cBSA-AuNCs: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (2.5 mol/L,5 mL) was added to purified BSA-AuNCs (10 mmol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 50mg of EDC was added and the reaction was continued with stirring at 1000rpm for 4 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared sample cBSA-AuNCs was stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 1:1. 15mg of BGN was dispersed in a small volume of deionized water by sonication thoroughly and slowly added to 10mL of cBSA-AuNCs (1.5 mg/mL), the reaction was stirred vigorously at room temperature for 48 hours, the reaction final solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
Example 7
1) Preparing BGN: 0.6g of urea and 1g of bromohexadecylpyridine were taken to be sufficiently dissolved in 30mL of deionized water for 20 minutes, and then 30mL of cyclohexane and 1.1mL of n-butanol were slowly added and vigorously stirred for 30 minutes to form a uniform emulsion. 2.7mL of TEOS was then added slowly dropwise, hydrolyzed for 30 minutes and then warmed to 70 ℃ and allowed to react for 13 hours with continued vigorous stirring. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed 3 times with acetone, ethanol, and water in that order. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparation of cBSA-AuNCs: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (3 mol/L,5 mL) was added to purified BSA-AuNCs (10 mmol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 60mg of EDC was added and the reaction was continued with stirring at 1000rpm for 5 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared sample cBSA-AuNCs was stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 1:1. 15mg of BGN was dispersed in a small amount of deionized water by sonication thoroughly and slowly added to 10mL of cBSA-AuNCs (1.5 mg/mL), the reaction was stirred vigorously at room temperature for 24 hours, the reaction solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
Example 8
1) Preparing BGN: 0.6g of urea and 1g of bromohexadecylpyridine were taken to be sufficiently dissolved in 30mL of deionized water for 20 minutes, and then 30mL of cyclohexane and 1.1mL of n-butanol were slowly added and vigorously stirred for 30 minutes to form a uniform emulsion. 2.7mL of TEOS was then added slowly dropwise, hydrolyzed for 30 minutes and then warmed to 70 ℃ and allowed to react for 8 hours with continued vigorous stirring. Finally, 0.7mg of calcium nitrate tetrahydrate was added and the reaction was continued for 4 hours. The final solution was washed with acetone, ethanol, and water 3 times each. The resulting precipitate was freeze-dried and calcined at 600 ℃ for 5 hours (rate of temperature rise 1 ℃/min). The prepared sample BGN is stored at room temperature for standby.
2) Preparation of cBSA-AuNCs: BSA-AuNCs are prepared by taking bovine serum albumin as a template agent and a reducing agent, and the specific preparation steps refer to: xie J, zheng Y, ying J Y. Protein-directed Synthesis of high throughput Fluorescent reagents [ J ]. Journal of the American Chemical Society,2009,131 (3): 888-889. Ethylenediamine hydrochloride solution (4 mol/L,5 mL) was added to purified BSA-AuNCs (10 mmol/L,5 mL) with stirring at 1000rpm, and after stirring for 2 minutes, 80mg of EDC was added and the reaction was continued with stirring at 1000rpm for 2 hours. The final reaction solution was subjected to 5 ultrafiltration centrifugation operations using a 30kDa ultrafiltration tube to purify the amino-modified cBSA-AuNCs. The prepared samples cBSA-AuNCs were stored at 4 ℃ until use.
3) Preparation of bioactive glass nanocomposite: the mass ratio of BGN to cBSA-AuNCs is 1:1. 15mg of BGN was dispersed in a small amount of deionized water by sonication slowly into 10mL of cBSA-AuNCs (1.5 mg/mL), stirred vigorously at room temperature for 24 hours, the final reaction solution was centrifuged at 8000rpm for 15 minutes and the precipitate was washed 3 times to remove inefficiently bound cBSA-AuNCs.
The bioactive glass nano composite material obtained in the preparation process is a faint yellow powder after being frozen and dried, and has obvious red fluorescence under an ultraviolet lamp.
The effective cBSA-AUNCs amount loaded in the bioactive glass nano composite material prepared by the invention has obvious dependency on the feeding ratio. FIG. 1 is a fluorescence spectrum of a bioactive glass nanocomposite, wherein samples BA1, BA2, BA3 and BA4 are successively BGN-AuNCs prepared with a BGN to cBSA-AuNCs mass ratio of 3:1 (example 1), 2:1 (example 2), 1.5. As can be seen from FIG. 1, when the mass ratio of BGN to cBSA-AuNCs is gradually reduced in the range of 3:1-1:1, i.e., the input amount of cBSA-AuNCs in the reaction system is gradually increased, the effective loading amount of BGN to cBSA-AuNCs is also continuously increased, which shows that the fluorescence intensity of BGN-AuNCs is continuously increased.
FIG. 2 is Zeta potential diagram of bioactive glass nano composite material, BGN shows electronegativity, zeta potential is-12.9 + -0.1 mV, with the increase of cBSA-AuNCs dosage in the reaction system, the amount of cBSA-AuNCs loaded by BGN also increases, therefore potential shows electropositivity and gradually increases, when the mass ratio of BGN to cBSA-AuNCs is 1:1, the potential of BGN-AuNCs bioactive glass nano composite material reaches 30.8 + -0.3 mV. Therefore, when the reaction mass ratio of BGN to cBSA-AuNCs is 1:1, the recombination efficiency of the two is optimal, and sample BA4 has excellent fluorescence properties and a surface with abundant positive charges.
FIG. 3 (a) is a transmission electron micrograph of the bioactive glass nanocomposite BA4, wherein FIG. 3 (a) is a transmission electron micrograph of the sample BA4, FIG. 3 (b) is an energy spectrum scanning region of the sample BA4, and FIGS. 3 (c), (d), (e), (f) and (g) are distribution diagrams of Si, O, ca, au and S elements, respectively. It can be seen that when the reaction mass ratio of BGN to cBSA-AuNCs is 1:1, the BGN has good monodispersity and has no damaged form and structure after being efficiently combined with the cBSA-AuNCs, and the cBSA-AuNCs can be judged to be uniformly distributed in the BGN through the distribution of Au and S elements.
Fig. 4 is a graph of the enzyme-like catalytic effect of bioactive glass nanocomposite BA 4. The catalase-like property of the BGN-AuNCs of the bioactive glass nanocomposite is detected by using a substrate 3,3',5,5' -Tetramethylbenzidine (TMB). 20 μ L of BGN-AuNCs (BA 4) with certain concentration and uniform ultrasonic dispersion was added into 150 μ L Phosphate Buffer Solution (PBS) with pH =6.0, and then 10 μ L of TMB (final concentration 800 μmol/L) and 20 μ L H were sequentially added 2 O 2 (final concentration 50 mmol/L), after incubation for 15 min at 37 deg.C, BGN-AuNCS was removed by centrifugation and the change in absorbance of each set of supernatants was recorded using UV-visible absorption spectroscopy. At H 2 O 2 BGN-AuNCs catalyze H in the presence of 2 O 2 The decomposition produces hydroxyl radicals, thereby oxidizing TMB to the oxidized state TMB, with distinct characteristic absorption peaks at 370nm and 650 nm. Therefore, as shown in fig. 4, BGN has no obvious catalytic performance, but the bioactive glass nanocomposite BGN-AuNCs of the present invention has obvious characteristic absorption peaks at 370nm and 650nm, which indicates that BGN has excellent catalase-like catalytic activity after being compounded with cBSA-AuNCs.
Fig. 5 is a graph of the antibacterial effect of the bioactive glass nanocomposite BA 4. By means of 10 -4 mol/L H 2 O 2 And BGN-AuNCs with different concentrations are used together with 10 6 CFU/mL of Escherichia coli or Staphylococcus aureus was incubated on a shaker for 6 hours (37 ℃,200 rpm), 100. Mu.L of the diluted co-incubation solution was applied to an agar plate, and incubated at 37 ℃ for 16 hours for colony counting. From the results of FIG. 5, it is understood that BGN-AuNCs are effective in killing 95.9% of E.coli (see (a) in FIG. 5) and 56.4% of Staphylococcus aureus (see (b) in FIG. 5) at a low concentration of 75. Mu.g/mL. Furthermore, BGN-AuNCs have excellent peroxidase-like activity and can be used for low-concentration H 2 O 2 Under the condition, the good synergistic antibacterial performance is exerted by generating active oxygen. At 10 -4 M H 2 O 2 And 75 mug/mL BGN-AuNCs, the effective killing rate of the material against escherichia coli is 100% (shown in figure 5 (a)), the effective killing rate against staphylococcus aureus is 81.6% (shown in figure 5 (b)), the bioactive glass nanocomposite material of the invention shows excellent antibacterial effect,can kill bacteria through various ways such as surface positive charge and generation of active oxygen.
The invention carries out amination modification on gold nanoclusters (BSA-AuNCs) synthesized in a green way in one step, then the gold nanoclusters (BSA-AuNCs) are reacted with BGN prepared by a microemulsion method in a certain proportion, cBSA-AuNCs which are not effectively combined are finally removed by centrifugation, and the obtained bioactive glass nanocomposite (BGN-AuNCs) are stored at room temperature after being freeze-dried. The positive electrochemical technology of BSA-AuNCs can promote the high-efficiency compounding of the BSA-AuNCs and bioactive glass nano particles on one hand, and is the key for improving the catalytic antibacterial performance of BGN-AuNCs on the other hand.
The preparation method of the bioactive glass nano composite material is simple, the reaction condition is mild, and complex instruments and equipment are not needed. Meanwhile, the bioactive glass nano composite material prepared by the invention has good biocompatibility and excellent enzyme-like catalysis and antibacterial activity, can greatly reduce the acting dosage of the bioactive glass nano material in the treatment of infectious diseases, and is widely applied to the biomedical fields of tissue repair, reconstruction and the like.
The invention is different from the traditional synthetic strategy (ion doping and antibiotic loading) of the antibacterial bioactive glass, thereby effectively eliminating the adverse factors of large potential toxicity of ions, uncontrollable release of antibiotics and the like and breaking through the application limit of the antibacterial bioactive glass in the field of regenerative medicine. According to the invention, gold clusters (cBSA-AuNCs) protected by aminated bovine serum albumin are efficiently loaded by mesoporous radial Bioactive Glass Nanoparticles (BGN), and the prepared nano-composite not only has good biocompatibility, but also has excellent enzyme-like catalytic antibacterial activity.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (8)

1. A preparation method of a bioactive glass nano composite material with catalytic antibacterial performance is characterized by comprising the following steps:
1) Preparing bioactive glass nanoparticles by a microemulsion method;
2) And reacting the bioactive glass nano particles with the positive-electrochemical gold nanoclusters in a reaction solvent to obtain the bioactive glass nano composite material with catalytic antibacterial property.
2. The method of claim 1, wherein the bioactive glass nanoparticles comprise elemental silicon and elemental calcium in a molar ratio of 4:1-4:2.
3. The method for preparing the bioactive glass nanocomposite with catalytic antibacterial properties of claim 1, wherein the gold nanoclusters for positive electrification are prepared by the following processes: and performing amination modification reaction on the gold nanocluster, ethylenediamine hydrochloride and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to obtain the positive electrochemical gold nanocluster.
4. The method for preparing the bioactive glass nanocomposite with catalytic antibacterial properties according to claim 3, wherein the ratio of the gold nanoclusters to the ethylenediamine hydrochloride is 10mmol:1-5mol.
5. The method for preparing the bioactive glass nanocomposite with catalytic antibacterial properties of claim 3, wherein the ratio of the gold nanoclusters to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide used is 10mmol:4-20mg.
6. The method for preparing a bioactive glass nanocomposite with catalytic antibacterial properties as claimed in claim 3, wherein the time for performing the amination modification reaction is 0.5 to 6 hours.
7. The method for preparing a bioactive glass nanocomposite with catalytic antibacterial properties as claimed in claim 1, wherein the mass ratio of bioactive glass nanoparticles to gold nanoclusters for positive electrification is 1:1-3:1.
8. The method for preparing a bioactive glass nanocomposite with catalytic antibacterial properties as claimed in claim 1, wherein the reaction solvent in step 2) is ultrapure water and the reaction time is 12-48 hours.
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