CN117285725A - Composition containing structural protein and preparation method and application thereof - Google Patents
Composition containing structural protein and preparation method and application thereof Download PDFInfo
- Publication number
- CN117285725A CN117285725A CN202310789201.3A CN202310789201A CN117285725A CN 117285725 A CN117285725 A CN 117285725A CN 202310789201 A CN202310789201 A CN 202310789201A CN 117285725 A CN117285725 A CN 117285725A
- Authority
- CN
- China
- Prior art keywords
- composition
- structural protein
- solution
- stabilizer
- fibroin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 125
- 101710172711 Structural protein Proteins 0.000 title claims abstract description 121
- 238000002360 preparation method Methods 0.000 title claims description 23
- 239000003381 stabilizer Substances 0.000 claims abstract description 92
- 239000000463 material Substances 0.000 claims abstract description 64
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 19
- 239000001257 hydrogen Substances 0.000 claims abstract description 19
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 185
- 108010022355 Fibroins Proteins 0.000 claims description 122
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims description 66
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- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 59
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- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 47
- 239000011259 mixed solution Substances 0.000 claims description 47
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical class CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 38
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- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 claims description 21
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- WVRNUXJQQFPNMN-VAWYXSNFSA-N 3-[(e)-dodec-1-enyl]oxolane-2,5-dione Chemical compound CCCCCCCCCC\C=C\C1CC(=O)OC1=O WVRNUXJQQFPNMN-VAWYXSNFSA-N 0.000 claims description 5
- IBVAQQYNSHJXBV-UHFFFAOYSA-N adipic acid dihydrazide Chemical compound NNC(=O)CCCCC(=O)NN IBVAQQYNSHJXBV-UHFFFAOYSA-N 0.000 claims description 5
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- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 4
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- 229940073490 sodium glutamate Drugs 0.000 claims description 4
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 2
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- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 6
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
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- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 description 4
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/05—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media from solid polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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Abstract
The invention relates to a composition containing structural proteins, which is prepared by contacting the structural proteins with a stabilizer in the presence of a special material which can destroy the secondary structure of the structural proteins, wherein the composition is in a solution state and comprises the structural proteins, the stabilizer and a solvent, the hydrophilicity and the hydrophobicity of the stabilizer are the same as or similar to those of the structural proteins, and the stabilizer can form hydrogen bonding with the structural proteins to influence the self-assembly behavior of the structural proteins. The composition containing the structural protein has good stability, the structural protein in the composition can keep the original structure and performance for a long time, and the effectiveness of the composition can be kept for a longer time under different pH, shearing force and temperature environments than the structural protein without the stabilizer.
Description
Technical Field
The invention relates to a composition containing structural proteins, a preparation method and application thereof.
Background
Numerous fundamental studies in life sciences and medicine have shown in recent years that structural protein materials (such as fibroin, collagen, fibrin, keratin, etc.) have excellent compatibility with cells and tissues and have unique properties, such as purified antibodies, whose secondary and tertiary folding patterns are the basis of their structure, have significant target specificity, and remain functional after introduction into patients. This specificity requires structural proteins to maintain amino acids and functional organization of amino acid conformations, forming higher order secondary (e.g., alpha helix, beta sheet), tertiary (3-dimensional shape) or quaternary (multiple protein subunit interactions) structures. These arrangements are guided by electrostatic interactions between amino acid residues, including covalent bonds (e.g., disulfide bonds) and non-covalent bonds (e.g., hydrogen bonds, hydrophobic bonds, ionic interactions), but in the development of structural protein materials, problems such as instability of fibroin solutions, easy self-assembly into gels, flocculation have not been solved.
In view of the above problems, a stable preparation of silk protein has been studied, such as CN114980839a, using polysorbate 80, a silk fibroin derivative protein composition, a penetrant and a buffer, and although the preparation can be stored for 12 weeks, the preparation has too many additives, and no basic rule for maintaining stability of structural proteins is found, which has a great limitation. In another chinese patent, CN102516777a, for example, a silk fibroin aqueous solution is disclosed, in which a strong polar polyol or a soluble salt that inhibits gelation of the silk fibroin aqueous solution is added, and then the silk fibroin aqueous solution is sterilized by high-temperature steam, so that the silk fibroin aqueous solution is obtained, which can be stored at room temperature for more than 3 months without gelation or deterioration, and has stability, but the strong polar polyol and the soluble salt have a certain influence on the performance of the silk fibroin aqueous solution. The stabilizer has no interaction with the silk fibroin, and only relies on soluble salts (often in high concentration) to maintain the silk fibroin in an aqueous solution in a 'structurally disrupted state' of the silk fibroin, so that the silk fibroin can quickly self-assemble into gel or flocculate once compounded into other compositions or processed into products.
Therefore, there is a need for a solution containing structural proteins with simple components and good stability, which gives structural proteins long-term stability and low particle count, and improves the situations of low development efficiency and poor practicality caused by, for example, the fact that the fibroin needs to be used immediately after extraction, and the extraction time is long, and the operations of stirring, separation, concentration, etc. cannot be performed with an instrument.
Disclosure of Invention
The invention aims to provide a composition containing structural proteins, which is simple in component and good in stability.
A second object of the present invention is to provide a method for preparing a composition comprising a structural protein.
A third object of the present invention provides the use of a composition comprising a structural protein.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a composition comprising a structural protein prepared by contacting a structural protein with a stabilizer in the presence of a specific material that disrupts the secondary structure of the structural protein, the composition being in solution and comprising the structural protein, the stabilizer having a hydrophilic-hydrophobic nature the same as or similar to that of the structural protein, and a solvent, the stabilizer being capable of forming hydrogen bonds with the structural protein.
Preferably, the stabilizer has a hydrophilic angle θ 1 The hydrophilic angle of the structural protein is theta 2 And (θ) 2 -25.5°)≤θ 1 ≤(θ 2 +25.5°)。
Preferably, the stabilizer is selected from one or more of celluloses, high molecular polymers, inorganic substances and modified materials.
According to some preferred embodiments, the celluloses comprise one or more of methylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, lignocellulose.
According to some preferred embodiments, the high molecular polymer comprises one or both of polyvinyl alcohol and sodium poly-L-glutamate.
According to some preferred embodiments, the inorganic species comprises carbon nanotubes.
According to some preferred embodiments, the modified material class comprises one or more of hydrophobically modified Hyaluronic Acid (HA), hydrophobically modified hydroxyethyl cellulose. Specifically, hydrophobically modified hyaluronic acid comprises hydrophobically modifying HA with adipic acid dihydrazide or methacrylic anhydride; the hydrophobically modified hydroxyethyl cellulose includes dodecenyl succinic anhydride hydrophobically modified hydroxyethyl cellulose.
Preferably, the mass of the stabilizer is 0.1% and more of the total mass of the structural protein and stabilizer.
Preferably, the concentration of structural protein in the composition is 0.01-40%.
Preferably, the composition has a shelf life of not less than 7 days and not more than 6 months, for example 7 days, 15 days, 1 month, 2 months, 3 months, 4 months, 5 months or 6 months, etc. During the shelf-life period, the structural protein does not spontaneously or progressively gel and the color or turbidity of the composition does not change visibly. The term "shelf life" refers to the shelf life of the composition at 4℃ unless otherwise specified.
Preferably, the structural proteins include one or more of collagen, fibrin, silk fibroin, sericin, keratin. The collagen may be, for example, one or more of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, and type VI collagen; the keratin may be, for example, one or both of wool keratin and feather keratin.
Preferably, the special material comprises one or more of lithium bromide, ionic liquid, urea, guanidine hydrochloride, SDS, and calcium chloride-ethanol-water.
In a second aspect the present invention provides a process for the preparation of a composition as described above comprising the steps of:
Step one: mixing a structural protein, a stabilizer and a special material capable of destroying the secondary structure of the structural protein in the presence of a solvent;
step two: removing special materials in the mixed solution of the step one, or diluting the mixed solution of the step one to the acceptable concentration of human tissues to obtain the composition.
Wherein the solvent includes, but is not limited to, water.
In some embodiments, the preparation method comprises the steps of:
s1, mixing one of structural protein and a stabilizer with a special material aqueous solution capable of destroying the secondary structure of the structural protein to obtain a first mixed solution;
s2, mixing the other one with the first mixed solution or mixing the other one with a special material aqueous solution capable of destroying the secondary structure of the structural protein and then mixing the mixed solution with the first mixed solution to obtain a second mixed solution;
s3, removing special materials in the second mixed solution, or diluting the second mixed solution to a concentration acceptable to human tissues to obtain the composition.
In the present invention, the order of adding the structural protein and the stabilizer is not specifically defined, and in some embodiments, the structural protein and the aqueous solution of the special material that can destroy the secondary structure of the structural protein may be mixed to obtain a first mixed solution, and then the stabilizer is directly added to the first mixed solution to obtain a second mixed solution. In other embodiments, the structural protein may be mixed with an aqueous solution of a specific material that may disrupt the secondary structure of the structural protein to obtain a first mixed solution, the stabilizer may be mixed with an aqueous solution of a specific material that may disrupt the secondary structure of the structural protein to obtain a second mixed solution, and the first mixed solution and the second mixed solution may be mixed. In still other embodiments, the stabilizing agent is mixed with an aqueous solution of a particular material that disrupts the secondary structure of the structural protein to provide a first mixed solution, and the structural protein is added to the first mixed solution. In still other embodiments, stabilizers may be added to the structural protein solution, to which specific materials that disrupt the secondary structure of the structural protein are added. In still other embodiments, the structural protein may be added to the stabilizer solution, to which a special material that disrupts the secondary structure of the structural protein may be added.
Preferably, the concentration of the aqueous solution of the special material is 0.05-10M.
In a third aspect the present invention provides the use of a composition as described above in a buffer, an ophthalmic surfactant formulation, a humectant composition or a hair care composition.
Preferably, the addition amount of the solution in the preparation of the ophthalmic surfactant is 0.5-5% of the total mass of the preparation of the ophthalmic surfactant.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
the composition containing the structural protein has simple components and good stability, can keep the original structure and performance for a long time, and can preserve the effectiveness of the composition for a longer time under different pH, shearing force and temperature environments than the structural protein without the stabilizer.
Drawings
FIG. 1 is a schematic diagram of self-assembly of fibroin to form a gel;
FIG. 2 is a schematic diagram of the formation of hydrogen bonds between the stabilizer and the interior of the fibroin molecule;
FIG. 3 shows the shelf life of cellulose fibroin solutions of different cellulose addition at 37 ℃;
FIG. 4 is a diagram showing the size of the hydrophilic angle of fibroin and various stabilizers.
Detailed Description
The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other.
The structural protein solution is unstable and cannot be maintained in a solution state for a long period of time. This is mainly due to the formation of beta-sheet structures between the internal molecules of the structural proteins through hydrogen bonding, as shown in fig. 1. The inventors have innovatively found that the stabilizer of the present invention can form hydrogen bonding with the inside of the structural protein molecule, and form physical barriers inside and between the structural protein molecules, so that the inside and between the structural protein molecules cannot form hydrogen bonding, see fig. 2, and thus cannot form a beta-sheet structure, so that the structural protein maintains the original performance, and further maintains the solution state for a longer time. The invention is further discussed below.
A composition comprising a structural protein, the composition being prepared by contacting the structural protein with a stabilizer in the presence of a specific material that disrupts the secondary structure of the structural protein, the composition being in solution and comprising the structural protein, the stabilizer and a solvent, the stabilizer having a hydrophilic-hydrophobic nature that is the same as or similar to that of the structural protein, the stabilizer being capable of forming hydrogen bonds with the structural protein. The stabilizer in the invention can form hydrogen bond action with structural proteins, thereby reducing or even avoiding the formation of beta-sheet structures, and further enabling the composition to maintain a solution state for a longer time while maintaining the original performance.
According to the invention, the stabilizer has a hydrophilic angle of θ 1 Structural protein having a hydrophilic angle of θ 2 And (θ) 2 -25.5°)≤θ 1 ≤(θ 2 +25.5°)。
Further, the stabilizer is one or more selected from celluloses, high molecular polymers, inorganic substances and modified materials.
Specifically, the cellulose includes one or more of methylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, and lignocellulose. The molecular weight of the cellulose is not particularly limited, and preferably the molecular weight of the cellulose is 100 to 1000kDa.
The high molecular polymer comprises one or two of polyvinyl alcohol and poly-L-sodium glutamate.
The inorganic species include carbon nanotubes.
The modified material comprises one or more of hydrophobically modified Hyaluronic Acid (HA) and hydrophobically modified hydroxyethyl cellulose. The hydrophobically modified hyaluronic acid may be adipic acid dihydrazide grafted HA (HA-aDH), HA modified with methacrylic anhydride to methacrylated (HA-MA), and the hydrophobically modified hydroxyethyl cellulose may be dodecenylsuccinic anhydride hydrophobically modified hydroxyethyl cellulose (HEC-DDSA), for example. The modification by means of hydrophobic modification or the modified materials, without any particular explanation, are commercially available according to conventional methods.
The inventors have found that even at low amounts of stabilizer added (e.g., 0.1% of the total mass of structural protein and stabilizer), the stability of the structural protein composition can be significantly improved, and that as the ratio of stabilizer is increased, the stability of the composition can be further improved. The stabilizer is preferably 0.1% or more, more preferably 0.1 to 50%, for example 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, 50% or the like based on the total mass of the structural protein and the stabilizer.
According to the invention, the structural proteins include one or more of collagen, fibrin, silk fibroin, sericin, keratin. The collagen may be, for example, one or more of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, and type VI collagen; the keratin may be, for example, one or both of wool keratin and feather keratin.
According to the present invention, the concentration of the structural protein in the composition is 0.1 to 40%, preferably, the concentration of the structural protein in the composition is 0.5 to 30%, for example, 0.5%, 1%, 5%, 10%, 20%, 30%, etc.
According to the invention, the composition containing the structural protein has a shelf life of not less than 7 days and not more than 6 months, during which the structural protein does not spontaneously or progressively gel and the color or turbidity of the composition does not change visually. In some embodiments, the composition is capable of being stored at 37 ℃ for 144 hours and above. The solutions of the present invention have not only long term stability but also low particle counts, even when stored for two weeks at a pH of 6.2 and 40 ℃, the number of particles per 0.5mL of solution is no higher than 240.10.+ -. 2.19.
The present invention also provides a process for preparing a composition as described above, which in some embodiments comprises the steps of:
s1, mixing structural protein, a stabilizer and a special material capable of destroying the secondary structure of the structural protein in the presence of a solvent to obtain a mixed solution; the adding sequence of the structural protein and the stabilizer is not definitely defined, the structural protein and the special material aqueous solution which can destroy the secondary structure of the structural protein can be mixed to obtain a first mixed solution, and then the stabilizer is directly added into the first mixed solution to obtain a second mixed solution; the structural protein and the special material aqueous solution capable of destroying the secondary structure of the structural protein can be mixed to obtain a mixed solution I, the stabilizer and the special material aqueous solution capable of destroying the secondary structure of the structural protein are mixed to obtain a mixed solution II, and finally the mixed solution I and the mixed solution II are mixed; the stabilizer and the special material aqueous solution which can destroy the secondary structure of the structural protein can be mixed to obtain a mixed solution I, and then the structural protein is added into the mixed solution I; the stabilizer can also be added into the structural protein solution, and then special materials which can destroy the secondary structure of the structural protein are added into the structural protein solution; structural proteins may also be added to the stabilizer solution, to which specific materials that disrupt the secondary structure of the structural proteins may be added.
S2, removing special materials in the S1 mixed solution, or diluting the S1 mixed solution to a concentration acceptable to human tissues to obtain the composition.
The means for removing the special material according to the present invention comprises dialysis using a dialysis bag, the molecular weight cut-off of which can be selected according to the nature of the special material or the like, to remove the special material. For example, the dialysis bag can have a molecular weight cut-off of 3500da.
The structural protein-containing solutions of the present invention have excellent properties and can be used in buffer, ophthalmic surfactant formulations, moisturizer compositions or hair care compositions.
In some embodiments, when the solution is used in an ophthalmic surfactant, the composition is added to the formulation of the ophthalmic surfactant in an amount of 0.5 to 5% of the total mass of the formulation of the ophthalmic surfactant.
Further, the formulation of the ophthalmic surfactant further comprises acetate buffer, magnesium chloride, dextrose, and poloxamer 188.
In some embodiments, the humectant composition comprises 70-99% by mass of water, a composition containing structural protein (composed of 0.1-5% by mass of the humectant composition of structural protein solution and 0.5-1.5% by mass of the humectant composition of stabilizer), 0-25% by mass of jojoba oil and rose hip oil, 0-5% by mass of vitamin E, 0.5-2% by mass of poplar bark and 0.1-1% by mass of sodium anisole, 0.1-0.5% by mass of sodium hydroxide, 0.5-1.5% by mass of hydrochloric acid, and 0.1-1.0% by mass of hyaluronic acid.
According to the present invention, the concentration detection method of fibroin is as follows, without particular explanation:
the concentration of the fibroin solution is determined by a weighing method, namely a weighing dish is taken to weigh and record as W, 1mL of fibroin solution is added into the dish and then weighed and marked as W 1 The dish containing the fibroin solution was dried in an oven for 24 hours and then weighed again and marked as W 2 . The concentration (w/w) of the fibroin solution was calculated according to the formula:
concentration= (W) 2 -W)/(W 1 -W)×100％。
According to the invention, the degummed silk is prepared by the following method without any particular description:
60g of raw silk and 25.44g of anhydrous sodium carbonate are weighed for standby, 12L of deionized water is weighed and poured into a stainless steel barrel, and the stainless steel barrel is heated by an electromagnetic oven. When deionized water is about to boil, adding weighed anhydrous sodium carbonate, continuously heating and stirring until the anhydrous sodium carbonate is boiled, fully dissolving the anhydrous sodium carbonate, adding weighed raw silk, keeping boiling for 10min (namely degumming for 10 min), and stirring once every 5min to dissolve sericin on the surface of the raw silk. And kneading the degummed raw silk with deionized water for 4 times to remove sericin on the surface of the raw silk, wringing out degummed silk (degummed silk), and drying overnight in a fume hood.
According to the invention, the fibroin solution is prepared by the following method without any particular explanation:
10g of the dried degummed silk was placed in a solution of lithium bromide of 40mL with 9.3M and stirred well with a glass rod. The lithium bromide solution and silk were mixed and heated in an oven at 60 ℃ for 4 hours to promote silk dissolution. Next, the completely dissolved silk fibroin solution was poured into a dialysis bag (molecular weight cut-off 3500 Da) and sealed. The dialysis bag carrying the silk fibroin solution was placed in a container containing 5L of deionized water, and the solution was continuously stirred at the bottom of the container using a magnetic stirrer to dilute the permeated lithium bromide, for a dialysis time of 3 days, and water was exchanged for 7 to 8 times in total. After complete desalting, the fibroin solution is placed in a centrifugal bottle and is repeatedly centrifuged twice under the conditions of 9000rpm and 4 ℃ low temperature, and finally the clean fibroin solution is obtained and is placed in a refrigerator at 4 ℃ for storage. The solvent in the solution of the present invention is water unless otherwise specified.
Comparative example 1
This example examines the stability of a solution containing a structural protein prepared from stabilizers of different systems in the presence of an aqueous solution of a particular material which does not disrupt the secondary structure of the structural protein
a. Cellulosic material system:
10mL of 5% fibroin solution is measured, 0.015g (3% of total solid content) of cellulose is gradually added into the fibroin solution in the stirring process, and the mixture is stirred at room temperature (25+/-5 ℃) until the mixture is completely dissolved, so as to obtain the cellulose fibroin solution, and the mixture is placed at 37 ℃ for standing.
The inventors examined the stability of the cellulose fibroin solution when the cellulose was Methyl Cellulose (MC), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC) or lignocellulose, respectively.
b. Polymeric material system
10mL of fibroin solution with the concentration of 5% is measured, 0.015g (3% of total solid content) of polymer is gradually added into the fibroin solution in the stirring process, and the mixture is stirred at room temperature until the polymer fibroin solution is completely dissolved, and then the mixture is placed at 37 ℃ for standing.
The inventors examined the stability of the polymer fibroin solution when the polymer was polyvinyl alcohol (PVA) or poly L-sodium glutamate (PGA) or Hyaluronic Acid (HA), respectively.
c. Inorganic material system:
10mL of fibroin solution with the concentration of 5% is measured, 0.015g (3% of total solid content) of inorganic material is gradually added into the fibroin solution in the stirring process, and the mixture is stirred at room temperature until the mixture is completely dissolved, so that the fibroin solution of the inorganic material is obtained, and the mixture is placed at 37 ℃ for standing.
The inventors have examined the stability of fibroin solution, an inorganic material, when the inorganic material is carbon nanotubes or graphene, respectively.
d. Modified material system:
10mL of fibroin solution with the concentration of 5% is measured, 0.015g (3% of total solid content) of modified material is gradually added into the fibroin solution in the stirring process, and the mixture is stirred at room temperature until the mixture is completely dissolved, so that the modified material fibroin solution is obtained, and the mixture is placed at 37 ℃ for standing.
The inventors examined the stability of fibroin solutions of the modified materials when the modified materials were adipic acid dihydrazide grafted HA (HA-ADH), methacrylic acid anhydride modified to methacrylic acid HA (HA-MA) or dodecenyl succinic anhydride hydrophobically modified hydroxyethyl cellulose (HEC-DDSA), respectively.
e. Control group (SF)
10mL of fibroin solution with concentration of 5% is measured, and the solution is placed at the temperature of 37 ℃ for standing.
Example 1
According to the experimental scheme of comparative example 1, the experimental scheme of example 1 was modified as follows: 10mL of 9.3M lithium bromide is measured, 0.06g (3% of total solid content) of stabilizer material is gradually added into the lithium bromide in the stirring process, the mixture is stirred at room temperature until the stabilizer material is completely dissolved, a stabilizer lithium bromide solution is obtained, 1.94g of degummed silk is respectively placed into the stabilizer lithium bromide solution for full infiltration, the solution is dissolved at 60 ℃ for 1.5h, and a mixed solution with the total concentration of 200mg/mL is obtained. The mixed solution was poured into a dialysis bag (molecular weight cut-off 3500 Da) and sealed, placed in a container containing 5L of deionized water, and stirred continuously at the bottom of the container using a magnetic stirrer to dialyze out lithium bromide for 3 days, changing water a total of 7-8 times. After the dialysis is completed, the mixed solution is placed in a centrifugal bottle and is repeatedly centrifuged twice under the conditions of the rotating speed of 9000rpm and the low temperature of 4 ℃, impurities are removed, and the composition containing structural protein is obtained and is placed at 37 ℃ for standing.
Example 2
The inventors listed CMC and HEC in cellulose as stabilizers for testing:
10mL of 9.3M lithium bromide is measured, 4 parts of each solution is measured, 0g (SF of a control group), 0.005g (1% of total solid content), 0.025g (5% of total solid content) and 0.05g (10% of total solid content) of CMC are gradually added into the lithium bromide in the stirring process, the solution is stirred at room temperature until the solution is completely dissolved, CMC lithium bromide solution is obtained, and 0.5g, 0.495g, 0.475g and 0.45g of degummed silk are respectively placed in the CMC lithium bromide solution for full infiltration and then dissolved at 60 ℃ for 1.5h, thus obtaining CMC lithium bromide solution with the total concentration of 50 mg/mL.
The CMC in this group was replaced with HEC and the same procedure was used to obtain HEC lithium bromide cellosolve at a total concentration of 50 mg/mL. Next, each group of solutions was desalted and purified by dialysis as in example 1, and left to stand at 37 ℃.
The polymers have different reaction rates according to different temperatures, and can be analyzed according to an accelerated experimental calculation formula, so that the low-temperature storage time can be deduced from the relatively high temperature.
The calculation formula of the acceleration experiment is as follows:
T 0 ＝T 2 /Q 10 (T 1 -T 3 )/10
t in the formula 0 To accelerate aging time, T 1 To accelerate the aging temperature, T 2 T is the life time of the temperature at which the polymer is 3 Q is the temperature at which the polymer is 10 Is the reaction rate coefficient (typically 2).
In general, when the absorbance of the solution is about 1.6, it means that the solution is in a gel state. The storage time of the solution prepared in example 2 is shown in FIG. 3, and the CMC fibroin solution in the graph can be maintained in a solution state for a longer time according to the observation of FIG. 3, and the CMC fibroin solution in the graph is in a gel state after less than 24 hours in a control group. The stability of the HEC fibroin solution is inferior to that of the CMC fibroin solution (under the condition of the same cellulose addition amount), and almost no stable effect can be achieved.
The storage time of the structural protein-containing solutions in example 2 and comparative example 1 is shown in table 1 below.
TABLE 1
From table 1 above, it is observed that when the stabilizer is Methylcellulose (MC), carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC) or lignocellulose; polyvinyl alcohol (PVA) or poly-L-sodium glutamate (PGA); a carbon nanotube; adipic acid dihydrazide grafted HA (HA-ADH), HA (HA-MA) modified by methacrylic anhydride to be methacrylated, or dodecenyl succinic anhydride hydrophobically modified hydroxyethyl cellulose (HEC-DDSA) and a stabilizer are contacted with fibroin in the presence of lithium bromide, the composition can be kept in a solution state for 144 hours or more at 37 ℃, and can be stored for more than 12 weeks in a 4 ℃ environment and more than 6 weeks in a room temperature environment according to an accelerated experimental calculation formula, so that the composition HAs a stable effect; whereas CMC-PAM in comparative example 1 and example 2 was gel-like already after 24 hours of holding, and thus had poor stability.
The inventors conducted experiments according to the experimental protocols of examples 1 and 2 on one or various combinations of substances having a secondary structure such as type i collagen, type ii collagen, type iii collagen, type iv collagen, type v collagen, type vi collagen, fibrin, fibroin, sericin, wool keratin, feather keratin, etc., and arrived at experimental phenomena and conclusions similar to examples 1 and 2.
The inventors conducted experiments according to the experimental scheme of example 2 on one or more of specific materials such as lithium bromide solution, ionic liquid solution, urea solution, guanidine hydrochloride solution, SDS solution, calcium chloride-ethanol-water solution, etc., which are capable of breaking down a substance having a secondary structure, and arrived at experimental phenomena and conclusions similar to those of example 2.
Example 3
The solution stabilizing effect can be achieved by adding various stabilizing agents
a. CMC was used as a stabilizer according to the experimental procedure of example 2.
b. 10mL of 9.3M lithium bromide is measured, 0.485g of degummed silk is placed in a lithium bromide solution for full infiltration, then dissolved at 60 ℃ for 1.5h, 0.015g (3% of total solid content) of CMC is gradually added into a silk dissolving solution in the stirring process, and the mixture is placed at room temperature for stirring until the mixture is completely dissolved, thus obtaining a mixed solution, and the CMC lithium bromide silk dissolving solution with the total concentration of 50mg/mL is obtained. Dialysis was then performed as in example 2.
c. 10mL of fibroin solution with the concentration of 10% is measured, 0.03g (3% of total solid content) of CMC is gradually added into the fibroin solution in the stirring process, then 10mL of lithium bromide solution is added, and the mixture is stirred at room temperature until the solution is completely dissolved, thus obtaining a mixed solution. Dialysis was then performed as in example 2.
d. The difference from the above c is that: lithium bromide is not added in the preparation process.
a. b, c were each placed in an oven at 37℃after dialysis and light transmittance was measured at a wavelength of 500nm using a Carry 5000 ultraviolet spectrophotometer (Agilent, USA).
The results are shown in Table 2 below:
TABLE 2
| Addition mode | a | b | c | d |
| Hold solution for a long period of time | 176 | 176 | 176 | 24 |
It was observed that in the presence of lithium bromide, the stabilizer had good stabilizing effect, either during the silk dissolving process or in the regenerated silk fibroin, whereas the stabilizer lacking lithium bromide did not produce better stabilizing effect.
Example 4 latent stabilizer contact Angle test-expected results to give satisfactory contact Angle with structural protein differences of less than or equal to 25.5 degrees
Preparing fibroin solution with the concentration of 50 mg/mL; CMC, high viscosity HPMC (H-H), medium viscosity HPMC (M-H), low viscosity HPMC (L-H), HEC, HA were prepared with deionized water to give 50mg/mL solutions, respectively.
And (3) respectively taking 4mL of the solution, placing the solution in a common culture dish with the diameter of 35mm, and naturally air-drying the solution in a fume hood to obtain a silk protein film, a CMC film, a high-viscosity HPMC film, a medium-viscosity HPMC film, a low-viscosity HPMC film, a HEC film and an HA film. The water contact angle of the film was observed for its size and was measured using a contact angle meter (DSA 100, kruss, germany).
As shown in fig. 4, the contact angles of silk protein film (SF), CMC film, high viscosity HPMC (H-H) film, medium viscosity HPMC (M-H) film, low viscosity HPMC (L-H) film are relatively close, 72.60 ±4.86°, 72.07 ±1.65°, high viscosity 56.63 ±3.79°, medium viscosity 58.00±2.82°, and low viscosity 55.53±6.90°, respectively; the HEC film and the HA film are 46.50 +/-3.21 degrees and 43.23+/-4.71 degrees respectively, and compared with the silk fibroin film, the HEC film and the HA film have smaller water contact angle and are more hydrophilic.
Meanwhile, a water contact angle test is also carried out on a material with relatively hydrophobicity, wherein the graphene water contact angle is 103.25 +/-3.23 degrees, and the carbon nanotube water contact angle is 97.81+/-3.45 degrees. When graphene (total solid content 5%) was used as a stabilizer, the silk fibroin solution was stored at 37 ℃ for 24 hours only, and when carbon nanotubes (total solid content 5%) were used as a stabilizer, the silk fibroin solution was stored at 37 ℃ for 192 hours.
Therefore, in combination with the above HEC, HA, and graphene, the results of the water contact angle as a stabilizer showed that materials having a hydrophilicity similar to that of silk fibroin (±25.5°) can be used as a stabilizer.
The inventors conducted experiments according to the experimental scheme of example 3 on one or various combinations of substances having a secondary structure such as type i collagen, type ii collagen, type iii collagen, type iv collagen, type v collagen, type vi collagen, fibrin, fibroin, sericin, wool keratin, feather keratin, etc., and arrived at experimental phenomena and conclusions similar to those of example 3.
Example 5 hydrophilic modification of materials which can be used as stabilizers, the effect of the stabilizers cannot be achieved
A mixed solution was prepared according to example 2, wherein the stabilizer was 0.015g (3% of total solids) of polyacrylamide modified carboxymethylcellulose (CMC-PAM), dialyzed to give CMC-PAM fibroin solution (50 mg/mL) and placed in an oven at 37℃for standing.
The prepared CMC-PAM fibroin solution was placed in an oven at 37℃and the absorbance of the gel at 500nm wavelength was observed every 8 hours, wherein a light transmittance test was performed using a Carry 5000 ultraviolet spectrophotometer (Agilent, USA).
It was observed that the non-gel state was maintained for less than 24 hours in an oven at 37℃and the CMC-PAM had a water contact angle of 41.17.+ -. 4.20 °, so that it was further confirmed that the excellent effect of the present invention could be achieved by the hydrophilic-hydrophobic properties of the stabilizer in the examples being similar to those of the material having the secondary structure.
EXAMPLE 6 concentration Range of specific Material that destroys substances with Secondary Structure
The inventor respectively measures 10mL lithium bromide (0.04M, 0.05M, 1M, 3M, 7M, 9.3M, 10M and 10.1M) with different concentrations in beakers, adds 0.06g (total solid content 3%) of carboxymethyl cellulose into each beaker, adds 1.94g of degummed silk after full dissolution, and places the beakers in a 60 ℃ oven for dissolution for 4 hours, and shakes once every 1 hour.
Next, the fully dissolved CMC lithium bromide silk solution was poured into dialysis bags (molecular weight cut-off 3500 Da) and sealed. The dialysis bag loaded with CMC lithium bromide solution silk liquid is placed into a container containing 5L of deionized water, and the magnetic stirrer is used for continuously stirring at the bottom of the container to dilute the penetrated lithium bromide, the dialysis time is 3 days, and the water is exchanged for 7-8 times in total. After complete desalting, the CMC fibroin solution is placed in a centrifugal bottle, and is repeatedly centrifuged twice under the conditions of 9000rpm and 4 ℃ low temperature, and finally the clean CMC fibroin solution is obtained and is placed in a refrigerator at 4 ℃ for storage. The fractions were placed in 96-well plates and placed in an oven at 37℃for standing. Absorbance was measured with an enzyme-labeled instrument (500 nm) every 16 h.
The results are shown in Table 3:
TABLE 3 Table 3
When the lithium bromide concentration is less than 0.05M, the solution storage time is similar to that of the solution without the stabilizer in the above examples, and the stabilizing effect cannot be obtained. When the lithium bromide concentration is higher than 10M, there is also no stabilizing effect.
The inventors conducted experiments according to the experimental scheme of example 6 on one or more of specific materials such as lithium bromide solution, ionic liquid solution, urea solution, calcium chloride-ethanol-water solution, etc. which are capable of breaking down a substance having a secondary structure, and arrived at experimental phenomena and conclusions similar to those of example 6.
Example 7 specific materials that disrupt substances having a secondary structure-expected results-the substances are all substances that disrupt hydrogen bonds-establish hydrogen bonding between stabilizers and structural proteins, hinder intermolecular hydrogen bonding within molecules of structural proteins, inhibit self-assembly, and achieve the purpose of stabilization.
Stabilizing agent: one or more of CMC, HPMC, carbon nanotube, PVA, graphene, etc.
Stabilizer addition amount: 0% (total solids), 1%, 3%, 5%, 10%, 50%, 99%.
Materials with secondary structure: one or more of fibroin, collagen, wool keratin, feather keratin, etc. Substances that disrupt the secondary structure: urea, guanidine hydrochloride, sodium hydroxide, DNA helicase, etc.
The inventor adopts CMC as stabilizer, and the addition amount is 1% (total solid content), fibroin and urea for principle verification.
8M urea solution, 8M urea/CMC 1% solution and CMC 1% solution are prepared for standby.
a. 10mL of fibroin solution with concentration of 5% is measured, a part of the fibroin solution is taken into a 96-well plate, and the fibroin solution is placed in a baking oven at 37 ℃ for standing after sealing.
b. A mixed solution was prepared according to example 2, wherein the stabilizer was 0.005g carboxymethyl cellulose, and the solution was dialyzed to obtain a cellulose fibroin solution (50 mg/mL), and a portion was taken into a 96-well plate, sealed, and then placed in an oven at 37℃for standing.
c. And (3) respectively adding the group a fibroin solution into an 8M urea solution, an 8M urea/CMC 1% solution and a CMC 1% solution to obtain a urea fibroin solution (c 1-1) and a urea/CMC fibroin solution (c 2-1), taking parts into a 96-well plate, sealing, and then placing in a baking oven at 37 ℃ for standing. Dialyzing the three solutions in deionized water with 3500kDa dialysis bag for 3 times
And (3) changing water every 6 hours, taking parts of the dialyzed solution urea fibroin solution (c 1-2) and the dialyzed solution urea/CMC fibroin solution (c 2-2) into a 96-well plate, sealing, and then placing the mixture in a baking oven at 37 ℃ for standing.
d. Adding the CMC fibroin solution of group b into 8M urea solution to obtain urea/CMC fibroin solution, dialyzing in deionized water for 3 days by adopting 3500kDa dialysis bag, changing water every 6h, taking part of the dialyzed solution into a 96-well plate, sealing, and standing in a baking oven at 37 ℃.
Group a is a control group; group b is a stabilizer addition group; group c is a dialysis validation group; group d is urea dialysis group; the time taken for the a, b, c, d group to stand in the gel state in the oven at 37℃was observed and determined by measuring the absorbance at 500nm every 8 hours. Absorbance was measured using a microplate reader (Synergy H1, usa, bioTek).
TABLE 4 Table 4
From Table 4, it is observed that group a is in gel state in the oven at 37 ℃ for 16 hours, the stability is poor, and group b can keep 192 hours of solution, has good stability, and shows that the stabilizer has excellent effect of stabilizing fibroin solution. The time for maintaining the solution state is 176h, 16h, 168h, respectively, observed from group c1-1, group c1-2, and group d, group c1-1 can maintain the solution state for a longer time due to the presence of urea, and group c1-2 can dialyze urea out due to dialysis, so beta-sheet is easily formed, and group d can maintain the solution state for a longer time even if urea is lost after dialysis due to the presence of CMC.
The observation from group c2-1 and group c2-2 shows that the time for maintaining the solution state is 176h and 168h respectively, the group c2-1 can maintain the solution state for a longer time due to the existence of urea/CMC, and the group c2-2 dialyzes urea out due to dialysis, but the urea is lost after dialysis due to the existence of CMC, but the solution state can still be maintained for a longer time.
Therefore, urea can destroy the hydrogen bonding action between fibroin solutions, so that the beta-sheet activity of the fibroin solutions is reduced, namely beta-sheets are difficult to form, the solutions are difficult to gel, but dialysis loss is realized, CMC can maintain the hydrogen bonding action and is difficult to dialyze, so that the stabilizer and the fibroin solutions directly form the hydrogen bonding action, and the solutions are difficult to gel.
Example 8
The inventor carries out orthogonal tests on different addition amounts of cellulose, fibroin concentration and different celluloses.
Different addition amounts of cellulose: 0.001% (total solids content 2 g), 0.05%, 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 30%, 50%, 80%, 99.9%.
Concentration of fibroin: 0.001%, 0.01%, 1%, 10%, 15%, 20%, 30%, 40%.
Different celluloses: CMC:175kDa, 300-450kDa, 500-600kDa; HPMC:400-500kDa, 550-600kDa, 650kDa; MC:200-400kDa, 450-600kDa
(1) The concentration of fibroin in the reaction system and the cellulose are fixed, and the different addition amounts of the fibers are changed.
a. The inventors listed a cellulose silk protein solution (total concentration 5%) prepared from 0g (control), 0.001g (0.05% of total solids), 0.002g (0.1%), 0.01g (0.5%), 0.02g (1%), 0.06g (3%), 0.1g (5%), 0.2g (10%), 0.3g (15%) of carboxymethyl cellulose (CMC) according to the procedure of example 2.
b. The difference from step a is that the cellulose is different. The cellulose in this step is hydroxypropyl methylcellulose (high viscosity, i.e., H-HPMC, medium viscosity, i.e., M-HPMC, low viscosity, i.e., L-HPMC), and the cellulose fibroin solutions (total concentration 5%) are prepared according to the method of step a.
Next, the completely dissolved cellulose fibroin solution in a and b was taken to be a partially clean cellulose fibroin solution in a 96-well plate, and the solution was placed in a 37 ℃ oven, and the absorbance at 500nm in the reaction system was observed every 8 hours, wherein absorbance at 500nm was measured using a Carry 5000 (Agilent) ultraviolet spectrophotometer, and when the absorbance was about 1.6, the solution was in a gel state, and the corresponding solution storage time was obtained according to the change of absorbance, and the results are shown in table 5.
TABLE 5
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From the above table it can be observed that the fibroin solution without added cellulose has a shelf life of less than 24 hours. When the cellulose addition is less than or equal to 0.05% of the total solids, the preservation time of the fibroin solution is not particularly significantly different, because too little cellulose is added, so that the sites for forming hydrogen bonds between cellulose molecules and fibroin molecules are too few, and the activity of the fibroin for forming beta-sheets is only partially reduced, and the beta-sheets can be formed quickly.
When the added amount of cellulose is more than 0.05% of the total solid content, the stability of fibroin is greatly improved, and especially when the added amount of cellulose accounts for 0.1% or more of the total solid content, the fibroin solution can be kept for 152h or more at 37 ℃. However, when the added amount of cellulose is 15% or more of the total solid content, the preservation time of the fibroin solution is still stable, and the inventors hypothesized that the reason is that the excessive cellulose has all satisfied the point of hydrogen bonding with fibroin molecules, but the excessive cellulose can form hydrogen bonding with cellulose, thereby slowing down the rate of fibroin to form gel.
(2) The concentration of fibroin is changed under the condition of fixing different adding amounts of fibers and cellulose in a reaction system.
The inventors have prepared a cellulose fibroin solution of 1mg/mL, 100mg/mL, 300mg/mL from the mixed solution when the cellulose (CMC) addition amount was 3% of the total solid content according to the protocol of example 2.
And (3) taking the partially clean cellulose fibroin solution prepared in the step (2), putting the partially clean cellulose fibroin solution into a 96-well plate, putting the 96-well plate into a 37 ℃ oven, and observing the light absorption value at 500nm in a reaction system every 8 hours. The change in absorbance was observed, and as shown in Table 3 below, the solution state was maintained for a long period of time regardless of the change in concentration of fibroin.
TABLE 6
(3) Different cellulose is changed under the condition of different addition amounts of fibers and fibroin concentration in a fixed reaction system.
The inventors listed CMC (175 kDa) and HPMC (650 kDa) according to the scheme of example 2, respectively taking 0.015g of the two celluloses, gradually adding the two celluloses into 10ml of 9.3M lithium bromide solution in the stirring process, obtaining a cellulose fibroin solution (total concentration is 5%) after dialysis, taking a part of clean cellulose fibroin solution into a 96-well plate, placing the 96-well plate into a baking oven at 37 ℃, and observing the light absorption value at 500nm in the reaction system every 8 hours. And observing the change of the absorbance value in the reaction system. The research shows that the fibroin solution added with CMC and HPMC can be preserved for 176 hours or more in a baking oven at 37 ℃, so the stabilizing effect is excellent.
Example 9
According to the embodiment 2 scheme, 0g (control), 0.025g (total solids content of 5%) CMC as stabilizer, dialysis to obtain CMC fibroin solution, using 96-well plate split charging and standing at 4deg.C, room temperature (20+ -5deg.C) and 37deg.C. The absorbance was measured every 8 hours.
The observation shows that the fibroin solution added with CMC can be preserved for 12 weeks at 4 ℃, for 5 weeks at room temperature (20+/-5 ℃) and for 152 hours at 37 ℃, and the three temperatures are calculated according to the calculation rule by the acceleration experiment calculation formula, and the control group can be preserved for 3 days at room temperature (20+/-5 ℃) for 1 week at 4 ℃ and for 24 hours at 37 ℃, so that the stabilizer can lead the fibroin solution to have more excellent preservation effect.
Example 10
Formulations containing citric acid buffer
A fibroin solution (control) and a composition containing structural proteins (containing 3% (total solids) CMC) prepared in the manner of example 1 were added to a citric acid buffer, respectively, to prepare a citric acid buffer-containing formulation. The citric acid buffer consists of citric acid and sodium citrate. By adding different proportions of citric acid and sodium citrate, the desired pH is obtained. The two sets of formulations were diluted with purified water to a final concentration of fibroin of 1.0% wt./wt., and CMC of 50mMC and fibroin of 1.0% wt./wt., respectively. The formulation was then filtered using a polyethersulfone filter and then aliquoted into 50mL polypropylene conical tubes. These conical tubes containing the formulation were placed in a stabilization chamber at 40 ℃.
After two weeks, particles in the sample were measured using a Coulter particle counter. The effect of pH on particle formation was observed and the effect of storage temperature on particle formation was shown in tables 7 and 8. These studies show that in the formulation with CMC citrate buffer, the physical stability of the fibroin solution decreases with increasing pH and increasing storage temperature, whereas the control group has gel-like after two weeks, no matter what the pH is, but is still in solution after two weeks of storage at 4 ℃, but the particle count has reached 313.89 ±1.28.
TABLE 7
TABLE 8
Example 11
Formulation development of ophthalmic surfactants:
additional formulation studies were conducted to explore whether other commonly used ophthalmic surfactants with known particle suppression adjuvants (magnesium chloride, dextrose, acetate) would produce results that inhibit particle formation. A surfactant is selected: poloxamer 188 and a set of controls with CMC added as stabilizer were set up.
The addition sequence of the preparation is as follows: adding 80% of required water, directly adding surfactant, magnesium chloride, dextrose, sodium acetate trihydrate and glacial acetic acid, mixing until all auxiliary materials are completely dissolved, adding CMC-containing fibroin solution (i.e. composition), and finally adding deionized water.
Note that: CMC-containing fibroin solutions were prepared as in example 1.
The formulation was then filtered using a polyethersulfone filter and dispensed into type I glass borosilicate vials (Prince Sterilization). The vials were placed in a stabilization chamber at 40 ℃ and 75% relative humidity and the microparticles were evaluated using visual appearance testing.
TABLE 9
As can be seen from table 9, when the stabilizer was added to the preparation of the ophthalmic surfactant and the amount of the composition containing the structural protein in the preparation was 0.5 to 5% of the total mass of the preparation, the formation of particles was effectively suppressed, and the preparation without the stabilizer or the solution in the preparation was added in an amount exceeding the limit, the preparation was stable for only 1 week, and the screening process was not passed.
The concentration of fibroin solution is in the range of 0.1-40% to reach the conclusion.
Example 12
Hair care compositions:
fibroin aqueous solution containing stabilizer is used as surfactant/shampoo active agent; the filaments provide foam when massaging into the hair, wash off well, and provide a clean, clear feel during application. Water (0.3 mL) is applied to the brown hair tress to wet it, and then a composition containing structural proteins (consisting of 0.0003g of stabilizer (1% of total solids) and 5% strength by weight aqueous fibroin solution (0.6 mL by volume) is applied to the tress, in a manner described with reference to reference example 1 or example 3); the tresses were combed 10 times with a fine comb and then high heat blown dry for 120 seconds. The tested surfactants provided some lather upon washing. The fibroin solutions containing the stabilizer feel especially clear during massage, indicating good surfactant performance. The sample was well blow dried, leaving no significant residue.
Example 13
A moisturizing composition that can provide a moisturizing agent comprises:
70-99% (w/v) of water, 0.1-5% (v/v) of 6% silk solution, 0-25% (v/v) of jojoba oil, 0-25% (v/v) of rose hip oil, 0-5% (v/v) of vitamin E, 0.5-2% (w/v) of aspen bark, 0.1-1% (w/v) of sodium anisoate, 0.1-0.5% (v/v) of 5N NaOH, 0.5-1.5% (v/v) of 2M HCl, 0.5-1.5% (w/w) of CMC and 0.1-1% (v/v) of hyaluronic acid.
Other ingredients for the moisturizing composition can include one or more of the following:
0-10% (v/v) of glycerin, 0-25% (v/v) of coconut oil, 0-25% (v/v) of lemon grass oil, 0-25% (v/v) of shea butter, 0-5% (w/v) of oat flour and 0-5% (w/v) of titanium oxide.
In preparing the moisturizing composition, an amount of RO/DI water may be added to a beaker or bowl. An amount of hyaluronic acid powder may be added to the RO/DI water. The mixture may be vigorously mixed at a power of 6-10 or 300-700rpm (in a laboratory mixer) until the hyaluronic acid is completely dissolved (about 1 to 3 hours).
The composition containing structural proteins (consisting of CMC in an amount of 0.5 to 1.5% by mass of the moisturizing composition and fibroin solution in an amount of 6% by volume of the moisturizing composition, prepared in the manner described in example 1 or 3) was then added to the prepared hyaluronic acid solution and gently mixed at a power of 2 or 50 to 80rpm (in a laboratory mixer) until the composition was uniformly mixed with the hyaluronic acid solution. The combined solution (hyaluronic acid/CMC/silk solution) was then stored in a refrigerator overnight. The solution may be transferred to another container.
The hyaluronic acid/CMC/silk solution was added to a beaker or bowl and jojoba oil, rose hip oil, vitamin E and 5N NaOH were added to the prepared hyaluronic acid/CMC/silk solution. The mixture was stirred at a power of 4-10 or 300-700rpm (in a laboratory mixer). The mixture was stirred until it provided a white, lotion-like, homogeneous mixture. The mixture was stirred for about 10 minutes.
2M HCl was added to the homogeneous mixture and the mixture was allowed to stir at a power of 4-10 or 300-700rpm (in a laboratory mixer) for at least 15 minutes. Aspen bark is then added to the mixture and stirred for at least 15 minutes at a power of 4-10 or 300-700rpm (in a laboratory mixer). Sodium anisoate may then be added to the mixture and stirred at a power of 4-10 or 300-700rpm (in a laboratory mixer) for at least 15 minutes to obtain a stable moisturizing composition.
Example 14
Artificial tear:
1g of tetrahydropyrimidine, 0.1g of oligomeric hyaluronic acid (5 kDa) and 60mL of a composition containing structural protein (composed of 0.001g of CMC and fibroin solution with concentration of 0.1 percent, the preparation method is referred to in example 1 or 3), are weighed, dissolved under the conditions of heating and stirring at 37 ℃, the pH of the solution is regulated to 7.0 by using sodium citrate, and the solution A is obtained by filtration; weighing 0.04g of vitamin A and 0.9g of soybean lecithin, adding 100mL of absolute ethyl alcohol for dissolution, and filtering to obtain a solution B; placing the solution B on a thin film evaporator, and removing the organic solvent under reduced pressure to obtain a lipid membrane; pouring the solution A into a lipid membrane, and crushing by an ultrasonic crusher under the incubation condition of 60 ℃ (the power is 100w, the ultrasonic time is 20min x 2 times, and the interval is 20 min) to obtain nano-liposome; 0.5g of high molecular weight hyaluronic acid (1600 kDa) was dissolved in 40mL of water to give a hyaluronic acid solution; and (3) dropwise adding the nano liposome into the hyaluronic acid solution under stirring to obtain the hyaluronic acid-coated nano liposome, adjusting the pH to 7.0, and filtering and sterilizing by using a 0.22 mu m filter membrane to obtain the stable artificial tear.
The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (13)
1. A composition comprising a structural protein, characterized in that: the composition is prepared by contacting a structural protein with a stabilizer in the presence of a special material that disrupts the secondary structure of the structural protein, the composition being in solution and comprising the structural protein, the stabilizer having a hydrophilic-hydrophobic nature the same as or similar to that of the structural protein, and a solvent, the stabilizer being capable of forming hydrogen bonding with the structural protein.
2. The composition of claim 1, wherein: the hydrophilic angle of the stabilizer is theta 1 The hydrophilic angle of the structural protein is theta 2 And (θ) 2 -25.5°)≤θ 1 ≤(θ 2 +25.5°)。
3. Composition according to claim 1 or 2, characterized in that: the stabilizer is one or more selected from celluloses, high molecular polymers, inorganic substances and modified materials, wherein the celluloses comprise one or more of methylcellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose and lignocellulose;
The high molecular polymer comprises one or two of polyvinyl alcohol and poly-L-sodium glutamate;
the inorganic species include carbon nanotubes;
the modified material comprises one or more of hydrophobically modified hyaluronic acid and hydrophobically modified hydroxyethyl cellulose.
4. A composition according to claim 3, characterized in that: the hydrophobically modified hyaluronic acid comprises one or two of adipic acid dihydrazide grafted hyaluronic acid and methacrylic acid hyaluronic acid; the hydrophobically modified hydroxyethyl cellulose comprises dodecenyl succinic anhydride hydrophobically modified hydroxyethyl cellulose.
5. The composition of claim 1, wherein: the mass of the stabilizer accounts for 0.1% or more of the total mass of the structural protein and the stabilizer.
6. The composition of claim 1, wherein: the concentration of structural protein in the composition is 0.01-40%.
7. The composition of claim 1, wherein: the composition has a shelf life of no less than 7 days and no more than 6 months, during which the structural protein does not spontaneously or progressively gel and the color or turbidity of the composition does not change visually.
8. The composition of claim 1, wherein: the structural protein comprises one or more of collagen, fibrin, silk fibroin, sericin and keratin; and/or the number of the groups of groups,
the special material comprises one or more of lithium bromide, ionic liquid, urea, guanidine hydrochloride, SDS and calcium chloride-ethanol-water.
9. A method of preparing a composition comprising a structural protein, characterized by: the composition is the composition of any one of claims 1 to 8, the preparation method comprising the steps of:
step one: mixing a structural protein, a stabilizer and a special material capable of destroying the secondary structure of the structural protein in the presence of a solvent;
step two: removing special materials in the mixed solution of the step one, or diluting the mixed solution of the step one to the acceptable concentration of human tissues to obtain the composition.
10. The composition of claim 9, wherein: the preparation method specifically comprises the following steps:
s1, mixing one of structural protein and a stabilizer with a special material aqueous solution capable of destroying the secondary structure of the structural protein to obtain a first mixed solution;
S2, mixing the other one with the first mixed solution or mixing the other one with a special material aqueous solution capable of destroying the secondary structure of the structural protein and then mixing the mixed solution with the first mixed solution to obtain a second mixed solution;
s3, removing special materials in the second mixed solution, or diluting the second mixed solution to a concentration acceptable to human tissues to obtain the composition.
11. The method of manufacturing according to claim 9, wherein: the concentration of the special material aqueous solution is 0.05-10M.
12. Use of a composition according to any one of claims 1 to 8, characterized in that: the composition is useful in a buffer, formulation of an ophthalmic surfactant, a humectant composition, or a hair care composition.
13. The use according to claim 12, characterized in that: the addition amount of the composition in the preparation of the ophthalmic surfactant is 0.5-5% of the total mass of the preparation of the ophthalmic surfactant.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310789201.3A CN117285725A (en) | 2023-06-30 | 2023-06-30 | Composition containing structural protein and preparation method and application thereof |
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| CN202310789201.3A CN117285725A (en) | 2023-06-30 | 2023-06-30 | Composition containing structural protein and preparation method and application thereof |
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| CN202310789201.3A Pending CN117285725A (en) | 2023-06-30 | 2023-06-30 | Composition containing structural protein and preparation method and application thereof |
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- 2023-06-30 CN CN202310789201.3A patent/CN117285725A/en active Pending
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