阅读说明：本技术 一种改性金纳米棒光热抑菌制剂及其制备方法与应用 (Modified gold nanorod photothermal bacteriostatic preparation as well as preparation method and application thereof ) 是由 孙大文 张翠云 黄伦杰 蒲洪彬 张道瑞 于 2021-08-18 设计创作，主要内容包括：本发明公开了一种改性金纳米棒光热抑菌制剂及其制备方法与应用；本制备方法采用金纳米棒溶胶、金属离子水溶液、多酚化合物水溶液以及Tris-HCl缓冲液在常温下振荡反应获得改性金纳米棒光热抑菌制剂,试验过程仅需约10分钟,所得改性金纳米棒光热抑菌制剂具有均一的核壳结构。本发明制备方法简单快速、绿色安全,不需要使用有机试剂和昂贵复杂的仪器设备,所用试剂易获得,且均无毒无污染,符合绿色化学理念,不会对环境造成污染。本发明通过高生物安全性的金属多酚网络置换单纯金纳米棒表面存在的高生物毒性的十六烷基三甲基溴化铵,获得具有良好生物相容性的改性金纳米棒光热抑菌制剂,使其在医药、食品、生物抗菌等领域显示出巨大优势。(The invention discloses a modified gold nanorod photothermal bacteriostatic preparation as well as a preparation method and application thereof; the preparation method adopts gold nanorod sol, metal ion aqueous solution, polyphenol compound aqueous solution and Tris-HCl buffer solution to carry out oscillation reaction at normal temperature to obtain the modified gold nanorod photothermal bacteriostatic preparation, the test process only needs about 10 minutes, and the obtained modified gold nanorod photothermal bacteriostatic preparation has a uniform core-shell structure. The preparation method is simple, rapid, green and safe, does not need organic reagents and expensive and complicated instruments and equipment, is easy to obtain the reagents, is nontoxic and pollution-free, conforms to the green chemical concept, and does not cause pollution to the environment. The invention replaces hexadecyl trimethyl ammonium bromide with high biological toxicity on the surface of a pure gold nanorod by a metal polyphenol network with high biological safety to obtain the modified gold nanorod photo-thermal antibacterial preparation with good biocompatibility, so that the modified gold nanorod photo-thermal antibacterial preparation has great advantages in the fields of medicines, foods, biological antibacterial agents and the like.)

1. A preparation method of a modified gold nanorod photothermal bacteriostatic agent is characterized by comprising the following steps:

adding water, gold nanorod sol, metal ion aqueous solution, polyphenol compound aqueous solution and Tris-HCl buffer solution into a 10mL centrifugal tube under the oscillation condition, and carrying out oscillation reaction at normal temperature;

and centrifuging after the reaction is finished, removing supernatant to obtain the gold nanorods modified by the metal polyphenol network, using the gold nanorods as a modified gold nanorod photo-thermal antibacterial preparation, and re-dispersing the obtained modified gold nanorod photo-thermal antibacterial preparation by using a Tris-HCl buffer solution and storing.

2. The preparation method of the modified gold nanorod photothermal bacteriostatic agent according to claim 1, wherein the concentrations of the gold nanorod sol, the metal ion aqueous solution, the polyphenol compound aqueous solution and the Tris-HCl buffer solution are sequentially as follows: (150-200) ppm, (1-2) mg/mL, (2-4) mg/mL, and (10-20) mM.

3. The method for preparing the modified gold nanorod photothermal bacteriostasis preparation according to claim 2, wherein the pH of the Tris-HCl buffer solution is 8.0.

4. The preparation method of the modified gold nanorod photothermal bacteriostatic agent according to claim 3, wherein the time intervals of adding the gold nanorod sol, the metal ion aqueous solution and the polyphenol compound aqueous solution are 1min in sequence.

5. The preparation method of the modified gold nanorod photothermal bacteriostasis preparation according to claim 4, wherein the metal ions in the metal ion water solution are Fe³⁺、Cu²⁺、Mn²⁺At least one of;

the polyphenol compound in the polyphenol water solution is: at least one of epigallocatechin gallate, tannic acid, and procyanidin.

6. The preparation method of the modified gold nanorod photothermal bacteriostasis preparation according to claim 5, wherein the Tris-HCl buffer is added and then the oscillation reaction is carried out for 5 min.

7. The preparation method of the modified gold nanorod photothermal bacteriostasis preparation according to claim 6, wherein the oscillation frequency is 600-800 rpm; the room temperature is 20-25 ℃; the rotating speed of the centrifugation is 8000-10000 rpm, the centrifugation time is 5-10min, and the temperature is 20-25 ℃.

8. A modified gold nanorod photothermal bacteriostatic agent, which is characterized by being obtained by the preparation method of any one of claims 1-7.

9. The application of the modified gold nanorod photothermal bacteriostasis preparation as claimed in claim 8, wherein the modified gold nanorod photothermal bacteriostasis preparation is used for photothermal antibiosis, and the steps are as follows:

mixing the bacterial solution and the modified gold nanorod bacteriostatic agent solution in a volume ratio of 99:1, uniformly mixing for 1-3min by using a vortex oscillator, and standing; then, the mixture was diluted 10 times with PBS buffer (1X) after irradiation with near infrared light²-10⁵And taking the diluted suspension for plate coating, culturing in an incubator at 37 ℃, and calculating the colony number so as to calculate the sterilization rate.

10. The application of the modified gold nanorod photothermal bacteriostasis preparation as claimed in claim 9, is characterized in that: the power of the laser irradiator of the near infrared light is 1.5W/cm²-3W/cm²The wavelength of the near infrared light is 808nm, and the irradiation time is 5-10 min.

Technical Field

The invention relates to a bacteriostatic preparation and a process preparation thereof, in particular to a modified gold nanorod photothermal bacteriostatic preparation as well as a preparation method and application thereof.

Background

Traditional approaches to combat bacteria have been dominated by antibiotics, metal ions and quaternary ammonium ions, but have the disadvantage of being expensive, toxic and environmentally unfriendly, limiting their further use and transformation.

Moreover, excessive use of antibiotics leads to the development of bacterial resistance, inevitably reducing the therapeutic effect, and even leading to the development of "superbacteria". Therefore, it is of great interest to explore a new therapeutic strategy to combat bacterial infections more effectively and safely without significant side effects and the resulting resistance to drugs.

Currently, with the development of nanotechnology, many nanomaterials are developed for antibacterial applications, including metal ion type nanomaterials, photocatalytic type nanomaterials, quaternary ammonium salt or quaternary phosphonium salt modified surface contact type nanomaterials, and nanocarrier antibiotic-loaded nanomaterials.

In addition, other novel antibacterial materials, antibacterial photodynamic therapy and photothermolysis methods have also been developed to solve the problem of bacterial resistance. Among them, photothermal therapy is a novel germicidal therapy which converts near infrared light into photothermal light using a photothermal agent to destroy various types of pathogens and microorganisms, and is considered as a novel germicidal therapy.

Due to its minimal invasiveness and high selectivity, it is the most promising therapeutic approach. Materials having good photothermal response have so far mainly included noble metal nanomaterials (gold, platinum, silver, etc.), carbon nanomaterials (graphene oxide, carbon nanotubes), transition metal chalcogenide nanomaterials (copper sulfide, zinc sulfide, etc.), and organic dye nanomaterials.

Gold Nanorods (GNRs) are excellent candidates for photothermal therapy because they can absorb laser wavelengths in the Near Infrared (NIR) region, are transparent windows in biological tissues, and can efficiently convert absorbed NIR light energy into heat.

However, the toxic surfactant cetyl trimethyl ammonium bromide is difficult to remove, so that the gold nanorods have high toxicity and poor biological safety.

In addition, the pure gold nanorods have low photothermal conversion efficiency and poor antibacterial effect, so that the application of the gold nanorods in various fields such as medical treatment, food, biological antibacterial and the like is limited. Therefore, functional modification of gold nanorods to improve photothermal conversion efficiency and biocompatibility and for efficient photothermal antibacterial of microorganisms are required. Phenolic compounds are a large class of natural molecules present in a variety of organisms, are considered to be very safe compounds, and can be added to food as food additives. These ubiquitous compounds have gained great research interest from fungi to bacteria, and from plants to animals due to their unique physicochemical properties.

The Metal Polyphenol Networks (MPNs) are supermolecular network structures formed by metal ions and polyphenols through coordination, the metal polyphenol networks have certain photothermal effect, when high-toxicity hexadecyl trimethyl ammonium bromide on the surfaces of the gold nanorods is replaced, the photothermal effect of the gold nanorods cannot be influenced because the gold nanorods are functionally modified on the surfaces of the gold nanorods, particularly, the gold nanorods are easy to coat on the surfaces of the gold nanorods due to high adhesiveness of the metal polyphenol networks, the gold nanorods and the metal polyphenol networks can play a synergistic photothermal effect, the photothermal conversion efficiency is greatly improved, and the metal polyphenol networks are obviously higher than that of the gold nanorods and the metal polyphenol networks alone.

Therefore, the modified gold nanorod antibacterial preparation prepared by functionally modifying the gold nanorods through the metal polyphenol network can overcome the defects of poor biological safety and high biological toxicity of the gold nanorods, expand the application range of the gold nanorods and can be widely used as a green safe antibacterial agent.

Secondly, the functional modification of the metal polyphenol compound can also play a role in the synergistic photothermal effect of the gold nanorods and the metal polyphenol network, so that the photothermal efficiency of the antibacterial preparation is obviously improved, and bacteria can be killed more efficiently.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides a simple, green and safe modified gold nanorod photothermal bacteriostatic agent as well as a preparation method and application thereof.

The modified gold nanorod photothermal bacteriostatic preparation provided by the invention utilizes a metal polyphenol network with high biological safety to replace cetyl trimethyl ammonium bromide with high biological toxicity on the surface of the gold nanorod, so that the modified gold nanorod photothermal bacteriostatic preparation with good biocompatibility is obtained.

By utilizing the photo-thermal antibacterial preparation based on the modified gold nanorods, the photo-thermal conversion efficiency of the modified gold nanorods is enhanced by 3-4 times by exerting the synergistic photo-thermal effect of the gold nanorods and the metal polyphenol network, and the purposes of efficient bacteriostasis and broad-spectrum sterilization are achieved.

The invention is realized by the following technical scheme:

a preparation method of a modified gold nanorod photothermal bacteriostatic agent comprises the following steps:

firstly, sequentially adding water, gold nanorod sol, metal ions, polyphenol compound aqueous solution and Tris-HCl buffer solution into a 10mL centrifugal tube under an oscillation condition, carrying out oscillation reaction under a normal temperature condition, centrifuging after the reaction is finished, removing supernatant to obtain metal polyphenol network modified gold nanorods (GNRs @ MPNs) serving as modified gold nanorod photo-thermal antibacterial agents, and re-dispersing the obtained modified gold nanorod photo-thermal antibacterial agents by using the Tris-HCl buffer solution and storing.

Preferably, the concentrations of the gold nanorod sol, the metal ions, the polyphenol compound aqueous solution and the Tris-HCl buffer solution are respectively as follows: (150-200) ppm, (1-2) mg/mL, (2-4) mg/mL, and (10-20) mM.

Preferably, the pH of the Tris-HCl buffer is 8.0.

Preferably, the time interval for adding the gold nanorod sol, the metal ion aqueous solution and the polyphenol compound aqueous solution is 1 min.

Preferably, the metal ions are: fe³⁺、Cu²⁺、Mn²⁺At least one of (1).

Preferably, the polyphenol compound in the aqueous polyphenol solution is: at least one of epigallocatechin gallate (EGCG), Tannic Acid (TA), and procyanidin (OPC).

Preferably, the Tris-HCl buffer is added and then the reaction is shaken for 5 min.

More preferably, 1-2 mL of ultrapure water, 1-2 mL of gold nanorod sol with the molar concentration of 200ppm, 300-500 uL of metal ion aqueous solution with the concentration of 1mg/mL, 300-500 uL of polyphenol compound aqueous solution with the concentration of 4mg/mL, and 1-2 mL of Tris-HCl buffer solution with the concentration of 20mM are sequentially added into a 10mL centrifuge tube.

Preferably, the oscillation frequency is 600-800 rpm; the room temperature is 20-25 ℃; the rotating speed of the centrifugation is 8000-10000 rpm, the centrifugation time is 5-10min, and the temperature is 20-25 ℃.

Preferably, the modified gold nanorod photothermal bacteriostasis preparation is stored below 4 ℃.

Compared with the prior art, the invention has the following advantages and effects:

(1) the preparation method is simple, the synthesis speed is high, the cost is low, and organic reagents and expensive and complicated instruments and equipment are not needed;

the used reagents are easy to obtain, are all nontoxic and pollution-free, accord with the green chemical concept, and have no pollution to the environment.

(2) The modified gold nanorod photothermal bacteriostatic preparation prepared by the invention utilizes the high-biosafety metal polyphenol network to replace the high-biotoxicity hexadecyl trimethyl ammonium bromide on the surface of the gold nanorod to obtain the modified gold nanorod photothermal bacteriostatic preparation with good biocompatibility, improves the disadvantages of poor toxicity and biocompatibility of the gold nanorod, improves the biosafety of the bacteriostatic preparation, widens the bacteriostatic application range, and has remarkable advantages in the fields of medicines, foods, biological antibacteria and the like.

(3) According to the preparation method of the modified gold nanorod photothermal antibacterial preparation, the gold nanorod surface is coated with the high adhesiveness of the metal polyphenol network to realize functional modification, the synergistic photothermal effect of the gold nanorod and the metal polyphenol network can be maximally exerted, the gold nanorod is modified by the metal polyphenol network, the photothermal conversion efficiency is greatly improved, compared with a pure gold nanorod, the photothermal conversion efficiency is improved by 3-4 times, bacteria can be efficiently killed, the preparation method has a broad-spectrum sterilization effect, and the generation of bacterial drug resistance is avoided.

(4) The modified gold nanorod photothermal bacteriostasis preparation prepared by the invention has a bacteriostasis rate of nearly 100% and an obvious bacteriostasis effect on gram-positive bacteria and gram-negative bacteria such as staphylococcus aureus and escherichia coli under the condition of trace use.

Drawings

Fig. 1 is a Transmission Electron Microscope (TEM) image of tannin-based modified gold nanorod bacteriostatic agent in an embodiment of the invention.

FIG. 2 is a photo-thermal conversion efficiency comparison effect diagram of gold nanorods and a modified gold nanorod photo-thermal bacteriostatic agent in the embodiment of the invention.

FIG. 3 is a histogram of the bactericidal effect of Escherichia coli of the modified gold nanorod photothermal bacteriostasis preparation in the embodiment of the invention.

FIG. 4 is a graph showing the comparative effect of biotoxicity of the gold nanorods and the modified gold nanorods photothermal bacteriostasis preparation in the embodiment of the invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Example 1

A modified gold nanorod photothermal bacteriostatic preparation and a preparation method thereof are disclosed, and the preparation method comprises the following steps:

under the condition of room temperature, adding 1mL of ultrapure water into a 10mL centrifugal tube, placing the centrifugal tube on an oscillator for oscillation, wherein the oscillation frequency of the oscillator is 600rpm/min, and sequentially adding 1mL of gold nanorod sol with the molar concentration of 200ppm and 500uL of MnSO with the concentration of 1mg/mL into the centrifugal tube₄The aqueous solution and 500uL of an aqueous proanthocyanidin solution with a molar concentration of 4mg/mL were mixed at intervals of 1min, and finally, 2mL of a 20mM Tris-HCl buffer (pH8.0) was further added thereto, and the reaction was carried out at room temperature for 5min with shaking. After the reaction is finished, taking down the centrifugal tube, centrifuging for 5min at the rotating speed of 8000rpm and the temperature of 25 ℃, removing the supernatant, adding 5mL of ultrapure water, and re-dispersing to obtain the modified gold nanorod photothermal bacteriostatic agent; and (4) storing the prepared antibacterial preparation in a refrigerator at 4 ℃.

Photo-thermal sterilization: 0.99mL of the Escherichia coli suspension and 0.01mL of the LGNRs @ Fe-OPC solution were mixed for 3min by a vortex oscillator, and then the mixture was allowed to stand. Using 1.5W/cm²Irradiating with 808nm near infrared laser for 10min, and diluting with PBS buffer (1X) for 10min²-10⁵And taking 0.1mL of the diluted suspension for plate coating, culturing in an incubator at 37 ℃ for 12-18h, and counting colonies to obtain the sterilization rate.

According to the embodiment 1, the whole test process of the synthesized modified gold nanorod photothermal bacteriostasis preparation only needs 10 minutes, and the polyphenol compounds (epigallocatechin gallate, tannic acid and procyanidine) are from plants, belong to natural compounds, are non-toxic and harmless, have no pollution to the environment, and accord with the concepts of green, environmental protection and safety.

FIG. 1 is a TEM image of a modified gold nanorod photothermal bacteriostatic agent based on tannic acid modification; as can be seen from FIG. 1, a shell layer with a certain thickness is formed on the surface of the gold nanorod, which means that the metal polyphenol network is successfully coated on the surface of the gold nanorod to replace the toxicity of cetyl trimethyl ammonium bromide, and the successful synthesis of the modified gold nanorod photothermal bacteriostasis preparation is shown.

FIG. 2 is a comparison graph of photo-thermal conversion efficiency of gold nanorods and modified gold nanorods photo-thermal bacteriostatic agent; as can be seen from figure 2, the photo-thermal conversion efficiency of the modified gold nanorod photo-thermal antibacterial preparation is greatly improved, and is improved by 3-4 times compared with the photo-thermal efficiency of a pure gold nanorod.

FIG. 3 is a bar graph of the respective bactericidal effects of the gold nanorods and the modified gold nanorods on escherichia coli; as can be seen from FIG. 3, the killing rate of the gold nanorods to escherichia coli is 32%, while the killing rate of the modified gold nanorod photothermal bacteriostasis preparation to escherichia coli reaches about 89-99%, and the antibacterial effect is significantly higher than that of a pure gold nanorod.

FIG. 4 is a graph showing the comparative effect of biotoxicity of the gold nanorods and the modified gold nanorods photothermal bacteriostatic agent in the embodiment of the invention; as can be seen from FIG. 4, after the cells are treated by the gold nanorods, the cell survival rate is only about 20%, which shows that the gold nanorods have very high biotoxicity, and after the modified gold nanorod photo-thermal antibacterial preparation is used for treating the cells, the cell survival rate is 99.1% -100%, which shows that the modified gold nanorod photo-thermal antibacterial preparation has very good biocompatibility.

Example 2

A modified gold nanorod photothermal bacteriostatic preparation and a preparation method thereof are disclosed, and the preparation method comprises the following steps:

under the condition of room temperature, 2mL of ultrapure water is added into a 10mL centrifuge tube and placed on an oscillator to oscillate, the oscillation frequency of the oscillator is 700rpm/min, 1mL of gold nanorod sol with the molar concentration of 200ppm and 500uL of FeCl with the concentration of 1mg/mL are sequentially added into the centrifuge tube₃Aqueous solution, 500uL of aqueous tannic acid solution at a concentration of 4mg/mL for 1min, and 1mL of 20mM Tris-HCl buffer (pH8.0) was added theretoThe reaction was carried out for 5min with warm shaking. After the reaction is finished, taking down the centrifugal tube, centrifuging at 9000rpm and 25 ℃ for 7min, removing the supernatant, adding 5mL of ultrapure water, and re-dispersing to obtain the modified gold nanorod photothermal antibacterial agent; and (4) storing the prepared antibacterial preparation in a refrigerator at 4 ℃.

Photo-thermal sterilization: 0.99mL of the Escherichia coli suspension and 0.01mL of the LGNRs @ Fe-TA solution were mixed for 2min by a vortex shaker, and then the mixture was allowed to stand. Using 2W/cm²Irradiating with 808nm near infrared laser for 6min, and diluting with PBS buffer (1X) for 10min²-10⁵And taking 0.1mL of the diluted suspension for plate coating, culturing in an incubator at 37 ℃ for 12-18h, and counting colonies to obtain the sterilization rate.

Example 3

A modified gold nanorod photothermal bacteriostatic preparation and a preparation method thereof are disclosed, and the preparation method comprises the following steps:

under the condition of room temperature, 1.4mL of ultrapure water is added into a 10mL centrifuge tube and placed on an oscillator for oscillation, the oscillation frequency of the oscillator is 800rpm/min, 1mL of gold nanorod sol with the molar concentration of 200ppm and 300uL of CuCl with the concentration of 1mg/mL are sequentially added into the centrifuge tube₂The time interval between the aqueous solution and 300uL of epigallocatechin gallate aqueous solution with the molar concentration of 4mg/mL is 1min, and finally, 1mL of Tris-HCl buffer solution (pH8.0) with the concentration of 20mM is continuously added into the mixture, and the reaction is carried out for 5min at room temperature by shaking. After the reaction is finished, taking down the centrifugal tube, centrifuging for 5min at the rotating speed of 10000rpm and the temperature of 20 ℃, removing the supernatant, adding 5mL of ultrapure water, and re-dispersing to obtain the modified gold nanorod photothermal bacteriostatic agent; and (4) storing the prepared antibacterial preparation in a refrigerator at 4 ℃.

Photo-thermal sterilization: taking 0.99mL of escherichia coli suspension and 0.01mLGNRs @ Fe-EGCG solution, mixing for 1min by using a vortex oscillator, and standing. Using 3W/cm²Irradiating with 808nm near infrared laser for 5min, and diluting with PBS buffer (1X) for 10min²-10⁵And taking 0.1mL of the diluted suspension for plate coating, culturing in an incubator at 37 ℃ for 12-18h, and counting colonies to obtain the sterilization rate.

Those who do not specify specific conditions in the examples of the present invention follow conventional conditions or conditions recommended by the manufacturer. The raw materials, reagents and the like which are not indicated for manufacturers are all conventional products which can be obtained by commercial purchase.

The preparation method disclosed by the invention is simple and rapid in preparation process, green and safe, does not need organic reagents and expensive and complicated instruments, is easy to obtain the reagents, is non-toxic and pollution-free, conforms to the green chemical concept, and does not cause pollution to the environment.

The invention replaces hexadecyl trimethyl ammonium bromide with high biological toxicity on the surface of a pure gold nanorod by a metal polyphenol network with high biological safety to obtain the modified gold nanorod photo-thermal antibacterial preparation with good biocompatibility, so that the modified gold nanorod photo-thermal antibacterial preparation has great advantages in the fields of medicines, foods, biological antibacterial agents and the like.

Secondly, the gold nanorod is coated with high adhesiveness of a metal polyphenol network to realize functional modification, the synergistic photothermal effect of the gold nanorod and the metal polyphenol network can be exerted to the maximum degree, the gold nanorod is modified by the metal polyphenol network, the photothermal conversion efficiency is greatly improved, compared with the pure gold nanorod, the photothermal conversion efficiency is improved by 3-4 times, bacteria can be killed efficiently, the broad-spectrum sterilization effect is achieved, and the generation of bacterial drug resistance is avoided.

In addition, the modified gold nanorod photothermal bacteriostasis preparation has a broad-spectrum bactericidal function, does not generate bacterial drug resistance, and has a good bacteriostasis effect on gram-positive bacteria, gram-negative bacteria and drug-resistant bacteria.

As described above, the present invention can be preferably realized.

The embodiments of the present invention are not limited to the above-described embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and they are included in the scope of the present invention.