Preparation method of long-acting antibacterial nano-enzyme turbid liquid capable of realizing spraying type rapid film forming
By optimizing the preparation process of nanoenzymes, including magnetic stirring, wet dispersion grinding and supercritical extraction, the problems of uneven distribution and low stability of nanoenzyme particles are solved, achieving efficient and safe antibacterial effects and long-term antibacterial inhibition. performance.
Patent Information
- Application Number
- CN202411208997.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2024-12-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
During the preparation process, existing nanoenzymes have problems such as uneven distribution of nanoparticles, large particle size, poor biocompatibility, low stability and difficult to evaluate the antibacterial effect, which affects their application effect and safety.
By preparing a mixture of aluminum oxide suspension and silver salt solution, magnetic stirring and rotary evaporation, coarse and fine grinding were carried out after calcination, nanoparticles were dispersed using a wet dispersion grinder, supercritical extraction and multiple centrifugal washing were performed. , the antibacterial effect and cytotoxicity of different silver contents were measured, and the preparation conditions were optimized to obtain uniformity and small particle size nanoenzyme suspension.
The uniformity and small particle size characteristics of nanoparticles are achieved, the antibacterial effect and biocompatibility are enhanced, the stability and long-term antibacterial properties of nanoenzyme suspension are ensured, and the preparation efficiency and product safety are improved.
Smart Images
Figure CN119184110A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of polymer nanozymes, and specifically to a method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension. Background Art
[0002] Traditional nanozymes often face problems such as uneven distribution of nanoparticles, large particle size and difficulty in rapid film formation during the preparation and application process, which not only affects the antibacterial effect of the nanozymes, but also has an adverse effect on their biocompatibility and stability. The polymer nanozymes of the present application can ensure that the nanoparticles are evenly distributed during the preparation process, avoiding the occurrence of particle agglomeration. At the same time, by optimizing the reaction conditions and parameters, the nanoparticles are reduced in size, so that the polymer nanozymes have a higher specific surface area and better biological activity, thereby promoting the rapid film formation of the polymer nanozyme suspension.
[0003] The defects of existing nanozymes are: 1. Patent document CN116173071B discloses a method for preparing MnNiPCN@polyvinylpyrrolidone nanozyme and its application. The document mainly considers how to solve the problems of poor biocompatibility and weak acid tolerance of nanozymes, but does not consider how to ensure the uniformity and small particle size of nanoparticles and optimize the preparation conditions to promote the rapid film formation of nanozyme suspension; 2. Patent document CN113318733B discloses a method for preparing a high-entropy nanozyme and its application. The document mainly considers how to prepare a high-entropy nanozyme by a new method, but does not consider how to solve the problems of low purity of nanoparticles, surface contamination and oxidation of nanoparticles, and poor activity and stability of nanozymes; 3. Patent document CN114917354B discloses the preparation and application of a Cu single-atom nanozyme. This document mainly considers how to use Cu to prepare single-atom nanozymes for the treatment of osteoarthritis, but does not consider how to scientifically evaluate the antibacterial effect of nanozymes, accurately determine the cytotoxicity of nanozymes, and optimize the balance between antibacterial and toxicity. 4. Patent document CN116212922B discloses a method for preparing and applying a nanozyme. The document mainly considers how to compound VOx with nitrogen-doped carbon nanomaterials to develop nanozymes with better performance, but does not consider how to ensure the quality of the nanozyme suspension, avoid problems during the spraying process, improve production efficiency and yield, and ensure the safety and effectiveness of the product. Summary of the invention
[0004] The object of the present invention is to provide a method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension to solve the problems raised in the above-mentioned background technology.
[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the preparation method of the nanozyme suspension is as follows: Step S1, preparing an aluminum oxide suspension and a silver salt solution, adding the silver salt solution dropwise into the aluminum oxide suspension and stirring, obtaining a mixed solution, performing magnetic stirring and rotary evaporation to obtain nanoparticles, and calcining the nanoparticles at a high temperature; Step S2, coarsely grinding and finely grinding the calcined nanoparticles, grinding the nanoparticles using a wet dispersion grinder to obtain a nano suspension; Step S3, measuring the particle sizes of nanoparticles with different solid contents ground under different conditions, and selecting nanoparticles with the smallest particle size; Step S4, filtering and washing the nano suspension to obtain nanoparticles; Step S5, using a supercritical extraction device to perform supercritical extraction on the nanoparticles, the supercritical fluid is carbon dioxide, and after the supercritical extraction is completed, nitrogen is used to purge the solution and supercritical fluid remaining on the surface of the nanoparticles to obtain a polymer nanozyme; Step S6, detecting the inhibition zone diameter and antibacterial effect of nanozymes with different silver contents on Escherichia coli and Staphylococcus aureus; Step S7, performing MTT quantitative detection on nanozymes with different silver contents to analyze their effects on the proliferation of L929 mouse fibroblasts within 24 hours; Step S8, preparing a polyvinyl pyrrolidone aqueous solution; Step S9, adding the nanozyme, sodium silicate solution and polyethylene glycol solution to the polyvinyl pyrrolidone aqueous solution in sequence; Step S10, adding hydroxyethyl cellulose to a mixed solution of polyvinyl pyrrolidone, nanozyme, sodium silicate solution and polyethylene glycol to obtain a polymer nanozyme suspension; Step S11: After the preparation is completed, the uniformity, stability and fluidity of the polymer nanozyme suspension are checked.
[0006] Preferably, step S1 further includes the following steps: Step S11: placing aluminum oxide and silver salt in deionized water respectively, and using a magnetic stirrer to fully stir the mixture of aluminum oxide and deionized water and the mixture of silver salt and deionized water until a uniform aluminum oxide suspension and silver salt solution are formed; Step S12: continuously stirring the mixed solution on a magnetic stirrer for 2 h, transferring the stirred mixed solution to a rotary evaporator and evaporating it to dryness for 3 h to obtain nanoparticles; Step S13: pour the nanoparticles out of the rotary evaporator, place them in a drying oven at 100° C., and dry them for 12 hours. Take the dried nanoparticles out of the drying oven and place them in a muffle furnace for calcination for 24 hours.
[0007] Preferably, step S2 further includes the following steps: Step S21: starting the grinding machine for coarse grinding to quickly remove impurities and coarse particles on the surface of the nanoparticles, and performing fine grinding when the average diameter of the nanoparticles is 500-1000nm, and stopping the fine grinding when the average diameter of the nanoparticles is 400-500nm; Step S22: placing the ground nanoparticles in a wet dispersion grinder, adding deionized water as a dispersion medium, adding an interface modifier, starting the wet dispersion grinder for high-speed stirring, and during the dispersion process, using a particle size analyzer to monitor the dispersion state and particle size distribution of the nanoparticles in real time, and obtaining a nanosuspension after the dispersion is completed.
[0008] Preferably, step S3 further includes the following steps: Step S31: measuring the particle sizes of the nanoparticles when the solid content is 1%, 2%, 5%, 10%, 20% and 30% respectively, when the grinding is performed at 300 r / min and 600 r / min.
[0009] Preferably, step S4 further includes the following steps: Step S41: Soak the filter in deionized water for 1 hour and then take it out, slowly add the dispersed nano suspension into the filter for filtration, transfer the filtered nano suspension into a centrifuge tube for centrifugation, after the centrifugation, the nanoparticles are deposited at the bottom of the centrifuge tube, and most of the impurities and washing liquid remain in the upper layer, after removing the upper layer of liquid, add new deionized water and centrifuge again for washing, repeating 6-10 times; Step S42: Take out the nanoparticles after centrifugal washing from the centrifuge tube, spread the nanoparticles evenly on a drying plate, and place the nanoparticles in a well-ventilated, temperature-appropriate and dust-free environment to dry naturally for 12-18 hours to obtain pretreated nanoparticles.
[0010] Preferably, step S8 further includes the following steps: Step S81: After adding deionized water to a clean and impurity-free glass container, slowly add polyvinyl pyrrolidone therein, use a stirrer to preliminarily stir the mixture of polyvinyl pyrrolidone and deionized water for 0.5 h, transfer the solution after preliminarily stirring to an ultrasonic dispersion device, start the ultrasonic dispersion device, and perform ultrasonic treatment for 3 h to form a polyvinyl pyrrolidone aqueous solution.
[0011] Preferably, step S9 further includes the following steps: Step S91: transfer the polyvinyl pyrrolidone aqueous solution to a stirrer, start stirring, slowly add the nanozyme to the polyvinyl pyrrolidone aqueous solution, and continue to stir the solution while adding the nanozyme. The stirring direction and speed are kept consistent, and the stirring time is 2 hours; Step S92: dissolving sodium silicate in deionized water to form a sodium silicate solution, slowly adding the sodium silicate solution to the solution containing the nanozyme and polyvinyl pyrrolidone, stirring while adding, and continuing to stir for 20-25 minutes after adding the sodium silicate solution; Step S93: dissolve polyethylene glycol in deionized water to form a polyethylene glycol solution, slowly add the polyethylene glycol solution to the solution containing nanozymes, polyvinyl pyrrolidone and sodium silicate, stirring while adding, and continue stirring for 20-25 minutes after adding the polyethylene glycol solution.
[0012] Preferably, step S10 further includes the following steps: Step S101: pre-disperse hydroxyethyl cellulose in high-speed stirring ethanol, and then slowly add it into high-speed stirring cold water, and gradually increase the temperature to promote dissolution, and slowly add the dissolved hydroxyethyl cellulose solution into the solution containing nanozymes, polyvinyl pyrrolidone, sodium silicate and polyethylene glycol, stirring while adding, and after adding hydroxyethyl cellulose, continue stirring for 45-50 minutes to obtain a polymer nanozyme suspension, and use a homogenizer to homogenize the polymer nanozyme suspension.
[0013] Preferably, step S11 further includes the following steps: Step S111: placing the prepared polymer nanozyme suspension in a glass test tube, leaving it to stand for 30 minutes, and then observing the distribution of solid particles in the polymer nanozyme suspension; Step S112: placing the prepared polymer nanozyme suspension in a glass test tube, sealing the tube mouth, and standing for 36 hours to observe whether the polymer nanozyme suspension shows stratification, precipitation, or solid particle aggregation during the standing process; Step S113: placing the prepared polymer nanozyme suspension in a glass test tube, slowly pouring it into another glass test tube, and observing its flow state; Step S114: If the inspection result shows that the polymer nanozyme suspension is uneven or stratified, repeat steps S8 to S11.
[0014] Preferably, the aluminum oxide is one of aluminum oxide and aluminum hydroxide, and the silver salt is one of silver nitrate, silver acetate and silver oxide.
[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention performs coarse grinding and fine grinding on the calcined nanoparticles, grinds the nanoparticles using a wet dispersion grinder, measures the particle sizes of nanoparticles with different solid contents grinded under different conditions, selects nanoparticles with the smallest particle size, and obtains a nano suspension. The coarse grinding and fine grinding are combined, and the nanoparticles are finely ground to an average diameter of 400-500nm, thereby greatly improving the uniformity and small particle size characteristics of the nanoparticles. The nanoparticles with small particle sizes have a higher specific surface area, thereby enhancing their surface activity, which is conducive to the antibacterial effect. Deionized water and an interface modifier are added to the wet dispersion grinder, and the nanoparticles are uniformly dispersed in the dispersion medium by high-speed stirring, and a particle size analyzer is used to monitor the dispersion in real time. The state and particle size distribution ensure the stability and long-term storage of the nanosuspension, and avoid uneven dispersion of nanoparticles or particle agglomeration affecting its antibacterial effect and film-forming performance. At the same time, by measuring the particle size of different nanoparticles with different solid contents ground at different speeds, the optimal preparation conditions can be screened out to obtain the nanosuspension with the smallest particle size and the most uniform dispersion. Due to the small particle size and high specific surface area of the nanoparticles, the nanozyme can more effectively contact with bacteria and exert an antibacterial effect, and can promote the rapid film formation of the nanozyme suspension. This optimization not only improves the preparation efficiency, but also reduces the production cost. Therefore, it can solve the problem of how to ensure the uniformity and small particle size of the nanoparticles, optimize the preparation conditions, and thus promote the rapid film formation of the nanozyme suspension.
[0016] 2. The present invention obtains nanoparticles by filtering and washing a nano suspension, and then uses a supercritical extraction device to perform supercritical extraction on the nanoparticles. The supercritical fluid is carbon dioxide. After the supercritical extraction, nitrogen is used to purge the residual solution and supercritical fluid on the surface of the nanoparticles to obtain nanozymes. Through multiple centrifugal washing processes, impurities, unreacted products, by-products and residues in the washing liquid on the surface of the nanoparticles can be effectively removed, which greatly improves the purity of the nanoparticles and provides a better foundation for their subsequent antibacterial performance. The residual solution on the surface of the nanoparticles can be effectively removed by supercritical extraction, because carbon dioxide has good solubility and diffusivity in a supercritical state, and can penetrate into the tiny pores on the surface of the nanoparticles to take out the residues. After supercritical extraction, Using nitrogen to purge the surface of the nanoparticles can further remove residual solution and supercritical fluid, making the surface of the nanoparticles cleaner. In addition, nitrogen purging may also have a certain activation effect on the surface of the nanoparticles, increase its surface activity, and be beneficial to the antibacterial performance. During the entire treatment process, especially in the drying stage, the nanoparticles are placed in a well-ventilated, temperature-appropriate and dust-free environment for natural drying. This can prevent the nanoparticles from being contaminated and oxidized, help maintain the stability and activity of the nanoparticles, ensure their long-lasting antibacterial effect, and enable the nanozyme to maintain its antibacterial effect for a longer time after spraying and film formation, thereby extending the service life of the substrate. Therefore, the problems of low purity of nanoparticles, surface contamination and oxidation of nanoparticles, and poor activity and stability of nanozymes can be solved.
[0017] 3. The present invention detects the diameter of the inhibition zone and the antibacterial effect of nanozymes with different silver contents on Escherichia coli and Staphylococcus aureus, performs MTT quantitative detection on nanozymes with different silver contents, and analyzes their effects on the proliferation of L929 mouse fibroblasts within 24 hours. By detecting the diameter of the inhibition zone and the antibacterial effect of nanozymes with different silver contents on Escherichia coli and Staphylococcus aureus, the inhibitory ability of nanozymes on these two common pathogens can be intuitively understood. This quantitative evaluation method provides a reliable basis for the antibacterial effect of nanozymes. It is determined through experiments that when the silver content is 100 μg / mL, the nanozyme can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus, and shows extremely low toxicity to mouse fibroblasts, with a cell survival rate of nearly 90%, which is conducive to achieving inhibition. It provides an important basis for a good balance between antibacterial effect and cytotoxicity. At the same time, the toxicity of nanozymes to mouse fibroblasts was evaluated by MTT quantitative detection, which proved that the nanozyme had no significant effect on cell survival at a specific concentration. It not only verified the biocompatibility of nanozymes, but also supported the conclusion that nanozymes had no significant effect on cell survival at a specific concentration through data. This provides strong data support for the safe application of nanozymes in biomedicine, food packaging, and environmental sanitation. By combining the nanozyme suspension with excellent antibacterial effect and good biocompatibility with the spray-type rapid film-forming technology, a film with long-lasting antibacterial function was prepared. Therefore, the problem of how to scientifically evaluate the antibacterial effect of nanozymes, accurately determine the cytotoxicity of nanozymes, and optimize the balance between antibacterial and toxicity can be solved.
[0018] 4. The present invention checks the uniformity, stability and fluidity of the nanozyme suspension after the nanozyme suspension is prepared. The quality of the nanozyme suspension directly affects its antibacterial effect and spray film performance. By checking the uniformity, stability and fluidity of the suspension, the poor quality of the suspension caused by improper operation or material problems in the preparation process can be discovered and eliminated in time to ensure the quality of the final product. If the nanozyme suspension is uneven, stratified or solid particles aggregate, then uneven spraying and poor film quality may occur during the spraying process, affecting the antibacterial effect and appearance. By checking and repeating The preparation of unqualified suspension can ensure the stability and consistency during the spraying process. At the same time, by conducting a quality inspection immediately after the preparation is completed, potential problems can be discovered and resolved in a timely manner, avoiding the use of unqualified suspension in subsequent production processes, thereby saving time and resources, and improving production efficiency and yield. Through strict quality inspections, it can be ensured that the nanozyme suspension will not cause harm to the substrate during use and achieve the expected antibacterial effect. Therefore, the problem of how to ensure the quality of the nanozyme suspension, avoid problems during the spraying process, improve production efficiency and yield, and ensure the safety and effectiveness of the product can be solved. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The antibacterial effect of nanozymes with different silver contents on Escherichia coli of the present invention; Figure 2 The antibacterial effect of nanozymes with different silver contents on Staphylococcus aureus of the present invention; Figure 3 is a scanning electron microscope image of the nanozyme of the present invention; Figure 4 This is the energy dispersion spectrum of the silver element on the nanozyme of the present invention; Figure 5 The survival rate of L929 mouse fibroblasts treated with nanozymes with different silver contents of the present invention Figure 6 The antibacterial effect of the long-acting antibacterial film with different nanozyme contents on Escherichia coli of the present invention; Figure 7 The figure shows the antibacterial effect of the long-acting antibacterial film with different nanozyme contents on Staphylococcus aureus. DETAILED DESCRIPTION
[0020] 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. Example 1
[0021] A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the preparation method of the nanozyme suspension is as follows: Step S1, preparing an aluminum oxide suspension and a silver salt solution, adding the silver salt solution dropwise into the aluminum oxide suspension and stirring, obtaining a mixed solution, performing magnetic stirring and rotary evaporation to obtain nanoparticles, and calcining the nanoparticles at a high temperature; Step S1 also includes the following steps: Step S11: placing aluminum oxide and silver salt in deionized water respectively, and using a magnetic stirrer to fully stir the mixture of aluminum oxide and deionized water and the mixture of silver salt and deionized water until a uniform aluminum oxide suspension and silver salt solution are formed; Step S12: continuously stirring the mixed solution on a magnetic stirrer for 2 h, transferring the stirred mixed solution to a rotary evaporator and evaporating it to dryness for 3 h to obtain nanoparticles; Step S13: pour the nanoparticles out of the rotary evaporator, place them in a drying oven at 100° C., and dry them for 12 hours. Take the dried nanoparticles out of the drying oven and place them in a muffle furnace for calcination for 24 hours.
[0022] Further, aluminum oxide is dispersed in deionized water to form a suspension, and silver salt is dissolved to form a solution, and a magnetic stirrer is used to ensure that aluminum oxide particles are evenly dispersed in the deionized water to avoid particle coagulation, and at the same time, the silver salt is fully dissolved in the deionized water to form a uniform solution; The mixed solution was stirred continuously for 2 hours on a magnetic stirrer to ensure full contact between the aluminum oxide and the silver salt, and then transferred to a rotary evaporator to remove water by rotary evaporation to avoid overheating and destruction of the nanoparticle structure. The structure of the nanoparticles was stabilized by calcination to promote crystallization of the nanoparticles and enhance their stability and performance. Example 2
[0023] A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S2, coarsely grinding and finely grinding the calcined nanoparticles, using a wet dispersion grinder to grind the nanoparticles to obtain a nano suspension; Step S2 also includes the following steps: Step S21: starting the grinding machine for coarse grinding to quickly remove impurities and coarse particles on the surface of the nanoparticles, and performing fine grinding when the average diameter of the nanoparticles is 500-1000nm, and stopping the fine grinding when the average diameter of the nanoparticles is 400-500nm; Step S22: placing the ground nanoparticles in a wet dispersion grinder, adding deionized water as a dispersion medium, adding an interface modifier, starting the wet dispersion grinder for high-speed stirring, and during the dispersion process, using a particle size analyzer to monitor the dispersion state and particle size distribution of the nanoparticles in real time, and obtaining a nanosuspension after the dispersion is completed.
[0024] Further, the mill is started to preliminarily crush the nanoparticles by mechanical force, quickly remove impurities and larger coarse particles on the surface of the particles, and preliminarily reduce the particle size, and control the average diameter of the nanoparticles to reach the range of 500-1000nm. When the average diameter of the nanoparticles reaches the preset coarse grinding range, it enters the fine grinding stage, and a finer grinding method is used in the fine grinding stage to continue to reduce the particle size until the desired particle size range of 400-500nm is reached; The ground nanoparticles are evenly dispersed in deionized water to form a stable nanosuspension, and the dispersibility and stability of the particles are improved by adding an interface modifier. The ground nanoparticles are transferred to a wet dispersion grinder, an appropriate amount of deionized water is added as a dispersion medium, and an interface modifier is added to the nanosuspension. The interface modifier can be adsorbed on the surface of the nanoparticles, changing its surface properties, thereby reducing the interaction between the particles and improving the dispersibility; Start the wet dispersion grinder to stir the nanosuspension at high speed. During the stirring process, the interfacial modifier will evenly cover the surface of the nanoparticles to promote the dispersion of the particles. At the same time, use a particle size analyzer to monitor the dispersion state and particle size distribution of the nanoparticles in real time to ensure that the dispersion effect meets the requirements. When the nanoparticles form a stable and uniform nanosuspension in deionized water and the particle size distribution meets the requirements, stop stirring and take out the nanosuspension for subsequent processing or application. Example 3
[0025] A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S3, measuring the particle sizes of nanoparticles with different solid contents ground under different conditions, and selecting nanoparticles with the smallest particle size; Step S3 also includes the following steps: Step S31: measuring the particle sizes of the nanoparticles when the solid content is 1%, 2%, 5%, 10%, 20% and 30% respectively, when the grinding is performed at 300 r / min and 600 r / min.
[0026] Further, the particle size measurement results of different nanoparticle solid contents milled under different conditions are as follows: The measurement results show that when the solid content of the nanoparticles is 30%, the particle size is 412d.nm when milled at 300r / min, which is the minimum particle size in the measurement data. Example 4
[0027] A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S4, filtering and washing the nano suspension to obtain nanoparticles; Step S4 also includes the following steps: Step S41: Soak the filter in deionized water for 1 hour and then take it out, slowly add the dispersed nano suspension into the filter for filtration, transfer the filtered nano suspension into a centrifuge tube for centrifugation, after the centrifugation, the nanoparticles are deposited at the bottom of the centrifuge tube, and most of the impurities and washing liquid remain in the upper layer, after removing the upper layer of liquid, add new deionized water and centrifuge again for washing, repeating 6-10 times; Step S42: Take out the nanoparticles after centrifugal washing from the centrifuge tube, spread the nanoparticles evenly on a drying plate, and place the nanoparticles in a well-ventilated, temperature-appropriate and dust-free environment to dry naturally for 12-18 hours to obtain pretreated nanoparticles.
[0028] Furthermore, the filter is first soaked in deionized water for 1 hour to remove impurities from the filter itself and fully wet it, and then the filter is taken out and prepared for filtering the nanosuspension, and the dispersed nanosuspension is slowly added to the filter to avoid excessive pressure that may cause damage to the nanoparticles. During the filtration process, the nanoparticles will be trapped on the filter, while most of the impurities and washing liquid will flow out through the filter; The filtered nano suspension is transferred to a centrifuge tube for centrifugation. During the centrifugation process, due to the effect of gravity, the nanoparticles will settle at the bottom of the centrifuge tube, while most of the impurities and washing liquid will remain in the upper layer. After the centrifugation, the upper layer of liquid is carefully removed to avoid disturbing the nanoparticles at the bottom. In order to completely remove impurities and washing liquid residues on the surface of the nanoparticles, new deionized water needs to be added for centrifugal washing again, and this process is repeated 6-10 times until the washing liquid is clear and transparent, and there is almost no impurity in the upper liquid after centrifugation.
[0029] The nanoparticles after centrifugal washing are taken out from the centrifuge tube and spread evenly on the drying tray. When spreading, avoid excessive accumulation of nanoparticles to ensure the drying effect. The drying tray with the spread nanoparticles is placed in a well-ventilated, temperature-appropriate and dust-free environment for natural drying. The drying time is 12-18 hours. Example 5
[0030] A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S5, using a supercritical extraction device to perform supercritical extraction on nanoparticles, the supercritical fluid is carbon dioxide, after the supercritical extraction is completed, using nitrogen to purge the solution and supercritical fluid remaining on the surface of the nanoparticles to obtain a polymer nanozyme; Further, prepare a supercritical extraction device, ensure that the supercritical extraction device is in good working condition, check whether all components are tightly connected and leak-free, prepare carbon dioxide as a supercritical fluid, and ensure that its purity and pressure meet the experimental requirements; Carefully add the pretreated nanoparticles into the extraction kettle of the supercritical extraction device, pay attention to controlling the loading amount to avoid the nanoparticles from being too densely piled up during the extraction process to affect the extraction effect, start the supercritical extraction device, adjust the temperature and pressure to above the critical point of supercritical carbon dioxide, the temperature and pressure are 31.1°C and 7.38MPa, so that carbon dioxide enters a supercritical state. In the supercritical state, carbon dioxide has a solubility similar to that of liquid and a diffusion speed similar to that of gas, and can penetrate deep into the nanoparticles for extraction; After the extraction is completed, the supercritical carbon dioxide is restored to a gaseous state and discharged from the extraction kettle by adjusting the temperature and pressure. At the same time, the extracted polymer nanozymes are collected. In order to completely remove the residual solution and supercritical fluid on the surface of the nanoparticles, nitrogen is used for purging. Nitrogen is an inert gas that will not chemically react with nanoparticles and is easy to obtain and purify. The nitrogen is passed into the container containing the nanoparticles to ensure that the surface of the nanoparticles is clean and free of residue. Example 6
[0031] See also Figure 1 , Figure 2 , Figure 3 and Figure 4 , an embodiment provided by the present invention: a method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S6, detecting the antibacterial zone diameter and antibacterial effect of nanozymes with different silver contents on Escherichia coli and Staphylococcus aureus; For further information, see Figure 1 , Figure 2 , Figure 3 and Figure 4 When the silver content is 10μg / mL, the polymer nanozyme suspension can completely kill Staphylococcus aureus and Escherichia coli within 20 minutes, and the antibacterial rate can reach 99.999% within 5 minutes of contact with bacteria, with good antibacterial effect. The carrier alumina without silver loading did not show antibacterial activity, indicating that the adsorption and inhibition of bacteria by the carrier alumina in this experiment can be ignored. The nanozyme suspension can show excellent antibacterial properties when the silver content is 10μg / mL, and its antibacterial effect is gradually enhanced with the increase of silver content. The bacterial suspension used in the experiment is 107CFU / mL, indicating that the effective substances released by the nanozyme suspension can quickly inactivate high-order bacteria, which further shows that the nanozyme suspension can show good antibacterial effect when used. Example 7
[0032] See also Figure 5 , an embodiment provided by the present invention: a method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S7, by performing MTT quantitative detection on nanozymes with different silver contents, analyzing their effects on the proliferation of L929 mouse fibroblasts within 24 hours; Furthermore, the diameters of the inhibition zones of nanozymes at different concentrations are as follows: The measurement results show that when the silver content is 100 μg / mL, the cell survival rates of the nanozyme suspension extract after being cultured with cells for 12 h and 24 h, respectively, were 92.612% and 89.863%. According to the cytotoxicity grade classification standard, it is considered that the toxicity to cells is very small at this concentration and the effect on cell survival is not significant. Example 8
[0033] A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S8, preparing a polyvinyl pyrrolidone aqueous solution; Step S8 also includes the following steps: Step S81: After adding deionized water to a clean and impurity-free glass container, slowly add polyvinyl pyrrolidone therein, use a stirrer to preliminarily stir the mixture of polyvinyl pyrrolidone and deionized water for 0.5 h, transfer the solution after preliminarily stirring to an ultrasonic dispersion device, start the ultrasonic dispersion device, and perform ultrasonic treatment for 3 h to form a polyvinyl pyrrolidone aqueous solution.
[0034] Further, first, a clean and impurity-free glass container is selected, and the inner wall of the container is ensured to be smooth and scratch-free to avoid affecting the purity of the subsequent solution. Then, deionized water is added to the container, and while slowly stirring the deionized water, the polyvinyl pyrrolidone powder is gradually added to the deionized water. The adding speed should not be too fast to prevent the polyvinyl pyrrolidone powder from agglomerating or causing a local excessive concentration. The stirrer is selected to be a type suitable for mixing low-viscosity liquids, including a magnetic stirrer and an electric stirrer, and it is ensured that the stirring blade can fully stir the polyvinyl pyrrolidone powder at the bottom of the container; Turn on the stirrer to preliminarily stir the mixture of polyvinyl pyrrolidone and deionized water for 0.5 hours. The purpose of the preliminarily stirring is to preliminarily disperse the polyvinyl pyrrolidone powder in the deionized water to form a relatively uniform mixture; The solution after preliminary stirring was transferred to an ultrasonic dispersion device. The ultrasonic dispersion device utilized the cavitation effect, mechanical effect, and thermal effect of ultrasonic waves to effectively disperse the particles or agglomerates in the solution into smaller particles and improve the uniformity and stability of the solution. The ultrasonic dispersion device was started, and the ultrasonic working time was set to 3 hours for ultrasonic treatment. After ultrasonic treatment, the polyvinyl pyrrolidone particles or agglomerates that may have existed originally will be effectively dispersed into smaller particles and evenly distributed in the deionized water to form a stable and transparent polyvinyl pyrrolidone aqueous solution. Example 9
[0035] A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S9, adding the nanozyme, sodium silicate solution and polyethylene glycol solution to a polyvinyl pyrrolidone aqueous solution in sequence; Step S9 also includes the following steps: Step S91: transfer the polyvinyl pyrrolidone aqueous solution to a stirrer, start stirring, slowly add the nanozyme to the polyvinyl pyrrolidone aqueous solution, and continue to stir the solution while adding the nanozyme. The stirring direction and speed are kept consistent, and the stirring time is 2 hours; Step S92: dissolving sodium silicate in deionized water to form a sodium silicate solution, slowly adding the sodium silicate solution to the solution containing the nanozyme and polyvinyl pyrrolidone, stirring while adding, and continuing to stir for 20-25 minutes after adding the sodium silicate solution; Step S93: dissolve polyethylene glycol in deionized water to form a polyethylene glycol solution, slowly add the polyethylene glycol solution to the solution containing nanozymes, polyvinyl pyrrolidone and sodium silicate, stirring while adding, and continue stirring for 20-25 minutes after adding the polyethylene glycol solution.
[0036] Furthermore, polyvinyl pyrrolidone is used as a dispersant to help the nanozyme particles disperse evenly in deionized water. Polyvinyl pyrrolidone has good water solubility and colloid protection ability, can effectively prevent the agglomeration of nanoparticles, and improve their stability in aqueous solution. The polyvinyl pyrrolidone aqueous solution is placed in a stirrer, and the nanozyme is slowly added after stirring. During this process, it is very important to keep the stirring direction and speed consistent to ensure that the nanozyme particles can be evenly and stably dispersed in the polyvinyl pyrrolidone aqueous solution. The stirring time is set to 2 hours to ensure that the nanozyme and polyvinyl pyrrolidone are fully mixed; Sodium silicate, as a stabilizer, can further enhance the stability of the nanozyme suspension. Sodium silicate forms silicate ions in water. These ions can be adsorbed on the surface of nanoparticles to form a protective layer to prevent mutual attraction and agglomeration between particles. After sodium silicate is dissolved in deionized water to form a solution, it is slowly added to the mixture containing nanozyme and polyvinyl pyrrolidone, stirring while adding to ensure that the sodium silicate is evenly distributed and effectively acts on the surface of the nanoparticles. After adding the sodium silicate solution, continue stirring for 20-25 minutes; As a film-forming aid, polyethylene glycol can improve the film-forming properties of the nanozyme suspension. Polyethylene glycol has good film-forming properties and water solubility, can increase the viscosity of the suspension, and helps to form a uniform and continuous film layer in the subsequent processing. After polyethylene glycol is dissolved in deionized water to form a solution, it is also slowly added to the mixed solution containing nanozymes, polyvinyl pyrrolidone and sodium silicate, stirring while adding to ensure that the polyethylene glycol is evenly distributed and fully exerts its role as a film-forming aid. After adding the polyethylene glycol solution, continue stirring for 20-25 minutes.
[0037] Embodiment 10: A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, the method for preparing the nanozyme suspension is as follows: step S10, adding hydroxyethyl cellulose to a mixed solution of polyvinyl pyrrolidone, nanozyme, sodium silicate solution and polyethylene glycol to obtain a polymer nanozyme suspension; Step S10 also includes the following steps: Step S101: pre-disperse hydroxyethyl cellulose in high-speed stirring ethanol, and then slowly add it into high-speed stirring cold water, and gradually increase the temperature to promote dissolution, and slowly add the dissolved hydroxyethyl cellulose solution into the solution containing nanozymes, polyvinyl pyrrolidone, sodium silicate and polyethylene glycol, stirring while adding, and after adding hydroxyethyl cellulose, continue stirring for 45-50 minutes to obtain a polymer nanozyme suspension, and use a homogenizer to homogenize the polymer nanozyme suspension.
[0038] Furthermore, hydroxyethyl cellulose, as a thickener, mainly plays the role of regulating viscosity and fluidity in the nanozyme suspension. By increasing the viscosity of the nanozyme suspension, the sedimentation rate of nanoparticles can be slowed down and the stability of the nanozyme suspension can be improved. At the same time, appropriate viscosity can also help improve the processing performance and film-forming properties of the nanozyme suspension.
[0039] Embodiment 11: A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension. The method for preparing the nanozyme suspension is as follows: Step S11, after the preparation is completed, check the uniformity, stability and fluidity of the polymer nanozyme suspension.
[0040] Step S11 also includes the following steps: Step S111: placing the prepared polymer nanozyme suspension in a glass test tube, leaving it to stand for 30 minutes, and then observing the distribution of solid particles in the polymer nanozyme suspension; Step S112: placing the prepared polymer nanozyme suspension in a glass test tube, sealing the tube mouth, and standing for 36 hours to observe whether the polymer nanozyme suspension shows stratification, precipitation, or solid particle aggregation during the standing process; Step S113: placing the prepared polymer nanozyme suspension in a glass test tube, slowly pouring it into another glass test tube, and observing its flow state; Step S114: If the inspection result shows that the polymer nanozyme suspension is uneven or stratified, repeat steps S8 to S11.
[0041] Furthermore, the aluminum oxide is one of aluminum oxide and aluminum hydroxide, and the silver salt is one of silver nitrate, silver acetate and silver oxide.
[0042] Embodiment 12: See also Figure 6 and Figure 7 The present invention provides an embodiment: a method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, wherein the nanozyme suspension is placed under natural light for 15 days to simulate actual use, and then a continuous antibacterial test is performed. Figure 6 and Figure 7 The antibacterial effects of long-lasting antibacterial films with different nanozyme contents on Escherichia coli and Staphylococcus aureus are shown in Figure 2. Figure 6 and Figure 7 a indicates that the nanozyme content is 2%, b indicates that the nanozyme content is 1%, c indicates that the nanozyme content is 0.5%, and d indicates the blank control; The results of the continuous antibacterial test showed that when the nanozyme content was 2%, the long-lasting antibacterial film made could reach more than 99.99% against Escherichia coli and Staphylococcus aureus within 30 minutes of reaction, indicating that after the nanozyme suspension forms a film, it can come into contact with bacteria, allowing the nanozyme suspension contained on the membrane surface to inactivate Escherichia coli and Staphylococcus aureus in a short time.
[0043] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.
Claims
1. A method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension, characterized in that: The preparation method of nanozyme suspension is as follows: Step S1, preparing an aluminum oxide suspension and a silver salt solution, adding the silver salt solution dropwise into the aluminum oxide suspension and stirring, obtaining a mixed solution, performing magnetic stirring and rotary evaporation to obtain nanoparticles, and calcining the nanoparticles at a high temperature; Step S2, coarsely grinding and finely grinding the calcined nanoparticles, grinding the nanoparticles using a wet dispersion grinder to obtain a nano suspension; Step S3, measuring the particle sizes of nanoparticles with different solid contents ground under different conditions, and selecting nanoparticles with the smallest particle size; Step S4, filtering and washing the nano suspension to obtain nanoparticles; Step S5, using a supercritical extraction device to perform supercritical extraction on the nanoparticles, the supercritical fluid is carbon dioxide, and after the supercritical extraction is completed, nitrogen is used to purge the solution and supercritical fluid remaining on the surface of the nanoparticles to obtain a polymer nanozyme; Step S6, detecting the inhibition zone diameter and antibacterial effect of nanozymes with different silver contents on Escherichia coli and Staphylococcus aureus; Step S7, performing MTT quantitative detection on nanozymes with different silver contents to analyze their effects on the proliferation of L929 mouse fibroblasts within 24 hours; Step S8, preparing a polyvinyl pyrrolidone aqueous solution; Step S9, adding the nanozyme, sodium silicate solution and polyethylene glycol solution to the polyvinyl pyrrolidone aqueous solution in sequence; Step S10, adding hydroxyethyl cellulose to a mixed solution of polyvinyl pyrrolidone, nanozyme, sodium silicate solution and polyethylene glycol to obtain a polymer nanozyme suspension; Step S11: After the preparation is completed, the uniformity, stability and fluidity of the polymer nanozyme suspension are checked.
2. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S1 also includes the following steps: Step S11: placing aluminum oxide and silver salt in deionized water respectively, and using a magnetic stirrer to fully stir the mixture of aluminum oxide and deionized water and the mixture of silver salt and deionized water until a uniform aluminum oxide suspension and silver salt solution are formed; Step S12: continuously stirring the mixed solution on a magnetic stirrer for 2 h, transferring the stirred mixed solution to a rotary evaporator and evaporating it to dryness for 3 h to obtain nanoparticles; Step S13: pour the nanoparticles out of the rotary evaporator, place them in a drying oven at 100° C., and dry them for 12 hours. Take the dried nanoparticles out of the drying oven and place them in a muffle furnace for calcination for 24 hours.
3. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S2 also includes the following steps: Step S21: starting the grinding machine for coarse grinding to quickly remove impurities and coarse particles on the surface of the nanoparticles, and performing fine grinding when the average diameter of the nanoparticles is 500-1000nm, and stopping the fine grinding when the average diameter of the nanoparticles is 400-500nm; Step S22: placing the ground nanoparticles in a wet dispersion grinder, adding deionized water as a dispersion medium, adding an interface modifier, starting the wet dispersion grinder for high-speed stirring, and during the dispersion process, using a particle size analyzer to monitor the dispersion state and particle size distribution of the nanoparticles in real time, and obtaining a nanosuspension after the dispersion is completed.
4. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S3 also includes the following steps: Step S31: measuring the particle sizes of the nanoparticles when the solid content is 1%, 2%, 5%, 10%, 20% and 30% respectively, when the grinding is performed at 300 r / min and 600 r / min.
5. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S4 also includes the following steps: Step S41: Soak the filter in deionized water for 1 hour and then take it out, slowly add the dispersed nano suspension into the filter for filtration, transfer the filtered nano suspension into a centrifuge tube for centrifugation, after the centrifugation, the nanoparticles are deposited at the bottom of the centrifuge tube, and most of the impurities and washing liquid remain in the upper layer, after removing the upper layer of liquid, add new deionized water and centrifuge again for washing, repeating 6-10 times; Step S42: Take out the nanoparticles after centrifugal washing from the centrifuge tube, spread the nanoparticles evenly on a drying plate, and place the nanoparticles in a well-ventilated, temperature-appropriate and dust-free environment to dry naturally for 12-18 hours to obtain pretreated nanoparticles.
6. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S8 also includes the following steps: Step S81: After adding deionized water into a clean and impurity-free glass container, slowly add polyvinyl pyrrolidone therein. The mixture of polyvinyl pyrrolidone and deionized water was preliminarily stirred using a stirrer for 0.5 h. The solution after the preliminarily stirring was transferred to an ultrasonic dispersion device, which was started and subjected to ultrasonic treatment for 3 h to form a polyvinyl pyrrolidone aqueous solution.
7. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S9 also includes the following steps: Step S91: transfer the polyvinyl pyrrolidone aqueous solution to a stirrer, start stirring, slowly add the nanozyme to the polyvinyl pyrrolidone aqueous solution, and continue to stir the solution while adding the nanozyme. The stirring direction and speed are kept consistent, and the stirring time is 2 hours; Step S92: dissolving sodium silicate in deionized water to form a sodium silicate solution, slowly adding the sodium silicate solution to the solution containing the nanozyme and polyvinyl pyrrolidone, stirring while adding, and continuing to stir for 20-25 minutes after adding the sodium silicate solution; Step S93: dissolve polyethylene glycol in deionized water to form a polyethylene glycol solution, slowly add the polyethylene glycol solution to the solution containing nanozymes, polyvinyl pyrrolidone and sodium silicate, stirring while adding, and continue stirring for 20-25 minutes after adding the polyethylene glycol solution.
8. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S10 also includes the following steps: Step S101: pre-disperse hydroxyethyl cellulose in high-speed stirring ethanol, and then slowly add it into high-speed stirring cold water, and gradually increase the temperature to promote dissolution, and slowly add the dissolved hydroxyethyl cellulose solution into the solution containing nanozymes, polyvinyl pyrrolidone, sodium silicate and polyethylene glycol, stirring while adding, and after adding hydroxyethyl cellulose, continue stirring for 45-50 minutes to obtain a polymer nanozyme suspension, and use a homogenizer to homogenize the polymer nanozyme suspension.
9. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: Step S11 also includes the following steps: Step S111: placing the prepared polymer nanozyme suspension in a glass test tube, leaving it to stand for 30 minutes, and then observing the distribution of solid particles in the polymer nanozyme suspension; Step S112: placing the prepared polymer nanozyme suspension in a glass test tube, sealing the tube mouth, and standing for 36 hours to observe whether the polymer nanozyme suspension shows stratification, precipitation, or solid particle aggregation during the standing process; Step S113: placing the prepared polymer nanozyme suspension in a glass test tube, slowly pouring it into another glass test tube, and observing its flow state; Step S114: If the inspection result shows that the polymer nanozyme suspension is uneven or stratified, repeat steps S8 to S11.
10. The method for preparing a spray-type fast-film-forming long-acting antibacterial nanozyme suspension according to claim 1, characterized in that: The aluminum oxide is one of aluminum oxide and aluminum hydroxide, and the silver salt is one of silver nitrate, silver acetate and silver oxide.