CN113827732A - Preparation method and application of self-assembled fibroin/polylactic acid block copolymer for drug delivery - Google Patents
Preparation method and application of self-assembled fibroin/polylactic acid block copolymer for drug delivery Download PDFInfo
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- CN113827732A CN113827732A CN202111090132.4A CN202111090132A CN113827732A CN 113827732 A CN113827732 A CN 113827732A CN 202111090132 A CN202111090132 A CN 202111090132A CN 113827732 A CN113827732 A CN 113827732A
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- polylactic acid
- fibroin
- solution
- silk fibroin
- block copolymer
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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
-
- 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
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/225—Mixtures of macromolecular compounds
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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Abstract
The invention discloses a preparation method of self-assembled fibroin/polylactic acid block copolymer for drug delivery, which comprises the following steps: adding the degummed silk fibroin fibers into ionic liquid to obtain silk fibroin solution; adding polylactic acid into N-N dimethylformamide to dissolve to obtain polylactic acid solution; mixing and stirring the silk fibroin solution and the polylactic acid solution to obtain uniform silk fibroin/polylactic acid solution; after the silk fibroin/polylactic acid solution is frozen and dried, the silk fibroin/polylactic acid solution is immersed in ethanol and is solidified in a self-assembly mode to form a hydrogel state, and the silk fibroin/polylactic acid composite material with the characteristic similar to a typical block copolymer is obtained after drying. The unique block-like structures of the present invention can be applied to the inclusion and delivery of drugs through hydrophobic or electrostatic interactions. The preparation method is environment-friendly and simple and convenient to operate, can regulate and control the shape, structure, physical property and biological property of the fibroin/polylactic acid composite material, and provides a new method and thought for the green production process of the biological composite material.
Description
Technical Field
The invention belongs to a processing method of a high polymer material, and particularly relates to a preparation method and application of a self-assembled fibroin/polylactic acid block copolymer for drug delivery.
Background
The Silk Fibroin (SF) is derived from protein fibers spun by arthropods in the nature, has the advantages of environmental protection, low cost, no toxicity, reproducibility, degradability, biocompatibility, minimum inflammatory reaction and the like, is one of the best choices for producing sustainable materials, and is particularly suitable for application in the fields of biotechnology and biomedicine. However, the molecular conformation of the single-component silk fibroin film is mainly in an unstable amorphous structure, so that the single-component silk fibroin film is fragile in a dry state and is not suitable for practical application. Also, polylactic acid (PLA) derived from grains is a non-toxic, non-irritating, biodegradable aliphatic polyester, having good biocompatibility and biodegradability, which is considered as a promising material for reducing environmental and ecological problems. But its hydrophobicity and lack of surface groups that specifically enhance cell adhesion and activity inhibit its wide use in the biomedical field.
Many recent studies have shown that blended materials based on Silk Fibroin (SF) and synthetic Polymers (PLA) have promising research prospects. The blending material can effectively overcome the defects of single-component materials, and simultaneously, the respective advantages of the single-component materials are exerted, so that an ideal composite material is obtained. Thus, silk fibroin and polylactic acid have been used to prepare biocomposites that can provide good performance in mechanical and biological terms. However, silk fibroin is composed of intermolecular and intramolecular hydrogen bonds, and the solvent resistance of the internal crystal structure causes it to be poorly soluble in general solvents. At present, strong acid, strong base, high concentration salt solutions such as LiBr-H are generally used2O solution, CaCl2-C2H5OH-H2O solution, CaCl2The FA solution and the like dissolve the fibroin, and the solvents have the defects of high toxicity, strong volatility, difficult recovery, easy damage to protein structure and the like, and are easy to cause the reduction of various mechanical and biological properties of the raw material.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a method for preparing a self-assembled fibroin/polylactic acid block copolymer with good mixing by adopting a brand-new green ionic liquid binary solvent system, overcomes the defects of strong toxicity, strong volatility, difficult recovery and the like of most solvents used in the prior art, avoids serious pollution to the environment, and can obtain a unique biological composite material similar to a block structure under the solvent system.
The invention also provides a self-assembled fibroin/polylactic acid block copolymer which is prepared and has a unique block structure and is used for drug delivery, and an application of the block copolymer material in drug inclusion, delivery and the like.
The technical scheme is as follows: in order to achieve the above objects, the present invention provides a method for preparing a self-assembled fibroin/polylactic acid-based block copolymer for drug delivery, comprising the steps of:
(1) adding the degummed silk fibroin fibers into ionic liquid, and heating and dissolving to obtain a silk fibroin solution;
(2) adding polylactic acid into N-N dimethylformamide for dissolving to obtain polylactic acid solution;
(3) mixing and stirring the silk fibroin solution and the polylactic acid solution under the condition of condensation and reflux to obtain uniform silk fibroin/polylactic acid solution;
(4) and (2) freeze-drying the fibroin/polylactic acid solution to solidify the solution, then immersing the solution into ethanol for solidification to form a composite membrane, continuously washing the composite membrane with deionized water to remove the ionic liquid remained on the surface, and drying in vacuum to obtain the fibroin/polylactic acid block copolymer composite material.
Further, in the step (1), the mass ratio of the silk fibroin fibers to the ionic liquid is 1: 11.5-1: 19, preferably the mass ratio of 1: 11.5; the ionic liquid comprises any one of 1-butyl-3-methylimidazole chloride salt, 1-allyl-3-methylimidazole chloride salt, 1-ethyl-3-methylimidazole chloride salt, 1-butyl-3-methylimidazole acetate and 1-butyl-3-methylimidazole bromine salt.
Further, in the step (1), the temperature under constant temperature is 95-100 ℃, and the heating time is 24-36 hours.
Further, the mass ratio of the polylactic acid in the step (2) to the silk fibroin fibers in the step (1) is 1: 5-5: 1; wherein the mass concentration of the polylactic acid in the polylactic acid solution is 5.0 wt.%.
Further, in the step (3), the temperature of condensation reflux is 90-95 ℃, the mixing and stirring time is 6-8h, and the stirring rotating speed is 10-20 r/s.
Further, in the step (4), the freeze drying time is 24-48 h, and the freezing temperature is-45 to-50 ℃; the ethanol is immersed for solidification for 5-7 h, the vacuum drying temperature is 20-25 ℃, and the drying time is 12-24 h
Further, in the step (1), the degumming treatment specifically includes: the silkworm cocoon is put into a boiled sodium bicarbonate aqueous solution to remove sericin and a small amount of grease, wax, hair, grass scraps and other impurities. The degumming treatment is adopted to obtain silk fibroin which is more beneficial to human body absorption and is beneficial to the subsequent process processing of silk fiber; preferably, the concentration of the aqueous sodium bicarbonate solution is 0.21 wt.%; the degumming time is 30-50 min.
The self-assembled fibroin/polylactic acid block copolymer for drug delivery, which has a unique block-like structure, is prepared by the method disclosed by the invention.
The invention relates to an application of a self-assembled fibroin/polylactic acid block copolymer for drug delivery in a drug inclusion and delivery system, tissue engineering or wound dressing.
In the preparation method, a specific binary solvent system of ionic liquid and N, N-Dimethylformamide (DMF) is adopted, wherein the ionic liquid dissolves Silk Fibroin (SF), and regarding the ionic liquid dissolving silk fibroin, intermolecular and intramolecular hydrogen bonds are destroyed mainly through the interaction of anions in ionic liquid such as halogen, carboxylic acid, acetic acid and the like and hydroxyl in silk fibroin. Meanwhile, the synergistic effect of the nucleophilicity, the electrophilicity and the charge polarity of the anion and the cation can also accelerate the destruction of the hydrogen bond between the beta-folded structures, and finally the effect of dissolving the beta-folded structures is achieved. In the invention, polylactic acid (PLA) is dissolved in N, N-Dimethylformamide (DMF) to form a solution, and then the obtained SF solution and PLA solution are stirred and mixed under the condition of condensation and reflux, and in the mixing process, the N, N-Dimethylformamide (DMF) solvent can be used as a diluent to reduce the viscosity of ionic liquid on one hand, so that the whole blending system has better fluidity and miscibility; on the other hand, the ionic liquid and the N-N dimethylformamide generate a synergistic effect, so that hydrogen bonds, static electricity and hydrophobic-hydrophobic interaction between the two polymers can accelerate the formation of the SF/PLA blending solution. The final performance of the blend generally depends on the miscibility of polymers, and in the invention, a specific SF/PLA blending solution can be obtained by using the ionic liquid and the N, N-Dimethylformamide (DMF), and the ionic liquid and the N-dimethylformamide generate a synergistic effect to promote the miscibility of two components of a blending system, so that the SF/PLA blending solution without layering phenomenon is obtained. Microphase separation between crystallizable and non-crystallizable regions occurs during the reflux heating and freeze-drying process. Further, the uniformly mixed SF/PLA solution was freeze-dried and treated with ethanol to form a film with complete coagulation. In the processing process, the freeze drying of the system is to freeze and dry the solution into a solid state after the mixed solution of the fibroin and the polylactic acid is formed, and then the ethanol is added, so that the mutual dissolution phenomenon of the solution and the ethanol caused by directly adding the ethanol is avoided, and the complete solidification and film forming of the whole mixed solution are ensured. And ethanol is added to induce the formation of a beta-sheet structure and simultaneously remove anions and cations in the blended membrane (the ionic liquid is a solvent consisting of anions and cations). Meanwhile, the hydrophobic effect of the molecular chain and the self-assembly capability of the molecule are enhanced. Thus, polymer chains aggregate in the absence of them. In this case, the ethanol rapidly substitutes for anions on or around the polymer segment, and reacts with the polymer segment to form a hydrogen bond. Finally, the fibroin and the polylactic acid are blended to obtain a composite material which shows a physically cross-linked block-like structure. The ionic liquid can be dissolved in ethanol and recovered and reused.
The invention forms a structure similar to a block type structure because of phase separation and ethanol addition, so that the hydrophobic effect of a molecular chain and the self-assembly capability of molecules are enhanced. At the same time, SF interacts with PLA and can form physical crosslinks to help promote the formation of block copolymer structures. The aim of the ethanol is to induce the formation of a beta-sheet structure in the membrane, remove the ionic liquid on the one hand, and also play an important role in solidifying the solution into a membrane, wherein the ionic liquid does not exist around and in the membrane after the ionic liquid is dissolved into the ethanol, and at the moment, the ethanol can quickly replace anions on or around a polymer chain segment and react with the polymer chain segment to form a hydrogen bond. Therefore, there is a competition between ethanol molecules and different polymers for forming hydrogen bonds, and at the same time, the physical cross-linking between SF and PLA finally forms a blocky structure.
In the invention, fibroin and polylactic acid are mixed together to form a macroscopic uniform solution through the action of ionic liquid and N-N dimethylformamide, and the macroscopic uniform solution is further heated and freeze-dried, so that micro-phase separation can be generated between a crystallizable region and a non-crystallizable region. Through ethanol treatment and deionized water washing, anions in the composite membrane are removed and form strong hydrogen bonds to induce the formation of beta-sheet crystals located in the phase separation domain thereof. Meanwhile, the hydrophobic effect of the molecular chain and the self-assembly capability of the molecule are enhanced. In addition, SF interacts with PLA and can form physical crosslinks to help promote the formation of block copolymer structures.
The ionic liquid/N, N-Dimethylformamide (DMF) binary solvent system adopted by the preparation method is a brand new method for preparing the fibroin/polylactic acid blend.
The preparation method of the invention blends fibroin and polylactic acid in an ionic liquid/N, N-Dimethylformamide (DMF) binary solvent system to form a self-assembled fibroin/polylactic acid block copolymer with a unique block structure. The self-assembly fibroin/polylactic acid segmented copolymer adopts a green solvent ionic liquid and an N, N-Dimethylformamide (DMF) binary solvent system, combines natural fibroin protein and synthetic polymer polylactic acid according to a certain proportion, improves the hydrophobicity and cell affinity of a polylactic acid material and the mechanical property of regenerated fibroin protein, and can obtain fibroin/polylactic acid materials with different shapes, structures and physical properties by changing the mass ratio of fibroin to polylactic acid.
The self-assembly fibroin/polylactic acid segmented copolymer has different block interaction and hydrophilicity and hydrophobicity degrees on the chemical structure, so that microphase separation and self-assembly can occur in aqueous solution to form spherical micelles. In the process, the hydrophobic drug can be automatically combined into the hydrophobic core of the micelle through non-covalent interactions such as hydrophobic interaction, electrostatic interaction, metal ion complexation and oxygen bond; meanwhile, under the protection of the hydrophilic chain segment, the effects of biological enzymes in the human body and other internal environments (blood plasma) of the human body and the medicament are avoided, the circulation practice of the medicament in the human body is prolonged, and the medicament can be slowly released at a pathological change part. Illustrating the important potential of the materials of the present invention in drug inclusion and delivery systems.
The invention provides a method for self-assembling protein-synthetic polymer material, and the whole mechanism is shown in figure 1. The first step is to dissolve the silk fibroin, and the ionic liquid BMIMCl has the function of weakening hydrogen bond networks in beta-folding among and in the silk fibroin molecules, so that the secondary conformation of the silk fibroin is changed. Generally, fibroin molecules are mainly in random coil and alpha-helix structures in solution, but the structures can be converted into beta-sheet structures by solvent, heat treatment or physical shearing and the like. In the invention, silk fibroin is dissolved in ionic liquid, and an amorphous structure chain and a partial crystal structure are formed (figure 1 a). Subsequently, blending with PLA of amorphous structure (FIG. 1b), the invention firstly proposes that a macroscopically homogeneous SF/PLA blending solution can be obtained by an ionic liquid and N-N dimethylformamide binary solvent system (FIG. 1 c). But a microphase separation exists between the crystallizable and non-crystallizable regions. While the miscibility (compatibility) of the components of the polymeric blend depends largely on the intermolecular interactions. In the invention, hydrogen bonds, static electricity and hydrophobic-hydrophobic interaction exist in the silk fibroin and the polylactic acid, the hydrogen bonds and the static electricity are mainly derived from enthalpy drive, the hydrophobic-hydrophobic interaction is driven by entropy, but the phase separation characteristics (for example, two glass transition in DSC) are shown from thermodynamics. The effect of microphase separation will be overcome due to strong interactions between polymer chains and a thermodynamically miscible system will be maintained on a macroscopic scale. In this case, a uniform morphology can still be shown on a macroscopic scale and very good homogeneous properties can be provided (see SEM results of fig. 2). The results of the present invention show that SF and PLA establish some interactions in the mixture, which results in improved physical as well as biological properties compared to the pure polymer. Further, a gel film was formed by ethanol treatment. In the treatment process, the ethanol induces the formation of beta-sheet, removes anions and cations in the blending membrane, and enhances the hydrophobic effect of molecular chains and the self-assembly capability of molecules. Thus, polymer chains aggregate in the absence of them. In this case, the ethanol rapidly substitutes for anions on or around the polymer segment, and reacts with the polymer segment to form a hydrogen bond. Thus, there is a competition between the ethanol molecule and the different polymers for hydrogen bond formation, eventually leading to the formation of stable spherical micelles consisting of hydrophobic and hydrophilic regions (fig. 1d), which is induced by the exchange between ethanol and anions, and the physical cross-linking between SF and PLA, eventually forming this block-like protein-synthetic polymer structure (fig. 1 e). The SEM image of fig. 8 of the present invention also confirms that the prepared SF/PLA blend film has a block-like structure. The block copolymer can connect the drug to an insoluble chain segment or embed the drug in a hydrophobic core of a copolymer micelle through non-covalent interactions such as hydrophobic interaction, electrostatic interaction, metal ion complexation, oxygen bond and the like, under the protection of a hydrophilic chain segment, the action of biological enzymes in a human body and other human internal environments (blood plasma) and the drug is avoided, the circulation practice of the drug in the body is prolonged, and the drug can be slowly released at a pathological change part. It has an important role in drug inclusion and delivery systems.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
(1) the invention adopts environment-friendly ionic liquid and an N, N-dimethylformamide binary solvent system, overcomes the defects of strong toxicity, strong volatility, difficult recovery and the like of the traditional solvent, and realizes an environment-friendly green preparation process;
(2) the preparation process is simple and convenient to operate, no harmful gas is discharged, the appearance, the micro-scale structure, the secondary structure, the physical and biological properties (hydrophilic and hydrophobic properties) and the like of the fibroin/polylactic acid composite material can be regulated and controlled by changing the blending ratio of fibroin and polylactic acid, so that the requirements on different properties of the material are met, and different micro-scale structures can provide surface properties which are beneficial to cell adhesion, growth and proliferation;
(3) according to the invention, after fibroin and polylactic acid are mixed into a solution, ethanol is used as a coagulant, so that the mixed solution is coagulated to form hydrogel, and meanwhile, the ionic liquid is dissolved in the ethanol, so that the ionic liquid can be recycled, and the energy-saving and environment-friendly concept is met;
(4) the invention adopts specific ionic liquid/N, N-Dimethylformamide (DMF) binary solvent to obtain the self-assembled fibroin/polylactic acid block copolymer with unique block structure.
(5) The self-assembled fibroin/polylactic acid block copolymer with a unique block-like structure, which is obtained by the invention, can play an important role in a drug inclusion and delivery system.
Drawings
Fig. 1 is a schematic diagram of the mechanism of the preparation process of the present invention, including dissolution and regeneration processes, wherein a.sf dissolves in ionic liquid to form amorphous structure chains and a portion of residual crystal structure; dissolving PLA in DMF to form an amorphous structure chain; blending process of SF and PLA under the interaction of hydrogen bond, hydrophobic-hydrophobic and static electricity; d. under the action of ethanol, the mixture is self-assembled to form a micelle structure; e. forming a SF/PLA blended material with a similar block structure;
FIG. 2 is SEM images of SF/PLA-based block copolymers of different mass ratios in examples;
FIG. 3 is a graph showing reversible heat capacity curves of SF/PLA-based block copolymers of different mass ratios measured at a heating rate of 2 ℃/min using TMDSC mode in the examples;
FIG. 4 is a TG curve of SF/PLA-based block copolymers of different mass ratios heated from room temperature to 550 ℃ at a heating rate of 10 ℃/min;
FIG. 5 is a DTG curve of SF/PLA-based block copolymers of different mass ratios heated from room temperature to 550 ℃ at a heating rate of 10 ℃/min;
FIG. 6 is water contact angles of SF/PLA-based block copolymers of different mass ratios;
FIG. 7 shows the survival rates of mouse fibroblasts cultured on SF/PLA block copolymers with different mass ratios for 6, 24 and 48h respectively measured by MTT method;
FIG. 8 is a typical SEM image after 24h of enzymatic degradation on SF/PLA-based block copolymers prepared at different mass ratios.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and examples.
The raw materials used in the following examples are all as follows:
the silkworm cocoons used were purchased from July trade Co., Ltd, Dandong; the polylactic acid is racemic polylactic acid and has a molecular weight of 10000; ionic liquids such as 1-butyl-3-methylimidazolium chloride (CAS:79917-90-1) were purchased from Shanghai Chengjie chemical Co., Ltd; N-N dimethylformamide was purchased from national pharmaceutical group chemical reagents, Inc.; the remaining solvents were analytically pure.
Example 1
(1) Placing 5.00g of cut mulberry silkworm cocoon into 2000mL of boiled sodium bicarbonate water solution with the concentration of 0.21 wt.% for treatment for 30min, removing sericin in the silkworm cocoon, fully washing with deionized water for three times, and drying in a ventilated place to obtain the degummed silk fibroin fiber.
(2) Adding 11.5g of ionic liquid 1-butyl-3-methylimidazolium chloride into a 50mL centrifuge tube, heating to melt, shearing 1g of degummed fibroin fibers, adding the degummed fibroin fibers into the ionic liquid for multiple times, and heating and dissolving in a constant-temperature water bath kettle at 100 ℃ for 36 hours to obtain 8 wt.% of clear amber fibroin protein solution.
(3) According to the mass ratio of the degummed silk fibroin fibers to the polylactic acid being 1: and 5, weighing 5g of racemic polylactic acid, wherein the molecular weight of the racemic polylactic acid is 1 ten thousand, dissolving the racemic polylactic acid in N-N dimethylformamide, and mechanically shaking the solution at room temperature for 5min to prepare a polylactic acid solution with the concentration of 5 wt.%. And (2) pouring the prepared silk fibroin solution and polylactic acid solution into a 250mL round-bottom flask, heating to 95 ℃ under the condition of condensation reflux, and magnetically stirring and mixing for 7 hours at the rotating speed of 20r/s to obtain the uniformly mixed silk fibroin/polylactic acid solution.
(4) Casting the fibroin/polylactic acid mixed solution in a glass culture dish, freeze-drying for 48h in a vacuum freeze-drying oven at the freezing temperature of-45 ℃ to obtain a solid-like fibroin/polylactic acid composite material, soaking in ethanol for 6h to achieve complete solidification, continuously washing with deionized water for 2-3 times to remove ionic liquid remained on the surface, controlling the vacuum drying temperature at 25 ℃ and drying for 12h to obtain the self-assembled fibroin/polylactic acid block copolymer composite membrane material.
Example 2
(1) Placing 5.00g of cut mulberry silkworm cocoon into 2000mL of boiled sodium bicarbonate water solution with the concentration of 0.21 wt.% for treatment for 30min, removing sericin in the silkworm cocoon, fully washing with deionized water for three times, and drying in a ventilated place to obtain the degummed silk fibroin fiber.
(2) Adding 11.5g of ionic liquid 1-butyl-3-methylimidazolium chloride into a 50mL centrifuge tube, heating to melt, shearing 1g of degummed fibroin fibers, adding the degummed fibroin fibers into the ionic liquid for multiple times, and heating and dissolving in a constant-temperature water bath kettle at 100 ℃ for 36 hours to obtain 8 wt.% of clear amber fibroin protein solution.
(3) According to the mass ratio of the silk fibroin fibers to the polylactic acid of 5: and 5, weighing 1g of racemic polylactic acid, wherein the molecular weight is 1 ten thousand, dissolving the racemic polylactic acid in N-N dimethylformamide, and mechanically shaking the solution at room temperature for 5min to prepare a polylactic acid solution with the concentration of 5 wt.%. And (2) pouring the prepared silk fibroin solution and polylactic acid solution into a 250mL round-bottom flask, heating to 95 ℃ under the condition of condensation reflux, and magnetically stirring and mixing for 7 hours at the rotating speed of 20r/s to obtain the uniformly mixed silk fibroin/polylactic acid solution.
(4) Casting the fibroin/polylactic acid mixed solution in a glass culture dish, freeze-drying for 48h in a vacuum freeze-drying oven at the freezing temperature of-45 ℃ to obtain a solid-like fibroin/polylactic acid composite material, soaking in ethanol for 6h to achieve complete solidification, continuously washing with deionized water for 2-3 times to remove ionic liquid remained on the surface, controlling the vacuum drying temperature at 25 ℃ and drying for 12h to obtain the self-assembled fibroin/polylactic acid block copolymer composite membrane material.
Example 3
(1) Placing 5.00g of cut mulberry silkworm cocoon into 2000mL of boiled sodium bicarbonate water solution with the concentration of 0.21 wt.% for treatment for 30min, removing sericin in the silkworm cocoon, fully washing with deionized water for three times, and drying in a ventilated place to obtain the degummed silk fibroin fiber.
(2) Adding 11.5g of ionic liquid 1-butyl-3-methylimidazolium chloride into a 50mL centrifuge tube, heating to melt, shearing 1g of degummed fibroin fibers, adding the degummed fibroin fibers into the ionic liquid for multiple times, and heating and dissolving in a constant-temperature water bath kettle at 100 ℃ for 36 hours to obtain 8 wt.% of clear amber fibroin protein solution.
(3) According to the mass ratio of the silk fibroin fibers to the polylactic acid of 5:1, weighing 0.2g of racemic polylactic acid with the molecular weight of 1 ten thousand, dissolving the racemic polylactic acid in N-N dimethylformamide, and mechanically shaking the solution at room temperature for 5min to prepare a polylactic acid solution with the concentration of 5 wt.%. And (3) pouring the prepared silk fibroin solution and polylactic acid solution into a 250mL round-bottom flask, heating to 95 ℃ under the condition of condensation reflux, and mixing for 7 hours by magnetic stirring. The rotating speed is 20r/s, and the evenly mixed fibroin/polylactic acid solution is obtained.
(4) Casting the fibroin/polylactic acid mixed solution in a glass culture dish, freeze-drying for 48h in a vacuum freeze-drying oven at the freezing temperature of-45 ℃ to obtain a solid-like fibroin/polylactic acid composite material, soaking in ethanol for 6h to achieve complete solidification, continuously washing with deionized water for 2-3 times to remove ionic liquid remained on the surface, controlling the vacuum drying temperature at 25 ℃ and drying for 12h to obtain the self-assembled fibroin/polylactic acid block copolymer composite membrane material.
Example 4
Example 4 was prepared identically to example 1, except that: the degumming time in the step (1) is 50 min.
In the step (2), the mass ratio of the silk cellulose to the ionic liquid is 1: 19; the ionic liquid is 1-allyl-3-methylimidazole chloride salt; heating to dissolve at 95 deg.C for 36 h.
The temperature of the condensation reflux in the step (4) is 95 ℃, the mixing and stirring time is 6h, and the stirring rotating speed is 20 r/s. The freeze drying time is 48h, and the freezing temperature is-45 ℃; the time for immersing in ethanol for solidification is 7h, the vacuum drying temperature is 25 ℃, and the drying time is 12 h.
Example 5
Example 5 was prepared identically to example 1, except that: in the step (2), the mass ratio of the silk cellulose to the ionic liquid is 1: 15; the ionic liquid is 1-ethyl-3-methylimidazole chloride salt; heating to dissolve at 100 deg.C for 24 hr.
The temperature of the condensation reflux in the step (4) is 90 ℃, the mixing and stirring time is 8h, and the stirring rotating speed is 10 r/s. The freeze drying time is 24h, and the freezing temperature is-50 ℃; the time for soaking in ethanol for solidification is 7h, the vacuum drying temperature is 20 ℃, and the drying time is 24 h.
Comparative example 1
(1) Placing 5.00g of cut mulberry silkworm cocoon into 2000mL of boiled sodium bicarbonate water solution with the concentration of 0.21 wt.% for treatment for 30min, removing sericin in the silkworm cocoon, fully washing with deionized water for three times, and drying in a ventilated place to obtain the degummed silk fibroin fiber.
(2) Adding 11.5g of ionic liquid 1-butyl-3-methylimidazolium chloride into a 50mL centrifuge tube, heating to melt, shearing 1g of degummed fibroin fibers, adding the degummed fibroin fibers into the ionic liquid for multiple times, and heating and dissolving in a constant-temperature water bath kettle at 100 ℃ for 36 hours to obtain 8 wt.% of clear amber fibroin protein solution.
(3) Weighing 5g of racemic polylactic acid, the molecular weight of which is 1 ten thousand, dissolving in ionic liquid 1-butyl-3-methylimidazolium chloride salt to prepare a polylactic acid solution with the concentration of 5 wt.%, and heating and dissolving for 36 hours in a constant-temperature water bath kettle at 100 ℃. Finally, it was found that polylactic acid could not be dissolved in ionic liquids.
Comparative example 2
(1) Placing 5.00g of cut mulberry silkworm cocoon into 2000mL of boiled sodium bicarbonate water solution with the concentration of 0.21 wt.% for treatment for 30min, removing sericin in the silkworm cocoon, fully washing with deionized water for three times, and drying in a ventilated place to obtain the degummed silk fibroin fiber.
(2) Adding 11.5g of ionic liquid 1-butyl-3-methylimidazolium chloride into a 50mL centrifuge tube, heating to melt, shearing 1g of degummed fibroin fibers, adding the degummed fibroin fibers into the ionic liquid for multiple times, and heating and dissolving in a constant-temperature water bath kettle at 100 ℃ for 36 hours to obtain 8 wt.% of clear amber fibroin protein solution.
(3) According to the mass ratio of the silk fibroin fibers to the polylactic acid of 1: and 5, weighing 5g of racemic polylactic acid, wherein the molecular weight of the racemic polylactic acid is 1 ten thousand, dissolving the racemic polylactic acid in dichloromethane, and mechanically shaking the solution at room temperature for 5min to prepare a polylactic acid solution with the concentration of 5 wt.%. Pouring the prepared silk fibroin solution and polylactic acid solution into a 250mL round-bottom flask, heating to 95 ℃ under the condition of condensation reflux, and mixing for 7 hours by magnetic stirring. The rotation speed is 20r/s, the layering phenomenon can occur, and the fibroin/polylactic acid solution which is uniformly mixed can not be obtained.
Comparative example 3
(1) Placing 5.00g of cut mulberry silkworm cocoon into 2000mL of boiled sodium bicarbonate water solution with the concentration of 0.21 wt.% for treatment for 30min, removing sericin in the silkworm cocoon, fully washing with deionized water for three times, and drying in a ventilated place to obtain the degummed silk fibroin fiber.
(2) Anhydrous calcium chloride is used as solute, formic acid is used as solvent, and calcium chloride-formic acid solution with 4.00 wt% is prepared. Weighing 11.5g of the solution, then weighing 1g of the degummed silk fibroin fiber and dissolving the degummed silk fibroin fiber into a calcium chloride-formic acid solution, wherein the calcium chloride-formic acid has strong dissolving capacity on silk fibroin, and the salt-acid dissolving system can quickly dissolve the silk fibroin at normal temperature, so that the dissolution is carried out at normal temperature.
(3) According to the mass ratio of the silk fibroin fibers to the polylactic acid of 1:5, weighing 5g of racemic polylactic acid, the molecular weight of which is 1 ten thousand, dissolving the racemic polylactic acid in N-N dimethylformamide, and mechanically shaking the solution at room temperature for 5min to obtain 5 wt.% polylactic acid solution. Pouring the prepared silk fibroin solution and polylactic acid solution into a 250mL round bottom flask, and under the condition of condensation reflux, selecting the heating temperature of 45 ℃ because formic acid is extremely volatile, and magnetically stirring and mixing for 1.5h at the rotating speed of 20 r/s.
And (3) casting the silk fibroin/polylactic acid mixed solution in a glass culture dish, and drying in vacuum at the temperature of 45 ℃ for 48 hours to form the silk fibroin/polylactic acid composite membrane. Repeatedly washing with deionized water for 2-3 times to remove residual solvent, moving to a ventilated drying place for natural air drying, and then treating the membrane with ethanol to induce the formation of a beta-folded structure (generally soaking for 20min), wherein the finally prepared composite membrane does not have the characteristics similar to a block structure.
Comparative example 4
(1) Placing 5.00g of cut mulberry silkworm cocoon into 2000mL of boiled sodium bicarbonate water solution with the concentration of 0.21 wt.% for treatment for 30min, removing sericin in the silkworm cocoon, fully washing with deionized water for three times, and drying in a ventilated place to obtain the degummed silk fibroin fiber.
(2) Anhydrous calcium chloride is used as solute, formic acid is used as solvent, and calcium chloride-formic acid solution with 4.00 wt% is prepared. 11.5g of the solution was weighed, and then 1g of degummed silk was weighed and dissolved in calcium chloride-formic acid solution to prepare 8.00 wt% silk fibroin solution.
(3) According to the mass ratio of the silk fibroin fibers to the polylactic acid of 1:5, weighing 5g of racemic polylactic acid, the molecular weight of which is 1 ten thousand, dissolving the racemic polylactic acid in dichloromethane, and mechanically shaking the solution at room temperature for 5min to obtain 5 wt.% polylactic acid solution. Pouring the prepared silk fibroin solution and polylactic acid solution into a 250mL round bottom flask, heating to 45 ℃ under the condition of condensation reflux, and magnetically stirring and mixing for 1.5h at the rotating speed of 20 r/s.
(4) And (3) casting the silk fibroin/polylactic acid mixed solution in a glass culture dish, and drying in vacuum at the temperature of 45 ℃ for 48 hours to form the silk fibroin/polylactic acid composite membrane. Repeatedly washing with deionized water for 2-3 times to remove residual solvent, moving to a ventilated drying place for natural air drying, and then treating the membrane with ethanol to induce the formation of a beta-folded structure (generally soaking for 20min), wherein the finally prepared composite membrane does not have the characteristics similar to a block structure.
In the embodiment 1 of the invention, in the preparation process, ethanol is added to completely solidify the blending solution into a film, the formation of a beta-folding structure can be induced, and the ionic liquid is removed, in the removal process of the ionic liquid, the anion and cation in the ionic liquid have ion exchange effect with the ethanol, and the addition of the ethanol enhances the hydrophobic effect of a molecular chain and the self-assembly capability of molecules. In addition, the physical crosslinking interaction of SF and PLA together promote the generation of block-like structures. In contrast, comparative examples 3 and 4, in which the membrane was prepared and then treated with ethanol, the simple purpose was to induce the formation of a β -sheet structure, and a block-like structure could not be formed. In fact, the solvent systems of comparative examples 3 and 4 can not obtain the gel-like or solid-like fibroin/polylactic acid composite materials, the solvents used in comparative examples 3 and 4 are very easy to volatilize, the solvent is only required to volatilize to form a film, a dried film is formed before ethanol treatment, and the ethanol treatment only induces beta-sheet formation and can not form a block-like structure.
Test example 1
Comparison of morphology structures of fibroin/polylactic acid block copolymer composite membrane materials
The SEM image of the fibroin/polylactic acid based block copolymer is shown in fig. 2: a to c: in examples 1 to 3, the mass ratio of fibroin to polylactic acid was 1:5,5: 5 and 5:1, surface morphology of the fibroin/polylactic acid block copolymer.
From comparison of electron micrographs of the fibroin/polylactic acid-based block copolymer composites in examples 1-3 of FIG. 2, it is clear that all composites exhibit their homogeneity and compatibility on a macroscopic scale. The composite material shows difference in topological morphology, and for sample a (SP1-5, i.e., the mass ratio of degummed silk fibroin fiber to polylactic acid in example 1 is 1: 5), it can be seen that a dense nano spherical particle structure is formed, and as the silk fibroin content increases, the composite material shows a more fibrous structure surface, and for sample b (SP5-5, i.e., the mass ratio of degummed silk fibroin fiber to polylactic acid in example 2 is 5: 5), the composite material surface shows a strip-like morphology with protruding nano particles. Continuing to increase the fibroin content, sample c (SP5-1, i.e., 5:5 mass ratio of degummed silk fibroin fiber to polylactic acid in example 3) exhibited a uniform strip topology. The various unique forms are mainly characterized in that the chain length ratio between hydrophilic/hydrophobic regions in the obtained block-like copolymer is different by changing the ratio of fibroin and polylactic acid. While different micro-scale structures may provide other surface properties to the composite material, such as improved cell attachment and propagation, etc. Therefore, the surface appearance and the structure of the composite material can be flexibly controlled by adjusting the mass ratio between the silk fibroin and the polylactic acid so as to meet different application fields.
Test example 2
Phase state analysis of fibroin/polylactic acid block copolymer composite membrane material
The SF/PLA-based block copolymer composites were tested at different ratios using RV differential scanning calorimeter (RVDSC, DSC7000X, JEOL Ltd., Japan) in a Temperature Modulation DSC (TMDSC) mode. The preparation method is carried out under the condition that the heating rate is 2 ℃/min, the dried SF/PLA composite material is packaged in an aluminum disc, the flow rate is 30mL/min, the modulation frequency is 0.02Hz, the modulation temperature amplitude is 3 ℃, and the temperature is increased from minus 20 ℃ to 220 ℃ at the heating rate of 2 ℃/min.
FIG. 3 shows the reversible heat capacity curve of the sample between-20 ℃ and 220 ℃. For the composite materials (SP 1-5-SP 5-1), the glass transition temperatures are respectively in the temperature ranges of 40-50 ℃ and 100-200 ℃, and the glass transition steps (delta C) contributed by the fibroin are increased along with the increase of the ratio of the fibroin in the composite materialp) Gradually becomes obvious and shifts to high temperature, and moves from 143.5 ℃ to 168.9 ℃, but the T of the polylactic acid is classified in the area of 40-50 DEG CgThe temperature will shift slightly towards the lower temperature. From the DSC results above, it can be seen that SF interacts with PLA. However, the 2 characteristic thermal transitions appearing on the TMDSC curve in turn suggest that microphase separation of the two-phase molecular chains may occur and have characteristics similar to those of typical block copolymers. The hydrophilic shell inside the block copolymer can reduce hydrophilic protein adsorption, enable various immune cells in blood to escape immune recognition, and lead the blood circulation time to be long, and the hydrophobic core can contain the drug through hydrophobic or electrostatic interaction and has important functions in a drug inclusion and delivery system.
Test example 3
Comparison of thermal stability of fibroin/polylactic acid block copolymer composite film material
Using Pyris 1TThermogravimetric analysis (TG) of GA (Perkin-Elmer, USA), N2The atmosphere was used as a shielding gas, and the flow rate of nitrogen gas was 50 mL/min-1In the case of (2), the temperature is 10 ℃ per minute-1The temperature ramp rate of (2) heats the sample from 20 ℃ to 550 ℃.
From fig. 4 and 5, which are graphs of TG and DTG of the SF/PLA composite materials of examples 1 to 3, it can be seen from fig. 4 that the thermal weight loss process of the SF/PLA composite material mainly occurs between 200 ℃ and 400 ℃, and there is one and only one significant weight loss step, which indicates that the decomposition process of the SF/PLA composite material is completed in one step, and shows that the two components of silk fibroin and polylactic acid have good thermodynamic compatibility under this novel binary solvent system. In this phase, the initial decomposition temperature T of the sample degradationonsetGradually shifts to low temperature with increasing silk fibroin content, and decreases from 335.21 ℃ (SP ═ 1-5) to 287.23 ℃ (SP ═ 5-1). Also, as can be seen in fig. 5, mass loss of all samples is rapid during the main degradation phase, and once the initial decomposition temperature is reached, the SP1-5 sample mass decreases significantly, with the fastest decomposition rate reaching about 19.57%/min. It is shown that the silk element addition can slow down the decomposition rate of the blended material at higher temperature. Temperature T corresponding to maximum degradation ratepFrom 364.45 ℃ (SP ═ 1-5) to 305.75 ℃ (SP ═ 5-1). Finally, as the silk fibroin content increased, the residual amount at 550 ℃ also increased. For example, sample SP1-5 has a residue of around 7.57% at 550 ℃ and SP5-1 sample is about 35.68%. This indicates that the residual amount increases with increasing SF content. These results again demonstrate the thermodynamic compatibility of the two materials.
Test example 4
Comparison of hydrophilic and hydrophobic performances of fibroin/polylactic acid block copolymer composite membrane material
The hydrophilic performance of SF/PLA block copolymer composite membrane materials with different proportions is tested by adopting a sitting drop method. And (3) dropping ultrapure water on the composite membrane material by using a needle, standing the water drop on the membrane for 5s, shooting the shape of the water drop by using an optical contact angle measuring instrument, calculating a static contact angle, measuring each sample for 3 times, and taking an average value.
In general, the static water contact angle of a material surface is one of important indicators for evaluating the hydrophilicity/hydrophobicity of a material. In the present invention, as shown in fig. 6, the water contact angles of the SF-PLA composite films at mass ratios of 1:5, 5:5, and 5:1 are 83.68 °, 73.48 °, and 68.52 °, respectively. With the increase of the SF component, the water contact angle of the composite membrane is gradually reduced, which shows that the hydrophilic property of the composite membrane is gradually improved, and the addition of the SF improves the hydrophilicity of the composite membrane. For an ideal tissue engineering material, besides a certain mechanical property, good plasticity and biodegradability, the most important point is that the material has good biocompatibility and a good material-cell interface, and good hydrophilic property is more favorable for cell adhesion, growth and proliferation.
Test example 6
Cytotoxicity research of fibroin/polylactic acid block copolymer composite membrane material
Mouse fibroblast (L929) is adopted to carry out in-vitro cytotoxicity experiments, and the biocompatibility and the cell activity of the SF/PLA block copolymer composite membrane materials with different mass ratios are evaluated. Before cell culture, all SF/PLA composite membrane materials were sterilized with Ultraviolet (UV) light for 1h, then soaked in 75% ethanol for 10min, and finally the samples were washed 3 times with sterile Phosphate Buffered Saline (PBS). Fetal Bovine Serum (FBS) was mixed with modified Eagle medium (DMEM, Sigma, USA) at a volume ratio of 1: 9 mixing to prepare a culture solution. Cells were briefly washed with sterile PBS, trypsinized, and plated at approximately 1X 10 in medium4Resuspend at individual cell/mL concentration. Different samples (composite membranes prepared in examples 1-3) were placed on the bottom of a 96-well culture plate (TCP). The fibroblasts were then placed on the composite membrane while a blank was added to the well plate without cells, with 5% CO at 37 deg.C2Incubate until cell fusion reaches 80%. By the MTT method (3- [4, 5-dimethylthiazole 0-yl)]Bromination of 0, 5-diphenyltetrazole) to detect cellular activity in composite membranes at different pressures. During the course of cell culture, the medium was changed daily and tetrazolium salt (MTT) solution (5mg/mL, 20. mu.L) was added. After further incubation for 4h, the supernatant was taken, replaced by 150. mu.L of dimethyl sulfoxide (DMSO), and then shaken horizontally for 10 min. Finally, the sample is removed and measured at 570nm using an ELISAAbsorbance at the long edge. At least 3 measurements were made for each sample.
FIG. 7 shows the survival rates of mouse fibroblasts cultured on SF/PLA composite materials with different mass ratios for 6, 24 and 48 h. All composite membranes showed a tendency to increase in cell viability with increasing culture time. Specifically, the cell viability of samples SP5-5 cultured for 6, 24, and 48h was 106.4%, 109.7%, and 111.8%, respectively. The cell viability of the sample is related to the silk fibroin content, and the cell viability of the sample with more silk fibroin content (SP5/1) is obviously better than that of the composite membrane sample with low silk fibroin content (SP1-5, SP 5-5). For example, after 24h of culture, when the mass ratio of SF to PLA is 5: at 1, the cell activity was 114.4%. Whereas the cell viability of samples SP1-5 and SP5-5 was 101.8% and 109.7%, respectively. After 48h of cell proliferation, the same rule was shown as that of 24h of culture. In summary, the presence of SF in the SF/PLA composite membrane contributes to the ability of the cells to adhere and grow. The improved surface proliferation and absence of cytotoxicity of complex SF/PLA may be attributed to the interaction between SF and the cells. At the same time, due to the hydrophilicity of SF and its ability to interact with the negatively charged surface of the cell membrane. In contrast, polylactic acid is poorly hydrophilic, lacking in biological activity and cell affinity. Therefore, the addition of SF to the SF/PLA composite film clearly increases the hydrophilicity relative to pure PLA. In general, hydrophilic surfaces have a better affinity than hydrophobic surfaces.
Test example 7
Enzymolysis of composite membrane material of fibroin/polylactic acid block copolymer
First, about 10mg of the composite membrane material prepared in examples 1 to 3 was weighed, and the initial mass of each sample was kept the same. Then, the film was immersed in a solution containing 3.1U mL–1Chymotrypsin (from Michelin, China) in 10mL PBS buffer (pH7.4) was treated in a hot environment at 37 ℃ for 24 h. Finally the specimens were removed, rinsed three times gently with distilled water to completely remove the enzyme and PBS residues, and placed in a vacuum freeze-drying oven for further study.
To examine the crystalline and amorphous structures in the SF/PLA blend films of the present invention, samples of examples 1-3 at different mass ratios were treated in chymotrypsin/PBS buffer solution for 24 h. Chymotrypsin is a proteolytic enzyme secreted by pancreas, and can be applied to degrading amorphous regions of biopolymer materials to obtain a highly-crystallized material structure. After 24h of enzymatic degradation, the surface morphology of the sample is observed by using SEM, and the blended film presents different fissures and porous structures (figure 8). After 24h of enzymatic degradation, the remaining composite membrane is mainly a crystal scaffold, and the amorphous domain is attacked. This porous structure by enzyme treatment demonstrates the properties of amorphous-crystalline block copolymers, demonstrating that the composite membrane material of the present invention is a biocomposite material with a unique block-like structure. In addition, during the preparation of the composite film material, the amorphous state and the crystalline state in the two-component SF/PLA can be self-assembled in a small micro-domain, so that microphase separation is caused, and the size and the appearance of the crystal are changed.
Claims (10)
1. A method for preparing a self-assembled fibroin/polylactic acid-based block copolymer for drug delivery, comprising the steps of:
(1) adding the degummed silk fibroin fibers into ionic liquid, and heating and dissolving to obtain a silk fibroin solution;
(2) adding polylactic acid into N-N dimethylformamide for dissolving to obtain polylactic acid solution;
(3) mixing and stirring the silk fibroin solution and the polylactic acid solution under the condition of condensation and reflux to obtain uniform silk fibroin/polylactic acid solution;
(4) and (2) freeze-drying the fibroin/polylactic acid solution, then immersing the solution into ethanol for solidification to form a hydrogel state, continuously washing the solution with deionized water to remove the residual ionic liquid on the surface, and drying the solution in vacuum to obtain the fibroin/polylactic acid block copolymer composite material.
2. The method for preparing a self-assembled silk fibroin/polylactic acid based block copolymer for drug delivery according to claim 1, wherein the mass ratio of the silk cellulose fiber to the ionic liquid in the step (1) is 1: 11.5-1: 19; wherein, the ionic liquid preferably comprises any one of 1-butyl-3-methylimidazole chloride salt, 1-allyl-3-methylimidazole chloride salt, 1-ethyl-3-methylimidazole chloride salt, 1-butyl-3-methylimidazole acetate and 1-butyl-3-methylimidazole bromine salt.
3. The method for preparing the silk fibroin/polylactic acid composite material based on the ionic liquid as claimed in claim 1, wherein the heating dissolution in the step (1) is 95-100 ℃ and the heating time is 24-36 h.
4. The method for preparing a self-assembled silk/polylactic acid-based block copolymer for drug delivery according to claim 1, wherein the mass ratio of the polylactic acid in the step (2) to the silk fiber in the step (1) is 1: 5-5: 1.
5. the method for preparing the silk fibroin/polylactic acid composite material based on the ionic liquid as claimed in claim 1, wherein the temperature of the condensation reflux in the step (3) is 90-95 ℃, the mixing and stirring time is 6-8h, and the stirring rotation speed is 10-20 r/s.
6. The method for preparing the self-assembled fibroin/polylactic acid-based block copolymer for drug delivery according to claim 1, wherein the freeze-drying time in the step (4) is 24-48 h, and the freezing temperature is-45 to-50 ℃; the ethanol is immersed for solidification for 5-7 h, the vacuum drying temperature is 20-25 ℃, and the drying time is 12-24 h.
7. The method for preparing a self-assembled silk/polylactic acid based block copolymer for drug delivery according to claim 1, wherein the degumming treatment in the step (1) is to remove sericin by putting bombyx mori cocoons in a boiled aqueous solution of sodium bicarbonate.
8. The method for preparing a self-assembled silk/polylactic acid based block copolymer for drug delivery according to claim 7, wherein the degumming time is 30-50 min.
9. A self-assembled fibroin/polylactic acid-based block copolymer having a unique block-like structure for drug delivery prepared by the method of any one of claims 1-8.
10. Use of the self-assembled silk/polylactic acid based block copolymer for drug delivery according to claim 9 in a drug inclusion and delivery system, tissue engineering or wound dressing.
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