CN113647409B - Silver-loaded silk fibroin nanoparticle antibacterial agent, preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of nano materials and high-molecular biomedical materials, and particularly relates to a silver-loaded silk fibroin nanoparticle antibacterial agent, a preparation method and application. The preparation method comprises the following steps: preparing silk fibroin solution with certain mass fraction concentration, then adding silver nitrate or nano silver into the silk fibroin solution according to a certain proportion, uniformly mixing, then placing the mixture into liquid nitrogen for freezing, and after complete freezing, carrying out annealing treatment to obtain the silver-loaded silk fibroin nanoparticles. The silver-loaded silk fibroin nanoparticle antibacterial agent provided by the invention has an obvious antibacterial effect, the preparation method has the advantages of simple raw material composition and simple preparation process, other chemical reagents are not required to be added in the preparation process, and the silver-loaded silk fibroin nanoparticle antibacterial agent is suitable for industrial production.

Description

Silver-loaded fibroin nanoparticle antibacterial agent, preparation method and application

technical field

The invention belongs to the technical field of nanomaterials and macromolecule biomedical materials, and in particular relates to a silver-loaded fibroin nanoparticle antibacterial agent, a preparation method and an application.

Background technique

Antimicrobial disinfection is indispensable in everyday life, medical technology and industry. There are many forms of antibacterial methods, such as light method, radiation method, disinfectant method, antibacterial method, high temperature method and so on. Metal ions such as silver and copper have effective antibacterial effects. Among them, silver ions have long been known as antibacterial agents. Silver ions from dissolved silver nitrate and silver sulfadiazine have a potent bactericidal effect on Gram-positive and Gram-negative bacteria. Due to its ultra-high specific surface area and activity, nano-silver has also been widely used as an antibacterial agent, but the antibacterial principle may still be the role of silver ions. Since silver salts or silver nanoparticles are unstable, suitable carriers are needed when they are used as antibacterial agents. Currently commonly used carriers are zeolite, calcium phosphate, silicon dioxide and oxides.

Silk fibroin is a natural polymer protein with good biocompatibility and degradability, and has been widely used as a biomaterial in the field of biomedical materials. The invention patent with the application number of 202011358899.6 proposes a silver-loaded silk fibroin antibacterial dressing on one side, its preparation method and application. Some of the silk fibroin dressings are loaded with nano-silver to provide a good antibacterial effect. However, it is difficult to provide stable and sustained release by directly loading silver onto materials such as membranes and sponges. Nanoparticles have been widely used as carriers of antibacterial agents due to their ultra-high specific surface area and high release efficiency. Loading silver-based antibacterial agents into degradable nanoparticle carriers is an effective way to prepare novel antibacterial agents.

The invention patent with the application number 201210108553.X proposes a silk protein-silver nanoparticle composite material and its preparation method. After mixing the silk protein solution and the silver nitrate solution, the silver precursor is irradiated by sunlight or simulated sunlight. The body is reduced into silver nanoparticles in situ, and then the material is shaped or prepared. The invention patent with the application number 202011335506.X proposes a method for preparing a silk fibroin-based bimetallic antibacterial coating. Silver nitrate, tetrachloroauric acid powder and silk fibroin solution are blended, and then reduced by ultraviolet irradiation to obtain silver nanometer coatings. particles and gold nanoparticles. However, the above-mentioned methods all use silk fibroin as a stabilizer, and prepare silk fibroin-silver nanocomposites by a reduction method. The preparation process involves chemical reactions and is uncontrollable. Moreover, only using silk fibroin as a stabilizer does not give full play to the advantages of silk fibroin as a degradable nanocarrier. The prepared silk fibroin-silver nanocomposite has poor dispersibility as an antibacterial agent and it is difficult to maintain long-term sustainable release. .

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a silver-loaded fibroin nanoparticle antibacterial agent, preparation method and application. agent and its preparation method. The invention adopts a freezing method to directly obtain silver-loaded fibroin nano particles.

The present invention is achieved in this way, a preparation method of silver-loaded silk fibroin nanoparticle antibacterial agent, comprising the following steps: adding silver salt or silver simple substance to the silk fibroin solution to obtain a blend solution, and subjecting the blend solution to ultra-low temperature freezing, Afterwards, heat-up annealing treatment is carried out to induce silk fibroin assembly and structural transformation, and obtain a water-insoluble silver-loaded silk fibroin nanoparticle frozen body; after the frozen body is thawed, obtain a silver-loaded silk fibroin nanoparticle antibacterial agent; or frozen body Freeze-dry to obtain silver-loaded silk fibroin nanometer freeze-dried powder.

Further, the silver salt includes silver nitrate, and the silver element includes nano silver.

Further, the nano-silver particle size: 10-200nm.

Further, the silk fibroin solution is obtained by degumming and dissolving silk; the mass fraction of the silk fibroin solution is 0.05-0.8%.

Further, the dosage of the silver salt or simple silver is 5-30% of the silk fibroin.

Further, the ultra-low temperature freezing includes liquid nitrogen freezing; the elevated annealing treatment temperature includes freezing at a temperature of -10 to 0°C for 12h to 96h; the thawing includes thawing at a temperature of 4 to 60°C.

Further, the size of the nanoparticles is 50-1000 nm.

Further, the silk fibroin solution is a regenerated silk fibroin solution obtained after degumming, dissolving and dialysis of silk or cocoons.

The present invention also provides the silver-loaded fibroin nanoparticle antibacterial agent prepared by the above-mentioned preparation method.

The invention also provides the application of the silver-loaded fibroin nanoparticle antibacterial agent as an antibacterial agent.

In summary, the advantages and positive effects of the present invention are:

(1) The present invention does not involve complex chemical reactions, the preparation process is controllable, and no by-products are produced;

(2) The reaction conditions in the preparation process of the present invention are mild, and are prepared based on an aqueous solution system, without the use of other chemical reagents;

(3) The preparation method of the present invention is simple, the preparation process is short, no special equipment and complicated process are required, and the macro-preparation of nanoparticles can be realized, which has industrial application value;

(4) At the same time, the different nanoparticles prepared by the present invention have good dispersibility and good antibacterial effect. Silk fibroin can be degraded slowly, which can realize the continuous release of silver.

Description of drawings

Fig. 1 is the optical microscope picture of the silver ion-loaded silk fibroin nanoparticle dispersion liquid prepared in embodiment 1;

Fig. 2 is the scanning electron micrograph of the silver-loaded fibroin nanoparticle prepared in embodiment 1;

Fig. 3 is an antibacterial experiment data diagram of silver-loaded fibroin nanoparticles prepared in Examples 1-5.

Detailed ways

In order to make the purpose of the present invention, technical solutions and advantages clearer, the present invention will be described in further detail below in conjunction with the examples. The equipment and reagents used in the various examples and test examples can be obtained from commercial sources unless otherwise specified. . The specific embodiments described here are only used to explain the present invention, not to limit the present invention.

From the information contained herein, various changes in the precise description of the invention will readily become apparent to those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the processes, properties or components defined, since these embodiments, as well as other descriptions, are only intended to illustrate certain aspects of the invention. In fact, various changes to the embodiments of the present invention that can be obviously made by those skilled in the art or related fields fall within the scope of the appended claims.

In order to better understand the present invention but not to limit the scope of the present invention, all figures representing dosage, percentage, and other numerical values used in this application should be understood as being modified by the word "about" in all cases. Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At a minimum, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5%, of a given value or range.

Unless otherwise specified, the freezing temperature of the silk fibroin solution described in the following embodiments of the present invention in liquid nitrogen is -196°C.

The invention discloses an antibacterial agent of silver-loaded fibroin nanoparticle, a preparation method and an application. The invention uses silk fibroin nanoparticles as a carrier, blends silk fibroin solution with silver nitrate powder or nano-silver, freezes and heats up and anneals to obtain the silver-loaded silk fibroin nanoparticle antibacterial agent. The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

Example 1

Step 1, prepare silk fibroin solution;

Specifically, the following process can be used: immerse 100g of mulberry silk in 5L of 0.1% sodium carbonate solution, boil at 98-100°C for 30min, repeat three times to degumming the silkworm cocoon, and obtain pure silk fibroin fiber after fully washing and drying. Immerse the treated silk fiber into the prepared CaCl ₂ /CH ₃ CH ₂ OH/H ₂ O ternary solution with a molar ratio of 1:2:8, and dissolve at 72°C for one hour. After cooling, pour the dissolved solution into a dialysis bag with a molecular weight cut-off of 9-14 kDa, and dialyze with deionized water. After the dialysis, the silk fibroin solution was obtained by filtering with gauze, stored in a refrigerator at 4°C for use, and its concentration was determined by the dry mass method. Pour the prepared silk fibroin solution into a centrifuge tube and centrifuge to remove aggregates and air bubbles. After centrifugation, the silk fibroin solution was diluted to 0.4 wt%.

Step 2, preparation of silk fibroin nanoparticles loaded with silver ions.

Specifically, silver nitrate powder is added to the prepared silk fibroin solution (the nitrate content accounts for 5% of the silk fibroin mass). The prepared blend was frozen under liquid nitrogen, and then transferred to a -4°C refrigerator for annealing treatment at elevated temperature for 48 hours to induce structural transformation of silk fibroin.

After thawing at a room temperature of 25° C., a well-dispersed silver ion-loaded silk fibroin nanoparticle suspension was obtained.

The finally prepared silver-ion-loaded silk fibroin nanoparticles were observed under a microscope, and the test results are shown in FIG. 1 . It can be seen from Figure 1 that the prepared silver ion-loaded silk fibroin nanoparticles are uniformly dispersed in the suspension.

The obtained silver-ion-loaded silk fibroin nanoparticle suspension was dried and then tested by scanning electron microscope. The test results are shown in FIG. 2 . It can be seen from Figure 2 that the prepared silver ion-loaded silk fibroin particles are regular spherical.

Example 2

The only difference between Example 2 and Example 1 is: in step 2, the nitrate content accounts for 10% of the silk fibroin; and after thawing and filtering at room temperature 25°C, impurities are removed to obtain silver-loaded silk fibroin with good dispersibility nanoparticle suspension.

Example 3

The only difference between Example 3 and Example 1 is: in step 2, the nitrate content accounts for 5% of the silk fibroin; the prepared blend is frozen under liquid nitrogen, and then transferred to a -4°C refrigerator to heat up Annealing treatment for 3 days induced structural transformation of silk fibroin.

Thaw and filter in an oven at 37°C to remove impurities and obtain a well-dispersed suspension of silver ion-loaded silk fibroin nanoparticles. protein nanoparticles.

Example 4

Step 1, prepare silk fibroin solution;

Specifically, the following process can be used: immerse 100g of mulberry silk in 5L of 0.1% sodium carbonate solution, boil at 98-100°C for 30min, repeat three times to degumming the silkworm cocoon, and obtain pure silk fibroin fiber after fully washing and drying. Immerse the treated silk fiber into the prepared lithium bromide solution (9.3mol/L) and dissolve at 60°C for one hour. After cooling, pour the dissolved solution into a dialysis bag with a molecular weight cut-off of 9-14 kDa, and dialyze with deionized water. After the dialysis, the silk fibroin solution was obtained by filtering with gauze, stored in a refrigerator at 4°C for use, and its concentration was determined by the dry mass method. Pour the prepared silk fibroin solution into a centrifuge tube and centrifuge to remove aggregates and air bubbles. After centrifugation, the silk fibroin solution was diluted to 0.4 wt%.

Step 2, preparation of silver-loaded silk fibroin nanoparticles;

Specifically, silver nitrate powder is added to the prepared silk fibroin solution (the nitrate content accounts for 10% of the silk fibroin mass). The prepared blend was frozen under liquid nitrogen, and then transferred to a -4°C refrigerator for annealing treatment at elevated temperature for 3 days to induce structural transformation of silk fibroin.

Thaw and filter at room temperature 25°C to remove impurities to obtain a well-dispersed suspension of silver-loaded silk fibroin nanoparticles, and then further freeze-dry the prepared silver-loaded silk fibroin nanoparticle suspension to prepare silver-loaded silk fibroin nanoparticles .

Example 5

The only difference between embodiment 5 and embodiment 4 is: in step 2, the content of nitrate accounts for 20% of the silk fibroin.

Thaw and filter in an oven at 37°C to remove impurities and obtain a well-dispersed suspension of silver-loaded fibroin nanoparticles, and then further freeze-dry the prepared suspension of silver-loaded silk fibroin nanoparticles particles.

Example 6

Step 1, prepare silk fibroin solution;

Specifically, the following process can be used: immerse 100g of mulberry silk in 5L of 0.1% sodium carbonate solution, boil at 98-100°C for 30min, repeat three times to degumming the silkworm cocoon, and obtain pure silk fibroin fiber after fully washing and drying. Immerse the treated silk fiber into the prepared lithium bromide solution (9.3mol/L) and dissolve at 60°C for one hour. After cooling, pour the dissolved solution into a dialysis bag with a molecular weight cut-off of 9-14 kDa, and dialyze with deionized water. After the dialysis, the silk fibroin solution was obtained by filtering with gauze, stored in a refrigerator at 4°C for use, and its concentration was determined by the dry mass method. Pour the prepared silk fibroin solution into a centrifuge tube and centrifuge to remove aggregates and air bubbles. After centrifugation, the silk fibroin solution was diluted to 0.4 wt%.

Step 2, preparation of silver-loaded silk fibroin nanoparticles;

Specifically, nano-silver dispersion (the content of nano-silver accounts for 10% of the mass of silk fibroin) is added to the prepared silk fibroin solution. The prepared blend was frozen under liquid nitrogen, and then transferred to a -4°C refrigerator for annealing treatment at elevated temperature for 3 days to induce structural transformation of silk fibroin.

Thaw and filter at room temperature 25°C to remove impurities to obtain a well-dispersed suspension of silver-loaded silk fibroin nanoparticles, and then further freeze-dry the prepared silver-loaded silk fibroin nanoparticle suspension to prepare silver-loaded silk fibroin nanoparticles .

Example 7

The difference between embodiment 7 and embodiment 6 is that in step 2, the content of nano silver accounts for 20% of the silk fibroin.

Thaw and filter in an oven at 37°C to remove impurities and obtain a well-dispersed suspension of silver-loaded fibroin nanoparticles, and then further freeze-dry the prepared suspension of silver-loaded silk fibroin nanoparticles particles.

Example 8

Step 1, prepare silk fibroin solution;

Specifically, the following process can be used: immerse 100g of mulberry silk in 5L of 0.1% sodium carbonate solution, boil at 98-100°C for 30min, repeat three times to degumming the silkworm cocoon, and obtain pure silk fibroin fiber after fully washing and drying. Immerse the treated silk fiber into the prepared CaCl ₂ /CH ₃ CH ₂ OH/H ₂ O ternary solution with a molar ratio of 1:2:8, and dissolve at 72°C for one hour. After cooling, pour the dissolved solution into a dialysis bag with a molecular weight cut-off of 9-14 kDa, and dialyze with deionized water. After the dialysis, the silk fibroin solution was obtained by filtering with gauze, stored in a refrigerator at 4°C for use, and its concentration was determined by the dry mass method. Pour the prepared silk fibroin solution into a centrifuge tube and centrifuge to remove aggregates and air bubbles. After centrifugation, the silk fibroin solution was diluted to 0.4 wt%.

Step 2, preparation of silver-loaded fibroin nanoparticles.

Specifically, nano-silver dispersion liquid (the content of elemental silver accounts for 5% of the mass of silk fibroin) is added to the prepared silk fibroin solution. The prepared blend was frozen under liquid nitrogen, and then transferred to a -4°C refrigerator for annealing treatment at elevated temperature for 48 hours to induce structural transformation of silk fibroin.

After thawing at a room temperature of 25° C., a well-dispersed silver-loaded fibroin nanoparticle suspension was obtained.

Example 9

The only difference between embodiment 9 and embodiment 8 is: in step 2, the content of nano-silver accounts for 10% of the silk fibroin.

Thaw and filter in an oven at 37°C to remove impurities and obtain a well-dispersed suspension of silver-loaded fibroin nanoparticles, and then further freeze-dry the prepared suspension of silver-loaded silk fibroin nanoparticles particles.

Antibacterial tests were carried out on the silver-ion-loaded silk fibroin nanoparticles prepared in Examples 1-5.

The detection steps of the antibacterial properties of the silk fibroin nanoparticle antibacterial solvent loaded with silver ions obtained are:

(1) Preparation of Escherichia coli bacteria liquid: take frozen Escherichia coli bacteria and resuscitate and culture them in LB liquid medium at 37°C and 120rpm constant temperature incubator for 24 hours, take the resuscitated bacteria liquid and culture them for 9-12 hours until they grow to the logarithmic phase access.

(2) Prepare sample solution: centrifuge the bacterial suspension in the rapid growth phase at 8000rpm for 3 minutes, wash it with PBS solution (pH=7.2-7.4) for 3 times, measure its OD600 value, and dilute the concentration of the bacterial solution to 10 ⁹ CFU /mL, then disperse the silver-loaded fibroin nanoparticle sample with bacterial solution, so that the sample concentration is 20mg/mL, measure its OD600 at 0h, 10min, 30min, 1h, 2h, 4h, 8h, 10h, 24h, 48h Observe the changes in the number of bacteria.

The data in Figure 3 shows the changes in the difference in the absorbance of bacteria measured at different concentrations over time. In this test, the antibacterial effect of the sample containing E. coli is reflected by the absorbance value. Within a certain range, the more the number of E. coli in the liquid, the greater the absorbance value of the liquid. It can be seen from Fig. 3 that the silver salt content reaches 5% and has a bacteriostatic effect, and reaches 10% and 20% and has a strong bactericidal effect.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (4)

1. The silver-loaded silk fibroin nanoparticle antibacterial agent is characterized in that the silver-loaded silk fibroin nanoparticle antibacterial agent can realize the sustained release of silver and has a sustained antibacterial effect, and the preparation method of the silver-loaded silk fibroin nanoparticle antibacterial agent comprises the following steps: adding silver salt or silver simple substance into the silk fibroin solution to obtain a blending solution, wherein the using amount of the silver salt or silver simple substance is 5-30% of the mass of the silk fibroin, freezing the blending solution at an ultralow temperature, then carrying out heating annealing treatment, and inducing the assembly and the structure transformation of the silk fibroin to obtain a water-insoluble silver-loaded silk fibroin nanoparticle frozen body; unfreezing the frozen body to obtain a silver-loaded silk fibroin nanoparticle antibacterial agent; or freeze-drying the frozen body to obtain silver-loaded silk fibroin nano freeze-dried powder;

the ultra-low temperature freezing is liquid nitrogen freezing; the temperature of the heating annealing treatment comprises freezing treatment for 12 to 96 hours under the condition that the temperature is-10 to 0 ℃; the unfreezing comprises the step of thawing at 4-60 ℃;

the silver salt is silver nitrate, and the silver simple substance is nano silver dispersion liquid;

the mass fraction of the silk fibroin solution is 0.05 to 0.8 percent;

the silk fibroin solution is regenerated silk fibroin solution obtained by degumming, dissolving and dialyzing silk or silkworm cocoons.

2. The silver-loaded silk fibroin nanoparticle antibacterial agent of claim 1, wherein: the particle size of the nano silver is as follows: 10-200nm.

3. The silver-loaded silk fibroin nanoparticle antibacterial agent of claim 1, wherein: the size of the nano-particles is 50 to 1000nm.

4. Use of a silver-loaded silk fibroin nanoparticle antimicrobial agent as defined in any one of claims 1-3 for the preparation of an antimicrobial agent.

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