CN106474482B - Silk fibroin nanoparticle and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the technical field of polymer biomedical material preparation, in particular to a silk fibroin nanoparticle and a preparation method and application thereof, comprising the following steps: S1. Adjust to 0.01-0.6%, and freeze to complete solidification in a liquid nitrogen environment to obtain a silk fibroin frozen body; S2. freeze-dry the silk fibroin body to obtain a silk fibroin nanoparticle powder; S3. The vegan protein nanoparticle powder is added to an alcohol solution with a mass concentration of 50-100%, followed by ultrasonic dispersion and centrifugation to obtain a mixed solution of upper and lower layers, and the upper layer of mixed solution is collected; S4. The upper layer of liquid is subjected to centrifugal treatment, After washing and ultrasonic dispersion, silk fibroin nanoparticles with good dispersibility are obtained. The preparation method of the invention is simple, no auxiliary reagent is added, and the prepared silk fibroin nanoparticles have excellent performance, which is of great significance for promoting the application and development of silk fibroin.

Description

A kind of silk fibroin nanoparticle and its preparation method and application

technical field

The invention relates to the technical field of polymer biomedical materials, in particular to a silk fibroin nanoparticle and a preparation method and application thereof.

Background technique

With the development of genetic engineering and regenerative medicine, the preparation technology of gene and drug carrier has attracted much attention. The degradable polymer particle carrier has good stability, high loading rate and sustainable release, and is widely used in the fields of gene carrier and drug sustained and controlled release. In particular, subcellular-sized nanoparticles with large specific surface area and high phagocytosis efficiency are one of the research hotspots in the field of drug release carriers.

Silk fibroin is a high-purity protein secreted by the endothelial cells of the silk gland in the rear of the silkworm, free of organelles and other biological impurities, and has high biological safety. Silk fibroin is composed of 20 kinds of amino acids such as acetyl, alanine and serine. It has good biocompatibility and can be biodegraded. The final degradation products are polypeptides and free amino acids, which are easily metabolized by the body. Therefore, silk fibroin nanoparticles have broad prospects as drug carriers.

At present, the commonly used methods for preparing silk fibroin carrier particles include salting out method, emulsification method, chemically induced assembly (silk fibroin assembly induced by ions, pH, alcohol, etc.), electrostatic spray method (coagulation bath collection, spray drying), etc. Most of these methods require the addition of chemical reagents such as organic solvents or salts, which limit the clinical application of silk fibroin particles. Patent No. ZL 200410016856.4 reported the preparation of silk fibroin nanospheres by freeze-thaw method, avoiding the use of initiators, surfactants and cross-linking agents, but still need to add a certain amount of organic solvent as a denaturant to induce silk fibroin protein assembly. Although the preparation of silk fibroin particles by electrostatic spraying can avoid the addition of chemical reagents, the production efficiency is low.

In view of the above defects, the inventors have designed a method for inducing silk fibroin to form nanoparticles by means of solution concentration regulation and ultra-low temperature rapid freezing through active research and innovation, so that it has more industrial application value.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the above technical problems, to provide a silk fibroin nanoparticle, a preparation method and application thereof, and to realize the preparation of silk fibroin nanoparticle under green and mild conditions.

In order to solve the above problems, the present invention adopts the following technical solutions:

A preparation method of silk fibroin nanoparticles, comprising the following steps:

S1. Take the purified silk fibroin solution, adjust the mass concentration of the silk fibroin solution to 0.01-0.6%, and freeze to complete solidification in a liquid nitrogen environment to obtain a frozen silk fibroin body;

S2. freeze-drying the silk fibroin frozen body to obtain silk fibroin nanoparticle powder;

S3. adding silk fibroin nanoparticle powder to the alcoholic solution with a mass concentration of 50-100%, carrying out ultrasonic dispersion and centrifugation successively to obtain a mixed solution of upper and lower layers, and collecting the upper layer mixed solution;

S4. The upper layer liquid is subjected to centrifugal treatment, washing and ultrasonic dispersion in sequence to obtain silk fibroin nanoparticles with good dispersibility.

Further, the silk fibroin is silk fibroin of Bombyx mori, tussah or cerebellum.

Further, in the step S3, the alcohol solution is at least one of methanol, ethanol, isopropanol, butanol, isobutanol, and tert-butanol.

Further, the alcohol solution is ethanol.

Further, the freezing time in the step S1 is 15-60min; the freeze-drying time in the step S2 is 24-48h; the ultrasonic dispersion time in the step S3 is 1-5min, and the centrifugation is performed at a rotating speed of 1000-2000rpm. /min process 5-10min.

Further, the step of step S4 is as follows: the upper layer liquid is subjected to centrifugal treatment, washing and ultrasonic dispersion in sequence to obtain silk fibroin nanoparticles.

Further, the centrifugal treatment is performed at a rotational speed of 10,000-20,000 rpm/min for 10-30 min.

A silk fibroin nanoparticle is prepared by the above-mentioned preparation method.

Further, the silk fibroin nanoparticles are spherical, with a diameter of 50-1000 nm, and have biodegradation properties.

The application of the above-mentioned silk fibroin nanoparticle, the silk fibroin nanoparticle is a delivery carrier, and is applied in the field of medicine or gene.

The principle of the invention is: in a relatively dilute silk fibroin solution, the silk fibroin macromolecules have a coil-like structure, and have a tendency to transform into a colloidal nano-particle-like structure, and the ice crystal effect during the ultra-low temperature rapid freezing of liquid nitrogen It will induce the further assembly of silk fibroin macromolecules into nanoparticle-like structures, and finally form nanoparticle frozen bodies. Nanoparticle powder can be formed after freeze-drying, and further alcohol and ultrasonic treatment can obtain water-insoluble and well-dispersed silk fibroin nanoparticles.

Compared with the prior art, a silk fibroin nanoparticle of the present invention and its preparation method and application have outstanding features and excellent effects as follows:

1. The present invention utilizes a simple preparation method of regulating the concentration of silk fibroin solution and freeze-drying to obtain silk fibroin nanoparticles with a particle size between 50 and 1000 nm; the method is simple in process and high in yield, and can meet the requirements of sustained drug release. , biopharmaceuticals, enzyme immobilization materials and other application requirements;

2. The silk fibroin nanoparticles prepared by the freeze-drying method in the present invention do not need to add any auxiliary reagents during the formation of the nanoparticles, do not cause the residue of chemical reagents, have higher biological safety, and show good performance in biomedicine. obvious advantage;

3. The preparation method of the present invention is simple, the preparation cost is low, and the prepared silk fibroin nanoparticles have excellent performance, which is of great significance for promoting the application and development of silk fibroin.

Description of drawings

Fig. 1 is the electron microscope image of silk fibroin nanoparticle powder in the preparation process of Example 1;

2 is a 5 μm electron microscope image of the silk fibroin nanoparticles obtained in Example 1;

FIG. 3 is a 1 μm electron microscope image of the silk fibroin nanoparticles obtained in Example 1. FIG.

Detailed ways

The present invention will be further described in detail below through specific embodiments and accompanying drawings, but it should not be understood that the scope of the present invention is limited to the following examples. Without departing from the above-mentioned method idea of the present invention, various substitutions or changes made according to common technical knowledge in the art and conventional means should all be included within the scope of the present invention.

Example 1

Immerse 100 g Bombyx mori raw silk in 5 L of 0.05% NaCO ₃ solution, boil at 98-100 °C for 30 min, repeat 3 times to degumming the silk, fully wash and dry to obtain pure silk fibroin fiber; add silk fibroin fiber to In a ternary solution of CaCl ₂ /CH ₃ CH ₂ OH/H ₂ O with a molar ratio of 1:2:8, stir and dissolve at 72 °C for 1 h to obtain a mixed solution of silk fibroin; into a dialysis bag, and dialyzed with deionized water for 3 days to obtain a purified silk fibroin solution;

Adjust the mass concentration of the silk fibroin solution to 0.1 wt%, pour it into a stainless steel plate, adjust the thickness of the solution to 4 mm, then place the stainless steel plate in liquid nitrogen, and quickly freeze for 30 min to form a silk fibroin frozen body;

Freeze-drying the silk fibroin frozen body to obtain silk fibroin nanoparticle powder;

The silk fibroin nanoparticle powder was added to absolute ethanol, and the nanoparticle suspension was obtained after ultrasonic dispersion, and centrifuged at 2000 r/min for 5 min to remove a small amount of flaky structures formed on the surface layer during the freeze-drying process;

The centrifuged supernatant was centrifuged at 13,000 r/min for 15 min, and the supernatant was discarded, washed with sterile deionized water, and repeated three times to obtain well-dispersed silk fibroin nanoparticles.

The silk fibroin nanoparticle powder obtained in the preparation process of Example 1 was detected by electron microscope, and the detection results were shown in Figure 1; the silk fibroin nanoparticles obtained in Example 1 were detected by electron microscope, and the detection results were shown in Figure 2 and Figure 1 3 shown.

Example 2

The degummed silk fibroin fibers were added to a 9.3 mol/L LiBr solution, and stirred and dissolved at 60°C for 2 h to obtain a mixed solution of silk fibroin; Dialyzed for 3 days to obtain purified silk fibroin solution;

The mass concentration of the silk fibroin solution was adjusted to 0.01 wt%, and after stirring evenly, it was poured into a stainless steel plate to adjust the thickness of the solution to 3 mm, and then the stainless steel plate was placed in liquid nitrogen and rapidly frozen for 20 min to form a silk fibroin frozen body. ;

Freeze-drying the silk fibroin frozen body to obtain silk fibroin nanoparticle powder;

The silk fibroin nanoparticle powder was added to 90% methanol solution, and the nanoparticle suspension was obtained after ultrasonic dispersion, and centrifuged at 1000 r/min for 10 min to remove a small amount of flake structure formed on the surface layer during the freeze-drying process, and the upper and lower fractions were obtained. The mixed solution of the upper layer is collected, and the mixed solution of the upper layer is collected;

The upper layer mixed solution was centrifuged at 20000rpm./min for 10min, layered, the upper layer solution was removed, washed, and ultrasonically dispersed, and the operation described in this section was repeated 5 times to obtain a silk fibroin nanoparticle suspension;

The silk fibroin nanoparticle suspension is freeze-dried to obtain nanoparticle powder.

Example 3

100 g tussah raw silk was immersed in 5 L of 0.25% Na ₂ CO ₃ solution, boiled at 98-100 °C for 2 h, degummed the silk, washed and dried to obtain pure silk fibroin; the degummed tussah silk fibroin was obtained. Add into molten Ca(NO ₃ ) ₂ solution, stir and dissolve at 100 °C for 3 h to obtain a mixed solution of silk fibroin; put the obtained mixed solution of silk fibroin into a dialysis bag, and dialyze with deionized water to obtain purified tussah silk vegan protein solution;

The mass concentration of the tussah silk fibroin solution was adjusted to 0.05 wt%, and after stirring evenly, it was poured into a stainless steel plate, and the thickness of the solution was adjusted to 3 mm. body.

Freeze-drying the silk fibroin frozen body to obtain silk fibroin nanoparticle powder;

The silk fibroin nanoparticle powder was added to 90% ethanol solution, and the nanoparticle suspension was obtained after ultrasonic dispersion, and centrifuged at 1500 r/min for 10 min to remove a small amount of flakes formed on the surface layer during the freeze-drying process, and the upper and lower fractions were obtained. The mixed solution of the layers was collected, and the mixed solution of the upper layer was collected.

The upper layer mixed solution was centrifuged at 10,000 rpm./min for 30 min, layered, the upper layer solution was removed, washed, and ultrasonically dispersed, and the operations described in this section were repeated 5 times to obtain a silk fibroin nanoparticle suspension;

The silk fibroin nanoparticle suspension is freeze-dried to obtain nanoparticle powder.

Example 4

The silk fibroin cocoon was degummed, and then the degummed natural silk fibroin fibers were added to a 9.0 mol/L LiSCN solution and dissolved with stirring at 45 °C for 1 h to obtain a silk fibroin mixed solution; the obtained silk fibroin mixed solution was put into a dialysis bag, Dialyzed with deionized water to obtain purified silk fibroin solution;

The mass concentration of the silk fibroin solution was adjusted to 0.2 wt%, and after stirring evenly, it was poured into a stainless steel plate, and the thickness of the solution was adjusted to 3 mm. body;

Freeze-drying the silk fibroin frozen body to obtain silk fibroin nanoparticle powder;

The silk fibroin nanoparticle powder was added into anhydrous methanol solution, and the nanoparticle suspension was obtained after ultrasonic dispersion. Centrifuge at 2000 r/min for 5 min to remove a small amount of flake-like structure formed on the surface layer during the freeze-drying process, and the upper and lower fractions were obtained. The mixed solution of the upper layer is collected, and the mixed solution of the upper layer is collected;

The upper layer mixed solution was centrifuged at 20,000 rpm./min for 10 min, layered, the upper layer solution was removed, washed, and ultrasonically dispersed, and the operations described in this section were repeated three times to obtain silk fibroin nanoparticles.

Claims (4)

1. A preparation method of silk fibroin nanoparticles is characterized by comprising the following steps:

s1, taking a purified silk fibroin solution, adjusting the mass concentration of the silk fibroin solution to be 0.01-0.6%, adjusting the thickness of the solution to be 3mm or 4mm, freezing in a liquid nitrogen environment until the solution is completely solidified to obtain a silk fibroin frozen body, wherein the freezing time in the step S1 is 15-60 min;

s2, carrying out freeze drying treatment on the silk fibroin frozen body to obtain silk fibroin nanoparticle powder; the freeze drying time in the step S2 is 24-48 h;

s3, adding the silk fibroin nanoparticle powder into an alcohol solution with the mass concentration of 50-100%, sequentially performing ultrasonic dispersion and centrifugation to obtain a vertically-layered mixed solution, and collecting an upper-layer liquid; the ultrasonic dispersion time in the step S3 is 1-5min, and the centrifugation is carried out for 5-10min at the rotation speed of 1000-;

s4, sequentially carrying out centrifugal treatment, washing and ultrasonic dispersion on the upper layer liquid to obtain silk fibroin nanoparticles with good dispersibility; the silk fibroin nano-particles are spherical, have the diameter of 50-1000nm and have biodegradability; the step of step S4 is: sequentially performing centrifugal treatment, washing and ultrasonic dispersion on the upper layer liquid, and repeating the operations for 3-5 times to obtain the silk fibroin nanoparticles; the centrifugal treatment is carried out at 10000-.

2. The method of claim 1, wherein the silk fibroin is silk fibroin of Bombyx mori, Antheraea pernyi, or Bombyx mori L.

3. The method as claimed in claim 1, wherein the alcohol solution in step S3 is at least one selected from methanol, ethanol, isopropanol, butanol, isobutanol, and tert-butanol.

4. The method as claimed in claim 3, wherein the alcohol solution is ethanol.

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