AU2021106313A4

AU2021106313A4 - Antibacterial hydrogel and preparation method and application thereof

Info

Publication number: AU2021106313A4
Application number: AU2021106313A
Authority: AU
Inventors: Yong Liu; Shanshan Wang; Xun YUAN
Original assignee: Qingdao University of Science and Technology
Current assignee: Qingdao University of Science and Technology
Priority date: 2021-08-21
Filing date: 2021-08-21
Publication date: 2021-11-04
Anticipated expiration: 2029-08-21

Abstract

The invention belongs to the technical field of antibacterial hydrogel, which discloses an antibacterial hydrogel and preparation method and application thereof. The method comprises the following steps: soaking bacterial cellulose in silver nitrate solution, adding with corresponding ligand lipoic acid, stirring the mixture at room temperature, adding with corresponding reducing agent sodium borohydride for reduction, stirring the mixture at room temperature, and finally synthesizing a hydrogel composite AgNCs based on silver nanocluster.

Description

Antibacterial hydrogel and preparation method and application thereof

TECHNICAL FIELD This invention relates to antibacterial hydrogel, in particular relates to antibacterial hydrogel and preparation method and application thereof

BACKGROUND Bacterial infection has been a great threat to public health since ancient times. Among all kinds of antibacterial strategies, the most commonly used means is the application of antibiotics to treat bacterial infections. However, the emergence of antibiotic-resistant pathogens has greatly restricted the application of antibiotics, thus forcing researchers to explore new antibacterial drugs. In recent years, silver-based nanomaterials have gradually attracted wide attention in the industry, and it has become an antibacterial agent with high efficiency, non-drug resistance and broad-spectrum sterilization. At present, silver-based nanomaterials have been widely used in the treatment of burns, wounds and other bacterial infections. Silver nanoclusters (AgNCs, with size less than 2nm and atomic number ranging from tens to thousands) have attracted much attention in biomedical fields such as the treatment of bacterial infections due to their advantages of ultra-small size, high antibacterial activity, and their rich controllable surface chemistry and good biocompatibility. However, due to the rapid decomposition of AgNCs after internalization by bacteria, its antibacterial durability is affected to some extent. Therefore, it is the key to realize the controlled release of Ag* without affecting its antibacterial performance.

SUMMARY In order to solve the technical problems of poor sustained antibacterial property and low biological toxicity of antibacterial hydrogel in the prior art, the invention provides preparation method of antibacterial hydrogel.

In order to solve the above technical problems, the invention adopts the following technical scheme Antibacterial hydrogel and preparation method, immersing bacterial cellulose in silver nitrate solution, adding with corresponding ligand lipoic acid, stirring at room temperature, adding with corresponding reducing agent sodium borohydride for reduction, stirring the mixture at room temperature, and finally synthesizing the hydrogel composite AgNCs@BC based on silver nanocluster. Silver nanoclusters (AgNCs) are a core-shell structure with silver as the metal core and thiol ligand as the outer layer. The size (metal core) is less than 2nm, which is composed of silver core and outer layer ligand. It has broad-spectrum bactericidal and biocompatibility characteristics, and has good dispersion performance in water phase. Compared with silver nanoparticles with the same bactericidal effect, silver nanoclusters have smaller size (less than 2nm), so they have more advantages. However, the stability of silver nanoclusters in complex organisms is not good, and it is easy to decompose, which is not conducive to long-term drug action. Therefore, the inventor tried to use natural hydrogel bacterial cellulose (BC) with three-dimensional network structure to realize the controlled release of silver nanoclusters to solve the stability problem of silver nanoclusters in organisms. Therefore, the inventor initially soaked bacterial cellulose BC in the synthesized silver nanoclusters AgNCs solution, but the hydrogel composite AgNCs@BC synthesized by this method is relatively toxic and the antibacterial effect is not good. With the progress of the experiment, the inventor was pleasantly surprised to find that bacterial cellulose was soaked in silver nitrate solution, and the corresponding ligand lipoic acid was added, which was stirred at room temperature, then the corresponding reducing agent sodium borohydride was added for reduction, and the hydrogel composite AgNCs@BC was obtained by stirring at room temperature. According to the invention, the hydrogel composite AgNCs@BC based on silver nanoclusters prepared by the in-situ synthesis method has the best antibacterial effect, the toxicity can be reduced to a lower level, and the chemical reaction is complex and changeable. The reason may be that: In the invention, natural hydrogel bacterial cellulose (BC) is used as a skeleton structure, silver ions are first introduced into bacterial cellulose, and then silver nanoclusters are synthesized, so in the synthesis process of the hydrogel composite AgNCs@BC, In-situ synthesis method makes the interaction between silver nanocluster silver core and bacterial cellulose three dimensional structure network stronger, so that the release rate of silver ions tends to be flat, which is more conducive to reducing biotoxicity, thus improving the biocompatibility of the system and being more conducive to its biomedical application. At the same time, the loss of silver nanoclusters in hydrogel was restrained to a certain extent by in-situ synthesis, which was beneficial to the improvement of antibacterial properties. Preferably, the concentration range of the silver nitrate solution is 4-12mmol/L, the concentration range of the lipoic acid is 20-60mmol/L, and the concentration range of the sodium borohydride is 8-24mmol/L. Preferably, the mass concentration ratio of silver nitrate, lipoic acid and sodium borohydride is 1:5:2. Bacterial cellulose is soaked in silver nitrate solution, preferably, the soaking time of the bacterial cellulose is 20h. Furthermore, in the traditional hydrogel synthesis process, crosslinking agents (such as glutaraldehyde, etc.) with certain biological toxicity are usually used, so that the biocompatibility of the system is reduced, which is not conducive to its biomedical application. According to the invention, crosslinking agent is not needed in the preparation method it provides, therefore, the toxicity of the composite hydrogel is further reduced. After the corresponding ligand lipoic acid is added, the stirring time at room temperature can be 15min. According to the invention, a corresponding reducing agent sodium borohydride is added for reduction, and the stirring time at room temperature can be 24h. Another purpose of the present invention is to provide an antibacterial hydrogel prepared by the above preparation method. Finally, the invention provides the application of the antibacterial hydrogel in preparing antibacterial and controlled release drugs. Preferably, the concentration of the drug is 10 mmol/l. According to the invention, bacterial cellulose is used as a hydrogel skeleton, and the hydrogel compound AgNCs@BC is synthesized in situ on the surface. According to that invention, the traditional antibacterial hydrogel based on silver nanoparticles or silver ions is improve into the antibacterial hydrogel based on silver nanoclusters, so that the controllable release of silver ions is realized, and the biotoxicity of the system is preliminarily reduced. Any toxic crosslinking agent is not used in the synthesis method, so that the biotoxicity is further reduced. Combining silver nanoclusters with bacterial cellulose hydrogels, the technical defect that AgNCs will decompose rapidly after internalization by bacteria is solved, and the super antibacterial type of silver nanoclusters and controllable release and water retention of hydrogels are brought into full play, so that the sustained antibacterial effect of hydrogel composite AgNCs@BC is the best, and its biological toxicity is reduced to a certain extent. According to the above technical scheme, the antibacterial hydrogel provided by the invention can give full play to the advantages of broad-spectrum antibacterial and high sterilization efficiency of AgNCs, and the silver nanoclusters obtained by the in-situ synthesis method play a synergistic antibacterial role, and the prepared composite material has high-efficiency continuous antibacterial activity and low biological toxicity.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a schematic diagram of synthesis of hydrogel composite AgNCs@BC. Fig. 2 shows the ultraviolet-visible absorption spectra of AgNCs and AgNCs@BC. Fig. 3 is a transmission electron micrograph of AgNCs@BC. Fig. 4 is a field emission scanning electron micrograph of AgNCs@BC. Fig. 5 is an elemental analysis diagram of AgNCs@BC. Fig. 6 is a comparison chart of antibacterial activity between AgNCs/BC and AgNCs@BC. Fig. 7 is an antibacterial activity diagram of AgNCs@BC against different bacteria. Fig. 8 is a statistical diagram of the diameter of bacteriostatic ring of different concentrations of AgNCs@BC (based on the concentration of silver) against staphylococcus aureus. Fig. 9 is a test chart of toxicity of AgNCs@BC and AgNO3 to mammalian cells. Fig. 10 is the slow release curve of Ag species from AgNCs@BC.

DESCRIPTION OF THE INVENTION The invention discloses an antibacterial hydrogel, a preparation method and application thereof, and technical personnel in that field can learn from the contents of the article and appropriately improve the realization of process parameters. In particular, all similar substitutions and modifications are obvious to those skilled in the art, and they are considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and it is obvious that relevant personnel can modify or appropriately modify and combine the method and application described herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the technology of the present invention. In order to enable those skilled in the art to better understand the present invention, the present invention will be further described in detail with reference to specific embodiments. Embodiment 1 Bacterial cellulose is soaked in silver nitrate solution for 20h, and the corresponding ligand lipoic acid is added, stirred at room temperature for 15min, then the corresponding reducing agent sodium borohydride is added for reduction, stirred at room temperature for 24h, the concentration of silver nitrate solution is 4mmol/L, the concentration of lipoic acid is 20mmol/L, and the concentration range of sodium borohydride is 8 mmol/L. The molar concentration ratio of AgNO3(silver nitrate):DHLA (lipoic acid): NaBH4 (sodium borohydride) is 1:5:2, and finally the silver nanocluster-based hydrogel composite AgNCs@BC is synthesized. The above synthesis schematic diagram is shown in Fig. 1. Detecting the ultraviolet-visible absorption spectra of AgNCs and AgNCs@BC prepared in embodiment 1, and the spectrogram is shown in Fig. 2. As shown in Fig. 2, the absorption spectra of the leaching solution of AgNCs@BC synthesized in situ with BC as skeleton show the same characteristic absorption peak as that of Ag NCs, which preliminarily proves that the AgNCs embedded in AgNCs@BC is Ag29. The AgNCs@BC prepared in embodiment 1 was scanned by projection electron microscope, and the transmission electron microscope image is shown in Fig. 3. In Fig.3, the characteristic structure of black polka-dot silver nanoclusters with size less than 2nm can be clearly observed, which further confirms the structure of silver nanoclusters. The field emission scanning of AgNCs@BC prepared in embodiment 1 is shown in Fig. 4. In Figure 4, it can be observed that BC skeleton has obvious fiber structure with a size of about 20-30 nm and abundant pores. Elemental analysis was carried out on AgNCs@BC obtained in embodiment 1. See Fig. 5 for details. Fig. 5 is an elemental analysis diagram of AgNCs@BC, which analyzes three elements: C, 0 and Ag. Fig. 5 shows that the three elements are uniformly dispersed in the hydrogel structure, thus proving that AgNCs is successfully embedded in BC structure. The AgNCs@BC prepared in embodiment 1 was scanned by projection electron microscope, and the transmission electron microscope image is shown in Fig. 3. In Fig. 3, the characteristic structure of black polka-dot silver nanoclusters with size less than 2nm can be clearly observed, which further confirms the structure of silver nanoclusters. Comparative embodiment 1 At room temperature, bacterial cellulose BC was soaked in the synthesized Ag NCs solution for 48h to obtain AgNCs/BC. The antibacterial properties of AgNCs/BC obtained in comparative embodiment 1 were compared with those of AgNCs@BC obtained in embodiment 1, and the antibacterial activities of AgNCs@BC and Ag NCs/BC against bacteria were determined with staphylococcus aureus as bacterial model. Staphylococcus aureus was cultured in fresh bacterial culture solution for 12h, then diluted to OD6OO=0.1, then 100 microliters of bacterial solution was taken out and evenly coated on the culture plate. AgNCs@BC and Ag NCs/BC were placed in the center of the plate, and the culture plate was cultured in a constant temperature box at 370 C for 12h. The size of bacteriostatic ring was observed. The results are shown in Fig.6 and Fig.6. The antibacterial activity of AgNCs@BC and Ag NCs/BC with the same concentration against staphylococcus aureus was compared. the bacteriostatic ring diameter of the bacterial culture plate (right) treated by AgNCs@BC was larger, and it had greater antibacterial activity compared with Ag NCs/BC. Embodiment 2 Bacterial cellulose is soaked in silver nitrate solution for 20h, then added with corresponding ligand lipoic acid, stirred at room temperature for 15min, then added with corresponding reducing agent sodium borohydride for reduction, stirred at room temperature for 24h, the concentration of silver nitrate solution is 8mmol/L, the concentration of lipoic acid is 40mmol/L, and the concentration range of sodium borohydride is 16mmol/L., The molar concentration ratio of AgNO3 (silver nitrate): DHLA (lipoic acid): NaBH4 (sodium borohydride) is 1:5:2, and finally the silver nanocluster based hydrogel composite AgNCs@BC is synthesized. the above synthesis schematic diagram is shown in Fig. 1. 2.1 Test the antibacterial activity of AgNCs@BC obtained in embodiment 2. the specific test is as follows: Antibacterial experiment of AgNCs@BC against staphylococcus, aureus. Staphylococcus aureus was cultured in fresh bacterial culture solution for 12h, then the bacterial solution was taken out and diluted to OD600 =0.1 (OD600 = 0.1, i.e. about 108 colonies), then 100 microliters of bacterial solution was taken out and evenly coated on the culture plate. AgNCs@BC and pure BC were placed in the center of the plate, and the culture plate was cultured in a constant temperature box at 370 C for 12h, and the size of bacteriostatic ring was observed. The results are shown in the figure. Antibacterial experiment of AgNCs@BC against Bacillus subtilis. Bacillus subtilis was cultured in fresh bacterial culture solution for 12h, and then diluted to ODoo=0.1. Then, 100 microliters of bacterial solution was taken out and evenly coated on the culture plate. AgNCs@BC and pure BC were placed in the center of the plate, and the culture plate was cultured in an incubator at 37 0C for 12h. the size of bacteriostatic ring was observed. the results are shown in Fig.7. Antibacterial experiment of AgNCs@BC against Bacillus subtilis. Bacillus subtilis was cultured in fresh bacterial culture solution for 12h, and then diluted to ODoo=0.1. then, 100 microliters of bacterial solution was taken out and evenly coated on the culture plate. AgNCs@BC and pure BC were placed in the center of the plate, and the culture plate was cultured in an incubator at 370 C for 12h. The size of bacteriostatic ring was observed. The results are shown in Fig.7. The antibacterial experiment of AgNCs@BC on Escherichia coli was cultured in fresh bacterial culture solution for 12h, then diluted to OD6oo=0.1, then 100 microliters of bacterial solution was taken out and evenly coated on the culture plate. AgNCs@BC and pure BC were placed in the center of the plate, and the culture plate was cultured in an incubator at 37 0C for 12h, and the size of bacteriostatic ring was observed. the results are shown in Fig.7. Staphylococcus aureus, Bacillus subtilis and Escherichia coli were treated with AgNCs@BC, and the results of bacteriostatic ring as shown in the figure were obtained after 12h of culture. Fig.7 proved that AgNCs@BC has broad-spectrum antibacterial effect; At the same time, BC control group treated by the same method did not show bacteriostatic ring, which indicated that BC itself had no antibacterial activity. 2.2 Antibacterial experiments of AgNCs@BC with different concentrations: With staphylococcus aureus as bacterial model, the antibacterial activity of AgNCs@BC with different concentrations against bacteria was determined. Staphylococcus aureus was cultured in fresh bacterial culture solution for 12h, and then diluted to OD6oo=0.1. Then, 100 microliters of bacterial solution was taken out and evenly coated on the culture plate. 4mM, 6mM, 8mM, 10mM and 12mM AgNCs@BC were placed in the center of the plate, and the culture plate was cultured in a constant temperature box at 37 0C for 12h. The size of bacteriostatic ring was observed. The results are shown in Fig. 8. The antibacterial activity was tested, and the results showed that when the concentration was 10mM, it had the best antibacterial activity. 2.3 AgNCs@BC is tested for cytotoxicity, and the test process and results are as follows: The extract (10mM) of AgNCs@BC was taken out, and the mammalian cells were treated in a 96-well plate for 12h. The cells were stained with fluorescent indicator carnitine homodimer and calcein, and incubated for 45min. the coloring of the cells was observed by cold color digital camera. the mammalian cells were treated with AgNCs@BC, AgNO3 and ultrapure water respectively, and cultured for 12h. The cells treated with ultrapure water were used as control group. The fluorescence photos as shown in the figure are obtained. as shown in Fig. 9, the cells treated with AgNCs@BC have no obvious difference compared with the control group, and the cell survival rate is high, which proves that AgNCs@BC has extremely low biotoxicity, while the corresponding AgNO3 treated cells have relatively low survival rate, thus proving that AgNCs@BC hydrogel system is superior in biocompatibility. 2.4 Ag sustained release experiment in AgNCs@BC:

Immerse 200mg of AgNCs@BC in 500mL of ultrapure water, place it in an environment of 37 0C and stir slowly, take out 50ml of solution at 30min, 1h, 2h, 4h, 8h, 12h, 18h, 24h, 36h and 48h respectively, and measure the content of Ag+ in the solution. the results are shown in Fig. 10. The slow release curve in Fig. 10 shows that Ag is released. However, the rate slows down after 2h of release, starts to release slowly after 8h, and reaches the release balance after 48h, thus proving the effect of sustained release of Ag in AgNCs@BC. Therefore, the AgNCs@BC provided by the present invention has better sustained sterilization due to its sustained release; Antibacterial hydrogel can be used in the preparation of antibacterial and controlled-release drugs. Embodiment 3 Bacterial cellulose is soaked in silver nitrate solution for 20h, then corresponding ligand lipoic acid is added, stirred at room temperature for 15min, then corresponding reducing agent sodium borohydride is added for reduction, stirred at room temperature for 24h, the concentration of silver nitrate solution is 12mmol/L, the concentration of lipoic acid is 60mmol/L, and the concentration range of sodium borohydride is 24mmol/L., The molar concentration ratio of AgNO3 (silver nitrate): DHLA (lipoic acid): NaBH4 (sodium borohydride) is 1:5:2, and finally the hydrogel composite AgNCs@BC based on silver nanocluster is synthesized. Embodiment 4 Bacterial cellulose was soaked in silver nitrate solution for 20h, then added with corresponding ligand lipoic acid, stirred at room temperature for 15min, then added with corresponding reducing agent sodium borohydride for reduction, stirred at room temperature for 24h, the concentration of silver nitrate solution was 10mmol/L, the concentration of lipoic acid was 48mmol/L, and the concentration range of sodium borohydride was 18 mmol/L. Finally, the hydrogel composite AgNCs@BC based on silver nanocluster was synthesized. The above is only the preferred embodiment of the present invention, and it should be pointed out that for ordinary people in the technical field, without departing from the principle of the present invention, several improvements and embellishments can be made, and these improvements and embellishments should also be regarded as the protection scope of the present invention.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. Antibacterial hydrogel and preparation method is characterized in immersing bacterial cellulose in silver nitrate solution, adding with corresponding ligand lipoic acid, stirring at room temperature, adding with corresponding reducing agent sodium borohydride for reduction, stirring the mixture at room temperature, and finally synthesizing the hydrogel composite AgNCs@BC based on silver nanocluster.
2. Antibacterial hydrogel and preparation method, according to claim 1, is characterized in that the concentration range of the silver nitrate solution is 4-12mmol/L, the concentration range of the lipoic acid is 20-60mmol/L, and the concentration range of the sodium borohydride is 8-24mmol/L.
3. Antibacterial hydrogel and preparation method, according to claim 2, is characterized in that the mass concentration ratio of silver nitrate, lipoic acid and sodium borohydride is 1:5:2.
4. Antibacterial hydrogel and preparation method, according to claim 1, is characterized in that adding with corresponding ligand lipoic acid, and stirring at room temperature for 15min.
5. Antibacterial hydrogel and preparation method, according to claim 1, is characterized in that adding with corresponding ligand lipoic acid, and stirring at room temperature for 15min.
6. Antibacterial hydrogel and preparation method, according to claim 1, is characterized in that adding with corresponding reducing agent sodium borohydride for reduction, and stirring at room temperature for 24h.
7. The antibacterial hydrogel prepared by the preparation method according to any from claim 1 to 6.
8. The antibacterial hydrogel, according to claim 7, applies for preparing antibacterial and controlled release drugs.
9. The application according to claim 8, is characterized in that the concentration of the drug is 10mmol/L.