Preparation method of biocompatible nano-silver

Abstract

The invention belongs to the technical field of silver particle preparation, and particularly relates to a preparation method of biocompatible nano-silver. Na [OSO3CH (C19H37N203) 2] is synthesized by using lysine and epichlorohydrin, the substance can be used as a reducing agent and a surfactant for preparing nano-silver, the obtained nano-silver particles are spherical, and the nano-silver particles stabilized by the novel surfactant have no influence on red blood cells of a human body and cannot cause apoptosis of the cells. According to the nano-silver prepared through the method, on the basis that the antibacterial performance of the nano-silver is guaranteed, the biocompatibility of the nano-silver is improved, and the nano-silver can be better applied to the antibacterial field.

Description

Technical Field

[0001] The invention belongs to the technical field of silver particle preparation, and in particular relates to a method for preparing biocompatible nano silver. Background Art

[0002] In the prior art, nanosilver, as an important nanomaterial, has broad application prospects in the fields of medicine, biology and materials science. Nanosilver has a bactericidal effect, and its mechanism of action is that the silver ions precipitated by nanosilver and the surface of pathogens are opposite in charge. When the two are close to each other, the force between the opposite charges will make the nanosilver and the cell membrane of the pathogen tightly bound, and then the nanosilver enters the cell through the cell wall, reacts chemically with the sulfhydryl group, prevents the synthesis of enzymes, and then makes the pathogen lose activity and die. After the bacteria die, the nanosilver is freed and circulates to kill bacteria continuously.

[0003] The invention patent with patent number CN115415538A discloses an antibacterial nano-silver and a rapid preparation method. This method uses agar to quickly prepare antibacterial nano-silver, and the prepared nano-silver has good antibacterial properties; the invention patent with patent number CN101223887B discloses a nano-silver antibacterial solution and a preparation method. This method prepares a nano-silver antibacterial solution containing nano-metal silver particles and carboxymethyl chitosan. The preparation process avoids the use of toxic chemicals and has good antibacterial properties; the invention patent with patent number CN103785857B discloses a nano-silver for antibacterial dressing and a preparation method. Roselle extract is used as a reducing agent, and medical grade polyethylene glycol and medical glycerin are used as dispersants to synthesize nano-silver. The nano-silver dressing has good antibacterial properties and can be directly applied to the medical field.

[0004] Due to the widespread use of nanosilver, human contact with nanosilver is inevitable. Nanosilver not only has a bactericidal effect, but also has certain toxicity to human cells. Therefore, biocompatibility has attracted more and more attention. Modifying nanosilver particles and improving their biocompatibility while retaining their antibacterial properties is a difficult problem that needs to be solved urgently.

[0005] The prior art does not consider the harm of nanosilver to the human body, which is not conducive to its application in the antibacterial field. Biocompatibility should be added as an application indicator, and the prepared nanosilver should have good biocompatibility. Therefore, how to propose a method for preparing biocompatible nanosilver is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention discloses a method for preparing biocompatible nanosilver.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing biocompatible nanosilver comprises the following steps:

[0009] (1) A certain amount of lysine is dissolved in 20 mL of acetic acid at room temperature, a certain amount of catalyst is added and a certain volume of epichlorohydrin is added dropwise, the mixture is reacted at a certain temperature for a certain time, deionized water and ethanol are used to wash the mixture after the reaction is completed, and then the mixture is dried to obtain a dry powder; a certain amount of the dry powder is dissolved in a certain volume of methanol, a certain volume of dodecanoyl chloride, a certain volume of chlorosulfonic acid and a certain amount of sodium hydroxide are added, a certain volume of isopropanol is added as a promoter, the mixture is reacted at room temperature for a certain time, and after the reaction is completed, the mixture is dried to obtain sample 1.

[0010] (2) A certain amount of sample 1 is dissolved in 45 mL of ethanol at room temperature, and a pH adjuster is added to the above solution to adjust the pH value of the solution to 8-11, thereby obtaining solution 1; a certain amount of silver salt is dissolved in 5 mL of ethanol at room temperature to obtain solution 2, solution 1 is heated to a certain temperature, and solution 2 is added dropwise under stirring conditions. After a period of reaction, the mixture solution is placed in a centrifuge bottle, centrifuged for 10 minutes, the supernatant is removed, ethanol is added, and the mixture is repeatedly washed three times, and dried to obtain nanosilver particles.

[0011] The specific reactions involved are as follows:

[0012]

[0013]

[0014] This substance has hydroxyl and carboxyl functional groups, which can provide hydrogen atoms to convert Ag into + It is reduced to silver element, so it can be used as a reducing agent. This substance forms a protective film on the surface of silver particles through its long-chain carbon, which prevents silver particles from agglomerating by reducing interfacial tension and maintains its dispersion, so it can be used as a surfactant for preparing silver powder.

[0015] Preferably, in step (1), the amount of lysine used is 1-5 g.

[0016] Preferably, in step (1), the catalyst is selected from concentrated sulfuric acid, p-toluenesulfonic acid or ammonium bisulfate, and the amount of the catalyst is 0.1%-1.0% of the mass of lysine.

[0017] Preferably, in step (1), the amount of epichlorohydrin used is 0.1-1 mL, the certain temperature is 0-60° C., and the certain time is 1-5 h.

[0018] Preferably, in step (1), the amount of the dry powder used is 1-5 g, a certain volume of methanol is 1-50 mL, a certain volume of dodecanoyl chloride is 1-5 mL, a certain volume of chlorosulfonic acid is 1-5 mL, and a certain mass of sodium hydroxide is 0.1-3 g.

[0019] Preferably, in step (1), the certain volume of isopropanol is 1-5 mL, and the reaction time at room temperature is 1-5 h.

[0020] Preferably, in step (2), the usage amount of the sample 1 is 1-5 g, and the pH adjuster is one of sodium carbonate, sodium hydroxide and sodium bicarbonate.

[0021] Preferably, in step (2), the silver salt is one of silver nitrate and silver carbonate, and the added mass is 50-120% of sample 1. The heating temperature of solution 1 is 30-70° C., and the reaction time is 1-6 h.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The present invention uses lysine and epichlorohydrin to synthesize Na[OSO3CH(C 19 H 37 N203)2], the substance can be used as a reducing agent and surfactant for the preparation of nanosilver, the obtained nanosilver particles are spherical, with a particle size of 9.66-34.31nm, and the nanosilver particles stabilized by the new surfactant have no effect on human red blood cells and will not cause cell apoptosis.

[0024] 2) The diameter of the antibacterial zone of the nanosilver prepared by the method of the present invention is 5.70 mm, the minimum inhibitory concentration of the prepared nanosilver is 156.25 mg / mL, and the minimum bactericidal concentration of the prepared nanosilver is 625 mg / L.

[0025] 3) The nanosilver prepared by the method of the present invention improves the biocompatibility of the nanosilver while ensuring the antibacterial performance of the nanosilver, so that the nanosilver can be better applied in the antibacterial field. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 This is a scanning electron microscope image of the nanosilver prepared in Example 1.

[0028] Figure 2 The MIC test results of the nanosilver prepared in Example 1 are shown.

[0029] Figure 3 This is the antibacterial zone of the nanosilver prepared in Example 1. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0031] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0032] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0033] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0034] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0035] The invention discloses a method for preparing biocompatible nano silver.

[0036] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0037] Example 1

[0038] 2 g of lysine was dissolved in 20 mL of acetic acid at room temperature, 20 mg of concentrated sulfuric acid was added and 0.3 mL of epichlorohydrin was added dropwise, and the mixture was reacted at 50°C for 2 h. After the reaction, the mixture was washed with deionized water and ethanol, and then dried to obtain a dry powder. 1 g of the dry powder was dissolved in 30 mL of methanol, 5 mL of dodecanoyl chloride, 1 mL of chlorosulfonic acid and 0.8 g of sodium hydroxide were added, and 3 mL of isopropanol was added as a promoter, and the mixture was reacted at room temperature for 2 h. After the reaction, the mixture was dried to obtain sample 1.

[0039] 2 g of sample 1 was dissolved in 45 mL of ethanol at room temperature, sodium carbonate was added to the above solution to adjust the pH value of the solution to 8-11, and solution 1 was obtained. 1.7 g of silver nitrate was dissolved in 5 mL of ethanol at room temperature to obtain solution 2. Solution 1 was heated to 70° C., and solution 2 was added dropwise under stirring. After reacting for 1 hour, the mixture solution was placed in a centrifuge bottle, centrifuged for 10 minutes, the supernatant was removed, ethanol was added, and it was repeatedly washed three times and dried to obtain nanosilver particles with a particle size of 14.69-28.15 nm.

[0040] Example 2

[0041] 3 g of lysine was dissolved in 20 mL of acetic acid at room temperature, 25 mg of p-toluenesulfonic acid was added, and 0.5 mL of epichlorohydrin was added dropwise, and the mixture was reacted at 50°C for 3 h. After the reaction, the mixture was washed with deionized water and ethanol, and then dried to obtain a dry powder. 1 g of the dry powder was dissolved in 30 mL of methanol, 5 mL of dodecanoyl chloride, 1 mL of chlorosulfonic acid and 0.7 g of sodium hydroxide were added, and 3 mL of isopropanol was added as a promoter, and the mixture was reacted at room temperature for 3 h. After the reaction, the mixture was dried to obtain sample 1.

[0042] 1 g of sample 1 was dissolved in 45 mL of ethanol at room temperature, sodium hydroxide was added to the above solution to adjust the pH value of the solution to 8-11, and solution 1 was obtained. 2.04 g of silver nitrate was dissolved in 5 mL of ethanol at room temperature to obtain solution 2. Solution 1 was heated to 50° C., and solution 2 was added dropwise under stirring. After reacting for 2 hours, the mixture solution was placed in a centrifuge bottle, centrifuged for 10 minutes, the supernatant was removed, ethanol was added, and it was repeatedly washed three times and dried to obtain nanosilver particles with a particle size of 11.77-23.93 nm.

[0043] Example 3

[0044] 4 g of lysine was dissolved in 20 mL of acetic acid at room temperature, 15 mg of ammonium bisulfate was added and 0.6 mL of epichlorohydrin was added dropwise, and the mixture was reacted at 45°C for 3 h. After the reaction, the mixture was washed with deionized water and ethanol, and then dried to obtain a dry powder. 1 g of the dry powder was dissolved in 30 mL of methanol, 5 mL of dodecanoyl chloride, 1 mL of chlorosulfonic acid and 1.2 g of sodium hydroxide were added, and 4 mL of isopropanol was added as a promoter, and the mixture was reacted at room temperature for 3 h. After the reaction, the mixture was dried to obtain sample 1.

[0045] 3 g of sample 1 was dissolved in 45 mL of ethanol at room temperature, sodium hydroxide was added to the above solution to adjust the pH value of the solution to 8-11, and solution 1 was obtained. 2.76 g of silver carbonate was dissolved in 5 mL of ethanol at room temperature to obtain solution 2. Solution 1 was heated to 40° C., and solution 2 was added dropwise under stirring. After reacting for 1 hour, the mixture solution was placed in a centrifuge bottle, centrifuged for 10 minutes, the supernatant was removed, ethanol was added, and it was repeatedly washed three times and dried to obtain nanosilver particles with a particle size of 10.38-20.72 nm.

[0046] Example 4

[0047] 5 g of lysine was dissolved in 20 mL of acetic acid at room temperature, 30 mg of concentrated sulfuric acid was added and 0.4 mL of epichlorohydrin was added dropwise, and the mixture was reacted at 60°C for 2 h. After the reaction, the mixture was washed with deionized water and ethanol, and then dried to obtain a dry powder. 1 g of the dry powder was dissolved in 30 mL of methanol, 5 mL of dodecanoyl chloride, 1 mL of chlorosulfonic acid and 0.9 g of sodium hydroxide were added, and 3 mL of isopropanol was added as a promoter, and the mixture was reacted at room temperature for 3 h. After the reaction, the mixture was dried to obtain sample 1.

[0048] 2 g of sample 1 was dissolved in 45 mL of ethanol at room temperature, sodium hydroxide was added to the above solution to adjust the pH value of the solution to 8-11, and solution 1 was obtained. 1.12 g of silver nitrate was dissolved in 5 mL of ethanol at room temperature to obtain solution 2. Solution 1 was heated to 30° C., and solution 2 was added dropwise under stirring. After reacting for 2 hours, the mixture solution was placed in a centrifuge bottle, centrifuged for 10 minutes, the supernatant was removed, ethanol was added, and it was repeatedly washed three times and dried to obtain nanosilver particles with a particle size of 9.66-14.79 nm.

[0049] Example 5

[0050] 1 g of lysine was dissolved in 20 mL of acetic acid at room temperature, 15 mg of p-toluenesulfonic acid was added, and 0.7 mL of epichlorohydrin was added dropwise, and the mixture was reacted at 50°C for 3 h. After the reaction, the mixture was washed with deionized water and ethanol, and then dried to obtain a dry powder. 1 g of the dry powder was dissolved in 30 mL of methanol, 5 mL of dodecanoyl chloride, 1 mL of chlorosulfonic acid and 1.5 g of sodium hydroxide were added, and 3 mL of isopropanol was added as a promoter, and the mixture was reacted at room temperature for 3 h. After the reaction, the mixture was dried to obtain sample 1.

[0051] 4 g of sample 1 was dissolved in 45 mL of ethanol at room temperature, sodium bicarbonate was added to the above solution to adjust the pH value of the solution to 8-11, and solution 1 was obtained. 1.7 g of silver nitrate was dissolved in 5 mL of ethanol at room temperature to obtain solution 2. Solution 1 was heated to 45° C., and solution 2 was added dropwise under stirring. After reacting for 3 hours, the mixture solution was placed in a centrifuge bottle, centrifuged for 10 minutes, the supernatant was removed, ethanol was added, and it was repeatedly washed three times and dried to obtain nanosilver particles with a particle size of 16.35-34.31 nm.

[0052] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the technical features of the nanosilver and its application disclosed in the present invention are further illustrated by the following experimental examples, but they should not be understood as limiting the present invention. Other improvements made by those skilled in the art based on the above invention content without creative work are also considered to fall within the scope of protection of the present invention.

[0053] Application Examples

[0054] This application example uses the nano silver particles obtained in Example 1 to conduct an experiment. The specific experimental contents are as follows:

[0055] (1) Inhibition zone test

[0056] The nanosilver solution prepared in Example 1 was subjected to ultraviolet irradiation for 2 hours, and the strain to be tested was cultured until visible plaques appeared. Then, an appropriate amount was picked from the mature colony, added to 5mL LB / TSA liquid culture medium, and then cultured at 37°C for 6-8 hours until the optical density (OD600) of the bacterial solution was about 0.6. LB plates were prepared, and 20mL LB culture medium was added to each plate. The test bacterial solution was accessed at a ratio of 0.1%, that is, 100μL of the bacterial solution with an OD600 of 0.6 was added to each plate. After mixing, the culture medium containing the bacterial solution was poured into a culture dish, and then cooled and solidified. When the bacterial culture medium solidified, it was cultured at 37°C for 24 hours, photographed and analyzed the size of the bacterial inhibition circle, and the diameter of the nanosilver inhibition ring was measured to be 5.70mm.

[0057] (2) Minimum inhibitory concentration (MIC) experiment

[0058] Take 2mL of the nanosilver solution prepared in Example 1, dry it and weigh it to obtain the concentration of the nanosilver stock solution. The minimum inhibitory concentration was determined by the two-fold dilution method. 100 μL of sterile water was added to each of the 1-12 wells in row A of the working 96-well plate, 100 μL of the nanosilver stock solution was added to well 1, 100 μL was added to well 2 after sufficient mixing, and 100 μL was taken out of well 12 after repeating the above operation to ensure that the volume of the system was consistent. The concentrations of the samples in wells 1-12 were 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256, 1 / 512, 1 / 1024, 1 / 2048, and 1 / 4096 of the stock solution concentration, respectively. Use a spray gun to absorb the prepared sample solution from wells 1-12 of row A of the working 96-well plate, and add it to wells 1-12 of rows A, B, C and D of the first test 96-well plate, adding 10 μL to each well; add the diluted bacterial solution of Escherichia coli ATCC25922 incubated by strains, and add it to rows A, B, and C of the test 96-well plate, 90 μL to each well, and add 90 μL of sterile water to row D, incubate at 37°C for 18 hours, observe and take pictures, and measure OD600. According to the test results, the OD600 at the sixth concentration gradient position is close to the lowest drug concentration of the drug negative control group, that is, the minimum inhibitory concentration, which is 156.25 mg / μL.

[0059] (3) Minimum bactericidal concentration (MBC) experiment

[0060] According to the MIC test results, the nanosilver solution prepared in Example 1 with MIC, 2×MIC, 4×MIC, and 8×MIC concentrations was taken, and 100 μL of each of the 4-well bacterial solution was applied to a solid plate, cultured at 37°C for 1 day, and the colony growth was observed. The number of Escherichia coli colonies on the culture medium was identified by a Scan1200 fully automatic colony counter. The colony number of nanosilver with a concentration of 4×MIC was 8 (<10), that is, the minimum bactericidal concentration was 625 mg / μL.

[0061] Table 1 Statistics of MBC experimental results

[0062]

[0063] (4) Effect of Nanosilver on Red Blood Cells (Biocompatibility Test)

[0064] Fresh blood from healthy volunteers was collected, anticoagulated with sodium citrate, and red blood cells were collected by centrifugation and washed 3 times with PBS. 20 μL of red blood cells were incubated with 1 mL of the silver nanoparticle solution prepared in Example 1 at a concentration of 50 μg / mL at room temperature for 30 min, then washed 3 times with PBS, fixed with 2.5% glutaraldehyde for 1 h, and the fixed red blood cells were dehydrated and sprayed with gold, and then observed with a scanning electron microscope. There was no obvious morphological change in the red blood cells, and each showed a normal round cake shape with both sides concave in the middle.

[0065] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing biocompatible nanosilver, characterized in that: The following steps are involved: (1) Lysine was dissolved in 20 mL of acetic acid at room temperature, a catalyst was added and epichlorohydrin was added dropwise for reaction, after the reaction was completed, the mixture was washed with deionized water and ethanol, and then dried to obtain a dry powder; the dry powder was dissolved in methanol, and then dodecanoyl chloride, chlorosulfonic acid, sodium hydroxide and accelerator isopropanol were added, the mixture was reacted at room temperature, and after the reaction was completed, the mixture was dried to obtain sample 1; (2) dissolving the sample 1 obtained in step (1) in ethanol at room temperature, and then adding a pH adjuster to adjust the pH value of the solution to obtain solution 1; dissolving a silver salt in ethanol at room temperature to obtain solution 2, heating solution 1 and adding solution 2 dropwise under stirring to react; The mixture solution is placed in a centrifuge bottle, centrifuged, the supernatant is removed, ethanol is added to wash repeatedly three times, and dried to finally obtain the nano silver particles.

2. The method for preparing biocompatible nanosilver according to claim 1, characterized in that: In step (1), the amount of lysine used is 1-5g.

3. The method for preparing biocompatible nanosilver according to claim 1, characterized in that: In step (1), the catalyst is selected from concentrated sulfuric acid, p-toluenesulfonic acid or ammonium bisulfate, and the amount of the catalyst is 0.1%-1.0% of the mass of lysine.

4. The method for preparing biocompatible nanosilver according to claim 1, characterized in that: In step (1), the amount of epichlorohydrin used is 0.1-1 mL, the reaction temperature is 0-60° C., and the reaction time is 1-5 h.

5. The method for preparing biocompatible nanosilver according to claim 1, characterized in that: In step (1), the amount of the dry powder is 1-5 g, the amount of methanol is 1-50 mL, the amount of dodecanoyl chloride is 1-5 mL, the amount of chlorosulfonic acid is 1-5 mL, and the amount of sodium hydroxide is 0.1-3 g.

6. The method for preparing biocompatible nanosilver according to claim 1, characterized in that: In step (1), the amount of isopropanol used is 1-5 mL, and the reaction time is 1-5 h at room temperature.

7. The method for preparing biocompatible nanosilver according to claim 1, characterized in that: In step (2), the amount of sample 1 used is 1-5 g, and the amount of ethanol used to dissolve sample 1 is 45 mL; the pH adjuster is sodium carbonate, sodium hydroxide or sodium bicarbonate, and the pH adjuster makes the pH value of the solution 8-11.

8. The method for preparing biocompatible nanosilver according to claim 1, characterized in that: In step (2), the silver salt is silver nitrate or silver carbonate, the amount of ethanol used to dissolve the silver salt is 5 mL, and the added mass of the silver salt is 50-120% of sample 1, the heating reaction temperature is 30° C.-70° C., and the reaction time is 1-6 h.

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