Preparation method of silk fibroin nanospheres

Abstract

The invention belongs to the technical field of silk fibroin microspheres, and discloses a preparation method of silk fibroin nanospheres. Belongs to the technical field of biopolymer microsphere preparation, and the preparation method comprises the following steps: 1) adding a crystallization inducer into a silk fibroin solution, uniformly mixing, adding a dispersant into the solution, and mixing to obtain a pretreatment liquid; 2) carrying out low-temperature solidification treatment on the pretreatment liquid, and unfreezing to obtain a silk fibroin nanosphere suspension; 3) performing ultrasonic dispersion on the silk fibroin nanosphere suspension to obtain a silk fibroin nanosphere solution; and 4) centrifuging and freeze-drying the silk fibroin nanosphere liquid to obtain the solid silk fibroin nanosphere. The silk fibroin nanosphere obtained by the invention has the advantages of excellent biocompatibility, controllable size and good dispersity. The preparation method is simple and high in yield.

Description

Technical Field

[0001] The invention relates to the technical field of silk fibroin microspheres, and in particular to an injectable silk fibroin nanosphere and a preparation method thereof. Background Art

[0002] Studies have shown that in clinical practice, carriers prepared from polymers with good biocompatibility, such as injectable micro / nanoparticles and hydrogels, can be used as delivery media for various drugs. They have the advantages of mild processing, high biocompatibility, and controllable release. Silk fibroin (SF), as a natural protein material, has a stable structure, is cheap and easy to obtain, has excellent mechanical properties, good degradability and biocompatibility, and is an excellent drug carrier material. Silk fibroin balls loaded with antibacterial and anticancer drugs have broad development prospects in the fields of biomedicine and other fields.

[0003] The size of drug-loaded microspheres has a significant impact on their application. For example, in cancer treatment, larger drug-loaded microspheres may be more suitable for embolizing larger tumor blood vessels, while smaller microspheres may be more suitable for embolizing smaller blood vessels or achieving more precise targeted treatment. Nanospheres are small in size and have an extremely high specific surface area. They can carry more drugs or active substances and provide a larger surface area for drug release, which is beneficial to the interaction between drugs and organisms. The preparation process of silk fibroin nanospheres is relatively complex and requires precise control of reaction conditions and parameters.

[0004] Currently, the preparation of silk fibroin nanospheres has been reported:

[0005] (1) In Chinese invention patent CN106474482B, "A kind of silk fibroin nanoparticles and its preparation method and application", a silk fibroin solution is rapidly frozen in liquid nitrogen and then freeze-dried, followed by alcoholization, centrifugation, washing, and ultrasonic dispersion, and finally silk fibroin nanoparticles with good dispersibility can be obtained. However, this method requires the use of a large amount of liquid nitrogen to freeze the silk fibroin solution, which will cause a large amount of liquid nitrogen waste and high cost.

[0006] (2) In Chinese invention patent CN102417733B, “A method for preparing silk fibroin nanospheres”, the silk fibroin nanospheres are prepared by an ultrasonic reverse microemulsion in-situ drying method. This method requires the use of cyclohexane, which is physiologically toxic. The biocompatibility of the prepared microspheres is poor. In addition, cyclohexane is extremely flammable and explosive, which greatly limits industrial mass production.

[0007] The preparation of injectable silk fibroin nanospheres with mild preparation conditions and good biocompatibility still needs further exploration. Summary of the invention

[0008] The present invention aims to provide a method for preparing silk fibroin nanospheres with a simple process and a high balling rate, and to prepare silk fibroin nanospheres without introducing other doping substances that are difficult to volatilize and remove.

[0009] The present invention provides a method for preparing silk fibroin nanospheres, comprising the following steps:

[0010] Step (1), obtaining a silk fibroin solution;

[0011] Step (2), mixing a polar solvent with the silk fibroin solution to obtain a mixed solution 1; the polar solvent is any one of methanol and ethanol;

[0012] Step (3), obtaining a gelatin solution, and mixing the gelatin solution with the mixed solution 1 to obtain a mixed solution 2;

[0013] Step (4), freezing the mixed solution 2, rewarming it and centrifuging it to obtain a silk fibroin nanosphere solution;

[0014] The centrifugation specifically includes: discarding the supernatant after centrifugation, adding ultrapure water to rinse and suspend, and repeating the centrifugation several times;

[0015] Step (5), freeze-drying the silk fibroin nanosphere solution to obtain solid silk fibroin nanospheres with a particle size range of 60nm to 300nm.

[0016] Freeze drying can retain the morphological characteristics of the material before drying. The morphology of the nanospheres obtained by this method is closest to that in the solution. Gelatin is a temperature-sensitive material. After cooling, it can quickly turn into a solid state. The three-dimensional network structure formed fills the container space and wraps the silk protein in it. The silk protein therefore aggregates in the pores of the gelatin network and self-assembles to form nanospheres. After rewarming, the gelatin is converted into a solution state. The gelatin can be removed during the cleaning process. Even if the cleaning process is not thorough and a small amount of gelatin remains, it will not have an adverse effect on the product performance because gelatin has good biocompatibility.

[0017] Preferably, in step (2), the polar solvent is mixed with the silk fibroin solution, specifically comprising: adding methanol dropwise to the silk fibroin solution, wherein the volume of methanol is 1 / 10 of the silk fibroin solution; and the mass concentration of silk fibroin in the silk fibroin solution is 1-2%.

[0018] Preferably, in step (3), the mass concentration of gelatin in the gelatin solution is 1% to 4%; the gelatin solution and the mixed solution 1 are mixed in a volume ratio ranging from 4:1 to 1:4.

[0019] Preferably, in step (4), the freezing temperature is 0 to -80 degrees Celsius.

[0020] Preferably, in step (4), when adding ultrapure water for suspension, the water temperature used is 37 to 80 degrees Celsius.

[0021] Preferably, in step (1), the method for preparing the silk fibroin solution comprises the following steps: obtaining silk, degumming the silk, washing and drying the silk to obtain pure silk fibroin fibers; the drying temperature is 60°C; using lithium bromide solution or ternary solution to dissolve the silk fibroin fibers, and dialyzing to obtain a silk fibroin solution.

[0022] Preferably, the specific process of degumming includes: cutting the silk into pieces, boiling the cut silk in a sodium carbonate solution with a concentration of 0.001 to 0.02 M for 30 to 60 minutes at a bath ratio of 1:100 to 1:1000, and rinsing with deionized water to obtain a treated product; repeating the following process for several times to complete the degumming of the treated product: boiling the treated product in a sodium carbonate solution with a concentration of 0.001 to 0.02 M for 30 to 60 minutes at a bath ratio of 1:100 to 1:1000, and rinsing with deionized water.

[0023] Preferably, in step (3), within the range of 4:1 to 1:4 of the volume ratio of the gelatin solution to the mixed solution 1, the particle size of the silk fibroin nanospheres is reduced by reducing the proportion of the gelatin solution.

[0024] The silk fibroin nanospheres prepared by the present invention have good suspension after ultrasound, exist stably in water, and spherical silk fibroin can be observed under a microscope; the dried nanospheres have aggregation phenomenon, which is a typical feature of nanoparticles, and the aggregated particles are still nanoscale with a particle size of 60-300nm.

[0025] The method for preparing silk fibroin nanospheres provided by the present invention comprises mixing a silk fibroin solution with a polar solvent, dispersing and curing the solution with gelatin at low temperature, and finally reheating and ultrasonicating the solution to obtain the silk fibroin nanospheres. The size of the silk fibroin nanospheres can be regulated by changing the ratio of the gelatin / silk fibroin ethanol mixed solution. The preparation process is simple, easy to operate, and has a significant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the specific implementation or the prior art description. Some specific embodiments of the present invention will be described in detail in an exemplary but not restrictive manner with reference to the drawings. The same reference numerals in the drawings indicate the same or similar components or parts. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings:

[0027] Figure 1 This is a flow chart of the method for preparing silk fibroin nanospheres provided by the invention described in Example 1.

[0028] Figure 2 This is a scanning electron microscope image of a silk fibroin ball prepared when the gelatin concentration is 1% and the ratio of the gelatin to silk fibroin ethanol mixture is 2:1 as described in Example 2.

[0029] Figure 3 This is a test chart of the silk fibroin ball prepared when the gelatin concentration is 2% and the ratio of the gelatin to silk fibroin ethanol mixture is 4:1 as described in Example 3.

[0030] Figure 4 This is a scanning electron microscope image of a silk fibroin ball prepared when the gelatin concentration is 4% and the ratio of the gelatin to silk fibroin ethanol mixture is 2:1 as described in Example 4.

[0031] Figure 5 This is a scanning electron microscope image of a silk fibroin ball prepared when the gelatin concentration is 8% and the ratio of the gelatin to silk fibroin ethanol mixture is 2:1 as described in Example 5.

[0032] A. The formation of silk fibroin balls; B. The enlarged image of the surface of a single ball.

[0033] Figure 6 It is a statistical histogram of the average diameter of silk fibroin balls prepared when the gelatin concentration is 2% and the mixing ratios of gelatin to silk fibroin are 4:1, 2:1, 1:1, 1:2, and 1:4, respectively, as described in Example 6. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0035] The idea of ​​the technical solution of the present invention is as follows:

[0036] 1. Obtain silk fibroin solution according to conventional methods;

[0037] 2. Mixing a polar solvent such as methanol or ethanol with the silk fibroin solution to obtain a mixed solution 1;

[0038] 3. Mixing gelatin solutions of different concentrations with the silk fibroin ethanol mixed solution 1 at a certain volume ratio to obtain a mixed solution 2;

[0039] 4. Freeze the entire solution at a certain temperature for 48 hours, then rewarm and centrifuge. After centrifugation, discard the supernatant, add ultrapure water to rinse and suspend, repeat centrifugation three times to obtain a silk nanosphere solution. Finally, freeze-dry to obtain solid silk protein nanospheres.

[0040] Embodiment 1:

[0041] Prepare silk fibroin solution. Specifically, 10 g of silk was dissolved in 0.02 M Na 2 CO 3 Boil the solution at 100°C for 30 minutes to remove the sericin on the outside of the silk, rinse with deionized water, repeat the above operation 3 times and dry the silk at 60°C. Weigh 27g of the degummed silk after the above treatment and dissolve it in 100mL of 9.3mol / L LiBr solution, and dissolve it at 60°C for 6 hours. Then use a dialysis bag with a molecular weight cutoff of 3500 to dialyze in deionized water for 3 days, during which the water is changed every two hours to remove the LiBr in the solution, thereby obtaining a silk fibroin solution. Dilute with deionized water and adjust its concentration to 3%.

[0042] Prepare 1% gelatin solution: Dissolve gelatin granules in hot water to make gelatin solution.

[0043] Add 1 / 10 volume of methanol to the silk solution, then mix it with gelatin solution, the volume ratio of gelatin to silk methanol mixture is 2:1, put the mixture into a centrifuge tube, put it into a -80 degree refrigerator, take it out after 48 hours, melt it in a 37 degree water bath, and obtain silk nanospheres by ultrasonication, centrifugation and washing. Silk nanospheres can also be dried, and the drying method is freeze-drying or blowing. Nanospheres have been obtained before drying, and the drying process is for preservation or convenience of testing. Freeze drying is required to obtain a large number of nanospheres. When testing, drop the nanosphere suspension on the silicon wafer and blow dry with an ear cleaning bulb.

[0044] See also Figure 1 , Figure 1 It is a flow chart of the method for preparing high-strength silk fibroin film of the present invention.

[0045] Embodiment 2:

[0046] The same method as in Example 1 was used to prepare a silk fibroin solution and a gelatin solution. Degummed silk fibroin was prepared. 1 / 10 volume of ethanol was added dropwise to the silk fibroin solution, and then mixed with the gelatin solution. The volume ratio of gelatin to silk fibroin methanol mixture was 2:1. The mixture was placed in a centrifuge tube, placed in a -20 degree refrigerator, taken out after 48 hours, dissolved in a 37 degree water bath, subjected to ultrasound, centrifugation, and washing, to obtain silk fibroin nanospheres.

[0047] See also Figure 2 , Figure 2 This is an image obtained under a scanning electron microscope after the silk fibroin ball suspension prepared when the gelatin concentration is 1% and the ratio of the gelatin to silk fibroin ethanol mixture is 2:1 as described in Example 2 is dropped on a silicon wafer and blown dry.

[0048] Embodiment three:

[0049] The same method as in Example 1 was used to prepare a silk fibroin solution and a gelatin solution. Degummed silk fibroin was prepared. 1 / 10 volume of ethanol was added dropwise to the silk fibroin solution, and then mixed with a 2% gelatin solution, the volume ratio of gelatin to silk fibroin methanol mixture was 4:1, the mixture was placed in a centrifuge tube, placed in a -20 degree refrigerator, taken out after 48 hours, dissolved in a 37 degree water bath, subjected to ultrasound, centrifugation, and washing, to obtain silk fibroin nanospheres.

[0050] See also Figure 3 , Figure 3 The SEM test image and particle size distribution statistics table of the silk fibroin ball suspension prepared when the gelatin concentration is 2% and the volume ratio of gelatin to silk fibroin ethanol mixture is 4:1 as described in Example 3 and then dropped on a silicon wafer and blown dry. 60 nanospheres in the image were randomly selected for size statistics, and the diameter distribution range was 150-370nm, with an average diameter of about 260nm.

[0051] Embodiment 4:

[0052] The same method as in Example 1 was used to prepare a silk fibroin solution and a gelatin solution. Degummed silk fibroin was prepared. 1 / 10 volume of ethanol was added dropwise to the silk fibroin solution, and then mixed with a 4% gelatin solution, the ratio of gelatin to silk fibroin methanol mixture was 4:1, the mixture was placed in a centrifuge tube, placed in a -20 degree refrigerator, taken out after 48 hours, dissolved in a 37 degree water bath, subjected to ultrasound, centrifugation, and washing, to obtain silk fibroin nanospheres.

[0053] See also Figure 4 , Figure 4 This is a scanning electron microscope image of a silk fibroin ball prepared when the gelatin concentration is 4% and the volume ratio of the gelatin to silk fibroin ethanol mixture is 2:1 as described in Example 4. When the gelatin concentration is increased to 4%, the spherical shape is good.

[0054] Embodiment five:

[0055] The same method as in Example 1 was used to prepare a silk fibroin solution and a gelatin solution. Degummed silk fibroin was prepared. 1 / 10 volume of ethanol was added dropwise to the silk fibroin solution, and then mixed with 8% gelatin solution, the volume ratio of gelatin to silk fibroin methanol mixture was 4:1, the mixture was placed in a centrifuge tube, placed in a -20 degree refrigerator, taken out after 48 hours, dissolved in a 37 degree water bath, subjected to ultrasound, centrifugation, and washing, to obtain silk fibroin nanospheres.

[0056] See also Figure 5 , Figure 5 This is a scanning electron microscope image of a silk fibroin ball prepared when the gelatin concentration is 8% and the ratio of the gelatin to silk fibroin ethanol mixture is 2:1 as described in Example 5. Figure 5A in the figure shows that the surface of the prepared silk fibroin ball is covered with a large amount of polymers and is difficult to clean, indicating that too high a concentration of gelatin is not conducive to the formation of silk fibroin balls. The surface of the microsphere has an uneven structure ( Figure 5 B) in the figure is beneficial to increase its specific surface area.

[0057] Embodiment six:

[0058] The same method as in Example 1 was used to prepare a silk fibroin solution and a gelatin solution. Degummed silk fibroin was prepared. 1 / 10 volume of ethanol was added dropwise to the silk fibroin solution, and then mixed with a 2% gelatin solution, the ratio of gelatin to silk fibroin methanol mixture was 4:1, the mixture was placed in a centrifuge tube, placed in a -20 degree refrigerator, taken out after 48 hours, dissolved in a 37 degree water bath, subjected to ultrasound, centrifugation, and washing, to obtain silk fibroin nanospheres.

[0059] See also Figure 6 , Figure 6 The figure is a statistical histogram of the average diameter of the silk fibroin balls prepared when the gelatin concentration is 2% and the mixing ratios of gelatin to silk fibroin are 4:1, 2:1, 1:1, 1:2 and 1:4 respectively in Example 6. As the gelatin ratio increases, the diameter of the silk fibroin nanospheres tends to increase.

[0060] The above descriptions are only some specific implementation methods of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any person familiar with the art within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing silk fibroin nanospheres, characterized in that: The following steps are involved: Step (1), obtaining a silk fibroin solution; Step (2), mixing a polar solvent with the silk fibroin solution to obtain a mixed solution 1; the polar solvent is any one of methanol and ethanol; Step (3), obtaining a gelatin solution, and mixing the gelatin solution with the mixed solution 1 to obtain a mixed solution 2; Step (4), freezing the mixed solution 2, rewarming it and centrifuging it; A silk fibroin nanosphere solution is obtained; The centrifugation specifically includes: discarding the supernatant after centrifugation, adding ultrapure water to rinse and suspend, and repeating the centrifugation several times; Step (5), freeze-drying the silk fibroin nanosphere solution to obtain solid silk fibroin nanospheres with a particle size ranging from 60 nm to 300 nm.

2. The method for preparing silk fibroin nanospheres according to claim 1, characterized in that: In step (2), the polar solvent is mixed with the silk fibroin solution, specifically comprising: adding methanol dropwise to the silk fibroin solution, wherein the volume of methanol is 1 / 10 of the silk fibroin solution; and the mass concentration of silk fibroin in the silk fibroin solution is 1-2%.

3. The method for preparing silk fibroin nanospheres according to claim 1, characterized in that: In step (3), the mass concentration of gelatin in the gelatin solution is 1% to 4%; The gelatin solution and the mixed solution 1 are mixed in a volume ratio ranging from 4:1 to 1:

4.

4. The method for preparing silk fibroin nanospheres according to claim 1, characterized in that: In step (4), the freezing temperature is 0 to -80 degrees Celsius.

5. The method for preparing silk fibroin nanospheres according to claim 1, characterized in that: In step (4), when the ultrapure water is added for suspension, the water temperature used is 37 to 80 degrees Celsius.

6. The method for preparing silk fibroin nanospheres according to claim 1, characterized in that: In the step (1), the method for preparing the silk fibroin solution comprises the following steps: The silk is obtained, and after degumming, the silk is washed and dried to obtain pure silk fibroin fibers; the drying temperature is 60° C. The silk fibroin fibers are dissolved with lithium bromide solution or ternary solution, and dialyzed to obtain a silk fibroin solution.

7. The method for preparing silk fibroin nanospheres according to claim 6, characterized in that: The specific process of degumming comprises: The silk is cut into pieces, and the cut silk is boiled in a sodium carbonate solution with a concentration of 0.001 to 0.02 M for 30 to 60 minutes at a bath ratio of 1:100 to 1:1000, and then rinsed with deionized water to obtain a treated product; The treated product is degummed by repeating the following process several times: The bath ratio is 1:100-1:1000. The treated product is boiled in a sodium carbonate solution with a concentration of 0.001-0.02M for 30-60 minutes and then rinsed with deionized water.

8. The method for preparing silk fibroin nanospheres according to claim 3, characterized in that: In step (3), within the range of 4:1 to 1:4 of the volume ratio of the gelatin solution to the mixed solution 1, the particle size of the silk fibroin nanospheres is reduced by reducing the proportion of the gelatin solution.

Images