WO2020200300A1

WO2020200300A1 - Method for stabilizing and enhancing silk fibroin microcapsule shell structure using nanoparticles

Info

Publication number: WO2020200300A1
Application number: PCT/CN2020/083113
Authority: WO
Inventors: 王晓沁
Original assignee: 苏州丝美特生物技术有限公司
Priority date: 2019-04-03
Filing date: 2020-04-03
Publication date: 2020-10-08
Also published as: CN111790322B; CN111790322A

Abstract

A core-shell microcapsule for stabilizing and enhancing a silk fibroin composite shell using nanoparticles. The shell of the microcapsule is a single-layer composite shell with the thickness of 50-1,000 nm, and comprises silk fibroin and nanoparticles randomly distributed in the single-layer composite shell, and the single-layer composite shell is optionally wrapped by other single-layer or multilayer shells. The other single-layer or multilayer shells each independently comprises silk fibroin and nanoparticles randomly distributed in the other shells, and the silk fibroin and nanoparticles can be the same or different between the shells, including between the single-layer composite shell and the other single-layer or multilayer shells. A method for preparing the core-shell microcapsule comprises: mixing and emulsifying internal phase materials and nanoparticle dispersion solution, and then incubating together with silk fibroin to form a stable silk fibroin/nanoparticle composite shell. The thickness of the composite shell can be controlled according to the type, particle size and density of nanoparticles located on an oil-water interface, and the concentration and molecular weight of silk fibroin.

Description

Method for stabilizing and enhancing shell structure of silk fibroin microcapsule by using nano particles

Technical field

The invention relates to various fields such as biomedicine, pharmaceutical preparations, food, beauty, skin care and hair care, textiles and the like. Specifically, the present invention relates to a method for stabilizing and enhancing the shell structure of core-shell microcapsules of natural degradable biological materials by using nanoparticles, and the microcapsules obtained therefrom and uses thereof.

technical background

Microcapsules are micro-scale or nano-scale core-shell micro-containers (ie, core-shell microcapsules) formed by coating a core in liquid, solid, or gas form with a shell material (ie, core-shell microcapsules), and active substances may exist in the core. Based on the responsiveness to the environment and controllable permeability, microcapsules can regulate the release of active substances (such as sustained release or controlled release), and can be used as activity in the fields of drug release, food, biosensors, biomedicine, and cosmetics. The carrier of the substance. The ideal microcapsule carrier should have good biocompatibility, degradability, environmental responsiveness, controllable permeability and core-shell stability. For the application of microcapsules, it should also have a high active material loading, the stability of the active material in the microcapsules, the simple preparation process of the microcapsules, the controllability and uniformity of the size of the microcapsules and the thickness of the shell wall And the modification of the outer shell. How to meet the above conditions and prepare microcapsule carriers with good biocompatibility, degradability, controllable wall thickness and permeability is still an urgent problem to be solved.

The materials used to prepare the shell of the microcapsule may include:

(1) Degradable or non-degradable synthetic organic polymer materials, such as polylactic acid (PLA), polymethyl methacrylate (PMMA), polyvinyl chloride, and polyurethane. The advantage of this type of material is that the resulting microcapsules have better stability, and the properties of the microcapsule shell can be optimized by changing the degree of polymerization of the material. The disadvantage of this type of material is that the acidic substances produced by the degradation of polymer materials in the body can easily cause inflammation in the body or the polymers are not conducive to the preservation of active substances.

(2) Degradable natural organic polymer materials, such as chitosan, gelatin, alginate, starch, etc., and proteins with emulsifying properties, such as soy protein, corn protein, whey protein, etc. The advantages of this type of material are its wide range of sources, non-toxicity, better film-forming properties and better ball-forming properties. The disadvantage of this type of material is that the prepared microcapsule shell has a network or porous structure, so the mechanical properties and stability of the microcapsule are poor, and it is not easy to be used for the embedding and controlled release of active substances. For example, hydrophobic zein is ethanolized and emulsified and dried with soybean oil to obtain microcapsules. During the molding process of microcapsules, zein is precipitated and stacked around the emulsion to form a shell, but the shell of the microcapsule has 0.1–1μm pores, and the larger Pores are not conducive to the sustained release of drugs. Compared with synthetic organic polymer materials, the use of biodegradable natural organic polymer materials to prepare microcapsules has significant advantages in biocompatibility, but how to effectively control the formation process of the shell to obtain ideal mechanical properties and stability , Transparency is the problem faced.

(3) Inorganic material particles, such as double metal hydroxide, calcium carbonate, phosphate, silicate, clay, etc. For example, inorganic and organic silica particles can be used to prepare an emulsion, and a microcapsule with a core-shell structure can be obtained through chemical crosslinking and curing steps, which has good thermal stability and mechanical properties, and a hard outer shell. However, due to its low permeability, a large external force must be used to destroy the shell in order to release the contained substances. At the same time, due to the use of polyurethane coating curing agents and organic synthesis intermediate isocyanates, the biocompatibility of the microcapsules will be reduced and the stability of the loaded active materials will be affected. Therefore, how to overcome the toxicity of the chemical crosslinking agent and curing agent used in the preparation process and how to effectively improve the rigidity and permeability of the shell are the main problems faced.

(4) Liposomes. After the outer layer is grafted with specific antibody molecules, it has cell targeting. As a carrier of active substances, it has great potential in the targeted drug delivery and treatment of malignant tumors, but its stability and permeability to polar molecules are poor, thus limiting It is widely used.

At present, the process of forming a microcapsule shell can be divided into a layer-by-layer self-assembly (LBL) process and a one-step cross-linking process.

Layer-by-layer self-assembly refers to the method of fabricating microcapsules by assembling materials layer by layer by electrostatic force or with a crosslinking agent, so as to achieve the purpose of controlling the thickness of the microcapsule shell.

For example, when the degradable small molecule polylactic acid is used to prepare microcapsules through the layer-by-layer self-assembly process, the wall thickness of the microcapsule shell is about 90nm after assembly 10 times, and only small molecules (molecular weight not exceeding 0.478KDa) can penetrate through the microcapsules. Shell: When assembled 20 times, the wall thickness of the microcapsule shell reaches 200nm, which can only allow smaller molecules to pass. Although the shell thickness of the microcapsules can be controlled in this way, the preparation method is cumbersome and time-consuming, and the polylactic acid molecules are degraded in the body to produce acidic substances, which easily cause inflammation.

For another example, silk fibroin can be used to prepare microcapsules through a layer-by-layer self-assembly process. Specifically, Chunhong Ye et al. repeatedly coated silk fibroin on a silica microsphere template, and used a crosslinking agent to crosslink and fix the layers, and then used a mixture of hydrofluoric acid and ammonium fluoride Remove the silica template (see Chunhong Ye et al., Biomacromolecules, 2011, 12(12): 4319-25). However, since hydrofluoric acid and ammonium fluoride are extremely corrosive and difficult to remove cleanly, they cause inconvenience to subsequent operations and mass production. Moreover, the crosslinking agent used will reduce the biocompatibility of the microcapsules and cause toxicity (for example, the survival rate of yeast cells co-cultured with the microcapsules is only 30%). In addition, although this method can be used to obtain a shell thickness of 19 ± 1nm (3 times of assembly) to 277 ± 11nm (9 times of assembly) of the microcapsule shell, but the literature results show that when the shell is assembled to about 150nm (fifth layer) The permeability has become uneven. In another study (Shchepelina O, Drachuk I, Gupta M K et al., Advanced Materials, 2011, 23(40): 4655-4660), the researchers used the same material to prepare microcapsules, but built the shell on the coating. No cross-linking agent (physical coating) is used between the layers in the wall, the thickness of each layer of the shell thus obtained is only about 2-5nm, and only 54±11nm thick shells are obtained after 12 assembly times. In addition, some researchers (Ye C, Combs Z A, Calabrese R et al., Small, 2014, 10(24): 5087-5097) modified silk fibroin (which is positively charged at this time) and the coating is The negatively charged silica microsphere template is then coated with fragments of negatively charged graphene oxide, the coated silk fibroin and graphene are recycled, and the layers are connected with EDC chemical crosslinking agent. The shell thickness of the microcapsules prepared by this method is only 5±0.5nm (1 layer) to 23±2nm (4 layers), and the average thickness of the single-layer shell is only 5.7nm, which does not have stable mechanical properties. Moreover, the poor dispersion of graphene makes the preparation process complicated. In addition, the cross/coupling agent introduced during the preparation process also deteriorates the compatibility of the microcapsules.

There are also studies using layer-by-layer self-assembly to prepare microcapsules, but without removing the core. For example, first prepare silk fibroin porous micron particles, soak them in an oily substance, so that the pores of the particles are filled with oil, and then physically coat the surface of the particles with silk fibroin/polyethylene oxide (PEO) alternately Cover the shell in a way to control the release of oily substances. The microcapsules prepared by this method have a low loading of oily substances, which is only 10-15% of the total weight of the capsule, and the effect of delaying the release of the shell coating is not obvious.

One-step cross-linking is to directly cross-link the emulsion through a cross-linking agent (or high-temperature spray drying) to form a shell on the surface of the emulsion to obtain microcapsules. Compared with the microcapsule shell prepared by the layer-by-layer self-assembly process, the shell prepared by the one-step cross-linking process has a uniform and single structure, but the mechanical properties, permeability and stability are usually poor and difficult to control. Moreover, the use of high-temperature spray drying has an impact on some temperature-sensitive drugs, and the yield of capsules prepared by the spray method is low.

To sum up, whether it is a layer-by-layer self-assembly process or a one-step cross-linking process, most of them use toxic chemical cross-linking agents, and in some preparations, it is necessary to use strong acids to dissolve the inorganic nuclei, leaving chemical residues in the final product. And low yield, poor mechanical properties, and affect the final active material loading, stability and release rate. Moreover, in the layer-by-layer self-assembly process, it is sometimes necessary to chemically modify the wall material, and the entire operation steps are cumbersome and difficult for industrial production.

In summary, the defects of the prior art method are:

1. Solvent residues caused by chemical cross-linking can cause toxicity, and the introduction of cross-linking agents/coupling agents, surfactants and other substances during the preparation process will reduce the biocompatibility of the microcapsules.

2. The operation steps are cumbersome, especially the layer-by-layer self-assembly process. In order to obtain a thicker-walled microcapsule, it needs to be coated layer by layer, which is time-consuming and labor-intensive, and has poor uniformity and repeatability, making it difficult to produce on a large scale.

3. The thickness and stability of the shell are poor. In literature reports, most of the microcapsules have thin shell walls and poor mechanical properties. Therefore, when oily substances are contained, the microcapsules are difficult to dry into dry powder.

4. The permeability of the shell is too high or too low. The inorganic or organic membrane formed on the surface of oil droplets by one-step cross-linking polymerization has strong permeability and cannot inhibit the diffusion of active substance molecules; while liposomes composed of amphiphilic phospholipid molecules have poor permeability in the outer lipid bilayer, and liposomes It is difficult for the active substance molecules embedded inside to diffuse through.

5. The thickness uniformity of the shell is poor. By adopting the layer-by-layer self-assembly method, the more the number of layers in the back, the more diverse the shape of the microcapsule, the different thickness of the shell, and the lower the yield.

6. The active substance loading and encapsulation efficiency of the microcapsules are low. Whether it is the layer-by-layer self-assembly method or the emulsion interface direct polymerization method, it is impossible to prevent the active substance from dissolving in the oil phase and diffusing outward. The diffusion rate is greatly affected by the shell formation speed, resulting in the active substance loading in the prepared capsule. The amount is low, and the encapsulation efficiency (the ratio of the mass of active substances loaded to the mass of total active substances input) is low.

7. The outer layer grafting specific targeting molecules is restricted. If chemically synthesized or natural-derived polymers are used to form microcapsules through chemical cross-linking methods, it is difficult to have reactive sites on the polymers for grafting specific targeting molecules for targeted drug delivery.

Therefore, how to overcome these shortcomings and obtain core-shell microcapsules with controllable shell wall thickness and permeability and excellent mechanical properties through physical crosslinking and one-step operation is a problem that needs to be overcome.

Summary of the invention

The invention successfully prepares microcapsules with a composite shell structure with ideal thickness and mechanical properties by utilizing the emulsifying properties of the nano particles and the interaction of the specific hydrophilic and hydrophobic fragments of silk fibroin at the oil-water interface, especially the synergistic effect. The microcapsule of the present invention has stable physical and chemical properties, and is an ideal active substance delivery carrier. The nanoparticles present in the shell of the microcapsule of the present invention can stabilize oil droplets in the initial stage of preparation, induce the aggregation and self-assembly of silk fibroin molecules at the oil-water interface, and adjust the mechanical properties, stability and stability of the composite shell together with silk fibroin. Permeability. The thickness of the shell can reach 1000nm, the mechanical elastic modulus value of the shell can reach 2665MPa, and the embedded drug can be released in vitro for more than 30 days.

In one aspect, the present invention provides a core-shell microcapsule that uses nanoparticles to stabilize and strengthen the composite shell of silk fibroin.

In another aspect, the present invention provides a method for preparing the core-shell microcapsules of the present invention. The method for preparing core-shell microcapsules of the present invention does not use chemical crosslinking, so the prepared microcapsules do not have the problems of crosslinking agent toxicity and organic solvent residues. In principle, the preparation method of the present invention is different from the layer-by-layer self-assembly method in the prior art. The method of the present invention is to mix and emulsify the internal phase substance and the nanoparticle dispersion and then incubate with silk fibroin to form a stable silk fibroin/nanoparticle composite shell. The thickness of the composite shell can be achieved by using the nanoparticle located on the oil-water interface. The type, size and density of particles and the concentration and molecular weight of silk fibroin are controlled.

In another aspect, the present invention also provides core-shell microcapsules obtained by or obtainable by the method of the present invention.

In another aspect, the present invention also provides products for biomedicine, pharmaceutical preparations, food, beauty, skin care, hair care, and textiles containing the core-shell microcapsules of the present invention.

In another aspect, the present invention also provides the use of the core-shell microcapsules of the present invention in the preparation of products for biomedicine, pharmaceutical preparations, food, beauty, skin care, hair care, and textiles.

More specifically, the present invention provides the following embodiments:

Embodiment 1. A core-shell microcapsule, characterized in that: the shell of the microcapsule is a single-layer composite shell with a thickness of 50nm-1000nm, comprising silk fibroin and nanometers randomly distributed in the single-layer composite shell. Particles, and the single-layer composite shell is optionally surrounded by one or more additional shells, each of the one or more additional shells independently containing silk fibroin and optionally randomly distributed in the The nanoparticle in the other shell; and wherein the silk fibroin and the nanoparticle are between the shells, including the single-layer composite shell and the one or more other shells Can be the same or different.

Embodiment 2. The core-shell microcapsule according to embodiment 1, wherein the nanoparticles are nanoparticles capable of forming a Pickering emulsion, preferably having a particle size range of 5-1000 nm, more preferably 5-500 nm. Preferably, the nanoparticles may be inorganic or organic nanoparticles. The shape of the particles is not limited, and can be flake, spherical or irregular in other forms.

Embodiment 3. The core-shell microcapsule according to

embodiment

1 or 2, wherein the silk fibroin has a molecular weight in the range of 5KDa-500KDa, preferably 10-400KDa.

Embodiment 4. The core-shell microcapsule according to any one of the preceding embodiments, wherein the core of the microcapsule is a biologically active organism or W/O type milk by oil phase, drug particles, bacteria, cells, etc. The drop is completely filled (ie, full) or partially filled or in the form of gas (ie, hollow).

Embodiment 5. The core-shell microcapsule according to embodiment 4, wherein the oil phase or the oil phase in the W/O emulsion droplets is derived from volatile oil, non-volatile oil and/or water-immiscible The non-oily fluid or solid stearin.

Embodiment 6. The core-shell microcapsule according to

embodiment

4 or 5, wherein the W/O type emulsion droplets are prepared by any method, and the size of the emulsion droplets is selected from 50nm-200um, preferably 200nm-100um; The /O type emulsion droplets are obtained by emulsifying the aqueous dispersion of nanoparticles with the oil phase or stabilized with commonly used small molecule emulsifiers.

Embodiment 7. A method for preparing the core-shell microcapsule of any one of embodiments 1 to 6, characterized in that it comprises the following steps:

(1) Mix and emulsify the aqueous dispersion of nanoparticles with the oil phase or W/O emulsion as the internal phase material to obtain a stable O/W Pickering emulsion or W/O emulsion containing oil droplets accordingly Drop's W/O/W double Pickering emulsion;

(2) Mix the Pickering emulsion prepared in step (1) with the silk fibroin aqueous solution;

(3) Incubating the obtained mixture to obtain an emulsion with a composite external phase composed of silk fibroin and nanoparticles;

(4) The emulsion of step (3) is cured by chemical and/or physical treatment of the composite external phase;

(5) Optionally, drying the mixture of step (4), such as freeze drying, spray drying or natural air drying, to obtain dry powdery microcapsules;

(6) Optionally, resuspend the microcapsules obtained in step (5) in a silk fibroin aqueous solution containing or not containing nanoparticles, and repeat steps (3) to (5) one or more times to obtain a multi-layer Shell microcapsules;

Wherein, the silk fibroin and the nanoparticle may be the same or different between each step.

According to the volatility of the internal phase substance used and the post-processing operation (such as the time of vacuuming), it can be obtained that the inner core is completely filled with biologically active organisms such as oil phase, drug particles, bacteria, cells, or W/O emulsion droplets ( That is, the microcapsules are filled) or partially filled or in the form of gas (for example, hollow).

Embodiment 8. The method according to embodiment 7, wherein in step (1), the aqueous dispersion of nanoparticles is obtained by uniformly dispersing nanoparticles in water. Further, the dispersion liquid contains nanoparticles with a concentration (weight/weight) of 0.1%-50%, preferably 0.5%-20%, more preferably 1%-10%.

Embodiment 9. The method according to embodiment 7, wherein in step (1), the aqueous dispersion of nanoparticles and the oil phase or W/O type emulsion as the internal phase substance are combined in a ratio of 0.1:9.9 to 9.9:0.1 , It is preferable to mix with a water-oil volume ratio of 9:1-7:3; in addition, the stable Pickering emulsion is to make the oil phase or W/O emulsion droplets uniformly dispersed into the nanoparticles under the condition of high energy provided by the outside Obtained in the aqueous dispersion. The high-energy dispersion method includes, but is not limited to, one or any combination of methods such as ultrasonic probe crushing, ultrasonic water bath shaking, high-speed homogenization, high-pressure homogenization, magnetic stirrer stirring, vortexing, etc., in order to obtain a stable Emulsion, among them, the so-called stable emulsion refers to the phenomenon of no oil-water separation after being left for more than 24 hours in order to complete subsequent operations.

Embodiment 10. The method according to embodiment 7, wherein in steps (2) and (6), the silk fibroin concentration of the silk fibroin aqueous solution is each independently 0.05 wt% to 45 wt%, preferably 0.5 wt%-35wt%, more preferably 1wt%-30wt%; in addition, preferably, in step (2), the Pickering emulsion and the silk fibroin aqueous solution are in a ratio of 50:1 to 1:50, preferably 10:1 to 1: 10. It is more preferable to mix at a volume ratio of 5:1 to 1:5.

Embodiment 11. The method according to embodiment 7, wherein in step (3), the incubation time is 0.5-120 hours, preferably 2-48 hours, more preferably 6-24 hours, and the incubation temperature is selected from 1°C-100 ℃, preferably 4℃-60℃.

Embodiment 12. The method according to embodiment 7, wherein in step (4), the chemical and/or physical treatment is a treatment method that can induce the silk fibroin structure to shift to a stable state, such as physical cross-linking ( For example, polyethylene glycol treatment, alcohol solvent treatment such as methanol treatment, salt ion treatment such as sodium chloride treatment, pH treatment, heat treatment, ultrasonic treatment, steam treatment, spray drying treatment, cycle freeze-thaw and any combination thereof) and One or any combination of chemical treatment (horseradish peroxidase-hydrogen peroxide (HRP-H ₂ O ₂ ), genipin).

Embodiment 13: The method according to Embodiment 12, wherein the polyethylene glycol treatment is performed with an aqueous solution of polyethylene glycol; preferably, the molecular weight of the polyethylene glycol is 200-20000 Daltons, And/or the polyethylene glycol concentration of the aqueous solution is 30% to 100% (weight/volume).

Embodiment 14: The method according to embodiment 12, wherein the methanol treatment is carried out with an aqueous methanol solution; preferably, the methanol concentration of the aqueous solution is 20-90% (volume/volume).

Embodiment 15: The method according to Embodiment 12, wherein the salt ion treatment is performed using an aqueous solution of salt ions; preferably, the salt ion concentration of the aqueous solution is 0.5 M to a saturated concentration.

Embodiment 16: The method according to embodiment 12, wherein in the pH treatment, the pH value is 1-14, preferably 1-5. Preferably, the pH value is adjusted with hydrochloric acid (for example, 1M hydrochloric acid) and sodium hydroxide (for example, 1M sodium hydroxide).

Embodiment 17. The method according to embodiment 7, wherein the internal phase substance in step (1) may contain an active substance; in particular, when the active substance is hydrophobic, the active substance is dissolved in and/or solid The form of particles is suspended in the oil phase as the internal phase material; when the active material is hydrophilic, the active material is suspended in the form of solid particles in the oil phase as the internal phase material, or dissolved in and/or The form of supersaturated solid particles is suspended in the water phase of the W/O type emulsion as the internal phase material.

The microcapsule of the present invention is a new type of microcapsule, which includes: (1) a core in the form of a solid, liquid or gas, (2) a single-layer composite shell with a thickness of 50nm-1000nm containing silk fibroin and nanoparticles, And (3) optionally one or more additional shells enclosing the single-layer composite shell, the one or more additional shells each independently comprising silk fibroin and optional nanoparticles. The present invention uses nano particles to stabilize and strengthen silk fibroin molecules, and prepares core-shell microcapsules with good biocompatibility, stable core-shell structure and controllable active substance release rate through physical cross-linking without chemical cross-linking. . The whole preparation process only needs to mix the nanoparticle emulsified Pickering emulsion with silk fibroin. After incubation, the silk fibroin molecules are fused with the nanoparticles bound at the oil-water interface, and then the silk fibroin structure is induced by chemical or physical methods. , Forming a stable, rigid or flexible, water-insoluble shell. The embedded substances in the microcapsules can be slowly released through the shell, or can be released by external force squeezing and crushing or changing pH or high temperature damage and other inducing factors. This technology can be used as active ingredients in hair care lotions and other beauty and skin care products The embedding, stabilization and release of The invention can also be applied to many fields such as biomedicine, pharmaceutical preparations, food, beauty and skin care, textiles and so on.

In the above preparation process, the nanoparticles as emulsifiers adsorb and accumulate on the oil-water interface in the initial stage of preparation, reducing the oil-water interface energy, through the stronger repulsion between the particles, greater steric hindrance and greater interface viscosity Plays the role of stabilizing oil droplets and makes the formed oil droplets irreversible in water. This kind of emulsion is called "Pickering emulsion". The preparation process of Pickering emulsion is convenient, and the oil droplet size is uniform and stable. However, the nanoparticle layer attached to the surface of the oil droplets in Pickering emulsion has high permeability and cannot be used as a carrier to control the release of active substances; and the mechanical properties are poor and cannot be dried into a solid powder. When the amphiphilic silk fibroin solution is mixed into the Pickering emulsion, the silk fibroin combines with the nanoparticles adsorbed and accumulated on the surface of the oil droplets to form a stable shell. In the subsequent preparation By changing the chemical or physical conditions of the solution (mixing methanol or polyethylene glycol, changing the pH value of the solution, applying shear force, etc.), the silk fibroin structure in the shell can be induced to change to a stable state, further stabilizing the core-shell structure, A composite shell with excellent mechanical properties and stability is formed, and then dried such as freeze-drying to obtain powdery microcapsules. Therefore, the nanoparticles not only stabilize the oil droplets in this process, but also induce the aggregation and self-assembly of silk fibroin at the oil-water interface, adjust the shell structure, and enhance the mechanical properties of the shell, which are not traditionally applied chemical surfactants. Replacement. The size of the microcapsule and the thickness and mechanical properties of the shell can be changed by changing the preparation conditions (such as emulsification temperature, rotation speed, silk fibroin and nanoparticle concentration, silk fibroin and nanoparticle ratio, mixed solution incubation time, adding polyethylene glycol The molecular weight, concentration, etc.) can be adjusted, and the multilayer shell can be prepared by repeating the above steps. The hydrophilic or hydrophobic active material can be added to the internal phase material before emulsification and then loaded into the core. The active substance can be distributed in the core in the form of single molecules (dissolved) or solid particles (suspended).

The present invention has the following beneficial technical effects:

1. The source of raw materials is green, the whole preparation process is simple and environmentally friendly, without any chemical crosslinking agent, with good repeatability, and large-scale production is possible.

2. The obtained microcapsules are stable in size, the thickness and permeability of the shell are controllable, and the core-shell structure is stable, which can ensure the stability and reliability of product quality.

3. The microcapsules of the present invention can efficiently load different types of active substances, increase the active substance loading and slow and controlled release effects, and can be used for slow or controlled release of active substances, targeted delivery, slow or controlled release of fragrances, in vitro Detection and other fields.

4. The microcapsules of the present invention can be used alone (as a drug suspension) or mixed with other biological materials such as gels and porous scaffolds to improve the application effect in the fields of tissue repair and tissue engineering.

5. The oil phase that can be used in the present invention can be all oily substances insoluble in water, especially volatile oily substances such as fragrances and essential oils; due to the simple and efficient preparation process, solids containing volatile oily substances can be prepared on a large scale Microcapsule powder is convenient for storage, transportation and use of such substances.

To illustrate the present invention, the following definitions are used, and when appropriate, terms used as the singular number also include the plural form, and vice versa. Moreover, unless otherwise indicated, the terms and operations used herein have meanings commonly understood by those skilled in the relevant art or can be routinely determined by those skilled in the art.

The "silk fibroin" (SF) of the present invention is a natural high molecular weight fibrin extracted from silk, which consists of a heavy chain (molecular weight of about 390kDa) and a light chain (molecular weight of about 26kDa) and glycoprotein P25 (molecular weight of about 25kDa). ) Composition, the ratio of heavy chain: light chain: glycoprotein P25 is 6:6:1 (based on molecular weight). The heavy chain includes a crystalline region and an amorphous region (amorphous region), and consists of 5263 amino acid residues. The light chain contains 3 cysteine residues, two of which form intramolecular disulfide bonds, and the third is located in the more hydrophilic region at the end of the chain and forms an interchain with the 5244th amino acid residue in the heavy chain Disulfide bond. Glycoprotein P25 controls the β-folding of silk fibroin through hydrophobic interaction and hydrogen bonding to form a more regular structure. At the same time, P25 is incorporated into the complex structure of heavy and light chains connected by disulfide bonds by non-covalent interaction to stabilize light and heavy Chain complex. Silk fibroin has highly repetitive hydrophobic and hydrophilic regions. The hydrophobic region contains short side chain amino acids such as glycine, alanine and serine. The hydrophilic region contains a large number of side chain amino acids and charged amino acids. The affinity of silk fibroin Water is conducive to the stability of the emulsion.

The silk fibroin of the present invention can be purified by any method. Preferably, the silk fibroin of the present invention is obtained by the following purification method: degumming raw silk to obtain cooked silk, dissolving the cooked silk, and then performing dialysis and centrifugation. More preferably, the silk fibroin of the present invention is obtained by the following purification method: raw silk is degummed with boiling sodium carbonate aqueous solution to obtain cooked silk, the cooked silk is dissolved in a solvent, and then dialysis and centrifugation are performed. The solvent used to dissolve the cooked silk is preferably a lithium bromide aqueous solution, a calcium chloride-ethanol-water ternary mixture, a formic acid/calcium chloride mixture, and the like. More preferably, the molar concentration of lithium bromide is 9.3Mol/L, and the bath ratio for dissolving cooked silk is 1g cooked silk corresponding to 4ml lithium bromide aqueous solution; calcium chloride-ethanol-water in the calcium chloride-ethanol-water ternary mixture The molar ratio is 1:2:8, and the bath ratio of dissolved cooked silk is 1g cooked silk corresponding to 10ml ternary mixture; formic acid and calcium chloride are prepared at a weight ratio of 22:1, and the dissolved cooked silk bath ratio is 1g cooked silk Corresponding to 11.5g of formic acid/calcium chloride mixture. Alternatively, the silk fibroin of the present invention can be purchased, for example, in the form of freeze-dried powder. Further, the silk fibroin of the present invention may have a molecular weight range of 5KDa to 500KDa, preferably 10KDa to 400KDa.

The "nanoparticle" described herein is any nanoparticle capable of forming a Pickering emulsion, including inorganic or organic nanoparticles, which may be hydrophilic or lipophilic. Inorganic nanoparticles include, but are not limited to, silica nanoparticles (such as ordinary silica nanoparticles and fumed silica nanoparticles, such as ordinary silica with a particle size of 7-500nm, a particle size of 7-500nm and a specific surface area of 50-400m ² / g hydrophilic fumed silica with a specific surface area of 50-400m ² / g hydrophobic fumed silica, the particle size of 7-20nm amino silica), zinc oxide nanoparticles ( For example, zinc oxide with a particle size of 20-500nm), titanium dioxide nanoparticles (for example, titanium dioxide with a particle size of 20-500nm), hydroxyapatite nanoparticles (for example, hydroxyapatite with a particle size of 50-500nm), nano silver (for example, particle size nanosilver is 25-100nm), gold nanoparticles (e.g., gold nanoparticles of a particle size of 1-100nm), Fe ₃ O ₄ magnetic nanoparticles (e.g., particle size of 5-100nm of Fe ₃ O ₄ magnetic nanoparticles), lipid nano Body and any combination of them. Organic nanoparticles, for example, include but are not limited to 50-500nm polylysine (PLL), polyethylene, polyvinyl chloride, polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polylactic acid ( PLA), silk fibroin nanoparticles, collagen, chitosan, starch, cellulose and any combination thereof.

As used herein, "random distribution" means that nanoparticles are randomly distributed on the inner, inner, and outer surfaces of a shell, such as a single-layer composite shell or the other shell.

The "internal phase substance" as used herein refers to a substance that can serve as the internal phase of Pickering emulsion, which can be in the form of an oil phase or a W/O type emulsion. According to the present invention, when the internal phase material is an oil phase, an oil-in-water (O/W) type Pickering emulsion is obtained; when the internal phase material is a W/O type emulsion, a water-in-oil-in-water (W/W) emulsion is obtained. O/W) Pickering emulsion. In one embodiment, the W/O type emulsion as the internal phase substance is a W/O type emulsion obtained by emulsifying a water phase and an oil phase. In another embodiment, the W/O type emulsion as the internal phase substance is a W/O type Pickering emulsion obtained by emulsifying an aqueous dispersion of nanoparticles with an oil phase.

The "oil phase" that can be used in the present invention, including the "oil phase" in the W/O emulsion droplets, can be any water-immiscible oil phase known to those skilled in the art, including volatile oils and non-volatile oils. Oils, non-oily fluids that are immiscible with water, and/or stearin solids. For example, the volatile oil is selected from linalyl acetate, geraniol, aceto-ionone, citral, ethyl acetate, myrcene, diphenylethanol, volatile silicone oil, essential oils and any combination thereof, so The non-volatile oil is selected from soybean oil, corn oil, camellia oil, argan oil, sweet almond oil, apricot kernel oil, wheat germ oil, jojoba oil, grape seed oil, avocado oil, macadamia oil, olive Oil, castor oil, fish oil and any combination thereof; said non-oily fluid immiscible with water is selected from solvents such as n-hexane, dodecane, n-hexanol and butyl butyrate and any combination thereof; said hard The lipid solids are selected from paraffin, cetyl stearin, shea butter, and any combination thereof.

The "solidification of the composite external phase" as used herein refers to the conversion of the silk fibroin structure from a random coiled structure in solution to a metastable α-helical structure or a more stable β-sheet structure by chemical and/or physical treatment. The chemical and/or physical treatments include, but are not limited to, polyethylene glycol treatment, alcohol solvent treatment (such as methanol treatment), salt ion treatment (such as sodium chloride treatment), pH value treatment, heat treatment, ultrasonic treatment, and water treatment. Steam treatment, spray drying treatment, horseradish peroxidase-hydrogen peroxide (HRP-H ₂ O ₂ ), genipin, cycle freeze-thaw and any combination thereof.

As used herein, "particle size" refers to the diameter of particles or droplets of the substance involved.

The inner core of the microcapsules of the present invention may be completely filled (that is, filled) or partially filled with oil phase or W/O emulsion droplets, or in the form of gas. The inner core contains non-volatile oil, volatile oil, and/or non-oily fluid or stearic solids that are immiscible with water. Moreover, the inner core may also contain any required substances, such as active substances. For example, according to specific applications, the core may also include: (1) Food field: additives, vitamins and ingredients, etc.; (2) Medical field: medicines, vaccines (such as bacteria, viruses and parasites), etc.; (3) Beauty Cosmetic field: active substances used in cosmetics and skin care, such as plant extracts, pigments, vitamins and antioxidants; (4) Textile field: heat-sensitive materials, dyes and fabric finishing auxiliaries.

In one embodiment, in the preparation method of the present invention, the active substance is present in the internal phase substance. In particular, when the active material is hydrophobic, the active material is dissolved or suspended in the form of solid particles in the oil phase as the internal phase material; when the active material is hydrophilic, the active material is solid particles It is suspended in the oil phase as the internal phase material, or dissolved and/or suspended in the water phase of the W/O emulsion as the internal phase material in the form of supersaturated solid particles.

In one embodiment, in the preparation method of the present invention, steps (3) to (5) can be repeated one or more times to obtain microcapsules with a multilayer shell. As such, the shell thickness of the microcapsules can be adjusted according to specific requirements. Moreover, the microcapsules of the present invention have good mechanical properties, a round shape, and still have a uniform shell thickness after multiple assembly.

"Wt%" as used herein means weight percentage. "%" as used herein also means percentage by weight, unless otherwise stated.

Description of the drawings

Figure 1 shows the hydrophilic fumed silica (SiO ₂ -S200) dispersion and corn with different concentrations (sequentially 1%, 1.5%, 2%, 2.5%, 3%, 5%) in Example ₂ The size and morphology of Pickering emulsion prepared from oil.

Figure 2 shows the dry powder photo and morphology of the microcapsule powder prepared with 3% SiO ₂ -S200 and corn oil in Example 2. The left image is the photo of the microcapsule powder obtained after freeze drying, the middle image and The picture on the right is the electron micrograph of the microcapsule powder obtained after freeze-drying under different scales.

Figure 3 shows the infrared spectrum of the microcapsule powder prepared with 3% SiO ₂ -S200 and corn oil in Example 2.

Figure 4 shows the percentage of corn oil in the total weight of the microcapsules in the microcapsules prepared with 3% and 1% SiO ₂ -S200 and corn oil respectively in Example 2.

Figure 5 shows the Pickering emulsion prepared with 3% SiO ₂ -S200 and corn oil in Example 2 (the two pictures on the left) and the core-shell microcapsules obtained (the two pictures on the right) with rhodamine Brightfield and fluorescence photos after B staining.

Fig. 6 shows the size and morphology of Pickering emulsion prepared with different internal phase substances observed by using an inverted fluorescence microscope in Example 3.

Fig. 7 shows the morphology of the emulsion before and after adding silk fibroin as observed with an inverted fluorescence microscope in Example 3.

Figure 8 shows the outer surface, cross-section and inner surface topography of the shell of the core-shell microcapsules prepared with 3% SiO ₂ -S200 and linalyl acetate in Example 3, wherein the top three figures correspond to the bottom three An enlarged view of a partial area of the image.

Fig. 9 shows the microscope pictures and centrifuge tube pictures of the

control

1 and 2 and the microcapsules obtained by different treatment methods in Example 4.

Figure 10 shows the SiO ₂ -S200 microcapsules and SiO ₂ -S400 nanoparticles prepared as shell thickness and a cross-sectional view of the embodiment 5 with SiO ₂ -d50,.

FIG. 11 shows a microscope photograph of microcapsules prepared with nano-zinc oxide and hydrophilic nano-titanium dioxide as nanoparticles in Example 5.

Figure 12 shows a statistical diagram of the shell thickness of silk fibroin microcapsules prepared by Chunhong Ye et al. using the LBL method and a cross-linking agent.

Figure 13 shows a schematic diagram of the mechanism and a statistical diagram of shell thickness of silk fibroin microcapsules prepared by Shchepelina O et al. using the LBL method.

Figure 14 shows the micrographs of the Pickering emulsion prepared in Example 6 under different conditions.

Figure 15 shows the microscope photographs of emulsions with complex external phases obtained with silk fibroin solutions of different concentrations and the electrons of microcapsule powders prepared from 7.5wt% silk fibroin solutions observed with an inverted fluorescence microscope in Example 7 Microscope photo illustration.

16 is a graph showing the in vitro cumulative release curve of curcumin drugs of the microcapsules and emulsion obtained in Example 8.

Fig. 17 is a microscopic photograph of the W ₁ /O/W ₂ double emulsion in which the hydrophilic drug is embedded in Example 10.

Figure 18 shows the in vitro drug release rate graphs of olanzapine-loaded microcapsules prepared as in Example 9 and Example 11, respectively.

Figure 19 shows the morphology of the microcapsules containing olanzapine prepared in Example 9 and Example 11 after being released in vitro for 27 days.

Figure 20 shows the morphology and the drug dissolution rate of the olanzapine-loaded microcapsules in Example 12 and the control microcapsules before and after the drug dissolution in 0.1M hydrochloric acid solution.

Figure 21 shows the microcapsules (silk fibroin / SiO ₂ -S200 / corn oil, left panel) prepared in Example 2 and the microcapsules prepared in Example 13 (SF / Tween 80 / corn oil, right panel) embodiment Electron microscopy photos.

Figure 22 shows the morphology of the microcapsules prepared by spray drying in Example 14.

Figure 23 shows the mechanical modulus diagram of the microcapsules prepared by spray drying in Example 14

Example

The following examples are used to further illustrate the present invention, but do not limit the scope of the present invention.

In the embodiment of the present invention, the instruments and parameters used are as follows:

-Ultrasonic cell crusher (JY92-IIDN, Ningbo Xinzhi Biological Technology Co., Ltd.), ultrasonic parameters are horn Φ6 and power 30%; used to prepare nanoparticle dispersion;

-Homogenizer (model T18, Aika (Guangzhou) Instrument Equipment Co., Ltd.), used for emulsification;

-Centrifuge (Avanti J-26SXP, Beckman Coulter, Inc. United States);

-Vacuum freeze dryer (CHRISR ALPHA 2-4 LSC plus, Germany, Martin Christ);

-Inverted fluorescence microscope (Axio Vert A1, Zeiss, Germany);

-Scanning electron microscope (s4800, Hitachi, Japan);

-Fourier transform infrared spectrometer (Nicolet 5700, Nicolet, USA);

-Microplate reader (Model Synergy H1, BioTeK, USA); and

-Atomic force microscope (Model Dimension Icon, Bruker, Germany).

Example 1: Purification of silk fibroin

(1) Degumming: Weigh 30g of raw silk (Zhejiang Shengzhou Xiehe Silk Co., Ltd., 6A grade) into a boiling 12L 0.02M sodium carbonate aqueous solution, boil for 30 minutes, stirring every 5 minutes during this period, and then put the degummed silk Wash with deionized water five times to remove the sericin and salt ions remaining on the surface of the silk, then dry it in an oven at 60°C to a constant weight, and seal the obtained cooked silk for use.

(2) Dissolving the silk: immerse the cooked silk obtained in step (1) in a 9.3M lithium bromide aqueous solution at a ratio of 1g:4ml, and place it in an oven at 60°C for 4 hours, during which time the solution is stirred every 1 hour, and it will be obtained after 4 hours Silk fibroin/lithium bromide mixture.

(3) Dialysis and centrifugation: the mixed solution obtained in step (2) is cooled to room temperature, transferred to a 3.5KDa dialysis bag, and dialyzed in deionized water at room temperature for 72 hours. The resulting dialysate (ie, silk fibroin solution) was then centrifuged (9000 r/min, 4°C) twice for 20 minutes each to remove other undissolved substances. Place the centrifuged silk fibroin solution in a refrigerator at 4°C for later use.

Determination of the concentration of silk fibroin solution: Measure 1ml of the silk fibroin solution after centrifugation, weigh and record the weight value. Dry it in an oven at 60°C to a constant weight. The percentage of the weight after drying to the weight before drying is the concentration (weight percentage) of the silk fibroin solution. After measurement, the concentration of the above-mentioned silk fibroin solution is 7wt%. Generally, the concentration of silk fibroin obtained by the above method is in the range of 6-10 wt%.

If a concentration greater than 10 wt% is required, the silk fibroin solution can be concentrated in the PEG aqueous solution. Specifically, transfer the centrifuged silk fibroin solution to a dialysis bag with a molecular weight cut-off of 3.5KDa, and perform dialysis in a 15% (weight/volume) PEG10K (polyethylene glycol with a molecular weight of 10,000) aqueous solution at room temperature. . For example, starting from a 7wt% silk fibroin solution, after about 12 hours of dialysis as above, the silk fibroin concentration is about 12wt%; after 24 hours of dialysis, the silk fibroin concentration is about 25wt%. Alternatively, the commercially available lyophilized silk fibroin powder (Suzhou Simeite Biotechnology Co., Ltd., 0.3 g/bottle) is dissolved in water to a desired high concentration, for example, 15 wt%.

Example 2: Preparation of core-shell microcapsules using silk fibroin and nanoparticles

Accurately weigh the hydrophilic fumed silica powder (particle size: 7-40nm, specific surface area: 200m ² /g, brand: Maclean, purchased from Suzhou Industrial Park Bomeida Reagent Instrument Co., Ltd.; denoted as SiO ₂ -S200) , Use deionized water to prepare nanoparticle dispersions of different concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 5%, 7%, weight/volume, ultrasonic dispersion time 60s) .

The obtained nanoparticle dispersion was mixed with corn oil (medical grade, Aladdin, Shanghai Aladdin Biochemical Technology Co., Ltd.) as the internal phase in a volume ratio of 7:3, and emulsified with a homogenizer at 16,000 rpm at room temperature for 1 minute. After standing for 2 hours, the Pickering emulsion in the upper layer and the excess nanoparticle dispersion liquid in the lower layer were obtained, and the height ratio of the two layers became a constant value. At this time, an inverted fluorescence microscope was used to observe the size and morphology of the upper layer of Pickering emulsion (Figure 1).

Then, the upper Pickering emulsion was mixed with an equal volume of 15wt% silk fibroin solution (as described in Example 1, obtained by dissolving commercially available silk fibroin lyophilized powder with water) and incubated at room temperature for 12 hours to obtain A composite external phase emulsion composed of nanoparticles and silk fibroin.

Then, PEG treatment is performed. Specifically, a 50% (weight/volume) PEG10K aqueous solution and the aforementioned emulsion with a composite external phase are mixed in equal volume and left for 12 hours. After centrifugation (12,000 rpm, 5 minutes), the mixture was divided into three layers. Suspend the top microcapsule layer in pure water (the volume of pure water is equal to the volume of silk fibroin solution + Pickering emulsion + PEG10K aqueous solution, that is, the volume before centrifugation) and centrifuge (12,000 rpm, 5 minutes), and take the upper layer The process of suspending and centrifuging the microcapsule layer with an equal volume of pure water was repeated twice. Then take the upper microcapsule layer and suspend it in pure water, pre-freeze it with liquid nitrogen or in a refrigerator at -80℃, and then vacuum it with a vacuum freeze dryer until the moisture is completely removed (vacuum degree: 0.01mbar, vacuum time: 48 hours), The microcapsule powder was obtained. All the above concentrations of hydrophilic fumed silica successfully obtained microcapsule powder. As the concentration of nanoparticles increases, the microcapsules become more complete, and the yield of microcapsules with complete shapes increases.

The microcapsule powder obtained by using 3% SiO ₂ -S200 was suspended in water, dropped onto the silicon wafer and dried, and sprayed with gold for 90 seconds. Observe the microcapsule's surface microstructure with a scanning electron microscope (Figure 2); use a Fourier transform infrared spectrometer to test the secondary structure of the microcapsule (Figure 3).

Figure 1 shows the results observed with an inverted fluorescence microscope using 1%, 1.5%, 2%, 2.5%, 3%, 5% SiO ₂ -S200 as the nanoparticle dispersion and corn oil as the oil phase as described above. The size and topography of the Pickering emulsion. It can be seen that all concentrations of hydrophilic fumed silica form Pickering emulsion. As the concentration of hydrophilic fumed silica increases, the size of Pickering emulsion becomes more uniform and the shape is regular round. shape. The nanoparticles attached to the surface of the oil droplets of Pickering emulsion achieve better stability through the stronger repulsive force between the particles, greater steric hindrance, or higher interfacial viscosity. However, the permeability of the nanoparticle layer attached to the surface of the oil droplets in Pickering emulsion is too high to be used as a carrier to control drug release, and its mechanical properties are poor, and it cannot be freeze-dried into a powder. By incubating with silk fibroin and performing physical or chemical treatment, microcapsules that can be freeze-dried are obtained.

Fig. 2 is a dry powder photograph and morphology diagram of microcapsule powder prepared with 3% hydrophilic fumed silica and corn oil. It can be seen from the figure on the left that the freeze-dried microcapsules are in powder form. The middle and right images are electron micrographs of the freeze-dried microcapsules at different magnifications, showing that a shell with excellent mechanical properties is formed, and the oil is successfully enclosed in the microcapsules.

Figure 3 is the infrared spectrum of the microcapsule powder prepared with 3% hydrophilic fumed silica and corn oil. The characteristic peaks of silicon dioxide and corn oil were observed from the infrared spectrum of the microcapsules, which proved that the microcapsules contained corn oil and silicon dioxide.

Determination of the content of corn oil in the microcapsules: The corn oil in the microcapsules was extracted with n-hexane (the corn oil was dissolved in n-hexane), and the percentage of corn oil in the microcapsules was calculated by the standard curve. Specifically, 2 mg of the microcapsule powder was accurately weighed, suspended in 2 ml of n-hexane, and the shell was destroyed by violent external forces such as vortexing ultrasound, so that the corn oil was completely dissolved in the n-hexane. Centrifuge (12,000 rpm, 10 minutes, room temperature), measure the UV absorption of the supernatant at 229 nm, calculate the oil content from the standard curve, and obtain the proportion of corn oil in the microcapsules. Fig. 4 is the percentage of the weight of corn oil in the microcapsule powder prepared with 3% and 1% hydrophilic fumed silica, respectively, to the total weight of the microcapsule. It can be seen from Figure 4 that the loading of corn oil can reach 75% by weight, which has a positive effect on the packaging of drugs. For the same weight of microcapsules, the loading of corn oil for microcapsules prepared with 3% hydrophilic fumed silica is lower than the loading of corn oil for microcapsules prepared with 1% hydrophilic fumed silica. The shell of the microcapsule prepared with 3% silica under the same weight is thicker, and the shell accounts for a larger proportion.

The distribution of silk fibroin in the microcapsule shell can be qualitatively judged by the fluorescent labeling of rhodamine B (molecular weight 479.01Da, purchased from Invitrogen). Rhodamine B is commonly used to label silk fibroin. Figure 5 is the Pickering emulsion prepared with 3% hydrophilic fumed silica and corn oil in this example (the two pictures on the left) and the core-shell microcapsules obtained (two pictures on the right) Brightfield and fluorescence photos of rhodamine B staining. The picture on the left is the control, that is, without silk fibroin treatment, the result is no fluorescence. It can be observed from the figure on the right that silk fibroin is evenly distributed in the outer shell of the microcapsule, forming a stable core-shell structure of the microcapsule.

Example 3: Preparation of microcapsules containing different internal phases

The microcapsule powder was prepared according to the method of Example 2, except that a 3% SiO ₂ -S200 dispersion (prepared as described in Example 2, the same below) was used, and one of the following substances was used as the internal phase: Corn oil (medical grade, Aladdin, Shanghai Aladdin Biochemical Technology Co., Ltd.); Linalyl Acetate (Shanghai Aladdin Biochemical Technology Co., Ltd.); Geraniol (Shanghai Aladdin Biochemical Technology Co., Ltd.); Myrcene ( Shanghai Aladdin Biochemical Technology Co., Ltd.); beta-ionone (Shanghai Aladdin Biochemical Technology Co., Ltd.); volatile silicone oil (skincare grade, Guangzhou Yuxuan Biotechnology Co., Ltd.).

Fig. 6 is a diagram of the size and morphology of Pickering emulsion prepared with different internal phase substances observed with an inverted fluorescence microscope. It can be seen that from the size of Pickering emulsion, the emulsion prepared with corn oil and linalyl acetate> the emulsion prepared with volatile silicone oil> the emulsion prepared with geraniol> the emulsion prepared with myrcene> the emulsion prepared with ethyl acetate Emulsion prepared by Ionone. Different oil phases can form Pickering emulsion, and then silk fibroin can be added for incubation, so that silk fibroin molecules are randomly distributed near the nanoparticles at the oil-water interface.

Figure 7 is the morphology of the emulsion before and after addition of silk fibroin observed under bright field using an inverted fluorescence microscope. The two images above are picograms obtained by using myrcene and ethylionone as the internal phases, respectively. The topography of Lin's emulsion. The following two pictures are the topography of the corresponding emulsion after adding silk fibroin. The comparison showed that the size of the emulsion did not change significantly after the silk fibroin was mixed into the Pickering emulsion. The emulsion maintained its original structure and stability in the presence of silk fibroin, which provided a basis for further processing.

Figure 8 shows the electron micrographs (obtained by scanning electron microscope) of the outer surface, cross section and inner surface of the shell of the microcapsule prepared with linalyl acetate as the internal phase. The upper three pictures correspond to the parts of the lower three pictures respectively. Area zoomed in. It can be seen that the shell of the microcapsule is a complex of silk fibroin and silica. Silica is randomly distributed on the outer surface, cross-section and inner surface of the shell, and forms the shell of the microcapsule together with silk fibroin, which is one of the contributions of the present invention. It is also found from the figure that the microcapsules are hollow. This is because the linalyl acetate used is volatile, which is sufficient to show that the hollow microcapsules can be successfully prepared by using volatile oil.

Example 4: Preparation of microcapsules using different processing methods

Using 3% SiO ₂ -S200 dispersion and corn oil, Pickering emulsion was prepared according to the method of Example 2 (control 1, compared with the experimental group with subsequent addition of silk fibroin).

Then, the above-mentioned Pickering emulsion was mixed with an equal volume of 15wt% silk fibroin solution and incubated at room temperature for 12 hours to obtain an emulsion with a composite external phase composed of nanoparticles and silk fibroin, and centrifuged (12,000 rpm, 5 minutes, After room temperature), take the upper emulsion and observe with an inverted fluorescence microscope (Control 2).

Then, the following different treatment methods (where the centrifugal operation and freeze-drying operation are the same as in Example 2):

Method (1). Polyethylene glycol treatment: mix 50% (weight/volume) PEG10K aqueous solution with the emulsion with the composite external phase in equal volume and leave it for 12 hours; after centrifugation, the mixture is divided into three layers, and the uppermost microcapsule layer Suspend with pure water (the volume of pure water is equal to the volume before centrifugation) and centrifuge, take the upper microcapsule layer and repeat the process of adding pure water to suspend and centrifuge twice; then take the upper microcapsule layer to suspend in pure water and freeze-dry;

Method (2). Methanol treatment: mix 50% (volume ratio) methanol aqueous solution with the same volume of the emulsion with the composite external phase, leave it for 10 minutes, centrifuge, take the bottom sediment and repeat the process of adding equal volume of pure water to suspend and centrifuge twice, then Suspend the bottom sediment in pure water and freeze-dry;

Method (3). Heat treatment: Place the emulsion with a composite external phase in an oven at 60°C for 12 hours and then centrifuge, take the bottom sediment and repeat the same volume of pure water suspension and centrifugation process twice, then take the bottom sediment and suspend it in pure water After freeze-drying;

Method (4). Salt ion treatment: Mix the 1M sodium chloride aqueous solution with the emulsion with the composite shell in equal volume, leave it for 24 hours and centrifuge, take the bottom sediment and repeat the process of adding equal volume of pure water to suspend and centrifuge twice, then take the bottom The precipitation is suspended in pure water and then freeze-dried;

Method (5). pH treatment: adjust the pH of purified water to 1.5 with 1M hydrochloric acid, mix the pH 1.5 solution with the emulsion with a composite external phase in equal volume, leave it for 12 hours, centrifuge, take the bottom sediment and add an equal volume of pure Water suspension and centrifugation process twice, and then take the bottom sediment to suspend in pure water and freeze-dry.

Method (6). Steam treatment: first spray-dry the emulsion to obtain microcapsule powder. The spray-drying parameters are: feed volume 60 ml/min, inlet air temperature 180°C, outlet temperature 80°C; then the obtained The microcapsule powder is placed in an environment with a temperature of 60 degrees Celsius and a humidity of 95% for 24 hours, and then air-dried at room temperature.

Under the influence of external factors (low pH, elevated temperature, salt ions, polymers, organic solvent induction, water vapor, etc.), the structure of silk fibroin changes from a metastable structure to a stable structure. Regulation of capsule preparation process and material properties. Based on the controllability of silk fibroin, shells with different stability and morphology can be obtained by different processing methods.

Figure 9 shows the microscopic photos and centrifuge tube photos of

Controls

1 and 2 and the microcapsules obtained by the above-mentioned different processing methods. Fig. 9A is a microscope photograph of control 1 (Pickering emulsion), showing that the emulsion is relatively uniform in size and round in shape. After mixing silk fibroin and incubating for 12 hours after centrifugation (Figure 9B, control 2), the emulsion is still round, and mixing silk fibroin will not damage the integrity of the emulsion. After treatment by different methods (Figures 9C-9F), the surface of the microcapsules became less rounded compared with the control (Figures 9A and 9B), indicating that the silk fibroin did constitute the shell of the microcapsules and had been stabilized. The bottom left part of Figure 9 shows the Pickering emulsion and its centrifuge tube photos after centrifugation, after adding pure water and centrifuging, and after adding silk fibroin and PEG10K treatment and centrifugation. Through the photos of these centrifuge tubes, you can intuitively understand the state of the emulsion before and after centrifugation and processing. The micrographs of A, B, and C in Fig. 9 correspond to the emulsions at A, B, and C marked on the centrifuge tube, respectively. After centrifugation (12000 rpm, 5 min) of the emulsion in the centrifuge tube A (control 1), it can be clearly found that the emulsion has demulsified, that is, oil-water separation, which is a manifestation of the unstable emulsion. It should be noted that centrifugation is an accelerated test to observe whether the emulsion is stable. After adding silk fibroin (SF), centrifuge at the same speed for 5 minutes. It is observed that the emulsion floats on the top layer (centrifuge tube B). There is no oil-water separation in the top layer. Stabilizing effect. The lower layer is a combination of some nanoparticles and silk fibroin. At the same time, as a control for adding silk fibroin, we added pure water to Pickering emulsion (control 1). After centrifugation at the same speed for 5 minutes, the separation of oil and water in the emulsion can be clearly observed. The stabilizing effect is silk fibroin instead of water. After treatment with PEG10K and centrifugation, it is divided into three layers (centrifuge tube C). The top layer is the microcapsule layer, and the bottom is the mixture of silk fibroin, PEG10K and nanoparticles. Take the top layer and repeat the steps of suspending and centrifuging with an equal volume of pure water twice, the obtained microcapsules are suspended in pure water and then freeze-dried to obtain powdered microcapsules.

In summary, the above treatment methods can stabilize the silk fibroin at the boundary of the emulsion, and obtain microcapsules with different mechanical properties and structures. For example, the shell obtained by methanol treatment is harder, the shell obtained by PEG treatment is tougher, the shell obtained by sodium chloride treatment is porous, and the shell structure obtained by temperature and low pH treatment is dense.

Example 5: Preparation of microcapsules with different nanoparticles

The microcapsules were prepared according to Example 2, except that linalyl acetate (Shanghai Aladdin Biochemical Technology Co., Ltd.) was used as the oil phase, and one of the following substances was used as the nanoparticles. Preparation 3 was described in Example 2. % (Weight/volume) nanoparticle dispersion:

(1) Hydrophilic fumed silica SiO ₂ -S200 (particle size: 7-40nm, specific surface area: 200m ² /g, brand: Maclean, purchased from Suzhou Industrial Park Bomeida Reagent Instrument Co., Ltd.);

(2) Hydrophilic fumed silica SiO ₂ -S400 (particle size: 7-40nm, specific surface area: 400m ² /g, brand: Maclean, purchased from Suzhou Industrial Park Bomeida Reagent Instrument Co., Ltd.);

(3) Silica SiO ₂ -d500 (particle size: 500nm, brand: Macleans, purchased from Suzhou Argon Krypton Xenon Trading Co., Ltd.);

(4) Silica SiO ₂ -d50 (particle size: 50nm, brand: Macleans, purchased from Suzhou Great Medical Technology Co., Ltd.);

(5) Hydrophilic nano titanium dioxide, particle size: 40nm, brand: Macleans, purchased from Suzhou Great Medical Technology Co., Ltd.;

(6) Nanometer zinc oxide, particle size: 30±10nm, brand: Macleans, purchased from Suzhou Argon Krypton Xenon Trading Co., Ltd.

FIG 10 is respectively SiO _₂ -d50, SiO ₂ -S200 and SiO ₂ -S400 microcapsules prepared as nanoparticles and shell thickness a cross-sectional view, respectively, FIG. 11 is a nano-zinc oxide nanoparticles prepared using titanium dioxide and a hydrophilic Microscopic photo of the microcapsules. The size of the emulsion with the composite external phase stabilized by zinc oxide particles is larger than the size of the emulsion with the composite external phase stabilized by the titanium dioxide particles, and the shells of the finally obtained microcapsules are relatively hard and easily dispersed in water. Shell thickness microcapsules prepared with SiO ₂ -d50 was 69.19 ± 17.6nm, the shell thickness of the microcapsules prepared with SiO ₂ -S200 is 405.2778 ± 67.1nm, the shell thickness of the microcapsules prepared with SiO ₂ -S400 is 65.14 ±21.09nm. The thickness of the single-layer shell of the microcapsules prepared by the present invention can reach more than 50nm, even up to 405nm, which is significantly higher than the thickness of the single-layer shell obtained by the current mature layer-by-layer self-assembly method (Figure 12 and Figure 13, respectively 32nm and 5nm). In the present invention, after mixing Pickering emulsion and silk fibroin, microcapsules with excellent mechanical properties can be obtained through processing, and shells with different thicknesses can be obtained by selecting different nanoparticles. Taking a 60nm shell as an example, the present invention can be implemented only once assembling, while the layer-by-layer self-assembly method requires repeated assembly 5 times. The simplicity of the present invention is incomparable to the layer-by-layer self-assembly method, which is the second significant advantage of the present invention.

Figure 12 shows the shell thickness statistics of silk fibroin microcapsules prepared by Chunhong Ye et al. (Ye C, Shchepelina O, Calabrese R et al., Biomacromolecules, 2011, 12(12): 4319-25) using the LBL method and a cross-linking agent Figure. In the study of Chunhong Ye et al., it is first necessary to modify the silk fibroin with two amino acids with opposite charges, and then use the negatively charged silica microspheres (4.0±0.2 microns) as a template and use electrostatic bonding. The outer layer is coated with positively charged silk fibroin (silk fibroin itself is negatively charged, and it is positively charged after being modified by polyglutamic acid), and then the negatively charged silk fibroin (or after polylysine) is coated. Acid-modified negatively charged silk fibroin), the cross-linking agent EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) is also needed for stabilization during the layer-by-layer assembly process . When the required number of layers is reached, the microcapsules are soaked in a mixture of hydrofluoric acid/ammonium fluoride overnight, and the silica cores are dissolved and removed after dialysis for 72 hours. This method can obtain microcapsules with a shell thickness of 19±1nm (3 times assembling) to 277±11nm (9 times assembling), and the average thickness of each layer is 6-32nm. The thickness of the monolayer shell of the microcapsule obtained by Chunhong Ye et al. is significantly lower than that of the microcapsule of the present invention. Moreover, from the modification of silk fibroin to multiple assembly coatings, the entire operation steps are cumbersome and complicated, and as the number of assembled layers increases, the uniformity of the shell thickness becomes worse (documents show that when assembled to the fifth layer, about 150nm The permeability of the shell has become uneven when it is thick), and the drug release rate will also be affected. At the same time, the introduction of EDC crosslinking agent also reduces the biocompatibility of the microcapsules.

Figure 13 is a schematic diagram of the mechanism and shell thickness of silk fibroin microcapsules prepared by Shchepelina O et al. (Shchepelina O, Drachuk I, Gupta M K et al., Advanced Materials, 2011, 23(40): 4655-4660) using the LBL method summary graph. Compared with the study of Chunhong Ye et al., the study of Shchepelina O et al. also uses the LBL method and uses silica microspheres as a template. The difference is that after coating silk fibroin, it is treated with methanol, and then the silk fibroin coating is repeated. In the process of layer and methanol treatment, no cross-linking agent is used in the process. Microcapsules with a shell thickness of 10±2nm (5 layers) to 54±11nm (12 layers) were obtained. The average thickness of each layer was about 2-5nm, which was significantly lower than that of the microcapsules prepared by Chunhong Ye et al. It is significantly lower than the shell thickness of the microcapsules prepared by the present invention.

The LBL methods used in Chunhong Ye and Shchepelina O have a common problem, that is, a mixture of hydrofluoric acid and ammonium fluoride must be used to remove silica nuclei. However, hydrofluoric acid and ammonium fluoride are extremely corrosive and difficult to remove cleanly, which brings inconvenience to subsequent operations and mass production. The thickness of the monolayer shell of the microcapsules prepared by the present invention is significantly greater than that of current literature reports, and there is no need to use a crosslinking agent during the preparation process, and there is no need to use a highly corrosive and difficult to remove a clean mixture of hydrofluoric acid and ammonium fluoride This is the third significant advantage of the present invention. The processing methods used in the present invention, such as temperature, pH, methanol and PEG, are safe and reliable methods. Among them, PEG is an FDA-approved material that can be used in the human body, and there will be no residue after methanol is processed and volatilized.

Example 6: Preparation of Pickering emulsion and microcapsules under different conditions

Using 3% SiO ₂ -S200 dispersion and corn oil, and using the following variables, respectively, Pickering emulsion was prepared according to the method of Example 2:

(1) Ultrasonic time of nanoparticle dispersion: 0s, 10s, 30s, 60s;

(2) The emulsification time of nanoparticle dispersion and oil phase: 30s, 1min, 2min, 5min;

(3) The emulsification rate of nanoparticle dispersion and oil phase: 10k, 15k, 20k;

(4) The emulsification temperature of the nanoparticle dispersion and the oil phase: 4°C, 20°C (room temperature), 37°C, 60°C;

(5) The volume ratio of oil phase and nanoparticle dispersion: 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1.

The combination method of test variables is to select one variable from each variable and conduct a full factor test. Figure 14 is a micrograph of Pickering emulsion prepared under different conditions. The first row of Figure 14 shows the 3% SiO ₂ -S200 dispersion and the Pickering emulsion obtained by corn oil emulsification (15,000 rpm, 30 seconds), where the ultrasound time of the nanoparticle dispersion is 0 s, 10 s and 30 s (ultrasound 60 s) See the middle figure in the second row of Figure 14). It can be seen from the figure that when the ultrasound time is 0s, it is found that the shape of part of the emulsion is not a regular circle; as the ultrasound time increases, the shape of the emulsion becomes regular. The second row of Figure 14 is a microscope photo of Pickering emulsion formed at different emulsification temperatures, in which the 3% SiO ₂ -S200 dispersion (ultrasound 60s) and corn oil were placed in a set temperature environment beforehand. After setting the temperature, mix and emulsify. The emulsion obtained by emulsification at low temperature (4°C) has poor stability and is easy to merge. The emulsion prepared at room temperature has a round shape. Some emulsions prepared at 37°C have irregular shapes and some emulsions at 60°C have irregular round shapes. Shape (photo not shown). The third row of Fig. 14 is a microscopic photograph of Pickering emulsion formed at different emulsification rates. As the emulsification rate increases, the morphology of the emulsion becomes more rounded and the stability of the emulsion improves. The emulsification time directly affects the shape and size of the emulsion. With the emulsification time from 30s to 2min, the size of the emulsion becomes regular. This is because the emulsification time increases and the energy obtained by the droplets also increases. The shape of the emulsion will become regular, but the size of the emulsion will become smaller, from 60um± 15 is reduced to 12um±5. There is no significant difference in the size of the emulsion when emulsifying for 2min and 5min, and the size is 2-15um. When the volume ratio of the oil phase to the nanoparticle dispersion is 1:9 to 6:4, the emulsion is an oil-in-water emulsion, and the size of the emulsion increases with the increase of the oil phase, from 3um±2um to 70um±13um . When the volume ratio of the oil phase to the nanoparticle dispersion is 7:3 to 9:1, the emulsion is a water-in-oil emulsion, and some oil-in-water emulsions appear. As the oil phase increases, the oil-in-water emulsion decreases. The size of the emulsion is between 20-100um.

Then, similar to Example 2, incubate with 15wt% silk fibroin solution and perform PEG treatment to obtain microcapsules with different shell thicknesses, and the shell thickness ranges from 50nm to 400nm.

Example 7: Preparation of core-shell microcapsules with different silk fibroin concentrations

Using 3% SiO ₂ -S200 dispersion and linalyl acetate (medical grade, Aladdin, Shanghai Aladdin Biochemical Technology Co., Ltd.), and using different concentrations of silk fibroin solutions (respectively 1wt%, 5wt%, 7.5wt% %, 10% by weight, 12.5% by weight, 15% by weight, and 20% by weight, obtained by dissolving commercially available silk fibroin lyophilized powder with water), and prepared microcapsules according to the method of Example 2. An inverted fluorescence microscope was used to observe the size and morphology of the obtained emulsion with a composite external phase, and a scanning electron microscope was used to observe the surface microscopic morphology of the obtained microcapsule powder.

Figure 15 shows the micrographs of the emulsions with composite external phases obtained by using 5wt%, 7.5wt%, 10wt%, 12.5%, and 15wt% silk fibroin solutions as described above with an inverted fluorescence microscope in Example 7 And the electron micrograph of the microcapsule powder prepared from the 7.5wt% silk fibroin solution. It can be seen that a stable emulsion with a composite external phase can be obtained with different silk fibroin concentrations. The emulsions obtained at the two concentrations of 12.5% and 15% have the best roundness, and the integrity of the capsules is better than that of capsules prepared with low concentrations of silk fibroin. After freeze-drying the emulsion obtained from the 7.5% silk fibroin solution, it can be seen that the microcapsules are basically intact.

Example 8: Preparation of drug-loaded microcapsules embedded with hydrophobic drug curcumin

The hydrophobic drug curcumin was taken as an example to prepare drug-loaded microcapsules. Accurately weigh a certain weight of curcumin powder and suspend it in corn oil, soybean oil, castor oil, mineral oil, silicone oil, dichloromethane, n-hexane, linalyl acetate, etc. as the oil phase. The suspended drug and oil The mass ratio is 1:2. Stir evenly with a magnetic stirrer (200rpm, 10min), so that the powder is fully infiltrated in the oil. Then it was mixed with the 3% SiO ₂ -S200 dispersion in a volume ratio of 3:7, and emulsified at 16,000 rpm for 2 minutes at room temperature. After standing for 2 hours, the drug-containing Pickering emulsion in the upper layer and part of the drug particles and nanoparticle dispersion liquid in the lower layer were obtained. Take the drug-containing Pickering emulsion of the upper layer and the same volume of silk fibroin solution (concentrations of 5wt%, 7.5w%, 10wt% and 15wt% respectively) and mix and incubate at room temperature for 12 hours to obtain a mixture of nanoparticles and silk fibroin An emulsion of a composite external phase composed of protein. Then, use the following two methods to treat separately: (1) mix an equal volume of 50% PEG10K and incubate for 12 hours, centrifuge at 12000 rpm for 5 minutes and divide into three layers, take the top microcapsule layer and wash twice with water; (2) Mix an equal volume of saturated sodium chloride solution and incubate for 12 hours, centrifuge at 12000 rpm for 5 minutes, and divide into two layers. Take the upper microcapsule layer and wash it twice with water. Then, the above-mentioned drug-loaded microcapsules were suspended in a small amount of water.

The 3% SiO ₂ -S200 dispersion was replaced with 1% (weight/volume) Tween 80 aqueous solution and the above process was repeated to prepare microcapsules stabilized with Tween 80 as a control. The emulsion obtained by mixing 3% SiO ₂ -S200 dispersion and 1% Tween 80 aqueous solution with the oil phase respectively was also used as a control. Take the curcumin microcapsules of the present invention, the curcumin microcapsules stabilized with Tween 80, the Pickering emulsion obtained with SiO ₂ -S200 and the emulsion obtained with Tween 80 (0.5 ml each) and 1.5 mg curcumin powder They were added to 1.5ml of anhydrous methanol solution, vortexed for 5 minutes, allowed to stand for 30 minutes, centrifuged (14,000rpm, 5 minutes), and the supernatant was extracted, diluted 200 times with 10mM PBS, and measured with a microplate reader. The UV absorption of the extract at 425 nm was substituted into the concentration-absorbance standard curve (which was made as described below) to obtain the drug content in the microcapsules and emulsion. Pickering emulsion with corn oil as the oil phase as the template, the microcapsules prepared by 15wt% silk fibroin solution and 50% PEG10K treatment accounted for 50.85% by weight of the microcapsules, and Tween 80 as the surface The active agent and the oil phase are the emulsion prepared by corn oil as the template. The drug contained in the microcapsules obtained by the same method accounts for 4.06% by weight of the microcapsules. This also fully highlights one of the advantages of this patent, that is, the ability to obtain Unexpectedly high active material load.

Dilute the drug-containing emulsion and drug-loaded microcapsules with 10 mM PBS buffer (pH 7.4) to a curcumin concentration of 4 mg/ml. In addition, 16 mg curcumin powder was suspended in 4 ml 10mM PBS buffer (pH 7.4). At ambient temperature, take 1ml of the 4mg/ml microcapsules, emulsion and drug solution obtained above and transfer them to a dialysis bag with a molecular weight cut-off of 3.5KDa. After sealing, place them in 20ml release solution (10mM pH 7.4PBS buffer, containing 3% methanol and 0.5% Tween). At regular intervals (2h, 6h, 12h, 24h, 48h, 72h, 120h, 168h, 240h, 360h) 10ml release solution was taken out and 10ml fresh release solution was added to the release system. Measure the absorbance of the taken-out release solution at 425 nm, and substitute it into the concentration-absorbance standard curve (which is made as described below) to calculate the drug concentration. After the release, calculate the cumulative release concentration of the drug and draw the drug release curve (see Figure 16).

Preparation of the standard curve: Dissolve curcumin powder in methanol with a drug concentration of 1 mg/ml. Use the release solution as described above for serial dilution, and the drug concentrations are 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml and 0.03125mg/ml respectively. Then use the microplate reader to measure the absorbance of the above concentration at 425nm, and use excel to draw the standard curve corresponding to the concentration and absorbance. The linear regression equation is calculated as Y=0.012X-0.0012, the degree of fit is R ² =0.9987, and X in the equation Is the absorbance value and Y is the concentration value.

16 is a graph showing the in vitro cumulative release curve of curcumin drugs of microcapsules and Pickering emulsion obtained by using corn oil as the oil phase in Example 8. Figure 16A shows the release curve of the microcapsules obtained with Pickering emulsion or Tween 80 emulsion as a template, treated with 15wt% silk fibroin solution and saturated sodium chloride; it can be seen that the Pickering emulsion is used as the template The drug release rate of the prepared microcapsules is slower than the drug release rate of the microcapsules prepared with Tween 80 emulsion as a template, which further illustrates that the shell of the microcapsules composed of silk fibroin and nanoparticles has a better slow-release effect on drugs. The shell composed of silk fibroin and Tween 80 has an obvious slow-release effect on the drug. Figure 16B shows the release curve of the microcapsules obtained by the 15wt% silk fibroin solution and PEG10K treatment with Pickering emulsion or Tween 80 emulsion as templates, respectively, which shows the same effect as Figure 16A, that is, from silk The microcapsule shell composed of protein and nanoparticles has a significantly better sustained-release effect. Figure 16C shows the release curve of Pickering emulsion and the microcapsules obtained by treatment with PEG10K and sodium chloride; it can be seen that the drug release rate of the microcapsules obtained by the two treatment methods is slower than that of Pickering without the shell Emulsion, which further shows that the shell of the microcapsule has a slow-release effect on the drug. Figure 16D shows the release curve of Pickering emulsion and microcapsules prepared from different silk fibroin concentrations treated with PEG10K. It can be clearly seen from the figure that as the concentration of silk fibroin increases, the drug in the microcapsules The release rate becomes slower, which further indicates that the shells of microcapsules prepared with high concentrations of silk fibroin are thicker and denser.

Example 9: Preparation of drug-loaded microcapsules embedded with hydrophobic drug olanzapine

Using 3% SiO ₂ -S200 dispersion as the nanoparticle dispersion, silicone oil as the oil phase, and silk fibroin solutions of different concentrations (concentrations of 1wt%, 5wt%, 7.5wt%, 10wt% and 15wt%), Olanzapine was used instead of curcumin, and an emulsion with a composite external phase composed of nanoparticles and silk fibroin was obtained according to the method of Example 8. Then, the PEG treatment and the methanol treatment as described in Example 4 were respectively performed and freeze-dried to obtain olanzapine-loaded microcapsule powder.

Standard curve drawing method:

A. Raw material: 0.1M sulfuric acid (preparation method: 5.434ml sulfuric acid is added to 900ml water, and the volume is set to 1000ml; 2M hydrochloric acid (preparation method: 16.6ml hydrochloric acid is added to 800ml of water, and the volume is set to 1000ml; 2M hydrochloric acid); 1*10 ^-3 Mol/L Chloramine T (molecular formula of Chloramine T: 281.64g/mol); Rhodamine B (concentration 0.02%); 0.5-10 μg/ml olanzapine drug (containing 0.1M sulfuric acid)

B. Standard solution configuration: take 0.5ml 0.5-10 microgram/ml olanzapine drug (containing 0.1M sulfuric acid) solution, add 0.1ml 2M hydrochloric acid solution and 0.05ml chloramine T solution (concentration 1*10 ^-3 Mol/L) ), vortex for 30s to mix well, and after incubating for 10 minutes, add 0.1 ml of 0.02% rhodamine solution, vortex to mix evenly, and incubate for 5 minutes.

C. Curve drawing: Use a microplate reader to measure the absorbance value of the above mixed solution at 550nm (96-well plate, 200ul per well). The obtained absorbance value and the corresponding known drug concentration of olanzapine are drawn to a standard curve of the olanzapine drug. Linear region range (olanzapine concentration: 0.5-10ul/ml). The equation of the obtained standard curve is: y=13.939x-0.2216, and the degree of fit R2=0.992.

Drug content determination:

A: Olanzapine drug particles are dissolved in 0.1Mol/L hydrochloric acid to ensure that the drug concentration is 1mg/ml, and the concentration is diluted with 0.1M sulfuric acid to 2ug/ml;

B: Weigh 1mg of the drug-loaded microcapsules respectively and suspend them in 1ml of 0.1M sulfuric acid. At this time, the concentration of the drug-loaded microcapsules is 1mg/ml. Vortex for 1-2min to extract the drug in the capsule completely. Dilute with 0.1M sulfuric acid to a concentration of 2ug/ml;

C: Take 0.5ml of 2ug/ml olanzapine drug and drug-loaded microcapsules (containing 0.1M sulfuric acid) solution, add 0.1ml 2M hydrochloric acid solution and 0.05ml chloramine T solution, vortex for 30s to mix well, and statically After incubating for 10 minutes, add 0.1ml 0.02% rhodamine solution, vortex to mix evenly, and incubate for 5 minutes.

D: Measure the absorbance value of the above mixed solution at 550nm with a microplate reader (96-well plate, 200ul per well). Bring the obtained absorbance value into the standard curve and compare with the standard to obtain the drug content of the microcapsule.

The microcapsules obtained by using silk fibroin of more than 5%wt and processed in different ways have different drug loadings. The drug loading of PEG-treated microcapsules (20%-40%) is 5%-10% lower than that of methanol-treated microcapsules. The microcapsules obtained with 1% wt silk fibroin have the lowest drug loading (less than 10%). This is because the obtained shell is very thin and part of the drug is lost during the preparation process. As the concentration of silk fibroin increases between 5%wt and 15%wt, the drug loading decreases.

Example 10: Preparation of drug-loaded microcapsules embedded with hydrophilic drugs

Rhodamine B (molecular weight 479.01Da, Invitrogen) was used as a hydrophilic drug model to prepare drug-loaded microcapsules.

First, a drug-containing nanoparticle dispersion liquid as the internal water phase (W ₁ ) is prepared. Specifically, accurately weigh the hydrophobic fumed silica (particle size: 7-40nm, specific surface area: 115m ² /g, brand: Macleans, purchased from Suzhou Industrial Park Bomeida Reagent Instrument Co., Ltd.; denoted as SiO _2- S115), nano titanium dioxide (particle size: 40nm, brand: Maclean, purchased from Suzhou Great Medical Technology Co., Ltd.) or nano zinc oxide (particle size: 30±10nm, brand: Maclean, purchased from Suzhou Argon Krypton Xenon Trading Co., Ltd. Company), each was prepared with deionized water to prepare a nanoparticle dispersion with a concentration of 3% (weight/volume) (ultrasonic dispersion for 1 minute). Then, accurately weigh 500 mg of rhodamine B crystals and dissolve them in 5 ml of the nanoparticle dispersion prepared above to obtain a drug-containing nanoparticle dispersion (W ₁ ).

Then, it was mixed with 5 ml of silicone oil and emulsified (16,000 rpm, 2 minutes) to obtain a drug-containing water-in-oil (W ₁ /O) emulsion. Then, it was mixed with 10 ml of 3% SiO ₂ -S200 dispersion (W ₂ ) and emulsified (13,000 rpm, 1 minute) to obtain a double emulsion W ₁ /O/W _{2. The} emulsion was uniformly dispersed without stratification. The volume of mixed double emulsion W ₁ / O / W ₂ with 15wt% silk fibroin solution or the like and incubated for 12 hours at room temperature to obtain an emulsion having a composite outer phase consisting of nanoparticles of SiO ₂ -S200 and silk fibroin. Then, use the following three methods to process: (1) Mix an equal volume of 50% PEG10K aqueous solution and incubate for 12 hours, centrifuge at 8,000 rpm for 5 minutes and divide into three layers, take the top microcapsule layer and wash twice with water; 2) Mix in an equal volume of 50% (v/v) methanol and incubate for 10 minutes, centrifuge at 8,000 rpm for 5 minutes and divide into 2 layers, take the bottom sediment and wash twice with water; (3) Mix in an equal volume of saturated sodium chloride solution Incubate for 12 hours, centrifuge at 8,000 rpm for 5 minutes, and divide into two layers. Take the upper microcapsule layer and wash it twice with water. Freeze-dry all the microcapsules after washing with water.

Take the prepared 1mg drug-loaded microcapsules and 1mg rhodamine B into 0.5ml of 9.3Mol/L lithium bromide aqueous solution, each group of 4 replicates, vortex for 5 minutes, and then place in a 60℃ oven for 30 After vortexing for 5 minutes, add 0.5ml PBS (pH 7.4), centrifuge for 5 minutes (speed 14,000 rpm), take the supernatant after centrifugation, dilute 10 times with 10 mM PBS (pH 7.4) and use enzyme labeling The instrument measures the fluorescence value of the extract at excitation 550 nm and emission 590 nm, and substitutes it into the concentration-fluorescence value standard curve (which is made as described below) to obtain the drug content in the microcapsules.

Suspend or dissolve the prepared drug-loaded microcapsules and rhodamine B in 1ml 10mM PBS (pH 7.4) respectively until the content of rhodamine B is 1mg/ml. At ambient temperature, transfer the above suspension and rhodamine aqueous solution to a dialysis bag with a molecular weight cut-off of 3.5KDa, and place it in 40ml PBS (pH 7.4) after sealing, and observe how fast the color of the released solution changes within 24 hours , The faster the color turns red, the faster the release of the embedded rhodamine B. From the results of the color change, the color of the microcapsule group is lighter, the pure drug is the reddest, and the emulsion prepared by the three kinds of nanoparticles is in the middle. This indicates that the release rate of the microcapsules is significantly lower than the release rates of the pure drugs and the drugs present in the emulsion prepared by the three kinds of nanoparticles.

Preparation of the standard curve: Rhodamine B particles were serially diluted in 10mM PBS, and the drug concentrations were 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml, 0.03125mg/ml. Then use a microplate reader to measure the fluorescence value at excitation 550nm and emission 590nm, and use excel to draw a standard curve corresponding to the concentration and fluorescence value. The linear regression equation is calculated as Y=X*0.0064+25.004, and the degree of fit is R ² =0.992.

Figure 17 shows the microscopic photograph of W ₁ /O/W ₂ double emulsion. The innermost layer (W ₁ ) contains hydrophilic drugs, the middle layer is the oil phase marked with lipophilic commonly used dye Oil Red O, and the outermost layer (W ₂ ) is a continuous water phase, which can be generally regarded as an oil-in-water emulsion. Fig. 17A is a double emulsion obtained with nano titanium dioxide and SiO2-S200, and Fig. 17B is a double emulsion obtained with nano zinc oxide and SiO2-S200. They show that a stable emulsion can be obtained by emulsification. Moreover, microcapsules can be obtained by the above three treatment methods. The microcapsules treated with PEG10K are relatively complete, followed by sodium chloride treatment, and finally methanol treatment.

Example 11: Preparation of multilayer shell microcapsules and drug-loaded multilayer shell microcapsules

The microcapsules of Example 2 and Examples 8-10 were respectively suspended in silk fibroin at concentrations of 30%, 15%, 10%, 7.5%, 5%, 3%, 1%, 0.5%, and 0.1%. The concentration of the microcapsules in the solution (with or without 3% SiO ₂ -S200) is 0.01mg/ml-2mg/ml. After standing for 1 hour, centrifuge (12,000 rpm, 10 minutes), remove the supernatant, mix in an equal volume of 50% PEG10K or 50% methanol aqueous solution and let stand for different time (2h, 6h, 12h), centrifuge at room temperature (14,000 rpm, 5 minutes) and then divide into two layers, remove the supernatant, suspend the bottom sediment in pure water and centrifuge twice. Freeze drying to obtain microcapsules with a double-shell structure. The preparation process is repeated several times to prepare microcapsules with a multilayer shell structure.

Figure 18 is a graph showing the drug release rate in vitro of olanzapine-containing microcapsules prepared as in Example 9 and Example 11, respectively. The different times in the figure represent the number of coatings, and 1 represents 1 time for coating, and 2 times. Represents the coating 2 times, and so on. The oil used to prepare the microcapsules in Figure 18 is silicone oil, the first layer of silk fibroin concentration is 10%, and the subsequent coating layer uses a silk fibroin concentration of 7.5%. The microcapsules are suspended in the silk fibroin and the hydrophilic gas phase. The concentration in the mixed solution of silica is 1 mg/ml. It can be clearly seen from the figure that the drug release rate of the drug-loaded microcapsules treated with PEG and methanol is significantly slower than that of simple drug particles (control), and as the number of treatments increases, the rate of drug release of olanzapine Slow down.

19 is a morphology diagram of the microcapsules containing olanzapine prepared as in Example 9 and Example 11 after being released in vitro for 27 days. In Figure 19, the oil used for preparing the microcapsules is silicone oil, the first layer of silk fibroin concentration is 15%, and the subsequent coating layer uses 5% silk fibroin concentration. The microcapsules are suspended in silk fibroin and hydrophilic gas phase. The concentration in the mixed solution of silica is 1 mg/ml. It can be seen that on the 27th day, most of the microcapsules treated with methanol once (single-layer shell) and twice (double-layer shell) kept the original shape, indicating that the shell of the methanol-treated microcapsules was more stable, which was similar to the shell. The secondary structure is related. Through chemical and physical treatment methods, the secondary structure and crystal structure of the silk fibroin in the shell are changed, and then the mechanical properties, permeability and degradability of the shell are adjusted.

Example 12: Drug dissolution rate of drug-loaded microcapsules

Since the hydrophobic olanzapine drug is easy to dissolve in acid, the drug-loaded microcapsules of Example 9 were suspended in a 0.1M hydrochloric acid solution, and the dissolution rate of the drug was observed. As a control, olanzapine was suspended in silicone oil, centrifuged to remove excess silicone oil, mixed with silk fibroin, and then treated with PEG similar to Example 8 to obtain olanzapine-loaded microcapsules. Suspend the drug-loaded microcapsules as a control in 0.1M hydrochloric acid solution and observe the dissolution rate of the drug.

Figure 20 shows the PEG-treated olanzapine-loaded microcapsules prepared by using 7.5wt% silk fibroin solution in Example 9 (right picture) and the control microcapsules prepared above (left picture) in 0.1M hydrochloric acid The morphology of the drug in the solution before and after dissolution and the drug dissolution rate. It can be seen from the figure that the presence of silicon dioxide slows down the dissolution rate of the drug from the microcapsules.

In addition, the microcapsules with the silk fibroin concentration of 7.5%, the oil phase being silicone oil, and the treatment methods being PEG treatment and methanol treatment in Example 9 were suspended in silk fibroin solutions of different concentrations (1-30wt%) and soaked Centrifuge for 2 hours, discard the supernatant, and treat the lower precipitate with different methods (PEG treatment, 50% methanol, salt ion treatment for 24h and steam treatment for 24h, the treatment method is the same as that described in Example 4. Repeat the process One or more times. Then, dry it in the air, place it in acid, observe the dissolution rate of the drug through a microscope and record the time (see Table 1 and Table 2).

Table 1 and Table 2 respectively show the time taken for the drug-loaded microcapsules to be completely dissolved out of the drug-loaded microcapsules that were further treated with methanol twice (three-layer shell) and PEG10K twice (three-layer shell) as described above. It is obvious from the table that with the protection of the outer shell of the microcapsule, the drug dissolution rate is obviously delayed. Based on the stability, ease of operation and stability of the core-shell structure of the prepared microcapsules, compound molecules or particles of different types and properties can be embedded in the core to achieve high loading and controlled release.

Table 1: The complete dissolution time of the drug-loaded microcapsules obtained after the methanol-treated microcapsules of Example 9 were further treated with methanol or saturated sodium chloride or steam for two times

Table 2: The complete dissolution time of the drug-loaded microcapsules obtained after the PEG-treated microcapsules of Example 9 were further treated with methanol or saturated sodium chloride or water vapor for two times

Example 13: Preparation of microcapsules using traditional small molecule surfactants

Taking the commonly used small molecule surfactant Tween-80 as an example, microcapsules were prepared in a similar manner to Example 2. Specifically, a 1% Tween 80 aqueous solution was prepared, mixed with corn oil in a volume ratio of 7:3, emulsified with a homogenizer at 16,000 rpm at room temperature for 1 minute, and an equal volume of 15wt% silk fibroin solution was added and mixed Incubate for 12 hours at room temperature. Then, it was mixed with an equal volume of 50% PEG10K, incubated for 12 hours, centrifuged (12,000 rpm, 5 minutes), and divided into three layers. Take the top layer and wash it twice with pure water, and then use a freeze dryer (vacuum degree: 0.01mbar) to vacuum for 48 hours to obtain microcapsule powder.

Figure 21 shows the microcapsules (silk fibroin / SiO ₂ -S200 / corn oil, left panel) prepared in Example 2 and the microcapsules prepared in Example 13 (SF / Tween 80 / corn oil, right panel) embodiment Electron microscopy photos. It can be seen that the microcapsules prepared with hydrophilic fumed silica nanoparticles SiO ₂ -S200 of Example 2 formed complete microcapsules after freeze-drying (left picture), while the surfactant Tween 80 could not Complete microcapsules are obtained, and oil cannot be encapsulated in microcapsules (right picture). Compared with microcapsules prepared with surfactants, the microcapsules of the present invention have better formability and stability, indicating that the advantages of the present invention are significantly higher than that of traditional microcapsules. Based on the high stability of the prepared capsules, it can be dried by freeze drying, spray drying or natural air drying to form a solid powder, which is the fourth significant advantage of the present invention.

Example 14: Preparation of microcapsules by spray drying

The 3% SiO ₂ -S200 dispersion was mixed with linalyl acetate and fish oil as internal phases in a volume ratio of 7:3, and emulsified at 16000 rpm for 1 minute at room temperature. After standing for 2 hours, the Pickering emulsion layer on the upper layer and the nanoparticle dispersion on the lower layer were obtained, and the height ratio of the two layers was a constant value. Then, the upper Pickering emulsion was mixed with an equal volume of 15 wt% silk fibroin solution and incubated at room temperature for 24 hours to obtain an emulsion having a composite external phase composed of nanoparticles and silk fibroin. Then it was filled into the spray dryer at a flow rate of 60ml/h, with an inlet temperature of 180°C and an outlet temperature of 80°C. The microcapsule powder is obtained by spray drying. Observe the morphology of the microcapsules by scanning electron microscope.

As a control, the small molecule surfactant Tween-80 was used instead of nanoparticles to prepare microcapsules in a similar manner to the above. Specifically, a 1% Tween 80 aqueous solution was prepared, mixed with linalyl acetate in a volume ratio of 7:3, emulsified with a homogenizer at 16,000 rpm at room temperature for 1 minute, and an equal volume of 15wt% silk fibroin was added The protein solution was incubated at room temperature for 24 hours. Then it was filled into the spray dryer at a flow rate of 60ml/h, with an inlet temperature of 180°C and an outlet temperature of 80°C. The microcapsule powder was obtained by spray drying. Observe the morphology of the microcapsules by scanning electron microscope.

Figure 22 shows the morphology of the microcapsules prepared by the spray drying process above. (1-4) followed by a _{2 -S200} microcapsules prepared linalyl acetate and SiO (1), the microcapsules prepared SiO _{2 -S200} and fish oil (2) and by a small molecule surfactants Tween 80, and Electron micrographs of microcapsules prepared by linalyl acetate (3) and microcapsules prepared by SiO ₂ -S200 and linalyl acetate after methanol extraction (4). It can be seen that the microcapsules obtained with fumed silica nanoparticles have a complete structure and a compact and flat surface structure than those obtained with traditional Tween small molecules, while the microcapsules prepared with Tween have holes on the surface and the structure is incomplete. After methanol extraction, we used fumed silica nanoparticles to obtain microcapsules and found that the oil in the microcapsules was extracted by methanol and the microcapsules became deflated. It can be seen from the figure that the wall thickness of the microcapsules is close to 1000nm. In addition, the yield of microcapsules stabilized with nanoparticles is significantly higher than that of microcapsules stabilized by Tween small molecules, and the yield is 4-5 times that of microcapsules stabilized by Tween small molecules. The nanoparticle-stabilized microcapsules successfully contained fish oil, so the microcapsules can also be used in food.

Figure 23 shows a graph of the mechanical modulus of the microcapsules. The left picture is the mechanical modulus diagram of the microcapsules prepared from SiO ₂ -S200 and linalyl acetate, and the right picture is the mechanical modulus diagram of the microcapsules prepared from the small molecule surfactant Tween 80 and linalyl acetate. The average modulus value in the white dashed box in the figure is obtained after the test of the atomic force microscope instrument and the simulation calculation. The test mode of the atomic force microscope is PeakForce peak force feedback mode, and the simulation calculation software selected is NanoScope 8. The result is that the mechanical modulus of the microcapsules prepared from SiO2-S200 and linalyl acetate is 2665Mpa, which is much higher than the mechanical modulus of the microcapsules prepared from the small molecule surfactants Tween 80 and linalyl acetate, 594Mpa. It shows that the shell composed of nanoparticles and silk fibroin has good mechanical properties.

It can be understood that although the specific embodiments and examples of the present invention have been described herein for the purpose of explanation, various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited except for the appended claims.

Claims

A core-shell microcapsule, characterized in that: the shell of the microcapsule is a single-layer composite shell with a thickness of 50nm-1000nm, containing silk fibroin and nanoparticles randomly distributed in the single-layer composite shell, and The single-layer composite shell is optionally surrounded by one or more layers of additional shells, and the one or more layers of additional shells each independently comprise silk fibroin and optionally randomly distributed in the additional shells. And wherein the silk fibroin and the nanoparticle may be the same or between the individual shells, including the single-layer composite shell and the one or more other shells different.
The core-shell microcapsule according to claim 1, wherein the nanoparticle is a nanoparticle capable of forming a Pickering emulsion, preferably having a particle size range of 5-1000nm, more preferably 5-500nm; preferably, the nanoparticle may be Inorganic or organic nanoparticles, such as inorganic nanoparticles including but not limited to silica nanoparticles (such as ordinary silica nanoparticles and fumed silica nanoparticles, such as ordinary silica with a particle size of 7-500nm, a particle size of 7 -500nm and a specific surface area of 50-400m 2 / g hydrophilic fumed silica with a specific surface area of 50-400m 2 / g hydrophobic fumed silica, the particle size of 7-20nm amino silica) , Zinc oxide nanoparticles (for example, zinc oxide with a particle size of 20-500nm), titanium dioxide nanoparticles (for example, titanium dioxide with a particle size of 20-500nm), hydroxyapatite nanoparticles (for example, hydroxyapatite with a particle size of 50-500nm) , nano silver (e.g., silver nano-particle size of 25-100nm), gold nanoparticles (e.g., gold nanoparticles of a particle size of 1-100nm), Fe 3 O 4 magnetic nanoparticles (e.g., particle size of 5-100nm of Fe 3 O 4 magnetic nano Particles), nano liposomes and any combination thereof; organic nanoparticles include but are not limited to 50-500nm polylysine (PLL), polyethylene, polyvinyl chloride, polylactic acid-glycolic acid copolymer (PLGA) , Polycaprolactone (PCL), polylactic acid (PLA), silk fibroin nanoparticles, collagen, chitosan, starch, cellulose and any combination thereof.
The core-shell microcapsule according to claim 1 or 2, wherein the molecular weight of the silk fibroin ranges from 5KDa to 500KDa, preferably 10-400KDa.
The core-shell microcapsule according to any one of the preceding claims, wherein the core of the microcapsule is completely filled with biologically active organisms such as oil phase, drug particles, bacteria, cells, or W/O type emulsion droplets ( That is, full or partially filled form or gas (ie hollow) form.
The core-shell microcapsules according to claim 4, wherein the oil phase or the oil phase in the W/O type emulsion is derived from volatile oil, non-volatile oil, non-oily fluid immiscible with water and/ Or stearic solids; for example, the volatile oil is selected from linalyl acetate, geraniol, aceto-ionone, citral, ethyl acetate, myrcene, diphenylethanol, volatile silicone oil, and essential oil And any combination thereof, the non-volatile oil is selected from soybean oil, corn oil, camellia oil, argan oil, sweet almond oil, apricot kernel oil, wheat germ oil, jojoba oil, grape seed oil, avocado Oil, macadamia oil, olive oil, castor oil, fish oil and any combination thereof; the non-oily fluid that is immiscible with water is selected from solvents such as n-hexane, dodecane, n-hexanol and butyl butyrate and their Any combination; the stearin solids are selected from paraffin, cetyl stearin, shea butter and any combination thereof.
The core-shell microcapsule according to claim 4 or 5, wherein the W/O type emulsion droplets are prepared by any method, and the size of the emulsion droplets is selected from 50nm-200um, preferably 200nm-100um; preferably, the W/O O-type emulsion droplets are obtained by emulsifying an aqueous dispersion of nanoparticles with an oil phase or are emulsion droplets stabilized with a small molecule emulsifier.
The method for preparing the core-shell microcapsules according to any one of claims 1 to 6, characterized by comprising the following steps:

(1) Mix and emulsify the aqueous dispersion of nanoparticles with the oil phase or W/O type emulsion as the internal phase material to obtain a stable O/W type Pickering emulsion or W/O type containing oil droplets accordingly W/O/W double Pickering emulsion of milk drops;

(2) Mix the Pickering emulsion prepared in step (1) with the silk fibroin aqueous solution;

(3) Incubating the obtained mixture to obtain an emulsion with a composite external phase composed of silk fibroin and nanoparticles;

(4) The emulsion of step (3) is cured by chemical and/or physical treatment of the composite external phase;

(5) Optionally, drying the mixture of step (4), such as freeze drying, spray drying or natural air drying, to obtain dry powdery microcapsules;

(6) Optionally, resuspend the microcapsules obtained in step (5) in a silk fibroin aqueous solution containing or not containing nanoparticles, and repeat steps (3) to (5) one or more times to obtain a multi-layer Shell microcapsules;

Wherein, the silk fibroin and the nanoparticle may be the same or different between each step.
The method according to claim 7, wherein in step (1), the aqueous dispersion of nanoparticles is obtained by uniformly dispersing nanoparticles in water; further, the dispersion contains 0.1%-50%, A concentration (weight/weight) of nanoparticles of 0.5%-20%, more preferably 1%-10% is preferred.
The method according to claim 7, wherein in step (1), the aqueous dispersion of nanoparticles and the oil phase or W/O type emulsion as the internal phase material are in a ratio of 0.1:9.9 to 9.9:0.1, preferably 9: 1-7:3 volume ratio mixing; in addition, the stable Pickering emulsion is to make the oil phase or W/O emulsion droplets uniformly dispersed into the aqueous dispersion of nanoparticles under the condition of high energy provided by the outside. Obtained; Preferably, the high-energy dispersion method includes, but is not limited to, one or any combination of methods such as ultrasonic probe pulverization, ultrasonic water bath shaking, high-speed homogenization, high-pressure homogenization, magnetic stirrer stirring, vortexing and the like.
The method according to claim 7, wherein in steps (2) and (6), the silk fibroin concentration of the silk fibroin aqueous solution is each independently 0.05 wt% to 45 wt%, preferably 1 wt% to 35 wt% , More preferably 3wt%-30wt%; in addition, preferably, in step (2), Pickering emulsion and silk fibroin aqueous solution are 50:1 to 1:50, preferably 10:1 to 1:10, more preferably 5 :1-1:5 volume ratio for mixing.
The method according to claim 7, wherein in step (4), the chemical and/or physical treatment is selected from physical crosslinking (for example, polyethylene glycol treatment, alcohol solvent treatment such as methanol treatment, salt ion treatment such as Sodium chloride treatment, pH treatment, heat treatment, ultrasonic treatment, steam treatment, spray drying treatment, cyclic freezing-thawing and any combination thereof) and chemical treatment (horseradish peroxidase-hydrogen peroxide (HRP-H 2 O 2 ), genipin) or any combination thereof.
The method according to claim 11, wherein the polyethylene glycol treatment is carried out with an aqueous solution of polyethylene glycol; preferably, the molecular weight of the polyethylene glycol is 200-20000 Daltons, and/or The polyethylene glycol concentration of the aqueous solution is 30% to 100% (weight/volume).
The method according to claim 11, wherein the methanol treatment is carried out with an aqueous methanol solution; preferably, the methanol concentration of the aqueous solution is 20-90% (volume/volume).
The method according to claim 11, wherein the salt ion treatment is carried out using an aqueous solution of salt ions; preferably, the salt ion concentration of the aqueous solution is 0.5M to a saturated concentration.
The method according to claim 11, wherein in the pH treatment, the pH value is 1-14, preferably 1-5; preferably, hydrochloric acid (such as 1M hydrochloric acid) and sodium hydroxide (such as 1M sodium hydroxide) are used Adjust the pH value.
The method according to claim 7, wherein the internal phase material in step (1) may contain an active material; in particular, when the active material is hydrophobic, the active material is dissolved in and/or suspended in the form of solid particles In the oil phase as the internal phase material; when the active material is hydrophilic, the active material is suspended in the oil phase as the internal phase material in the form of solid particles, or is dissolved and/or supersaturated in the solid The form of particles is suspended in the water phase of the W/O type emulsion as the internal phase material.