A tri-drug core-shell structure nanofiber and a preparation method and application thereof

Abstract

The application provides a kind of three drug-loading core-shell structure nanofiber and its preparation method and application, and the colloidal particles are prepared by self-assembly of polysaccharide, protein and first hydrophilic drug under weak interaction;With the colloidal particles as particle emulsifier, gelatin as spinning aid, the second hydrophilic drug is added in the water phase, and the oil phase is prepared by adding hydrophobic drug in food oil to prepare oil-in-water Pickering emulsion;The emulsion is electrospun under high pressure to obtain three drug-loading nanofiber with core-shell structure for drug sustained release.The material provided by the application is biodegradable, and the core-shell nanofiber membrane has good sustained release performance for hydrophilic-hydrophobic drugs, which can be applied in drug sustained release coating, drug release and drug tissue engineering scaffold fields.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical polymer material preparation, and particularly relates to a three-drug-loaded core-shell structure nanofiber as well as a preparation method and application thereof. The three-drug-loaded core-shell structure nanofiber is prepared based on emulsion electrospinning, and can be used for drug sustained release. BACKGROUND

[0002] Drug controlled release is to combine drugs with carriers, so that the drugs are released into the environment through diffusion and other ways in a certain time period and at a certain rate. By using the controlled release system, the drug release time can be prolonged, the blood drug concentration can be kept uniform for a long time, the drug utilization efficiency can be improved, so as to reduce the frequency of drug taking of patients; at the same time, the side effects caused by drug overdose can be avoided, and the pain of patients can be reduced. In addition, with the improvement of medical level and the needs of pathology, controlling the release of a single drug cannot meet the requirements of clinical treatment, and fibers loaded with multiple drugs are widely studied, and controlling the burst release of multiple drugs and the differential release have become the research focus at present.

[0003] In recent years, electrospun fibers have been widely used as drug carriers for local medication due to their large specific surface area, multiple pore structures and good stability. By using coaxial electrospinning or emulsion electrospinning technology, core-shell structure nanofibers can be prepared, and the shell layer and the core layer of the fiber can simultaneously load multiple drugs and control the slow release of the drugs. However, coaxial electrospinning requires special equipment, needs to control the molding process and the viscosity of the two phases, and needs to optimize the experimental conditions, which increases the complexity and repeatability of the experimental operation.

[0004] Compared with coaxial electrospinning, emulsion electrospinning has good controllability. Commonly used emulsion types include water-in-oil (W / O) and oil-in-water (O / W). After emulsion electrospinning, the water phase W of the W / O emulsion is the core layer of the fiber, and the oil phase O is the shell layer of the fiber; for the O / W emulsion, the oil phase O is the core layer of the fiber, and the water phase W is the shell layer of the fiber. Generally, the drugs located in the shell layer of the fiber are prone to burst release. It may be because the shell layer polymer and the drug are only simply mixed, and the interaction between the two is weak. Therefore, W / O is often used for sustained release of hydrophilic drugs, and O / W is often used for sustained release of hydrophobic drugs. Therefore, it is of great significance to use emulsion electrospinning technology to prepare a multi-drug-loaded core-shell structure nanofiber which is simple to operate, has good biocompatibility and controllable sustained release effect. SUMMARY

[0005] The present application aims to provide a kind of three drug core-shell structure nanofiber and its preparation method, to biological macromolecule polysaccharide, protein etc. and first hydrophilic drug is self-assembled to prepare colloidal particles under weak interaction;With this colloidal particle as particle emulsifier, gelatin as assistant spinning agent, second hydrophilic drug is added in water phase, and oil phase is prepared with hydrophobic drug-containing food oil to prepare oil-in-water Pickering emulsion;The emulsion is electrospun under the action of high pressure, and the three drug core-shell structure nanofiber for drug sustained release is obtained.

[0006] The present application also aims to provide a kind of three drug core-shell structure nanofiber and its application, the three drug core-shell structure nanofiber has good sustained release performance to hydrophilic-hydrophobic multiple drugs, and can realize the differential release of multiple drugs. And this nanofiber has good biocompatibility and biodegradability, and has good application prospect in the field of biological coating, drug release and tissue engineering.

[0007] The technical scheme of the present application is:

[0008] A kind of preparation method of three drug core-shell structure nanofiber, comprising the following steps:

[0009] 1) preparation first hydrophilic drug / biological macromolecule self-assembly colloidal particles;

[0010] 2) the solution containing first hydrophilic drug / biological macromolecule colloidal particles and second hydrophilic drug is as water phase, and water phase is mixed with oil phase containing hydrophobic drug, homogenization emulsification, and Pickering emulsion is prepared;

[0011] 3) Pickering emulsion is electrospun, and three drug core-shell structure nanofiber is prepared.

[0012] Step 1) is specifically:

[0013] 1-1) first hydrophilic drug is dissolved in biological macromolecule solution to obtain drug-containing mixed solution;

[0014] 1-2) to polysaccharide solution, dropwise add drug-containing mixed solution, at room temperature, self-assemble into first hydrophilic drug / biological macromolecule colloidal particles between three through weak interaction force, and the whole process needs about 1-2h.

[0015] The solvent of drug-containing mixed solution and polysaccharide solution in step 1-1) and step 1-2) is water, preferably distilled water.

[0016] The concentration of biological macromolecule in step 1-1) is 0.5-2.5mg / ml;

[0017] Preferably, the biological macromolecule in step 1-1) is selected from protein or polysaccharide;

[0018] Preferably, the protein in step 1-1) is selected from lysozyme, abbreviated as Lys; and the polysaccharide is selected from hyaluronic acid (HA);

[0019] The first hydrophilic drug in step 1-1) is selected from diclofenac sodium, vitamin B (VB 12 ) or doxorubicin hydrochloride; the concentration of the first hydrophilic drug in the mixed drug solution is 1-5 mg / ml;

[0020] The concentration of the polysaccharide solution in step 1-2) is 0.5-2 mg / ml; the polysaccharide used is sodium carboxymethyl cellulose (CMC) or chitosan (CS);

[0021] The volume ratio of the polysaccharide solution to the mixed solution containing the drug in step 1-2) is 1:1-1:3;

[0022] Step 2) is specifically:

[0023] 2-1) The solution containing the first hydrophilic drug / biopolymer colloidal particles is added dropwise to the mixed solution of the spinning aid and the second hydrophilic drug as the water phase;

[0024] 2-2) The hydrophobic drug is dissolved in oil as the oil phase;

[0025] 2-3) The water phase and the oil phase are mixed and homogenized to prepare a Pickering emulsion.

[0026] The concentration of the first hydrophilic drug / biopolymer colloidal particles in the solution containing the first hydrophilic drug / biopolymer colloidal particles in step 2-1) is 0.25-1.25 mg / ml;

[0027] The mass concentration of the spinning aid in the mixed solution of the spinning aid and the second hydrophilic drug in step 2-1) is 15%-45%, and the mass concentration of the second hydrophilic drug is 2.5%-10%;

[0028] The volume ratio of the mixed solution of the spinning aid and the second hydrophilic drug to the solution containing the first hydrophilic drug / biopolymer colloidal particles in step 2-1) is 4-7:6-3;

[0029] The spinning aid in step 2-1) is gelatin;

[0030] The second hydrophilic drug in 2-1) is riboflavin, amlodipine or chloroquine hydrochloride.

[0031] The first hydrophilic drug and the second hydrophilic drug can be interchangeable.

[0032] The preparation method of the mixed solution of the auxiliary spinning agent and the second hydrophilic drug in step 2-1) is as follows: the auxiliary spinning agent is dissolved in a pH 4.5 HAc solution to obtain an auxiliary spinning agent solution, the second hydrophilic drug is added to the auxiliary spinning agent solution, and stirring and dissolution are performed to obtain a mixed solution of the auxiliary spinning agent and the second hydrophilic drug.

[0033] In step 2-2), the hydrophobic drug is an oil-soluble drug selected from vitamin D3 (VD3) or levofloxacin.

[0034] In step 2-2), the oil is selected from edible oil, preferably corn oil, white oil or soybean oil.

[0035] In step 2-2), the concentration of the hydrophobic drug in the oil phase is 1-5 mg / mL.

[0036] In step 2-3), the volume ratio of the oil phase to the water phase is 0.1-0.4:1.

[0037] In step 2-3), the oil-in-water Pickering emulsion is obtained by homogenization and emulsification using a homogenizer at a speed of 5000-15000 r / min for 2-5 min.

[0038] In step 3), the Pickering emulsion is placed in a syringe, a syringe pump is connected to a receiving device, power is turned on, and the tri-drug core-shell structure nanofiber is obtained by electrospinning technology.

[0039] In step 3), the electrospinning voltage is 15-26 kV, the pushing speed is 0.0001-0.01 mm / s, and the distance between the receiving device and the syringe needle is 4-19 cm.

[0040] The tri-drug core-shell structure nanofiber provided by the application is prepared by the above method, the hydrophobic drug is in the core layer of the fiber, the first hydrophilic drug and the second hydrophilic drug are in the shell layer of the fiber, and the first hydrophilic drug is loaded in the colloidal particles.

[0041] The tri-drug core-shell structure nanofiber provided by the application is used for biological coating, drug release or tissue engineering.

[0042] In the drug release application, the release rates of the first hydrophilic drug, the second hydrophilic drug and the hydrophobic drug are different, and the drug release rate is: the second hydrophilic drug is the fastest, the hydrophobic drug is the second, and the first hydrophilic drug is the slowest, that is, the second hydrophilic drug > the hydrophobic drug > the first hydrophilic drug, so that the differential release of the three drugs can be realized.

[0043] Biological macromolecules are non-toxic and harmless, and have biodegradability. Macromolecular self-assembly refers to a process that macromolecules spontaneously form ordered aggregates under the action of weak intermolecular forces such as molecular interaction, hydrophilic / hydrophobic interaction, hydrogen bonding, electrostatic interaction, etc. The self-assembled colloidal particles are widely studied in the field of Pickering emulsion because of their controllable structure and amphiphilic property. In the present application, the colloidal particles are prepared by self-assembly of polysaccharides, biological macromolecules (protein or hyaluronic acid) and a first hydrophilic drug under weak interaction; the drug-loaded colloidal particles are used as a particulate emulsifier, gelatin is used as a spinning aid, a second hydrophilic drug is added in the aqueous phase, and an oil phase containing a hydrophobic drug is used to prepare an oil-in-water emulsion; the prepared emulsion is electrospun under high pressure to obtain three-drug-loaded nanofibers with a core-shell structure. The material provided by the present application is biodegradable, and the core-shell nanofiber membrane has good slow-release performance for various hydrophilic / hydrophobic drugs, and can be applied to the fields of drug slow-release coating, drug release and drug tissue engineering scaffold, etc.

[0044] Compared with the prior art, the main advantages of the present application are:

[0045] 1. The raw materials used in the present application are safe, non-toxic, have excellent biocompatibility and biodegradability, and have advantages that synthetic macromolecules cannot match; the raw materials are easy to obtain and can be recycled, which meets the sustainable development point of view, and has good economic value and application prospect.

[0046] 2. In the present application, the maximum initial concentration of biological macromolecules is 2.5 mg / mL. The larger the concentration of biological macromolecules, the larger the particle size of the colloidal particles formed in the self-assembly process. The larger the particle size of the colloidal particles, the more unstable they are, and the more prone to precipitation. Therefore, they cannot be used as particulate emulsifiers to prepare emulsions, and further ideal nanofibers with a core-shell structure cannot be obtained. Therefore, the present application controls the initial concentration of biological macromolecules to be 0.5-2.5 mg / ml to obtain colloidal particles with stable properties, which can be used to prepare emulsions and obtain nanofibers with a core-shell structure, and achieve the goal.

[0047] 3. In the present application, the self-assembled colloidal particles are used as a hydrophilic drug carrier to prepare Pickering emulsion with the colloidal particles as a particulate emulsifier, which has small toxic and side effects.

[0048] 4. The mass concentration of the spinning aid gelatin in the present application is 15%-45%. When the concentration is lower than 15%, the emulsion obtained by emulsion electrospinning cannot obtain good fibers, and therefore cannot form fibers with a core-shell structure; when the concentration of gelatin is greater than 45%, the emulsion viscosity is too large due to the too large concentration of gelatin, the needle is easily blocked during spinning, normal spinning cannot be carried out, and nanofibers with a core-shell structure cannot be obtained.

[0049] 5. Emulsion electrospinning is a key technology for preparing core-shell nanofibers. Drugs located in the core and shell layers of the fiber exhibit different interaction forces with the core and shell layers due to their different locations, enabling differential drug release. This invention further prepares core-shell nanofibers using Pickering emulsion electrospinning. The prepared fibers exhibit a certain sustained-release effect on three drugs: hydrophilic and hydrophobic, enabling differential release of multiple drugs. This invention controls the volume ratio of the oil phase to the water phase to be 0.1-0.4:1, which is beneficial for achieving sustained drug release. An unsuitable oil-water ratio is detrimental to sustained release.

[0050] 6. The core-shell structured nanofibers prepared by this invention have specific advantages in applications such as biocoating, drug release, and tissue engineering. Attached Figure Description

[0051] Figure 1 VB prepared in Example 1 12 - Particle size distribution, Tyndall effect (a) and SEM image (b) of Lys / CMC colloidal particles;

[0052] Figure 2 The images shown are SEM, TEM, and microscopic images of the nanofibers prepared in Example 1.

[0053] Figure 3 The sustained-release curves of three hydrophilic and hydrophobic drugs for core-shell structured nanofibers in Example 1 are shown.

[0054] Figure 4 The sustained-release curves of three hydrophilic and hydrophobic drugs for core-shell structured nanofibers in Example 2 are shown.

[0055] Figure 5 VB prepared in Example 3 12 - Particle size distribution, Tyndall effect (a), and SEM image (b) of HA / CS colloidal particles;

[0056] Figure 6 SEM and TEM images of the nanofibers prepared in Example 3;

[0057] Figure 7 The sustained-release curves of three hydrophilic and hydrophobic drugs for core-shell structured nanofibers in Example 3 are shown.

[0058] Figure 8 Example 4 shows the sustained-release curves of three drugs (one hydrophilic and one hydrophobic) produced by core-shell structured nanofibers.

[0059] Figure 9 VB prepared for Comparative Example 2 12 - Particle size distribution of HA / CS colloidal particles. Detailed Implementation

[0060] The design concept of this invention is as follows: First, colloidal particles are prepared by self-assembly of sodium carboxymethyl cellulose / chitosan, lysozyme / hyaluronic acid, and a hydrophilic drug under weak interactions; then, these colloidal particles are used as particle emulsifiers, gelatin is used as a spinning aid, a second hydrophilic drug is added to the aqueous phase, and an oil-in-water Pickering emulsion is prepared using food oil containing a hydrophobic drug as the oil phase; finally, the prepared emulsion is electrospun under high pressure to obtain core-shell structured drug-loaded nanofibers, which are intended for use in fields such as biocoatings, drug release, and tissue engineering.

[0061] The present invention will be further described below with reference to embodiments, but the present invention is not limited thereto.

[0062] Example 1

[0063] A method for preparing a three-drug-loaded core-shell nanofiber includes the following steps:

[0064] 1) Preparation of the first hydrophilic drug / protein / polysaccharide self-assembled colloidal particles: Prepare 1.5 mg / mL lysozyme aqueous solution (hereinafter referred to as Lys aqueous solution) and 0.75 mg / mL carboxymethyl cellulose sodium aqueous solution (hereinafter referred to as CMC aqueous solution); add vitamin B... 12 (VB 12 Dissolved in Lys aqueous solution, the obtained VB 12 -VB in Lys mixture 12 The concentration is 3 mg / mL; add 2 mL of VB 12 - The Lys mixture was added dropwise to 2 mL of CMC aqueous solution and mixed thoroughly; CMC, Lys, and VB 12 VB is formed in solution through self-assembly via weak interaction forces. 12 -Lys / CMC colloidal particles, the whole process takes about 1.5 hours.

[0065] The specific process of colloidal particle formation is as follows: Lys carries a positive charge over a relatively wide pH range, therefore Lys and VB... 12 Mixed solution VB 12 -Lys carries a positive charge, CMC carries a negative charge; when VB is... 12 During the dropwise addition of the Lys mixed solution to the CMC solution, primary hydrophobic complex fragments VB are generated under electrostatic interactions. 12 -Lys / CMC, hydrophobic complex fragments aggregate due to further interactions. VB 12 -Lys / CMC segments exhibit good flexibility and high charge density. Besides electrostatic interactions, VB also benefits from intermolecular / intramolecular hydrogen bonding and hydrophobic interactions. 12 -Lys / CMC segments intertwine and coil in solution, ultimately forming a three-dimensional cross-linked network structure, which is the hydrophobic core. VB12 uncomplexed COO on the Lys / CMC segment - surrounding the periphery of the core to form a hydrophilic shell of the colloidal particle to maintain the stability of the colloidal particle. The formed colloidal particle has the characteristics of a core-shell structure.

[0066] Figure 1 Lys-VB 12 Particle size distribution, Tyndall phenomenon (inset in a) and SEM image of the Lys-VB Figure 1 As can be seen from a in FIG. 4, the VB 12 The particle size of the Lys-VB Figure 1 As shown in b in FIG. 4, the colloidal particle has a spherical structure with a particle size of about 200 nm. This is because the SEM image shows the colloidal particle in a shrunken dry state after the water is evaporated.

[0067] 2) Preparation of Pickering emulsion:

[0068] 2-1) Dissolve gelatin in a pH = 4.5 HAc solution to obtain a gelatin solution with a mass concentration of 35%, and then add amlodipine to the solution, so that the mass concentration of amlodipine is 5%, and then stir and dissolve to obtain a mixed solution; add 0.375 mg / mL VB 12 -Lys / CMC colloidal particle solution to the mixed solution containing gelatin and amlodipine dropwise while stirring, so that the mixed solution and the VB 12 -Lys / CMC colloidal particle solution are mixed at a volume ratio of 7:3 to obtain an aqueous phase for preparing an emulsion;

[0069] 2-2) Dissolve VD3 in corn oil to obtain an oil phase for preparing an emulsion, and the concentration of VD3 is 3 mg / mL.

[0070] 2-3) Mix 4.3 mL of the aqueous phase and 1.7 mL of the oil phase, and then homogenize the mixture at a speed of 10000 r / min for 3 min, and the oil-water ratio is 0.4, to obtain a stable Pickering emulsion.

[0071] 3) Preparation of nanofiber:

[0072] Place the Pickering emulsion prepared in step 2) in a 5 mL syringe, connect the syringe pump with the receiving device, and prepare a core-shell structure nanofiber under the conditions of a voltage of 26 kV, a pushing rate of 0.0001 mm / s, and a distance between the receiving plate and the syringe needle of 12 cm.

[0073] Figure 2SEM, TEM and fluorescence microscope pictures of the fiber prepared in Example 1. From the SEM picture, it can be seen that the diameter of the fiber is relatively uniform, about 500 nm; from the TEM picture, it can be seen that the prepared fiber is a core-shell structure fiber, as indicated by the arrow in the picture. In addition, the fluorescence microscope picture after the Nile red labeled oil phase shows that the fiber is a green line, indicating that the fluorescence labeling is successful.

[0074] Test of drug sustained release:

[0075] A series of different concentrations of drugs were prepared in advance, and the absorbance values of the drugs at a specific wavelength were measured to draw a standard curve of drug concentration and absorbance. A certain amount of nanofiber was removed and placed in three dialysis bags, which were then placed in 90 mL of phosphate buffer PBS (pH 7.0) at 37°C for dynamic dialysis for 170 h. Every certain time, 5 mL of dialysate was taken out to measure the absorbance value of the drug at a specific wavelength, and an equal volume of buffer was supplemented to draw the cumulative release curve of the drug.

[0076] Figure 3 The sustained release curves of the core-shell structure nanofiber in Example 1 for the three drugs of hydrophilic and hydrophobic. From the curves, it can be seen that the release of amlodipine is faster, because amlodipine and colloidal particles are simply mixed in the aqueous phase, and the interaction force is weaker; the release speed of VD3 in the core layer is faster than that of VB 12 in the shell layer, possibly because the drug concentration of VD3 is larger. In summary, the nanofiber system has good sustained release effect for amlodipine, VD3 and VB 12 , and can achieve differential release of the three drugs.

[0077] Example 2

[0078] A preparation method of a three-drug core-shell structure nanofiber, comprising the following steps:

[0079] 1) The specific steps for preparing self-assembled colloidal particles are as follows: prepare 1.5 mg / mL lysozyme (Lys) aqueous solution and 0.75 mg / mL carboxymethyl cellulose sodium (CMC) aqueous solution. Dissolve VB 12 in the Lys solution, so that the concentration of VB 12 in the VB 12 -Lys mixed solution is 3 mg / mL. Add 2 mL of the VB 12 -Lys mixed solution dropwise into 2 mL of the CMC solution and mix uniformly; under the weak interactions of electrostatic force and hydrogen bond, the CMC, Lys and VB 12 self-assemble to form VB 12 -Lys / CMC colloidal particles, and the whole process takes about 1.5 h.

[0080] 2) The specific steps for preparing the Pickering emulsion are as follows:

[0081] 2-1) Dissolve gelatin in a pH = 4.5 HAc solution to obtain a gelatin solution with a mass concentration of 35%. Add amlodipine to the solution, stir to dissolve, and obtain amlodipine with a mass concentration of 5%. Add the 0.375 mg / mL colloidal particle solution prepared in step 1) dropwise to the mixed solution of gelatin and amlodipine, stirring while adding, so that the volume ratio of the mixed solution to the colloidal particle solution is 6:4, to obtain the water phase for preparing the emulsion.

[0082] 2-2) Dissolve VD3 in corn oil to obtain the oil phase for preparing the emulsion, and the concentration of VD3 is 3 mg / mL.

[0083] 2-3) Mix 5.0 mL of the water phase and 1.0 mL of the oil phase, and then homogenize the mixture, at a homogenization speed of 10,000 r / min, a homogenization time of 3 min, and an oil-water ratio of 0.2, to obtain a stable Pickering emulsion.

[0084] 3) The specific steps for preparing the nanofiber are as follows:

[0085] Place the emulsion prepared in step 2) in a 5 mL syringe, connect the syringe pump to a receiving device, and prepare the core-shell structure nanofiber under the following conditions: an electric voltage of 26 kV, a pushing rate of 0.0001 mm / s, and a distance between the receiving plate and the syringe needle of 14 cm.

[0086] Test the drug release:

[0087] Take a certain amount of nanofiber and place it in three dialysis bags, and then place the dialysis bags in 90 mL of phosphate buffer PBS (pH 7.0) at 37°C for dynamic dialysis for 170 h. At certain time intervals, take out 5 mL of dialysate to measure the absorbance value of the drug at a specific wavelength, and supplement the same volume of buffer to draw the cumulative release curve of the drug.

[0088] Figure 4 The cumulative release curves of the core-shell structure nanofiber for the three drugs in Example 2 are shown in the figure. It can be seen from the curves that the release curves of the three drugs have the same trend as Example 1. However, careful observation shows that the oil-water ratio in this example is smaller, the hydrophilicity of the fiber is stronger, and the release rate of the drug is faster. Compared with Example 1, the cumulative release amount of the drug reaches a relatively high value in a shorter time.

[0089] Example 3

[0090] A method for preparing a three-drug core-shell structure nanofiber, comprising the following steps:

[0091] 1) Prepare VB 12The specific steps for preparing the HA / CS colloidal particle solution are as follows: Prepare a 2 mg / mL hyaluronic acid (HA) aqueous solution and a 2 mg / mL chitosan (CS) aqueous solution; add VB... 12 Dissolves in HA solution, allowing VB to... 12 -VB in HA mixture 12 The concentration is 2 mg / mL. Add 2 mL of VB. 12 - The HA mixed solution was added dropwise to 2 mL of CS solution and mixed thoroughly. Under the weak interactions such as electrostatics and hydrogen bonding, CS, HA and VB... 12 Self-assembly forms VB 12 -HA / CS colloidal particles, the whole process takes about 1.5 hours.

[0092] Figure 5 For example, VB in Example 3 12 - Particle size distribution, Tyndall effect (inset in a), and SEM images of HA / CS colloidal particles. Figure 5 As can be seen from a, VB in aqueous solution 12 -HA / CS colloidal particles have a particle size of 280nm. Figure 1 As shown in b, the colloidal particles also have a spherical structure with a particle size of about 240 nm. This may be because the SEM image shows the colloidal particles in a contracted, dry state after the water has evaporated.

[0093] 2) The specific steps for preparing Pickering emulsion are as follows:

[0094] 2-1) Dissolve gelatin in a pH 4.5 HAc solution to obtain a 35% gelatin solution. Add amlodipine to the solution and stir to dissolve, bringing the amlodipine concentration to 5%. Add the 1 mg / mL colloidal particle solution prepared in step 1) dropwise to the mixed solution of gelatin and amlodipine while stirring, so that the volume ratio of the mixed solution to the colloidal particle solution is 6:4, obtaining the aqueous phase of the prepared emulsion.

[0095] 2-2) Dissolve VD3 in corn oil to obtain the oil phase for preparing the emulsion. The concentration of VD3 is 3 mg / mL.

[0096] 2-3) Take 3.8 mL of aqueous phase and 1.2 mL of oil phase, mix them and homogenize them. The homogenization speed is 10000 r / min, the homogenization time is 3 min, and the oil-water ratio is 0.3 to obtain a stable Pickering emulsion.

[0097] 3) The specific steps for preparing nanofibers are as follows:

[0098] The emulsion prepared in step 2) was placed in a 5 mL syringe, and a syringe pump was connected to a receiving device. The nanofiber with core-shell structure was prepared under the conditions of a voltage of 26 kV, a pushing rate of 0.0001 mm / s, and a distance between the receiving plate and the syringe needle of 12 cm.

[0099] Figure 6 SEM and TEM were performed on the fiber prepared in Example 3. It can be seen from the SEM that the diameter of the fiber is relatively uniform, about 250 nm. It can be seen from the TEM image that the prepared fiber is a fiber with core-shell structure, and the core layer and the shell layer have a clear boundary.

[0100] Drug release test was performed:

[0101] An amount of nanofiber was taken and placed in three dialysis bags, which were then placed in 90 mL of phosphate buffer PBS (pH 7.0) at 37°C for dynamic dialysis for 170 h. Every certain time, 5 mL of dialysate was taken out to measure the absorbance value of the drug at a specific wavelength, and an equal volume of buffer was supplemented to draw the cumulative release curve of the drug.

[0102] Figure 7 The drug release curves of the core-shell structure nanofiber in Example 3 for three kinds of hydrophilic and hydrophobic drugs were drawn. It can be seen from the curves that this system can also achieve differential release of the three kinds of drugs.

[0103] Example 4

[0104] A preparation method of a three-drug core-shell structure nanofiber, comprising the following steps:

[0105] 1) Preparation of VB 12 The specific steps of the HA / CS colloidal particle solution are as follows: 2 mg / mL hyaluronic acid (HA) aqueous solution and 2 mg / mL chitosan (CS) aqueous solution were prepared; VB 12 was dissolved in the HA solution, so that VB 12 in the HA mixture had a concentration of 2 mg / mL; 2 mL of the VB 12 HA mixed solution was added dropwise into 2 mL of the CS solution and mixed uniformly; under the weak interactions of electrostatic force and hydrogen bond, the CS, HA and VB 12 self-assembled to form VB 12 HA / CS colloidal particles, and the whole process took about 1.5 h. 12 HA / CS colloidal particles, and the whole process took about 1.5 h.

[0106] 2) The specific steps of the Pickering emulsion preparation are as follows:

[0107] 2-1) Dissolve gelatin in HAc solution with pH = 4.5 to obtain a gelatin solution with a mass concentration of 35%, and then add amlodipine into the solution and stir to dissolve, so that the concentration of amlodipine is 5%; add the colloidal particle solution prepared in step 1) dropwise into the mixed solution of gelatin and amlodipine, and stir while adding, so that the volume ratio of the mixed solution to the colloidal particle solution is 7:3, to obtain an aqueous phase for preparing an emulsion.

[0108] 2-2) Dissolve VD3 in corn oil to obtain an oil phase for preparing an emulsion, and the concentration of VD3 is 3 mg / mL.

[0109] 2-3) Mix 4.3 mL of the aqueous phase and 1.7 mL of the oil phase, and then homogenize the mixture, the homogenization speed is 10,000 r / min, the homogenization time is 3 min, and the oil-water ratio is 0.4, to obtain a stable Pickering emulsion.

[0110] 3) The specific steps for preparing the nanofiber are as follows:

[0111] Place the emulsion prepared in step 2) in a 5 mL syringe, connect the syringe pump with a receiving device, and prepare the nanofiber with a core-shell structure under the conditions that the voltage is 26 kV, the pushing rate is 0.0001 mm / s, and the distance between the receiving plate and the syringe needle is 12 cm.

[0112] Test the drug release:

[0113] Take a certain amount of the nanofiber, place it in three dialysis bags, and then put the dialysis bags into 90 mL of phosphate buffer PBS (pH 7.0) at 37°C, and dialyze dynamically for 170 h. Take out 5 mL of the dialysate every certain time to measure the absorbance value of the drug at a specific wavelength, and then supplement the buffer with the same volume, to draw a cumulative release curve of the drug.

[0114] Figure 8 The cumulative release curves of the three drugs in the core-shell structure nanofiber in Example 4 are shown in the figure. It can be seen from the curves that, compared with Example 3, the burst release phenomenon of the three drugs in the initial stage is obviously inhibited, and the cumulative release amount of the three drugs is also reduced. It indicates that the drug release effect of this system is better. It may be because the oil-water ratio of this system is large, the hydrophobicity of the fiber is strong, and the drug release rate is slow.

[0115] Comparative Example 1

[0116] A preparation method of a three-drug core-shell structure nanofiber, comprising the following steps:

[0117] 1) Preparation of VB 12 The specific steps for preparing the Lys / CMC mixed solution are as follows: prepare a 1.5 mg / mL lysozyme (Lys) aqueous solution and a 0.75 mg / mL carboxymethyl cellulose sodium (CMC) aqueous solution;12 VB is dissolved in the Lys solution to make a VB-Lys mixed solution 12 VB is dissolved in the Lys solution to make a VB-Lys mixed solution 12 The concentration is 3 mg / mL; 2 mL of VB is added dropwise into 2 mL of CMC solution 12 VB is dissolved in the Lys solution to make a VB-Lys mixed solution 12 VB is dissolved in the Lys solution to make a VB-Lys / CMC mixed solution.

[0118] 2) The specific steps for preparing the emulsion are as follows:

[0119] 2-1) Gelatin is dissolved in a pH 4.5 HAc solution to obtain a 50% gelatin solution. Amlopidine is added to the solution and stirred to dissolve, and the mass concentration is 5%. The colloidal particle solution prepared in step 1) is added dropwise into the mixed solution of gelatin and amlopidine, and stirred while adding, so that the volume ratio of the mixed solution and the colloidal particle solution is 7:3, to obtain an aqueous phase for preparing the emulsion.

[0120] 2-2) VD3 is dissolved in corn oil to obtain an oil phase for preparing the emulsion, and the concentration of VD3 is 3 mg / mL.

[0121] 2-3) 4.3 mL of the aqueous phase and 1.7 mL of the oil phase are mixed and homogenized, the homogenization speed is 10,000 r / min, the homogenization time is 3 min, and the oil-water ratio is 0.4, to obtain a stable emulsion.

[0122] 3) The specific steps for preparing the nanofiber are as follows:

[0123] The emulsion prepared in step 2) is placed in a 5 mL syringe, and a syringe pump and a receiving device are connected. Under the conditions of a voltage of 26 kV, a pushing rate of 0.0001 mm / s, and a distance between the receiving plate and the syringe needle of 16 cm, a core-shell structure nanofiber is prepared.

[0124] The results show that when the concentration of the auxiliary spinning agent gelatin is 50%, the viscosity of the emulsion is too large because the concentration of gelatin is too large, the needle is easily blocked during later spinning, and normal spinning cannot be performed. According to a large number of experimental explorations, the concentration of gelatin in the system of the application cannot be greater than 45%.

[0125] Comparative Example 2

[0126] A preparation method of a three-drug core-shell structure nanofiber, comprising the following steps:

[0127] 1) Preparation of VB 12 The specific steps for preparing the HA / CS colloidal particle solution are as follows: 2 mg / mL of a hyaluronic acid (HA) aqueous solution and 3 mg / mL of a chitosan (CS) aqueous solution are prepared; VB 12 VB is dissolved in the Lys solution to make a VB-Lys mixed solution 12HA mixed solution was added dropwise into 2 mL CS polysaccharide solution, and mixed evenly. Under the weak interactions of electrostatic force, hydrogen bond, etc., CS, HA and VB 12 concentration was 2 mg / mL; 2 mL VB 12 HA mixed solution was added dropwise into 2 mL CS polysaccharide solution, and mixed evenly. Under the weak interactions of electrostatic force, hydrogen bond, etc., CS, HA and VB 12 self-assembled to form VB 12 HA / CS colloidal particles.

[0128] It was determined that the particle size of the obtained colloidal particles reached more than 1000 nanometers, as shown in Figure 9 The concentration of chitosan was too large, the particle size of the obtained colloidal particles was too large, which led to poor stability and precipitation. It could not be further used to prepare emulsion, so it could not further obtain the core-shell structure nanofiber.

[0129] Comparative Example 3

[0130] A preparation method of a three-drug core-shell structure nanofiber, comprising the following steps:

[0131] 1) Preparation of VB 12 The specific steps of the HA / CS colloidal particle solution were as follows: 2 mg / mL hyaluronic acid (HA) aqueous solution and 2 mg / mL chitosan (CS) aqueous solution were prepared; VB 12 was dissolved in the HA solution, so that VB 12 The concentration of VB in the HA mixed solution was 2 mg / mL; 2 mL VB 12 The specific steps of the HA / CS colloidal particle solution were as follows: 2 mg / mL hyaluronic acid (HA) aqueous solution and 2 mg / mL chitosan (CS) aqueous solution were prepared; VB 12 HA mixed solution was added dropwise into 2 mL CS solution, and mixed evenly; under the weak interactions of electrostatic force, hydrogen bond, etc., CS, HA and VB 12 self-assembled to form VB 12 HA / CS colloidal particles.

[0132] 2) The specific steps of Pickering emulsion preparation were as follows:

[0133] 2-1) Gelatin was dissolved in a pH=4.5 HAc solution to obtain a gelatin solution with a mass concentration of 35%, and amlodipine was added to the solution and stirred to dissolve, so that the mass concentration of amlodipine was 5%; the colloidal particle solution prepared in step 1) was added dropwise into the mixed solution of gelatin and amlodipine, and stirred while adding, so that the volume ratio of the mixed solution to the colloidal particle solution was 7:3, to obtain an aqueous phase for preparing emulsion.

[0134] 2-2) VD3 was dissolved in corn oil to obtain an oil phase for preparing emulsion, and the concentration of VD3 was 3 mg / mL.

[0135] 2-3) Take 5.5 mL of the water phase and 0.5 mL of the oil phase, mix them and homogenize the emulsion, the homogenization speed is 10000 r / min, the homogenization time is 3 min, the oil-water ratio is 0.1, and a stable Pickering emulsion is obtained.

[0136] 3) The specific steps for preparing the nanofiber are as follows:

[0137] The emulsion prepared in step 2) is placed in a 5 mL syringe, a syringe pump is connected to a receiving device, and under the conditions of a voltage of 22 kV, a pushing rate of 0.0001 mm / s, and a distance between the receiving plate and the syringe needle of 12 cm, a nanofiber with a core-shell structure is prepared.

[0138] Test of drug sustained release:

[0139] Because the oil-water ratio is 0.1 when preparing the emulsion, the volume of the oil phase is too small. The low oil content leads to weak hydrophobicity and strong hydrophilicity of the fiber. Therefore, in the sustained release experiment, the fiber dissolves quickly and good sustained release data cannot be obtained.

Claims

1. A method for preparing a tri-drug core-shell structure nanofiber, characterized in that, The preparation method comprises the following steps: 1) preparing first hydrophilic drug / biomacromolecule self-assembled colloidal particles; 2) mixing a solution containing the first hydrophilic drug / biomacromolecule self-assembled colloidal particles and a second hydrophilic drug as an aqueous phase with an oil phase containing a hydrophobic drug, homogenously emulsifying to prepare a Pickering emulsion; 3) electrospinning the Pickering emulsion to obtain the tri-drug-loaded core-shell structure nanofiber; Step 1) specifically comprises: 1-1) dissolving the first hydrophilic drug in a biomacromolecule solution to obtain a drug-containing mixed solution; 1-2) adding the drug-containing mixed solution dropwise to a polysaccharide solution to obtain the first hydrophilic drug / biomacromolecule self-assembled colloidal particles; the concentration of the polysaccharide solution is 0.5-2 mg / ml; In step 1-1), the concentration of the biomacromolecule solution is 0.5-2.5 mg / ml; In step 1-1), the biomacromolecule is selected from lysozyme, and in step 1-2), the polysaccharide is selected from sodium carboxymethyl cellulose; or, in step 1-1), the biomacromolecule is selected from hyaluronic acid, and in step 1-2), the polysaccharide is selected from chitosan; Step 2) specifically comprises: 2-1) adding the solution containing the first hydrophilic drug / biomacromolecule self-assembled colloidal particles dropwise to a mixed solution of a second hydrophilic drug and a spinning aid as an aqueous phase; the spinning aid is gelatin; 2-2) dissolving the hydrophobic drug in oil as an oil phase; 2-3) mixing the aqueous phase and the oil phase, homogenously emulsifying to prepare a Pickering emulsion; In step 2-1), the volume ratio of the mixed solution of the spinning aid and the second hydrophilic drug to the solution containing the first hydrophilic drug / biomacromolecule self-assembled colloidal particles is 4-7:6-3; in the mixed solution of the spinning aid and the second hydrophilic drug, the mass concentration of the spinning aid is 15%-45%, and the mass concentration of the second hydrophilic drug is 2.5%-10%; In step 2-3), the volume ratio of the oil phase to the aqueous phase is 0.1-0.4:1; The drug release rate in the tri-drug-loaded core-shell structure nanofiber is: second hydrophilic drug > hydrophobic drug > first hydrophilic drug.

2. The production method according to claim 1, characterized by, In step 1-1), the concentration of the first hydrophilic drug in the drug-containing mixed solution is 1-5 mg / ml.

3. A tri-drug-loaded core-shell structure nanofiber prepared by the preparation method of any one of claims 1-2.

4. Use of the tri-drug-loaded core-shell structure nanofiber prepared by the preparation method of any one of claims 1-2 in preparing a biological coating, a drug release carrier or a tissue engineering scaffold.

Images