WO2005085327A1

WO2005085327A1 - A method of producing nanosize fibroin particle

Info

Publication number: WO2005085327A1
Application number: PCT/CN2005/000192
Authority: WO
Inventors: Yuqing Zhang
Original assignee: Yuqing Zhang
Priority date: 2004-03-04
Filing date: 2005-02-16
Publication date: 2005-09-15
Also published as: CN1243059C; CN1560136A

Abstract

The present invention discloses a method of producing nanosize fibroin particle from silk. A water soluble fibroin solution is mixed with excess organic solvent, causing rapid beta folding of fibroin molecule structure, forming crystalline microparticle, then removing the organic solvent, and after dispersing treatment the high purity crystalline nanosize fibroin particle is obtained. The fibroin particle has sphere shape, is water insoluble has an average particle size of from 30 to 60 nanometer, shows beta-folding structure, has a crystallinity of from 18 to 25 percent, which is about half of natural fiber, is harmless and atoxic to human body, there is no immune response of human body, has good biocompatibility, can effectively shield ultraviolent radiation, can be extensively used as cosmetic, skin protection products, synthetic material, surface modifying material as well as enzyme, polypeptide and delayed release carrier for drug and so on.

Description

Technical field for preparing nanometer silk granules

The present invention relates to the field of processing natural molecular materials, in particular to a method for manufacturing nano silk fibroin particles from silk. Background technique

Silk fibroin produced by the silkworm is a natural polymer protein with a molecular weight of up to 370kDa. Because this protein has good biocompatibility, it is non-toxic, harmless and non-immune to the human body. It has been used as a medical surgical suture for a long time. line. In recent years. The development and utilization of silk fibroin in artificial skin, cosmetics and nutritious food materials or as a carrier for immobilized cells, enzymes and antibodies has attracted much attention. Among them, the manufacture and application of silk fibroin are very active, and the silk fibroin powder is mainly prepared by physical or chemical methods. At present, silk fibroin powders are mainly divided into two categories. One is water-soluble silk fibroin powders with different molecular weight ranges, which are used in nutritional foods, surfactants, health products, medical materials and other fields; the second category is insoluble Water crystalline silk fibroin powder is widely used in cosmetics, skin care products, coatings, industrial materials, synthetic materials, printing inks and other fields. In practical applications, crystalline powder accounts for 70 ~ 80% of the total silk fibroin powder.

The preparation of crystalline silk fibroin powders is mostly made into water-insoluble silk fibroin powders by inducing agglomeration, drying, and pulverization of water-soluble silk fibroin proteins or by repeatedly mechanically pulverizing silk fibroin fibers. The former is mainly made of silk fibroin by degumming silk, and then made into a water-soluble silk fibroin solution, and then the silk fibroin is induced to aggregate by chemical or physical methods. For example, a monohydroxy, dihydroxy or trihydroxy fatty alcohol is mixed with silk fibroin solution in a volume ratio of 0.01 to 1.5: 1, so that silk fibroin aggregates into a gel, and then dehydrated, dried and mechanically pulverized into a powder. And then subjected to 50 ° C saturated steam wet heat treatment to obtain crystalline silk fibroin powder, in which more than 50% of the silk fibroin powder has a β-sheet structure and is insoluble in hot water (Japanese Patent, JP 55-66929; US Patent, US4233211). Silk fibroin aggregation was induced by methods such as salting out, ultrasonication, aeration, and high-speed stirring or adjustment of the isoelectric point to produce silk fibroin powders with similar characteristics to those described above (Japanese Patent, Japanese Patent Laid-Open No. 55-139427; US Patent, US4233212). The silk fibroin solution was energized to induce silk fibroin aggregation or precipitation, and then dried and pulverized to produce silk fibroin powder (Japanese Patent, Tetsuhei 4-26361 1). The silk fibroin solution is evaporated to form a film after dehydration, and then a high temperature and high pressure treatment is performed to form a water-insoluble silk fibroin shaped product. This shaped product can also be mechanically pulverized to produce silk fibroin powder (Japanese This patent, Japanese Patent Application Laid-Open No. 04-264137). The silk fibroin powders prepared by the above methods are not widely used in commercial production due to the large particle size, large particle size range, and different crystallinity. Some preparation costs are relatively high. Therefore, a method of directly pulverizing the silk fibroin mechanically was also developed. The degummed silk fibroin is immersed in water and placed in a container. After heating and pressure treatment or heat and pressure puffing treatment, it is dried and mechanically pulverized to produce 30-50 μm silk fibroin powder (Japanese Patent, Special Publication No. 58 -046097, 58-045232). Directly pulverize the silk fiber mechanically to make a powder of 100 ~ 250 μm, and then mix it with resin, adhesion promoter and water for surface coating (Japanese patent, Japanese Patent Application Laid-Open No. 06-306772). Silk fibroin is pulverized by dry, ball milling and air-flow three-step milling (Japanese patent, Japanese Patent Laid-Open No. 6-339924; European patent, EP0875523) or multistage crushing after reducing the strength of the fiber by alkaline solution treatment (Japanese patent, Japanese Patent Application Laid-Open No. 08-198970, US Patent No. 5,853,764, Chinese Patent No. CN1150438; Japanese Patent Application Laid-Open No. 2001-048989) are made into a particle size of 10 μm or 3 μη! Crystalline silk fibroin powder. Or the silk fibroin first 95. After the C treatment, mechanical pulverization is performed, followed by water pulverization to produce silk fibroin powder (Japanese patent, JP 11-100100510) or wet pulverization by adding organic solvents (Japanese patent, JP 1-293142) to 6 μπι Silk fibroin powder. Silk fibroin was irradiated to reduce its strength, and then ball milled (Hidefurai Takeshi ta, Kazushige I shi da, Youi chi Kami i shi, Fumio Yoshi i, Tamikazu Kume, Macromol. Mat er. Eng. 2000, 283, 126-131 ), Can also be directly degraded silk fibrin with high temperature and high pressure treatment directly into small molecular silk fibroin powder (Gyung-Don Kang, Ki-Hoon Lee, Bong-Seob Shin, Joong-Hee Nahm, Journal of Appl i ed Polymer Science. 85, 2890-2895, 2002). These mechanical pulverization methods are used to make crystalline ultra-fine silk fibroin powders ranging from 1 to 100 μm. They are widely used in cosmetics, skin care products, surface modification materials, polymer synthetic materials, super absorbent materials, coatings and printing inks. application. Since the above-mentioned existing methods for producing silk fibroin powders are mostly mechanically pulverized, it is difficult to produce ultra-micron silk fibroin particles. Therefore, there are shortcomings such as large crystalline silk fibroin particles, various shapes, wide particle size distribution range, particle surface activity, and poor dispersibility in solution, and the application range is limited to a certain extent. Technical solutions

The purpose of the present invention is to overcome the shortcomings of the prior art and provide a simple and effective method for manufacturing crystalline nanofilament particles without chemically harmful substances.

The technical solution to achieve the purpose of the present invention is: a method for manufacturing silk fibroin nanoparticles, which is water-soluble The silk fibroin solution is mixed with a protic organic solvent or a polar aprotic organic solvent that is miscible with water. The volume ratio of the silk fibroin solution and the organic solvent is' :: 2.3 or more. The milky white spherical particles are dispersed in the organic In the solvent system, a nano silk fibroin particle mixture or suspension is obtained, and the organic solvent is removed to obtain a crystalline silk fibroin nano particle suspension or silk fibroin powder.

The water-soluble silk fibroin described in the above technical solution includes degumming, refining or purifying silkworm or wild silk, tussah silk, castor silk, and spider silk, or purifying silk fibroin-like protein produced by genetic engineering; 1〜20%。 Silk fibroin molecular weight 200kDa; the silk fibroin solution concentration is 0. 1 ~ 20%.

The water-miscible organic solvents are methanol, ethanol, propanol, and isopropyl alcohol; and the water-miscible polar aprotic organic solvents are acetonitrile, acetone, and butyl. Ketone, tetrahydrofuran.

In the method for manufacturing silk fibroin nanoparticles, the working environment temperature is 5 ~ 50 ° C.

The organic solvent mixture or suspension of the nanofilament particles is subjected to repeated centrifugal dehydration treatment, or repeatedly filtered and washed until the organic solvent is completely removed.

Pure silk or aqueous solution was added to the obtained silk fibroin nanoparticles and then subjected to ultrasonic treatment for 1 to 10 minutes to prepare a nano silk fibroin solution.

The organic solvent mixture or suspension of the obtained silk fibroin nanoparticles was vacuum freeze-dried to prepare nano silk fibroin powder.

The silk fibroin particles prepared according to the above technical scheme are spherical, insoluble in water, an average particle size of 30 to 60 _nra , a structure of β-sheet, and silk fibroin nanoparticles of 18 to 25%.

Compared with the prior art, the advantages of the present invention are:

1. Since silk fibroin is mixed with an excessive amount of protonic organic solvents or polar aprotic organic solvents, the soluble silk fibroin molecules are instantly transformed from random coils and α-helical structures to insoluble anti-parallel β-sheet structures. Therefore, the obtained silk fibroin particles are about 60 nm in average, spherical in electron microscope observation, compact in structure, 15 to 20% in crystallinity, stable in performance, difficult to be decomposed by protease, and strong in blocking ultraviolet radiation.

2. No toxic chemical reagents are used in the manufacturing process. Therefore, the nanofilament obtained is non-toxic, harmless, non-immune to human body, and has good biocompatibility. It is a green environmental protection product.

3. The manufacturing process is simple, the cost is low, the efficiency is high, and it has a broad market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an ultraviolet absorption spectrum of the nano silk fibroin particles obtained by the method of Embodiment 1 of the present invention; FIG. 2 is a fluorescence emission spectrum of the nano silk fibroin particles obtained by the method of Embodiment 1 of the present invention; FIG. 3 is an embodiment of the present invention The infrared absorption spectrum of the nano silk fibroin particles obtained by the method 1. FIG. 4 is a ¹³ C CP / MAS nuclear magnetic resonance spectrum of the nano silk fibroin particles obtained according to the method of Example 1 of the present invention;

Fig. 5 is an X-ray diffraction pattern of nano silk fibroin particles obtained by the method of Example 1 of the present invention; Fig. 6 is a DSC thermal analysis curve of nano silk fibroin particles obtained by the method of Example 1 of the present invention; Transmission electron micrograph of nano silk fibroin particles obtained by the method of the first embodiment of the invention; FIG. 8 is a scanning electron micrograph of nano silk fibroin particles obtained by the method of the first embodiment of the invention; FIG. 9 is a nanometer obtained by the method of the first embodiment of the invention Atomic force microscopy of silk fibroin particles; FIG. 10 is a particle size distribution diagram of nano silk fibroin particles obtained by the method of Example 1 of the present invention; FIG. 11 is an electron scanning electron microscope image of tussah silk fibroin nanoparticles obtained by the method of Example 6 of the present invention;

FIG. 12 is an X-ray diffraction pattern of tussah silk fibroin nanoparticles obtained according to the method of Example 6 of the present invention;

13 is an electron scanning electron micrograph of ricin silk nanoparticles obtained by the method of Example 7 of the present invention;

Fig. 14 is an X-ray diffraction pattern of ricin silk nanoparticles obtained by the method of Example 7 of the present invention. detailed description

Example 1:

5% Sodium carbonate aqueous solution or other alkaline solution after adding 30 times the amount of 0.5% sodium carbonate aqueous solution or other alkaline solution after washing the silkworm, cocoon production and reeling, textile production, cocoon clothing, waste silk or waste silk cloth, etc. Boil the surfactant for 1 hour, change the solution once, and boil for another 1 hour to ensure that the sericin is completely removed. The silk fibroin from which the sericin was removed was repeatedly washed with water and then dried at 105 ° C for future use.

Take the above-mentioned silkworm-removed silkworm fibroin fibers and a 20-fold (W / V) 9M lithium bromide aqueous solution or a lithium bromide methanol water ternary mixed solvent, and dissolve at 50 ° C for 5 hours; or dissolve silk fibroin The fiber was mixed with 20 times (W / V) calcium chloride / ethanol / water ternary mixed solvent (molar ratio 1: 2: 8), and dissolved at 70 ° C for 2 hours; or the above silk fibroin was dissolved in other Concentrated salt solution, copper ammonium solution or organic solvent. The various silk fibroin solutions obtained above were dialyzed, desalted, and purified to prepare a water-soluble silk fibroin solution. 5% 〜 About Next, the silk fibroin solution was subjected to ultrafiltration using an ultrafiltration device with a cut-off molecular weight of 50 kDa, thereby obtaining a silk fibroin solution with a molecular weight of 50 kDa, with a concentration of 0.5 to 15%, preferably concentrated or diluted to 2.5% or so. Water soluble silk fibroin solution.

Take the purified water-soluble silk fibroin solution above, at 5 ° C environmental conditions, preferably at 25 ° C environmental conditions, in a volume ratio of 1: 2.3 or more with acetone (that is, the amount of acetone added is the final volume 70% or more), and the silk fibroin is rapidly denatured into ultrafine particles suspended in an organic solvent, centrifuged at 15000 rpm, the supernatant is removed, added with water and stirred well, and then centrifuged. This operation is repeated until acetone is removed. The above-mentioned silk fibroin organic solvent suspension can also be filtered through qualitative filter paper, washed repeatedly with water, and then filtered to remove acetone. After the silk fibroin obtained by the above purification is added to water or an aqueous solution, the nano silk fibroin particles can be well dispersed in the water or the aqueous solution and are not easily precipitated after being subjected to ultrasonic treatment at 10 min. The nano silk fibroin particles thus prepared are between 30 and 100 nm, with an average particle size of about 60 nm, and the crystallinity is half that of natural silk fibroin.

The silk fibroin precipitate or filter before or after the above-mentioned ultrasonic treatment can be directly made into nano silk fibroin by vacuum freeze-drying, but this nano silk fibroin will agglomerate due to dehydration drying, and its particle distribution range will be greater than 100n / n, average particle size is greater than 60nm.

Referring to FIG. 1, in the ultraviolet absorption spectrum chart of silk fibroin nanoparticles, the absorption curve indicated by the dashed line is the ultraviolet absorption spectrum of a water soluble silk fibroin solution with a concentration of 1.80mg / ml on a Hitachi U-3000 visible ultraviolet spectrophotometer There is a maximum absorption peak at 275nm. When the silk fibroin was nanonized (the nanofibroin content was O. SOmg / ml), the typical maximum absorption peak basically disappeared, and the ultraviolet absorption spectrum of the solid line part in the figure appeared, and its concentration was 20 times lower than the former.

Referring to FIG. 2, the fluorescence emission spectrum of a water-soluble silk fibroin solution and a nano silk fibroin particle aqueous suspension measured on a Hitachi fluorescence spectrophotometer (F- 4500 FL Spectrophotometer). Measurement conditions: excitation wavelength 290 nm, excitation slit 10 0 nm, emission slit 5.0 nm, scanning speed 240 nm / rain, sensitivity 0.5 s. It can be seen from the figure that after silk fibroin is nano-sized, the fluorescence emission spectrum undergoes a blue shift of about 10 nm.

Referring to FIG. 3, the water-soluble silk fibroin solution lyophilized powder and nano silk fibroin lyophilized powder were tabletted with KBr a little The sample was prepared and measured on a Magna 550 spectrophotometer (Nicolet Instrument Corp. USA) with a scanning range of OO SOOcnf ^ The infrared absorption spectrum of water-soluble silk fibroin in the figure (shown in the 2F curve) belongs to random curl With a-helical or crank-like structure (Silk I) characteristics, the four bands are 1654.8 (amide I), 1554.1 (amide II), 1242.1 (amide III), and 669.3 (amide V). When silk fibroin was nanosized, its absorption band (as shown in the 2NF curve) shifted, and antiparallel β-sheet (Silk II) structures appeared, namely 1635.8 (amide I), 1520 (amide II), and 1265 (amide III). ) And 682.9 (amide V)

Referring to figures 4, ¹³ C CP-soluble fibroin lyophilized powder and freeze-dried powder of nanowire pixel / MAS (BL7) Nuclear magnetic resonance spectrum (400.13 MHz), the water-soluble fibroin ¹³ C CP / MAS NMR spectra (Shown in the 3F curve) The ¹³ C signals of Gly, Ala, Ser residues can be distinguished well. The Cp and Ca carbon signals of Ala residues are 16.97 and 50.67ppni respectively _; the Cp carbon signal of Ser residues is 61.31ppm; and the Cot carbon signal of Gly residues is 50.67ppm; when silk fibroin is nanosized (shown in 3NF curve) ), ^'3 C carbon signal of these amino acid residues undergone significant chemical shift, Cp Ala residue, Ca and the carbon signal are 20.16 _49.25ppni; Ca Ser residues, and carbon signals are 62.19ppm ₀ 54.52 This is the obvious anti-parallel β-sheet (Silk II) structure of silk fibroin.

Referring to FIG. 5, the silk fibroin sample analysis was performed on a MERCURY CCD- AFC8 CCD single crystal X-ray diffractometer (Nikkei Denki Co., Ltd.), the tube voltage was 4.0 kV, the tube current was 35 mA, and the scanning speed was 2 ° / min. Ni filtering. Scanning from 2Θ = 5 ° ~ 45 °, we obtained natural silk fibroin (shown as F curve), water-soluble silk fibroin lyophilized powder (shown as C curve), and nano silk fibroin lyophilized powder (shown as NF curve). X-ray diffraction pattern. The water-soluble silk fibroin lyophilized powder was confirmed to have a completely amorphous structure, and the peak top of the scattering peak was 2Θ = 20.3 °. After nanofilamentization of silk fibroin, Silk II diffraction peaks appeared at 2Θ at 9.5 °, 20.0 °, and 24.0 °, indicating that the silk fibroin molecular conformation was transformed during the nanocrystallization, and its molecular The degree of alignment and order of the polyacrylamide increased, thereby increasing the crystallinity. The silk fibroin molecular conformation was transformed from random curl to Silkll, and its crystallinity was 18.9%. X-ray diffraction analysis of the natural silk fibroin fiber cut into a sample of very short fibers showed Silkll diffraction peaks at 2Θ of 9.5 ° and 24.5 °, and its crystallinity was 38.7 ° /. .

Referring to Fig. 6, the DSC thermal analysis curve of natural silk fibroin, water-soluble silk fibroin, and silk fibroin lyophilized powder were measured by a DuPont thermal analyzer (2960 SDTV3.0F). Measurement method: to600 @ 10K / min.

Referring to Figure 7, the lyophilized nanofilament powder was enlarged on a Hitachi H600A-II transmission electron microscope Morphology and size of nano silk fibroin particles at 10,000 times.

Referring to Figure 8, the appearance of the nanofilament particles was spherical when magnified 10,000 times on a Hitachi S-570 scanning electron microscope. Due to freeze-drying during the preparation of the powder, agglomeration occurs, so many small nanoparticles are aggregated into larger spheres.

Referring to Figure 9, the Nanoscope II Atomic Force Microscope (Digital Instruments,

Atomic force microscopy of nano silk fibroin particles measured on CA). Silk fibroin particles were distributed between 30 and 50, with an average of about 40 nm.

Referring to FIG. 10, a certain amount of nanofilament solution was diluted with water, sonicated directly into a sample cup, and the particle size distribution was measured on a Zetasizer 3000HSa laser particle size analyzer (Malvern Instruments Ltd, Malvern UK). Silk fibroin particles are distributed between 30 and 100 nm, with an average of about 60 nm.

The silk fibroin nanoparticles prepared by the method of the above embodiment were prepared into an aqueous solution, and a bacterial culture comparison test was performed with a common bacterial culture solution (beef extract peptone medium). The composition of common bacterial culture solution is 10 grams of peptone, 3.0 grams of beef extract, 5.0 grams of NaCl, and the volume is adjusted to 1000 ml with deionized water, and the _PH value is 7.0 to 7.2. The culture solution prepared according to the requirements was filled into test tubes, each tube was 5ral, 1.05 kg / cm ² , and 121.3 ° C was steam sterilized for 20 minutes.

Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli were cultured in a common bacterial culture medium at 37 ° C for 18 to 20 hours, and then 0.1 ml of the culture medium was added to 5ml of sterile water to prepare a bacterial suspension for use. The experiment was divided into two groups. One group was a control area. Two kinds of bacteria were cultured in the normal beef extract peptone medium. The other group was an equal volume of silk fibroin aqueous solution with a nanofibroin content of 1.0 mg / ml. There are 2 test areas in each test group (respectively 2 bacteria), and the samples in each zone are repeated 3 times and each sample is repeated 3 times. A blank experiment was performed using a non-sterilized beef extract peptone culture solution and a silk fibroin nanoparticle suspension. After sterilization of the culture medium or silk fibroin nanoparticle solution under sterile conditions, use a sterile pipette to take the test bacterial suspension quantitatively into the test tube for inoculation, and inoculate the E. coli and Bacillus subtilis shake at 36 ° C and shake Incubate for 24 hours. U-3000 spectrophotometer was used to determine the absorbance of various samples at 560 nm. Table 1 shows the growth and proliferation of bacteria in a standard culture medium, and Table 2 shows the growth and proliferation of bacteria in a nanofilament aqueous solution (nanofilin concentration: 0.1 mg / ml), see Table 1 and Table 2. The comparison results of culture experiments show that in common bacterial culture fluids, the growth and proliferation of E. coli and Bacillus subtilis are normal; in the nanofilament aqueous solution, these bacteria Can not grow and proliferate normally.

The amount of ε-amino groups on the surface of water-soluble silk fibroin or silk fibroin nanoparticles provided by the above examples was determined by the trinitrobenzene sulfonic acid method (TNBS) (AFSA Habeeb, Determination of free amino groups in protein by trini trobenzenesulfonic acid, Analytical Biochemistry 14, 328-336, 1966). Take 1mL of a 0.5mg / mL water-soluble silk fibroin solution or an equivalent amount of nanofibroin particle suspension, add 1mL «sodium bicarbonate solution (pH8.5) and 1mL 10% sodium dodecylsulfonate solution, Let stand for 20rain, then add 1mL of 0.1% TNBS (2, 4, 6-trinitrobenzenesulfonic acid) solution, incubate at 40 ° C for 2h, and stop the reaction with 0.5mL of 1mL / L hydrochloric acid. The absorbance value was measured on a Hitachi U-3000 visible UV spectrophotometer with 325mn. The ratio of the absorbance value of the protein solution to the nanoparticle suspension before and after modification was the percentage of residual ε-amino group. The results show that the number of ε-amino groups on the surface of the nanofilament particles is about half that of the water-soluble silk fibroin.

Example 2:

The water-soluble silk fibroin solution was prepared in the same manner as in Example 1. A 2.5% water-soluble silk fibroin solution was prepared at a temperature of 5 ° C (preferably at 25 ° C), at a temperature of 1: 2.3 or more. The volume ratio is mixed with methanol (that is, the amount of methanol added is more than 70% of the final volume), so that the silk fibroin is rapidly denatured into ultrafine particles and suspended in an organic solvent, centrifuged at 15000 rpm, the supernatant is removed, and the mixture is stirred, and then centrifuged. This operation is repeated until the methanol is removed. The above-mentioned silk fibroin organic solvent suspension may also be filtered through qualitative filter paper, repeatedly washed with water, and then filtered to remove methanol. After the purified silk fibroin obtained is added to water or an aqueous solution, After lOmin ultrasonic treatment, the nano silk fibroin particles can be well dispersed in water or aqueous solution. However, the nano silk fibroin particles prepared with methanol are easy to aggregate and sink, and the remaining properties are similar to those of nano silk fibroin prepared from acetone.

Example 3:

The water-soluble silk fibroin solution was prepared in the same manner as in Example 1. A 2.5% water-soluble silk fibroin solution was used at a temperature of 5 ° (:, preferably at 25 ° C, under an environmental condition of 1: 2.3. The above volume ratio is mixed with ethanol (that is, the amount of ethanol added is more than 70% of the final volume), so that the silk fibroin is rapidly denatured into ultrafine particles and suspended in an organic solvent, centrifuged at 15000 rpm, the supernatant is removed, and the mixture is stirred. Centrifuge and repeat the operation until acetone is removed. The above-mentioned silk fibroin organic solvent suspension can also be filtered through qualitative filter paper, washed repeatedly with water, and then removed by filtration. Ethanol. After the silk fibroin obtained by the above purification is added to water or an aqueous solution, the nano silk fibroin particles can be well dispersed in water or an aqueous solution and not easily precipitated after being subjected to ultrasonic treatment at 10 min. Its performance is similar to nanofilament prepared by acetone.

Example 4-The preparation of a water-soluble silk fibroin solution is the same as the preparation method of Example 1. A 2.5% water-soluble silk fibroin solution is used at an environmental condition of 5 ° C, preferably at 25 ° C. , Mixed with isopropanol (that is, the amount of isopropanol added is more than 70% of the final volume) in a volume ratio of 1: 2.3 or more, so that the silk fibroin is rapidly denatured into ultrafine particles suspended in an organic solvent, and centrifuged at 15000rpm After removing the supernatant, add water and stir well, then centrifuge, and repeat the operation until acetone is removed. The above-mentioned silk fibroin organic solvent suspension can also be filtered through qualitative filter paper, washed repeatedly with water, and then filtered to remove isopropyl alcohol. After adding the purified silk fibroin to water or an aqueous solution, the nano silk fibroin particles can be well dispersed in water or an aqueous solution after 10 min ultrasonic treatment, and its performance is similar to that of nano silk fibroin prepared by acetone.

Example 5:

The water-soluble silk fibroin solution was prepared in the same manner as in Example 1. A 2.5% water-soluble silk fibroin solution was subjected to an environmental condition of 5 ° C, preferably 25 ° C, to 1: 2.3 The above volume ratio is mixed with tetrahydrofuran (that is, the amount of tetrahydrofuran added is more than 70% of the final volume), and the silk fibroin is rapidly denatured into ultrafine particles suspended in an organic solvent, centrifuged at 15000 rpm, the supernatant is removed, and the mixture is stirred with water Then, centrifuge again and repeat the operation until the tetrahydrofuran is removed. The above-mentioned silk fibroin organic solvent suspension can also be filtered through qualitative filter paper, washed repeatedly with water, and then filtered to remove tetrahydrofuran. After the silk fibroin obtained by the above purification is added to water or an aqueous solution, the nano silk fibroin particles can be well dispersed in water or an aqueous solution and are not easily precipitated after ultrasonic treatment at 10 min. Its performance is similar to nanofilament prepared by acetone.

Example 6:

After taking tussah silkworm cocoons, cocoon coats or tussah silk or waste silk cloth and washing, add 30 times the amount of 0.5% sodium carbonate aqueous solution or other alkaline solutions or add surfactants, etc., and boil for 1 hour, then change the solution Once, boil for an additional hour to ensure that all sericin is removed. The silk fibroin from which the sericin has been removed is repeatedly washed with water and then dried for use.

Take the above-mentioned sericin-free tussah silk fiber and 20 times the amount (W / V) of 7M calcium nitrate aqueous solution Mix and dissolve at 80 ° C for 2 hours. The dissolved tussah silk fibroin solution is dialyzed with water, desalted, and purified to prepare a water-soluble tussah silk fibroin solution. 5% Water-soluble, using an ultrafiltration device with a cut-off molecular weight of 50kDa to perform ultrafiltration on the silk fibroin solution, thereby obtaining a silk fibroin solution with a molecular weight of 50kDa, the concentration of which is 0.5 to 15%, preferably concentrated or diluted to 2.5% water solubility Silk fibroin solution.

The 2.5% water-soluble tussah silk fibroin solution is mixed with acetone (that is, the amount of acetone) in a volume ratio of 1: 2.3 or higher under the environmental conditions of 5 ° C, preferably at 25 ° C. (The final volume is more than 70%) Mix, quickly denature silk fibroin protein into ultra-fine particles and suspend in organic solvent, centrifuge at 15000 rpm, remove the supernatant, stir with water, and centrifuge again. Repeat this operation until acetone is removed. . The above-mentioned silk fibroin organic solvent suspension can also be filtered through qualitative filter paper, washed repeatedly with water, and then filtered to remove acetone. After the silk fibroin obtained by the above purification is added to water or an aqueous solution and subjected to ultrasonic treatment for 10 minutes, the nano silk fibroin particles can be dispersed in water or an aqueous solution, but because the particles are large, they are liable to agglomerate and precipitate.

Referring to FIG. 11, the appearance of the silk fibroin particles when the nanoparticle suspension prepared from tussah silk fibroin was magnified 10,000 times on a Hitachi S-570 scanning electron microscope. Tussah silk fibroin particles prepared with acetone have an average particle size larger than that of silkworm nano silk fibroin particles, and the distribution range is between 150 and 350 mn.

Referring to FIG. 12, silk fibroin sample analysis was performed on a Mercury CCD-AFC8 CCD single crystal X-ray diffractometer (Nikkei Denki Co., Ltd.). The tube voltage was 4.0 kV, the tube current was 35 mA, and the scanning speed was 2 ° / mi. n, Ni filtering. Scanning from 2Θ = 5 ° ~ 45 °, X-ray diffraction patterns of water-soluble silk fibroin lyophilized powder and nano silk fibroin lyophilized powder were obtained.

Example 7:

Take the castor silkworm cocoons, cocoon coats, or tussah waste silk or waste silk cloth and wash them, add 30 times the amount of 0.5% sodium carbonate aqueous solution or other alkaline solution, or add surfactants, etc., and boil for 1 hour, then Change the solution once and boil for another hour to ensure that the sericin is completely removed. The silk fibroin from which the sericin has been removed is repeatedly washed with water and then dried for use.

The above-mentioned sericin-free silk fiber was mixed with a 20-fold (W / V) 7M calcium nitrate aqueous solution and dissolved at 80 ° C for 2 hours; the dissolved castor silk fiber solution was dialyzed with water, desalted, Purified to make a water-soluble ricin silk fibroin solution. Next, the silk fibroin solution was subjected to ultrafiltration using an ultrafiltration device with a cut-off molecular weight of 50 kDa, thereby obtaining a silk fibroin solution with a molecular weight of 50 kDa, with a concentration of 0.5 to 15%, preferably concentrated or diluted to 2.5% water soluble Sex silk solution. Add a 2.5% water-soluble ricin silk fibroin solution to acetonitrile (ie, acetonitrile) at a volume ratio of 1: 2.3 or higher under the environmental conditions of 5 ° C, preferably at 25 ° C. The amount is more than 70% of the final volume) mixed, and the ricin silk protein is rapidly denatured into ultrafine particles suspended in an organic solvent, centrifuged at 15,000 rpm, the supernatant is removed, and the mixture is stirred with water, and then centrifuged. Repeat this operation until it is removed. So far acetonitrile. The above-mentioned silk fibroin organic solvent suspension can also be repeatedly washed with water through qualitative filter paper filtration, and then filtered to remove acetonitrile. After the silk fibroin obtained by the above purification is added to water or an aqueous solution, the nano silk fibroin particles can be well dispersed in the water or the aqueous solution after being treated with ultrasonic waves, but the particles are easily aggregated and precipitated because the particles are large. '

Referring to FIG. 13, the appearance of the silk fibroin particles when the nanoparticle suspension prepared by ricin silk fibroin was magnified 10,000 times on a Hitachi S-570 scanning electron microscope. The castor silk fibroin particles prepared with acetonitrile have an average particle size larger than that of tussah silk fibroin particles, and the distribution range is between 200 and 450 nm.

Referring to FIG. 14, the silk fibroin sample analysis was performed on a MERCURY CCD- AFC8 CCD single crystal X-ray diffractometer (Nikkei Denki Co., Ltd.). The tube voltage was 4.0 kV, the tube current was 35 mA, and the scanning speed was 2 ° / min. , Ni filtering. From 2Θ = 5 ° ~ 45. Scanning was performed to obtain X-ray diffraction patterns of castor silk fibroin fibers, water-soluble silk fibroin lyophilized powder, and nano silk fibroin lyophilized powder.

China is a large silk-producing country. A large number of cocoons, cocoons, waste silk, and waste silk are available during the production of silkworms, reeling, refining, printing and dyeing, and textile processing. The present invention mainly uses nano materials produced from silkworm cocoon production and silk processing to produce nano silk fibroin, with an average particle diameter of 30 to 60 nm, a crystallinity of about half (18 to 25%) of natural fibers, and ε- The number of amino groups is about half that of water-soluble silk fibroin. This nano silk fibroin has good biocompatibility, is non-toxic, harmless, non-immunity to human body, and is not easily hydrolyzed by protease. This water-insoluble crystalline nanofilament particle has a small particle size, good dispersibility, and a strong function of blocking ultraviolet radiation. It can be made into various cosmetics, skin care products, especially sunscreens, etc .; it can also be used with other resins. It can be mixed with adhesion promoter to make surface modification materials, coating materials, coatings, etc .; it can be made into other synthetic materials with other polymer materials, and it can be used to make human biomedical materials; it can be combined with various dyes to make natural pigment materials. Used in cosmetics, pigments, printing inks, etc. This kind of particle has good biocompatibility, large specific surface, and active performance. There are many active groups on the surface, such as hydroxyl and amino groups. The biomolecules can be conjugated with a large capacity. It can be used as a carrier of biological linkers, enzymes, peptides, and drugs. And other combinations, because nanoparticles are much smaller than red blood cells and can run freely in the blood, they can be used as injections in the diagnosis and treatment of diseases. O / iiAVu / D / isId slooo

Claims

A method for producing nano silk fibroin particles, characterized in that: a water-soluble silk fibroin solution is mixed with a protonic organic solvent or a polar aprotic organic solvent that is miscible with water, and a volume ratio of the silk fibroin solution to the organic solvent is 1: 2.3 or more, milky white spherical particles are dispersed in the organic solvent system.

A nanofilament particle mixture or suspension is obtained, and then the organic solvent is removed to obtain a crystalline silk fibroin nanoparticle suspension or silk fibroin powder.

2. The method for producing nano silk fibroin particles according to claim 1, wherein the water-soluble silk fibroin includes degumming or purification from domestic silk or wild silk, tussah silk, castor silk, and spider silk. Or silk fibroin-like protein produced by genetic engineering; the molecular weight of silk fibroin is 200kDa.

3. The method for producing a book of nano silk fibroin particles according to claim 1, wherein: the silk fibroin solution has a concentration of 0.1 to 20%.

4. The method for producing nanofilament particles according to claim 1, wherein the protonic organic solvent miscible with water is methanol, ethanol, propanol, isopropanol.

5. The method for producing nanofilament particles according to claim 1, wherein the polar aprotic organic solvent miscible with water is acetonitrile, acetone, methyl ethyl ketone, and tetrahydrofuran.

6. The method for manufacturing nanofilament particles according to claim 1, wherein the working environment temperature is 5 ~ 50 ° C.

7. The method for producing nanofilament particles according to claim 1, wherein the organic solvent mixture or suspension of nanofilament particles is subjected to repeated centrifugal dehydration treatment until the organic solvent is completely removed.

8. The method for manufacturing nano-filament particles according to claim 1, wherein the organic solvent mixture or suspension of nano-filament particles is repeatedly filtered and washed until the organic solvent is completely removed.

The method for producing nano silk fibroin particles according to claim 1, characterized in that: adding pure water or an aqueous solution to the obtained silk fibroin particles and performing ultrasonic treatment for 1 to 10 min to prepare a nano silk fibroin solution.

10. The method for producing nano silk fibroin particles according to claim 1, wherein the organic solvent mixture or suspension of the obtained silk fibroin particles is vacuum freeze-dried to prepare nano silk fibroin powder.