Preparation method of hydrogel with photo-thermal and antibacterial functions
By synthesizing palladium nanoparticles and Ag@Pd composite structures in hydrogels, the ability to convert light and heat into heat is used to solve the problem of antibacterial resistance, achieving efficient bacterial inhibition and killing, and providing a safe, Effective antibacterial solution.
Patent Information
- Application Number
- CN202310889058.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-19
- Publication Date
- 2024-12-13
AI Technical Summary
Existing antibacterial methods such as antibiotics and antimicrobial peptides have increased bacterial resistance due to abuse, lack of effective alternative solutions, and infectious diseases threaten global health.
Using a hydrogel preparation method with photothermal and antibacterial functions, the ability of photothermal conversion into heat is used to achieve targeted selection by synthesis of palladium nanoparticles and Ag@Pd composite structures, and the ability to convert photothermal into heat is used to combine the combination of hydrogel and nanoparticles. and broad-spectrum antibacterial properties.
This method effectively inhibits and kills bacteria, has good biosafety and antibacterial effects, avoids unnecessary diffusion of nanoparticles, and improves the safety and efficiency of treatment.
Smart Images
Figure CN119113103A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of antibacterial drugs, and in particular to a method for preparing a hydrogel with photothermal and antibacterial functions. Background Art
[0002] In recent years, with the changes in the natural environment, the adaptability of pathogens has gradually increased, and they have strong reproductive ability, resulting in an increasing number of infectious diseases caused by bacterial infections, posing a great threat to public health and hygiene. Therefore, bacterial infections have become one of the global diseases and have received widespread attention from humans. Researchers point out that if the most efficient way to deal with bacterial and viral infections is not found, the number of deaths each year will reach 10 million, and the total economic losses will exceed one hundred trillion US dollars.
[0003] Traditional antibacterial methods include antibiotics, antimicrobial peptides and quaternary ammonium compounds. Among them, antibiotics are the most widely used common drugs and have a significant effect on inhibiting pathogenic bacteria infections. However, with the uncontrolled use and even abuse of antibiotics in antibacterial treatment, multidrug-resistant bacteria have resulted, which has brought great challenges to the treatment of pathogenic bacteria infections. At present, antibiotics have poor therapeutic effects on multidrug-resistant bacteria infections, or even completely fail. Bacterial resistance caused by improper use of antibiotics has become a very serious problem facing today's medical field and human living environment. Antimicrobial peptides are new and highly effective antibacterial drugs that have been studied recently, but the cumbersome means of synthesis, complicated purification methods and high pharmaceutical costs of the drug have limited the large-scale use of the drug in the treatment of cell infections. Quaternary ammonium compounds have convenient and effective characteristics in the treatment of bacterial infections, but their defects are similar to those of antibiotics and can cause bacterial resistance. In order to solve the global disease of bacterial infection, it is urgent to find new alternative solutions to inhibit and kill bacteria. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing a hydrogel with photothermal and antibacterial functions to solve the problem of finding new alternative solutions for inhibiting and killing bacteria in order to solve the global disease of bacterial infection proposed in the above background technology.
[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing a hydrogel with photothermal and antibacterial functions, the method for preparing a hydrogel with photothermal and antibacterial functions comprising the following steps:
[0006] S1: Synthesis of palladium nanoparticles: 1 mL of potassium chloropalladate solution, 2.5 mL of hexadecyltrimethylammonium bromide solution and 17.5 mL of secondary water were weighed and placed in a three-necked flask, heated in an oil bath to condense and reflux, and 160 μL of ascorbic acid solution was slowly added dropwise after heating for 10 min, and the heating and stirring were continued for a period of time. The solution was taken out and allowed to stand and cool to room temperature, and then placed in a centrifuge tube for centrifugation, and finally the supernatant in the centrifuge tube was removed, and secondary water was continued to be added to disperse the palladium nanoparticles with ultrasound, and the above operation was continued to be repeated, and the solution was washed 2-3 times for standby use;
[0007] S2: Synthesis of Ag@Pd composite structure: The palladium nanoparticles prepared in step S2 were washed three times with 20mM hexadecyltrimethylammonium chloride solution, and the obtained sample was diluted to 1.5mL by adding 10mM hexadecyltrimethylammonium chloride solution, and then 100uL of the diluted solution was taken, and 5mL of 20mM hexadecyltrimethylammonium chloride solution was added, and the solution was heated in a water bath, and 150uL of ascorbic acid solution and 50uL of silver nitrate solution were added after 10min of timing, and the solution was condensed and refluxed, and the solution was taken out after stopping the heating, and the solution was allowed to stand and cool to room temperature, and the operation of step S1 was repeated, and the solution was washed 2-3 times, and the supernatant in the centrifuge tube was finally removed, and water was added twice to disperse the palladium nanoparticles by ultrasound, and the washed samples were finally collected for standby use;
[0008] S3: Preparation of hydrogel: Weigh 976 mg MES and dissolve it in 100 mL 40% ethanol solution to prepare a 50 mmol / mL MES solution. Take 4 mL of the above MES solution into a centrifuge tube, weigh 80 mg EDC and 24 mg NHS and dissolve them therein to prepare a 20.6 mg / mL EDC / NHS solution for standby use;
[0009] Take 0.8 g of gelatin and add 2 mL of secondary water, heat in a water bath and stir continuously, add 2 mL of LEDC / NHS solution to the prepared gelatin solution, stir for 2 min to fully mix, and finally add the cross-linked gelatin solution into 2 well plates with a pipette while it is still hot, let it stand to eliminate bubbles, and cross-link at 4°C for 24 h to obtain a cross-linked gel prepared by the "one-step method".
[0010] Preferably, the oil bath temperature in step S1 is 80°C.
[0011] Preferably, the heating temperature in step S1 is maintained at 80° C. and the heating and stirring time is 2 h.
[0012] Preferably, the centrifugal speed is 12000 r / min and the centrifugal time is 10 min.
[0013] Preferably, the water bath temperature in step S2 is 60°C.
[0014] Preferably, the condensation reflux temperature is 60° C. and the reflux time is 6 hours.
[0015] Preferably, the molar concentration of the EDC / NHS solution in step S3 is 2:1.
[0016] Preferably, the water bath temperature in step S3 is 60°C.
[0017] Preferably, the well plate in step S3 is a 12-well plate, with 2 mL in each well.
[0018] Compared with the prior art, the present invention has the following beneficial effects: the preparation method of the hydrogel with photothermal and antibacterial functions utilizes the ability of Ag@Pd surface plasmon resonance to convert light into heat and applies it to photothermal antibacterial, while utilizing the combination of hydrogel and nanoparticles to ensure the functionality of Ag@Pd nanomaterials while limiting the nanoparticles in situ to prevent unnecessary diffusion, thereby ensuring the safety of the material, and having good targeting selectivity, broad-spectrum antibacterial properties and biosafety. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a schematic diagram of the Ag@Pd ultraviolet absorption spectrum of the present invention;
[0020] Figure 2 It is a transmission electron microscope scanning schematic diagram of palladium of the present invention;
[0021] Figure 3 Schematic diagram of transmission electron microscopy of Ag@Pd composites of the present invention at different magnifications;
[0022] Figure 4 This is a schematic diagram of the energy spectrum analysis of the Ag@Pd composite of the present invention;
[0023] Figure 5 A schematic diagram of the light-to-heat conversion efficiency of the present invention;
[0024] Figure 6 Schematic diagram of the Escherichia coli plate coating with different treatments of the present invention;
[0025] Figure 7 Schematic diagram of colony count statistics of different groups of the present invention. DETAILED DESCRIPTION
[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0027] The present invention provides a method for preparing a hydrogel with photothermal and antibacterial functions. The ability of Ag@Pd surface plasmon resonance to convert light into heat is applied to photothermal antibacterial. The hydrogel and nanoparticles are combined to ensure the functionality of the Ag@Pd nanomaterial while limiting the nanoparticles in situ to prevent unnecessary diffusion, thereby ensuring the safety of the material. The hydrogel has good target selectivity, broad-spectrum antibacterial properties and biosafety. The method for preparing the hydrogel with photothermal and antibacterial functions comprises the following steps:
[0028] S1: Synthesis of palladium nanoparticles: 1 mL of potassium chloropalladate solution, 2.5 mL of hexadecyltrimethylammonium bromide solution and 17.5 mL of secondary water were weighed and placed in a three-necked flask, heated in an oil bath at 80°C for condensation and reflux, and 160 μL of ascorbic acid solution was slowly added dropwise after heating for 10 min, and the mixture was heated at 80°C and stirred for 2 h, and the solution was taken out and allowed to stand and cool to room temperature, and centrifuged at 12000 r / min for 10 min, and finally the supernatant in the centrifuge tube was removed, and secondary water was added to disperse the palladium nanoparticles with ultrasound, and the above operation was repeated, and the mixture was washed 2-3 times for standby use;
[0029] S2: Synthesis of Ag@Pd composite structure: The palladium nanoparticles prepared in step S2 were washed three times with hexadecyltrimethylammonium chloride solution (20mM), and the obtained sample was diluted to 1.5mL by adding hexadecyltrimethylammonium chloride solution (10mM), and then 100uL of the diluted solution was taken, and 5mL of hexadecyltrimethylammonium chloride solution (20mM) was added, and the solution was heated to 60°C in a water bath, and 150uL of ascorbic acid solution and 50uL of silver nitrate solution were added after timing for 10min, and the solution was condensed and refluxed at 60°C for 6h, and the solution was taken out after stopping heating, and the solution was cooled to room temperature by standing, and the operation of step S1 was repeated, and the solution was washed 2-3 times, and the supernatant in the centrifuge tube was removed, and water was added twice to disperse the palladium nanoparticles by ultrasound, and the washed samples were collected for standby use;
[0030] S3: Preparation of hydrogel:
[0031] Preparation of cross-linking agent: Weigh 976 mg MES and dissolve it in 100 mL 40% ethanol solution to prepare a 50 mmol / mL MES solution. Take 4 mL of the above MES solution in a centrifuge tube, weigh 80 mg EDC and 24 mg NH and dissolve it therein to prepare a 20.6 mg / mL EDC / NHS solution (molar concentration 2:1) for standby use;
[0032] Blank control group: Take five groups of samples with the same concentration gradient of 0.2-1.0g gelatin, add 2mL ultrapure water at 60℃ to each group and stir continuously on an electromagnetic stirrer until the gelatin particles are completely dissolved into a solution, add 2mL of crosslinker solution to each group of gelatin solution to mix the two thoroughly, take 2mL of the final prepared solution in a 12-well plate, divide each group of solutions into two groups, let it stand to eliminate bubbles, place it in a 4℃ refrigerator for 24h to wait for molding, and finally select the most suitable crosslinking concentration according to the crosslinking strength and shaping degree of each group;
[0033] According to the results obtained in the blank control group of the test group, 0.8 g of gelatin was added with 2 mL of secondary water, heated in a water bath at 60°C with continuous stirring, 2 mL of cross-linking agent was added to the prepared gelatin solution, stirred for 2 min to fully mix, and finally the cross-linked gelatin solution was added to 2 well plates (12-well plates, 2 mL per well) with a pipette while still hot to eliminate bubbles, and cross-linked at 4°C for 24 h to obtain a cross-linked gel prepared by the "one-step method".
[0034] Study on photothermal conversion performance
[0035] Take a small amount of the prepared nano-Ag@Pd hydrogel on the filter paper, fix the laser irradiation part with an iron stand and a test tube clamp, keep the distance between the laser muzzle and the sample at 2 cm, turn on the laser switch, and adjust the knob to keep the current at 2.00A and the power at 1.388W. When the laser is irradiated at the photothermal conversion hydrogel sample, use an infrared temperature imager to measure and take pictures of the center of the sample every one minute. Finally, draw and analyze the recorded data. Repeat the above operations for the blank control group and Ag@Pd nanomaterials, and draw a chart for comparison of the three groups of recorded data.
[0036] Antimicrobial efficacy studies
[0037] Before the experiment, all test tubes and culture medium were sterilized by high temperature and high pressure at 115℃ for 30min. E. coli liquid was taken out from the -80℃ refrigerator. After thawing, 1μL of E. coli was taken out and placed in a test tube containing 5mL of lysogenic broth (LB) medium. Then, the E. coli was activated by incubation in a shaking incubator at 37℃ and 120r / min for 12h and then taken out for use. The E. coli incubated overnight was diluted ten times with liquid lysogenic broth medium and continued to be incubated in a shaking incubator at 37℃ and 200r / min for two hours. Then, the bacteria were taken out and diluted with PBS buffer. The Ag@Pd hydrogel was diluted 105 times, and then 1mL of Escherichia coli suspension was added to the centrifuge tube with 1mL of Ag@Pd hydrogel at the bottom. After irradiating the Ag@Pd hydrogel with a laser (current 2.00A, power 1.388w) for 10 minutes, 40μL of the upper suspension was spread on it and cultured at 37℃ for 24 hours. The number of grown colonies was counted. According to the above treatment method, the other three experimental groups were set up: ① blank control group, ② laser irradiation group, and ③ Ag@Pd hydrogel group. Three parallel samples were set up in each group. The antibacterial efficacy of the material was judged by comparing the final number of colonies in the four experimental groups.
[0038] Experimental results and data analysis
[0039] Ultraviolet visible absorption spectrum (UV-Vis)
[0040] Figure 1 The figure shows the UV absorption spectrum of Ag@Pd. It can be seen that there is a broad UV-visible absorption spectrum at a wavelength of λ=420nm. The Ag@Pd composite material has absorption between 350-650nm. It is speculated that it is the SPR resonance absorption of silver nanoparticles, which makes the UV-Vis transverse absorption peak broaden and the longitudinal absorption peak red-shift, confirming that the composite material is a rod-like structure with a certain aspect ratio.
[0041] Transmission electron microscopy (TEM)
[0042] Figure 2 This is a TEM image of palladium nanoparticles. The shape and size of the palladium nanoparticles can be observed through the transmission electron scanning image. It is not difficult to see that the morphology of the palladium nanoparticles prepared by chemical synthesis is clearly discernible, all showing a regular cubic structure, and the size distribution is uniform, about 15-20nm.
[0043] Figure 3 The transmission electron microscope images of the Ag@Pd composite at different magnifications, where a is a low-resolution TEM image of the Ag@Pd composite, b is a local magnification image, and c is a high-resolution TEM image of the Ag@Pd composite. Figure 3 In the transmission electron microscope image, in order to observe the general morphology of the prepared photothermal conversion material Ag@Pd nanocomposite, a low-magnification TEM test was carried out. Figure 3-a, it can be seen that the top of the silver nanorods is densely loaded with a large number of palladium particles, and the wrapped sample still maintains a rod-like structure. The particle size of the nano-Ag@Pd composite is concentrated around 15-20nm, and there is a certain accumulation phenomenon in the material, such as Figure 3 -b, silver nanorods can be clearly seen, with an average rod length of about 150-200nm, and an aspect ratio of 10:1 for Ag@Pd, which is a "matchstick" structure with palladium particles loaded on both ends, such as Figure 3 -c, the top structure of the Ag@Pd particles can be clearly observed. A large number of palladium particles are enriched at the head of the silver nanorods, and most of them have a regular three-dimensional structure.
[0044] Energy dispersive spectroscopy (EDS)
[0045] Figure 4 Energy spectrum analysis diagram of Ag@Pd complex, where a is the high-resolution TEM image of Ag@Pd nanoparticles, b is the EDS image of palladium, c is the EDS image of silver, and d is the element content spectrum of Ag@Pd complex. Figure 4 -a The overall structure of the "matchstick" of the nanomaterial can be seen. The material has different degrees of lightness and darkness, and the "match head" part is the darkest in color compared to other parts. The composite structure composed of two or more elements is analyzed. Figure 4 -b and Figure 4 -c can observe the distribution of palladium and silver in the material. It is not difficult to see that most of the palladium is enriched in the top part of the rod-like structure, and there is still a small amount of palladium dispersed in the middle of the rod, while the silver is basically concentrated in the rod part, confirming that the synthesis method of palladium nanoparticles as seeds for in-situ growth of silver nanorods is Figure 4 -d sample's EDS spectrum further characterized the elements of the Ag@Pd nanocomposite material. It can be seen from the energy spectrum that some silver and palladium elements responded in the same position. It is speculated that the response was caused by the distribution of a small amount of palladium and silver elements in the rod-shaped part. In summary, the Ag@Pd nanocomposite material was successfully prepared.
[0046] Photothermal conversion performance analysis
[0047] according to Figure 5The photothermal conversion efficiencies of the hydrogel, Ag@Pd photothermal conversion material, and Ag@Pd hydrogel composite material can be compared. When the current is 2.00A and the power is maintained at 1.388W, the temperature of the hydrogel increases from room temperature (to ensure the accuracy of the comparison, the room temperature is selected at 25.0℃) to 38.6℃ within 10 minutes, and the temperature change is small. The temperature of the Ag@Pd hydrogel composite material increases from room temperature to 55.2℃ within 30 minutes, and the temperature of the Ag@Pd photothermal conversion material increases from room temperature to 61.3℃ within 10 minutes. The temperature change is the largest among the three. Compared with the temperature change of the Ag@Pd hydrogel composite material, the overall amplitude is not much different, but the temperature stability value is higher. Analysis shows that the Ag@Pd material has a higher photothermal conversion efficiency, and the Ag@Pd hydrogel composite can localize heat at the interface.
[0048] Antimicrobial activity analysis
[0049] In order to better compare the photothermal antibacterial efficacy of Ag@Pd hydrogel, the colonies of E. coli were counted 24 h after plating on the plates, as shown in Figure 2. Figure 6 and Figure 7 As shown, the number of colonies in the blank control group was 268. The second group of Ag@Pd hydrogels showed a certain antibacterial effect due to the presence of nanosilver, but the effect was not significant, and the number of colonies was only reduced to 231. The third group was irradiated with NIR for ten minutes. Compared with the blank control group, the number of colonies was 245. The number of Escherichia coli colonies was not much different, and there was no obvious change. The number of colonies observed in the fourth group of Ag@Pd hydrogels under laser irradiation for 10 minutes was significantly reduced, and the number of Escherichia coli colonies was reduced to 14, indicating that the photothermal antibacterial effect of the composite material is very significant. In summary, it can be concluded that the prepared Ag@Pd hydrogel is a more ideal photothermal antibacterial material.
[0050] In summary, the photothermal antibacterial efficacy of composite materials such as precious metals and polymers can be combined with photothermal therapy to cause irreversible damage such as bacterial cell membrane or protein denaturation, thereby enhancing the thermal antibacterial effect. In addition, biocompatible hydrogels can be used as delivery carriers for photothermal conversion materials and injected into the body to achieve sterilization of specific parts.
[0051] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced with equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed by the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.
Claims
1. A method for preparing a hydrogel with photothermal and antibacterial functions, characterized in that: The preparation method of the hydrogel with photothermal and antibacterial functions comprises the following steps: S1: Synthesis of palladium nanoparticles: 1 mL of potassium chloropalladate solution, 2.5 mL of hexadecyltrimethylammonium bromide solution and 17.5 mL of secondary water were weighed and placed in a three-necked flask, heated in an oil bath to condense and reflux, and 160 μL of ascorbic acid solution was slowly added dropwise after heating for 10 min, and the heating and stirring were continued for a period of time. The solution was taken out and allowed to stand and cool to room temperature, and then placed in a centrifuge tube for centrifugation, and finally the supernatant in the centrifuge tube was removed, and secondary water was continued to be added to disperse the palladium nanoparticles with ultrasound, and the above operation was continued to be repeated, and the solution was washed 2-3 times for standby use; S2: Synthesis of Ag@Pd composite structure: The palladium nanoparticles prepared in step S2 were washed three times with 20mM hexadecyltrimethylammonium chloride solution, and the obtained sample was diluted to 1.5mL by adding 10mM hexadecyltrimethylammonium chloride solution, and then 100uL of the diluted solution was taken, and 5mL of 20mM hexadecyltrimethylammonium chloride solution was added, and the solution was heated in a water bath, and 150uL of ascorbic acid solution and 50uL of silver nitrate solution were added after 10min of timing, and the solution was condensed and refluxed, and the solution was taken out after stopping the heating, and the solution was allowed to stand and cool to room temperature, and the operation of step S1 was repeated, and the solution was washed 2-3 times, and the supernatant in the centrifuge tube was finally removed, and water was added twice to disperse the palladium nanoparticles by ultrasound, and the washed samples were finally collected for standby use; S3: Preparation of hydrogel: Weigh 976 mg MES and dissolve it in 100 mL 40% ethanol solution to prepare a 50 mmol / mL MES solution. Take 4 mL of the above MES solution into a centrifuge tube, weigh 80 mg EDC and 24 mg NHS and dissolve them therein to prepare a 20.6 mg / mL EDC / NHS solution for standby use; Take 0.8 g of gelatin and add 2 mL of secondary water, heat in a water bath and stir continuously, add 2 mL of LEDC / NHS solution to the prepared gelatin solution, stir for 2 min to fully mix, and finally add the cross-linked gelatin solution into 2 well plates with a pipette while it is still hot, let it stand to eliminate bubbles, and cross-link at 4°C for 24 h to obtain a cross-linked gel prepared by the "one-step method".
2. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 1, characterized in that: The oil bath temperature in step S1 is 80°C.
3. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 2, characterized in that: In step S1, the heating temperature is maintained at 80° C. and the heating stirring time is 2 h.
4. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 3, characterized in that: The centrifugal speed is 12000 r / min, and the centrifugal time is 10 min.
5. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 4, characterized in that: The water bath temperature in step S2 is 60°C.
6. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 5, characterized in that: The condensation reflux temperature is 60° C., and the reflux time is 6 h.
7. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 6, characterized in that: The molar concentration of the EDC / NHS solution in step S3 is 2:
1.
8. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 7, characterized in that: The water bath temperature in step S3 is 60°C.
9. The method for preparing a hydrogel with photothermal and antibacterial functions according to claim 8, characterized in that: The well plate in step S3 is a 12-well plate, with 2 mL per well.