CN106729982A - A kind of preparation method of silk fibroin nanosphere - Google Patents

Abstract

The invention discloses a method for preparing silk fibroin nanospheres. Degumming mulberry silk or tussah silk to obtain silk fibroin, then dissolving and dialyzing the silk fibroin to form a certain concentration of silk fibroin solution, mixing absolute ethanol and silk fibroin aqueous solution in a certain proportion, freezing, and then centrifuging , washing and drying to obtain silk fibroin nanospheres. Functional silk fibroin microspheres can be prepared by mixing silk fibroin aqueous solution with functional protein molecules such as bFGF‑2, IGF‑1, EGF, BMP‑2, OP‑1, etc. The preparation process is safe, convenient and less polluting, and can stably prepare silk fibroin nanospheres with uniform particle diameters, which are suitable for injection and can be used as drug carriers, sustained-release systems and other fields.

Description

A kind of preparation method of silk fibroin nanosphere

(1) Technical field

The invention relates to a preparation method of silk fibroin-based nanospheres suitable for controlling drug sustained release, and belongs to the technical field of biomedicine.

(2) Background technology

At present, from the perspective of the development trend of biomedical materials, non-degradable materials such as synthetic polymers or materials with poor biocompatibility of degradation products will gradually decrease, while natural materials with good biocompatibility and in vivo absorbability will gradually decrease. Biomaterials will be favored. Natural biomaterials have great potential for clinical applications with controllable structure, mechanics, and biodegradation properties.

Among many natural biomaterials, silk fibroin derived from natural silk has been favored in the field of tissue engineering in recent years due to its advantages of easy processing, good biocompatibility, high mechanical strength, slow degradation rate, and degradation products of amino acids. Research extensively. For example, mesh-like scaffold materials can be prepared by weaving for soft tissue reinforcement, porous sponge-like scaffold materials can be prepared for bone tissue repair by freeze-drying technology, and microsphere-structured scaffold materials can be prepared for drug sustained release by high-voltage electrospray technology. Wait. Among them, among the slow-release drug carriers, silk fibroin provides a new choice due to its slow degradation, and the degradation products are amino acids, which can be absorbed by the human body and have no toxic and side effects. At the same time, the silk fibroin and drugs are compounded into a spherical shape suitable for injection, and the special molecular structure of the silk fibroin molecule is conducive to stabilizing the structure of sensitive molecules and protecting the biological activity of protein functional molecules, which is beneficial to clinical application.

In the previous preparation process of silk fibroin microspheres and nanospheres, methods such as high-pressure electrospray method, inverse emulsion method, and spray drying method were mostly used. Among them, the high-voltage electrospray method needs to be carried out in a high-voltage electrostatic field, which requires high equipment and low yield; the inverse emulsion method is easy to introduce organic impurities into the material; the uniformity of the silk fibroin balls prepared by the spray drying method needs to be improved. Improvement, and the process is complicated and the equipment requirements are high. Here, the present invention provides a simple method for preparing silk fibroin nanospheres, which can stably prepare silk fibroin nanospheres with uniform particle size.

(3) Contents of the invention

The purpose of the present invention is to provide a simple method for preparing silk fibroin nanospheres, which can quickly and stably obtain silk fibroin nanospheres with uniform particle size and good dispersion, and at the same time minimize the application of organic reagents in the preparation process, so as to Reduce contamination during production while improving product biosafety.

The technical scheme adopted in the present invention is:

The invention provides a method for preparing silk fibroin nanospheres. The method is as follows: fully mix silk fibroin aqueous solution with a mass concentration of 0.1% to 5% and absolute ethanol (preferably precooled at 4°C for 1 hour), and store at -25°C Freeze at ~-18°C for 12-24 hours, then thaw at 4-40°C, centrifuge (preferably 4000-15000rpm), take the precipitate and wash it with deionized water (preferably the volume of deionized water is 5-20 times the volume of the precipitate ), repeated centrifugation and water washing (preferably repeated 3-10 times), and dried the precipitate after the last water washing (preferably one of the following methods for drying: 20-25 ° C room temperature drying, 40 ° C vacuum heat drying or -30 ° C freezing drying), that is, silk fibroin nanospheres; the volume ratio of the absolute ethanol to the silk fibroin aqueous solution is 1-10:10.

Further, the silk fibroin aqueous solution is prepared as follows: cut silk cocoons into pieces and immerse them in 0.2% anhydrous sodium carbonate aqueous solution (preferably the volume dosage of anhydrous sodium carbonate aqueous solution is 0.1 g/L based on the mass of silkworm cocoons), boil for 60 ~120 minutes, filter, take the filter cake and wash it with deionized water, dry at 50°C to obtain silk fibroin fiber; dissolve the silk fibroin fiber in 9.3mol/L lithium bromide aqueous solution or ternary solution (preferably dissolved at 65-70°C , the lithium bromide aqueous solution or the ternary solution volumetric dosage is as the criterion that can be dissolved, preferably 5-10ml/g based on silkworm cocoon mass), dialyze with deionized water as the dialysate, and take the permeate to prepare with deionized water A silk fibroin aqueous solution with a mass concentration of 0.1% to 5% is prepared; the ternary solution is formed by mixing calcium chloride, ethanol, and water at a volume ratio of 1:2:8.

Further, functional drugs are added to the silk fibroin aqueous solution, and the functional drugs are growth factor bFGF-2, growth-promoting factor IGF-1, epidermal growth factor EGF, recombinant human bone morphogenetic protein BMP2, osteogenic protein OP- 1. One of doxorubicin or gemcitabine hydrochloride.

Furthermore, the added amount of the functional drug is 10-100 μg/L, preferably 10-75 μg/L, based on the volume of the silk fibroin aqueous solution.

Further, the functional drug is added to the silk fibroin aqueous solution, and mixed thoroughly for 5-6 minutes at 2-8° C. and 30-150 rpm to obtain the silk fibroin aqueous solution containing the functional drug.

Further, the method is prepared according to the following steps: (1) Cut the cocoons of mulberry silkworm or tussah silkworm into pieces, add them to an aqueous solution of anhydrous sodium carbonate with a mass concentration of 0.2%, boil for 60 to 120 minutes, filter, wash the filter cake with deionized water, and wash it with deionized water for 50 °C drying to obtain silk fibroin fibers;

(2) Dissolve silk fibroin fiber in 9.3mol/L lithium bromide aqueous solution or ternary solution (preferably dissolved at 65-70°C), add it into a dialysis bag, and dialyze with deionized water for 2-4 days, and take The permeated liquid is prepared with deionized water to prepare an aqueous silk fibroin solution with a mass concentration of 0.1% to 5%; the molecular weight cut-off of the dialysis bag is 3500;

(3) Dissolve the functional drug in the silk fibroin aqueous solution in step (2), and mix thoroughly at 2-8°C and 30-150rpm for 5-6 minutes to obtain the drug-containing silk fibroin aqueous solution; the functional drug is the growth factor bFGF- 2 or recombinant human bone morphogenetic protein BMP2;

(4) Mix silk fibroin aqueous solution or drug-containing silk fibroin aqueous solution with absolute ethanol at a volume ratio of 1-3:1, freeze at -20°C for 12-24 hours; then thaw at 20-35°C, After centrifugation at 15000rpm, take the precipitate and wash it with 5-20 times the volume of deionized water, repeat the centrifugation and water washing process for 3-10 times; take the precipitate after the last water washing, and dry it at room temperature at 20-25°C, vacuum heat drying at 40°C or freeze-drying at -30°C to obtain the silk fibroin nanospheres.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that the previous preparation methods are mostly prepared by high-pressure electrospray method or inverse emulsion method, the operation is relatively complicated, and a large amount of organic reagents are used. The preparation process of the present invention is easy to operate, does not involve high pressure, extreme temperature and other conditions, and the equipment is simple and easy to obtain, which is suitable for stable batch production; secondly, the reagents used have high safety and little pollution, and have little impact on the environment of the preparation process, and the residual reagents in the materials can be It is removed by washing with water, which is beneficial to improve the biological safety of the product.

(4) Description of drawings

Fig. 1 SEM images of silk fibroin nanospheres.

Figure 2. Silk fibroin nanospheres were injected subcutaneously in rats. (a) Modeling by subcutaneous injection; (b) 1 week after subcutaneous injection; (c) 3 months after subcutaneous injection.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1:

Cut 50g of silkworm cocoons into pieces, put them into 5L of boiling 0.2% anhydrous sodium carbonate aqueous solution, continue boiling for 90 minutes for degumming treatment, filter, collect filter cake, and wash repeatedly with deionized water 5 times to remove surface residues Sodium carbonate, after drying at 50°C, it is silk fibroin fiber. Dissolve the dried silk fibroin fibers in 250mL 9.3mol/L lithium bromide aqueous solution (65°C). After complete dissolution, put the silk fibroin solution into a dialysis bag (molecular weight cut-off: 3500), and dialyze in deionized water for 3 days , Change the water 2 times a day to remove lithium bromide. The concentration of the silk fibroin aqueous solution obtained after dialysis was adjusted to a mass concentration of 1% with deionized water.

Anhydrous ethanol was pre-cooled in a refrigerator at 4°C for 1 hour. Take 10mL of absolute ethanol and add it to 100mL of 1% silk fibroin aqueous solution, mix quickly and evenly, seal the container and put it in a -20°C refrigerator for 18 hours, take out the mixed solution, and thaw it at 20°C to obtain a suspension After the solution was centrifuged at 12000rpm for 30 minutes in a centrifuge, the supernatant was removed, and the precipitate was washed with 100mL deionized water. The washing and centrifugation steps were repeated 5 times, and finally the precipitate was dried at 20°C to obtain silk fibroin nano ball. Observing the obtained silk fibroin nanospheres ( FIG. 1 ) with a scanning electron microscope, they are well dispersed and have a diameter of 100 nm to 200 nm.

Example 2:

Cut 50g tussah silkworm cocoons into pieces, put them into 5L of boiling 0.2% anhydrous sodium carbonate aqueous solution, boil for 120 minutes for degumming treatment, filter, collect filter cake, and repeatedly wash the obtained silk fibroin fibers with deionized water 5 After three times, the residual sodium carbonate on the surface is removed, and then dried at 50°C to obtain silk fibroin fibers. Dissolve the dried silk fibroin fibers in 500ml, 70°C ternary solution (the volume ratio of calcium chloride/ethanol/water is 1:2:8), after completely dissolving, put the silk fibroin solution into a dialysis bag ( MWCO: 3500), dialyze in deionized water for 4 days, change the water twice a day to remove residual solvent. The concentration of the silk fibroin aqueous solution obtained after dialysis was adjusted to a mass concentration of 5% with deionized water.

Dissolve 5 μg of bFGF-2 in 100 mL of an aqueous solution of 5% silk fibroin in mass concentration, and mix at 4° C. for 30 minutes at a speed of 30 rpm to obtain a silk fibroin solution containing bFGF-2.

Anhydrous ethanol was pre-cooled in a refrigerator at 4°C for 1 hour. Add 30mL of absolute ethanol to 100mL of silk fibroin solution containing bFGF-2, mix quickly and evenly, seal the container and put it in a -20°C refrigerator for 24 hours, take out the mixture, and thaw it at 25°C to obtain a suspension The liquid was centrifuged at 12000rpm for 30 minutes in a centrifuge, the supernatant was removed, the precipitate was washed with 100mL deionized water, the cleaning and centrifugation steps were repeated 5 times, and finally the precipitate was dried at 25°C to obtain silk fibroin containing bFGF2 The protein nanosphere has a diameter of 50nm to 150nm.

Example 3:

Put 50g of mulberry silkworm silk into 5L of boiling 0.2% anhydrous sodium carbonate aqueous solution, boil for 60 minutes for degumming treatment, filter, take the filter cake, and wash it repeatedly with deionized water for 5 times to remove surface residues. Sodium carbonate, after drying at 50°C, it is silk fibroin fiber. Dissolve the dried silk fibroin fibers in 500ml, 70°C ternary solution (the volume ratio of calcium chloride/ethanol/water is 1:2:8), after completely dissolving, put the silk fibroin solution into a dialysis bag ( MWCO: 3500), dialyze in deionized water for 4 days, change the water twice a day to remove residual solvent. The concentration of the silk fibroin aqueous solution obtained after dialysis was adjusted to a mass concentration of 2% with deionized water.

Dissolve 1 μg of BMP-2 in 100 mL of silk fibroin aqueous solution with a mass concentration of 2%, and mix at 4° C. for 30 minutes at a speed of 30 rpm to obtain a silk fibroin solution containing BMP-2.

Anhydrous ethanol was pre-cooled in a refrigerator at 4°C for 1 hour. Take 30mL of absolute ethanol and add it to 100mL of BMP-2 silk fibroin protein solution, mix quickly and evenly, seal the container and put it in a -20°C refrigerator for 12 hours, take out the mixed solution, and thaw it at 35°C to obtain a suspension Use a centrifuge to centrifuge at 12000rpm for 30 minutes, remove the supernatant, wash the precipitate with 100mL deionized water, repeat the cleaning and centrifugation steps 5 times, and finally dry the precipitate in vacuum at 40°C to obtain BMP-2-containing Silk fibroin nanospheres with a diameter of 100nm to 200nm.

Example 4:

Put 50g tussah silkworm silk into 5L boiling mass concentration 0.2% anhydrous sodium carbonate aqueous solution, continue to boil for 90 minutes to carry out degumming treatment, filter, take filter cake, wash repeatedly 5 times with deionized water and deionized water, remove surface residual Sodium carbonate, after drying at 50°C, it is silk fibroin fiber. Dissolve the dried silk fibroin fiber in 250mL 9.3mol/L lithium bromide aqueous solution (65°C). After completely dissolving, put the silk fibroin solution into a dialysis bag (molecular weight cut-off: 3500), and dialyze in deionized water for 4 Change the water twice a day to remove residual solvent. The concentration of the silk fibroin aqueous solution obtained after dialysis was adjusted to a mass concentration of 0.1% with deionized water.

Dissolve 7.5 μg of BMP-2 in 100 mL of silk fibroin aqueous solution, and mix for 30 minutes at 4° C. at a speed of 30 rpm to obtain a silk fibroin solution containing BMP-2.

Anhydrous ethanol was pre-cooled in a refrigerator at 4°C for 1 hour. Add 100mL of absolute ethanol to 100mL of silk fibroin aqueous solution, mix quickly and evenly, seal the container and put it in a -20°C refrigerator for 24 hours, take out the mixed solution, thaw at 4°C, and use a centrifuge to obtain the suspension After centrifuging at 12000rpm for 30 minutes, remove the supernatant, wash the precipitate with 100mL deionized water, repeat the cleaning and centrifugation steps 5 times, and finally freeze-dry the precipitate at -30°C in a low-temperature vacuum to obtain silk fibroin containing BMP-2 The protein nanosphere has a diameter of 50nm to 250nm.

Example 5

Soft tissue repair and filling: the silk fibroin nanospheres prepared in Example 2 were dispersed in saline for injection, and injected subcutaneously into rats to promote soft tissue regeneration. After the rats were anesthetized by abdominal cavity, the abdominal skin was prepared, and 2 injection areas were marked on the left and right sides of the abdominal midline (4 injection sites for each mouse), and the distance between two adjacent points was more than 2 cm. Lift the skin at the injection area, use a 1mL syringe to insert the needle into the deep dermis, avoid blood vessels, and inject 200 μL of the sample (a in Figure 2). One week later, the rats were sacrificed, and the distribution of silk fibroin nanospheres at the injection site and the condition of the surrounding soft tissues were observed (Fig. 2 b). One week after the injection site, the implanted silk fibroin nanospheres could still be clearly observed to bulge, but the volume of the bulge was reduced by about 16% compared with the day of injection, and there was no inflammation such as redness and swelling in the surrounding tissues, which indicated that the silk fibroin nanospheres had completely recovered. It is partially absorbed, and implanted in the body will not cause obvious adverse reactions, and has good biocompatibility. After 3 months of implantation, the appearance was natural, and no adverse reactions such as suppuration occurred. A small amount of nanospheres could still be observed (c in Figure 2), and the implantation site was filled with new fibrous tissue, indicating that it was a good drug carrier.

Claims (7)

1. a kind of preparation method of silk fibroin nanosphere, it is characterised in that methods described is：By mass concentration 0.1%~5% Silk fibroin water solution be sufficiently mixed with absolute ethyl alcohol, freezed 12~24 hours at -25 DEG C~-18 DEG C, then at 4~40 DEG C Thaw, centrifugation takes precipitation and clean with deionized water, repeated centrifugation and washing, take the precipitation drying after last time is washed, as Silk fibroin nanosphere；The absolute ethyl alcohol is 1~10 with silk fibroin water solution volume ratio:10.

2. the preparation method of silk fibroin nanosphere as claimed in claim 1, it is characterised in that the silk fibroin water solution is pressed It is prepared by following method：The Carbon Dioxide sodium water solution of mass concentration 0.2% is immersed after silk cocoon is shredded, 60~120 minutes, mistake are boiled Filter, takes filter cake and is cleaned with deionized water, 50 DEG C of dryings, obtains fibroin fiber；Fibroin fiber is dissolved in 9.3mol/L In lithium bromide water solution or ternary solution, dialysed by dialyzate of deionized water, take permeate and be configured to deionized water The silk fibroin water solution of mass concentration 0.1%~5%；The ternary solution by calcium chloride, ethanol, water with volume ratio be 1:2: 8 mix.

3. the preparation method of silk fibroin nanosphere as claimed in claim 1, it is characterised in that the absolute ethyl alcohol is 4 DEG C of precoolings Absolute ethyl alcohol.

4. the preparation method of silk fibroin nanosphere as claimed in claim 1, it is characterised in that in the silk fibroin water solution Function medicament is added with, the function medicament is growth factor bFGF-2, somatomedin IGF-1, epithelical cell growth factor One kind in EGF, recombinant human bone morphogenesis protein BMP2, BMP OP-1, adriamycin or gemcitabine hydrochloride.

5. the preparation method of silk fibroin nanosphere as claimed in claim 4, it is characterised in that the function medicament addition with Silk fibroin water solution volume is calculated as 10-100 μ g/L.

6. the preparation method of silk fibroin nanosphere as claimed in claim 4, it is characterised in that the function medicament is added to silk In the fibroin aqueous solution, 2-8 DEG C, 30-150rpm be sufficiently mixed 5-6 minutes, that is, obtain the fibroin albumen containing function medicament water-soluble Liquid.

7. the preparation method of silk fibroin nanosphere as claimed in claim 4, it is characterised in that methods described is made as follows It is standby：(1) 60~120 points are boiled in the silk cocoon of silkworm or tussah being shredded into the addition Carbon Dioxide sodium water solution of mass concentration 0.2% Clock, filtering, filter cake is washed with deionized, 50 DEG C of dryings, obtains fibroin fiber；

(2) fibroin fiber is dissolved in 9.3mol/L lithium bromide water solutions or ternary solution, add bag filter in, with go from Sub- water is that dialyzate is dialysed 2-4 days, takes the fibroin albumen water that permeate deionized water is configured to mass concentration 0.1%~5% Solution；The bag filter molecular cut off is 3500；

(3) function medicament is dissolved in step (2) silk fibroin water solution, is sufficiently mixed 5-6 minutes in 2-8 DEG C, 30-150rpm, Obtain pastille silk fibroin water solution；The function medicament is growth factor bFGF-2 or recombinant human bone morphogenesis protein BMP2；

(4) by pastille silk fibroin water solution and absolute ethyl alcohol with volume ratio 1~3:1 mixing, freezes 12~24 small at -20 DEG C When；Thawed at 20~35 DEG C, after 4000-15000rpm centrifugations, take precipitation and cleaned with the deionized water of 5-20 times of volume, weight Multiple centrifugation and water-washing process 3-10 times；The precipitation after last time washing is taken, after drying, the silk fibroin nanosphere is obtained.