CN101721377A - Method for preparing microspheres with solid-in-oil-in-hydrophilic oil-in-ethanol - Google Patents

Abstract

The invention discloses a method for preparing hydrophilic oil-in-ethanol-oil-in-hydrophilic oil-in-oil solid microspheres in the field of pharmaceutical technology. It includes the following steps: ①Add the drug particles into the organic solution of the sustained-release or controlled-release material, that is, the oil phase, and stir or vortex; ②Add the suspension to another hydrophilic organic solution, that is, the hydrophilic oil phase, and stir for 0.1-5min to form 1-500 μm balls; ③ Transfer the microsphere suspension containing the completed step ② to ethanol for solidification for 1-4 hours; ④ Freeze-dry the sample completed step ③ to obtain dry microspheres. The particle size of the microspheres prepared by the present invention can be controlled and does not pollute the environment; the surface of the particles is smooth and round, the particles are regular and non-adhesive, the particle size can be adjusted from 1 μm to 500 μm according to the needs, and the freeze-dried powder is white and delicate , loose, no collapse, no adhesion, good re-dispersibility. It can be applied to the preparation of slow-release or controlled-release microspheres of various drugs and adjuvants of vaccines.

Description

Method for preparing hydrophilic oil in ethanol-oil-in-hydrophilic-oil-in-oil solid microspheres

technical field

The invention relates to a preparation method in the field of pharmaceutical technology, in particular to a method for preparing hydrophilic oil-in-ethanol-oil-in-oil-oil-in-oil solid (S/O/hO/E) microspheres.

Background technique

In the pharmaceutical industry, from drug discovery to clinical application, the last link is drug preparation. Some of the drugs need long-term administration to be cured; others need targeted and other local administration. To achieve these goals, raw materials must be prepared into corresponding dosage forms. For example, drugs that require long-term administration but have a short half-life in the body should be prepared as sustained-release dosage forms; for the treatment of some tumors, some drugs need to be targeted to the disease, such as embolization microsphere preparations that target tumor blood vessels; Since recombinant technology has been used in the expression and production of therapeutic proteins for more than 20 years, so far, more than 30 protein drug products have been put into clinical use, and nearly 200 are in the process of approval and development. ), Gene Technology (Genentech) and a number of new large pharmaceutical companies. Compared with the rapid development of protein macromolecular drugs, their dosage form technology progresses slowly. On the one hand, protein macromolecular drugs are not absorbed orally and have a short half-life in the body, so they need to be administered by injection; on the other hand, many hormone and cytokine protein drugs have a long treatment cycle, and long-term and frequent injections are necessary, which also affects patients. The main reason for compliance. In the research and development of dosage forms of sustained-release protein drugs, microspheres prepared by the W/O/W method, S/O/W method and S/O/O method with low encapsulation efficiency are easy to lose due to the loss of activity during the preparation of microparticles. Disadvantages such as sudden release. It is imperative to develop methods for preparing protein microspheres with active protection and improving encapsulation efficiency and burst release. So far, there is no report on the preparation of microspheres by the method of hydrophilic oil in ethanol-solid in oil in oil (S/O/hO/E).

[Lee E.S., Kwon M.J., Lee H., and Kim J.J., Stabilization of protein encapsulated in poly(lactide-co-glycolide) microspheres by novel viscous S/W/O/W method was found by searching the prior art literature , International Journal ofPharmaceutics 331(2007) 27-37], (Lee E.S., Kwon M.J., Lee H., and Kim J.J., reported the use of new viscosity S/W/O/W method to stably encapsulate protein in PLGA micro Balli, International Journal of Pharmacy, 2007, 331: 27-37). Lee E.S. et al. reported in the literature that microspheres were prepared using the S/W/O/W method. This document utilizes protein cyclodextrin to be freeze-dried and then ground, and the ground protein cyclodextrin is added to a high-viscosity polysaccharide solution and a dichloromethane solution of PLGA to emulsify, and finally to the water phase to harden the microspheres and only target proteins Drugs, no other drugs have been reported. However, protein cyclodextrin particles are inevitably insoluble in contact with polysaccharide aqueous solution, and the dissolved protein aqueous solution is easy to contact with PLGA's dichloromethane, and there is an oil-water interface, which leads to aggregation. Also cause encapsulation efficiency is not high, there is incomplete release. [Morita T., Sakamura Y., Horikiri Y., Suzuki T., Yoshino H., Protein encapsulation into biodegradable microspheres by a novel S/O/W emulsion method using poly(ethyleneglycol) as a protein micronization adjuvant, Journal Rease Control 69(2000) 435-444], (Morita T. et al. have reported in this literature that utilizes new S/O/W emulsification method to prepare protein-carrying microspheres. Just changed surfactant and reported more is to use PVA before, In this document, PEG is used instead. But it still cannot overcome the shortcomings of low encapsulation efficiency, burst release and incomplete release.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing ethanol-in-hydrophilic-oil-hydrophilic-oil-in-oil-in-oil solid microspheres. The surface of the prepared particles is smooth and round, the uniformity is good, the particles are regular and non-adhesive; the encapsulation rate is high, the burst release is small, and the drug loading capacity is high.

The present invention is achieved through the following technical solutions. The present invention disperses the drug particles in an organic solution with slow-release or controlled-release materials, and stirs, vortex or ultrasonically disperses them to form a suspension; then the suspension Add to the outer oil phase, then stir or vortex to form microspheres, and finally transfer it to the large water phase to solidify for 1-4 hours; then collect the microspheres by centrifugation, freeze-dry and store.

The present invention comprises the following steps:

① Add the drug particles to the organic solution of the sustained-release or controlled-release material, that is, the oil phase (O), and stir or vortex to make them uniformly dispersed to form a suspension;

The weight percentage of the drug granules, slow-release or controlled-release materials is 0-50%, and the slow-release or controlled-release materials are 50-100%.

②Add the suspension to another hydrophilic organic solution, that is, the hydrophilic oil phase (hO), which is insoluble in the organic solvent of the slow-release or controlled-release material, and stir for 0.5-5min to form 1-500μm balls.

③Transfer the microsphere suspension containing the completed step ② into ethanol (E) and solidify for 1-4 hours.

④ Freeze-drying the sample completed in step ③ to obtain dry microspheres.

The drug particles refer to water-soluble drug particles, or particles prepared by loading drugs into excipients, or oil-soluble particles prepared to be insoluble in organic solvents through preparation methods;

The drugs include small molecule drugs and macromolecular drugs;

The small molecular drugs refer to chemical drugs, and the macromolecular drugs mainly refer to biological macromolecular drugs, especially protein macromolecular drugs, vaccines, antibodies, nucleic acid or liposome drugs.

The protein macromolecular drugs are: erythropoietin (EPO), recombinant human granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), vaccine, interferon ( INF), growth hormone (GH), insulin (Insulin), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-β), insulin-like growth factor (IGF), vascular endothelial cells Growth factor (VEGF), platelet growth factor (PDGF), endothelial growth factor (ECGF), nerve growth factor (NGF), bone-derived growth factor (BDGF), bone morphogenic protein (BMP), tissue polypeptide antigen (TPA), Antibody, coagulation factor VIII (VIII), or genetic factor of coagulation factor IX;

The nucleic acid is: antisense nucleotide (anti-RNA), small molecule RNA (RNAi) or gene (DNA);

Described adjuvant refers to mainly injectable grade, especially small sugars (sucrose, trehalose, glucose, maltose, or lactose), and polyhydroxy compounds (mannitol, sorbitol, glycerol, 1,2- Propylene glycol, erythritol, polyethylene glycol, polyvinyl alcohol, polyethylene oxide, or polypyrrolidone;), polysaccharides (dextran, sodium alginate, chitosan, starch, cellulose, PEI, polyarginine or cyclodextrin substances), amino acid compounds (glycine, lysine, arginine, glutamic acid or histidine;), or inorganic salts (zinc salts, calcium salts, copper salts, One or any combination of magnesium salt or molybdenum salt);

The drug particles have a particle size of 0.2-10 μmm, preferably 1-5 μmm.

The drug particles account for 0-50% of the total weight of the microspheres;

The material of described sustained release or controlled release is: polylactic acid (PLA), polylactic acid-polyglycolic acid (PLGA), any combination of PLA and PLGA, silicone rubber, polytetrafluoroethylene, polyvinyl chloride, polyethylene , polypropylene, polystyrene, polyethylene terephthalate, polycarbonate, carbomer, hyaluronic acid, gelatin, collagen, polycyanoacrylate, polyphosphazene, polyphosphate, fiber Proteinogen, fibrin, polyethylene glycol-polylactic acid (PLA-PEG), polyethylene glycol-polyglycolic acid (PLGA-PEG), polyglycolic acid-polyethylene glycol-polyglycolic acid (PLGA-PEG- PLGA), polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA), polyglycolide, polyethylene glycol-polycaprolactone (PEG-PCL), polycaprolactone poly-ethylene glycol - polycaprolactone (PCL-PEG-PCL), or polycaprolactone (PCL);

The slow-release or controlled-release material accounts for 50-100% of the total weight of the microspheres;

The oil phase (O) is: an organic solution of slow-release or controlled-release materials;

The organic solution of the slow-release or controlled-release material is: polylactic acid (PLA), polylactic acid-polyglycolic acid (PLGA), polyglycolic acid (PGA), polylactic acid (PLA) and polylactic acid-polyglycolic acid (PLGA), hyaluronic acid, gelatin, collagen, polycyanoacrylate, polyphosphazene, polyphosphate, fibrinogen, fibrin, polylactic acid-polyethylene glycol (PLA-PEG), Polylactic acid-polyglycolic acid-polyethylene glycol (PLGA-PEG), polylactic acid-polyglycolic acid-polyethylene glycol-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA), polylactic acid-polyethylene glycol -polylactic acid (PLA-PEG-PLA), polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLA-PEG), polyethylene glycol-polylactic acid-polyglycolic acid-polyethylene glycol (PEG- PLGA-PEG), polycarbonate, carbomer, hyaluronic acid, gelatin, collagen, polyethylene glycol-polycaprolactone (PEG-PCL), polycaprolactone poly-polyethylene glycol-polycaprolactone Lactone (PCL-PEG-PCL), polycaprolactone, silicone rubber, polytetrafluoroethylene, polyvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polycarbonate Dichloromethane, ethyl acetate, acetonitrile, heptane, chloroform or acetone organic solution of ester or carbomer, preferably the organic solution of dichloromethane, ethyl acetate, acetonitrile or any combination thereof;

The weight percent concentration of the organic solution of the slow-release or controlled-release material is: 2-30%;

The hydrophilic oil phase (hO) is: surfactant, glycerin, ethanol, propylene glycol, ethylene glycol solution, liquid polyethylene glycol or their combination;

Described their composition is: the aqueous solution of glycerin and ethanol plus surfactant, the aqueous solution of ethanol plus surfactant, the aqueous solution of glycerol and propylene glycol plus surfactant, the aqueous solution of propylene glycol plus surfactant, glycerol and ethylene glycol Aqueous solution with surfactant, aqueous solution of ethylene glycol with surfactant, aqueous solution of glycerin and liquid polyethylene glycol with surfactant, or liquid polyethylene glycol with surfactant;

The water-soluble of described tensio-active agent is: the mixed aqueous solution of salts such as polyvinyl alcohol (PVA) and sodium chloride, and their weight percentage concentration is respectively 1-10% and 0-10%; Polyethylene glycol (PEG) The mixed aqueous solutions of salts such as polyvinylpyrrolidone (PVP) and sodium chloride, their weight percentage concentrations are respectively 1-10% and 0-10%; the mixed aqueous solutions of polyvinylpyrrolidone (PVP) and sodium chloride etc. 1-10% and 0-10%; mixed aqueous solutions of salts such as poloxmer and sodium chloride, whose weight percent concentrations are 1-10% and 0-10% respectively; polysorbate and sodium chloride The mixed aqueous solution of salt such as, their weight percent concentration is respectively 1-10% and 0-10%; Or the mixed aqueous solution of salt such as ethyl cellulose (EC) and sodium chloride, their weight percent concentration is respectively 1- 10% and 0-10%;

The suitable ratio is: 0-50% by weight of glycerin, 0-30% by weight of surfactant aqueous solution, and 50-100% by weight of ethanol, propylene glycol, ethylene glycol, or liquid polyethylene glycol; 20-30% by weight of glycerin, 70-80% ethanol, propylene glycol, ethylene glycol, or liquid polyethylene glycol 50-100% is better, and the weight percentage of surfactant aqueous solution is 5-15% ;

The ethanol phase (E) is ethanol, propylene glycol or any mixed solution thereof that can dissolve the oil phase and the hydrophilic oil phase;

The particle size of the microspheres is 1-500 μm, preferably 10-100 μm;

The present invention selects suitable oil phase (O), hydrophilic oil phase (hO) and ethanol phase (E) and suitable controlled-release or slow-release materials, so that water-soluble drug particles or oil-soluble drugs pass through the preparation method Prepare particles insoluble in organic solvents to avoid the encapsulation efficiency of conventional W/O, W/O/W, S/O/W is not high, and the sudden release of S/O/O is serious, and the environmental pollution caused The shortcomings of microspheres; using this method to prepare microspheres, the size of the particle size can be controlled according to different needs, without polluting the environment; it can avoid the effect on the treatment of drugs, especially those whose physical and chemical properties are unstable, and the oil-water interface Sensitive drugs, such as biomacromolecular drugs such as protein macromolecular drugs, DNA, RNA or siRNA drugs. The surface of the particles is smooth and round, the particles are regular and non-adhesive, and the particle size can be adjusted from 1 μm to 500 μm according to the needs. The lyophilized powder is white, fine, loose, non-collapsing, non-adhesive, and has good redispersibility. It can be applied to the preparation of various drug sustained-release microspheres and the preparation of vaccine adjuvants.

Description of drawings

Figure 1 SEM image of prepared microspheres

Detailed ways

The following examples of the present invention are described in detail: the following examples are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and processes are provided, but the protection scope of the present invention is not limited to the following examples.

Conditions for the implementation of the following examples:

1. Preparation of blank microspheres

1. Preparation of oil phase (O) and hydrophilic oil phase (hO)

Preparation of the oil phase (O): the controlled-release or slow-release material is dissolved in an organic solvent to prepare a concentration of 1-50% by weight to form the oil phase (O).

The preparation of hydrophilic oil phase (hO): formula 1: use surfactant and sodium chloride etc. salt mixed aqueous solution, their weight percentage concentration is respectively 1-10% and 0-10%, accounts for the weight percentage of hydrophilic oil phase 0-40%; ethanol, ethylene glycol, propylene glycol or normal temperature liquid polyethylene glycol is 60-100%; or formula 2: mixed aqueous solution with salts such as surfactant and sodium chloride, and their weight percentage concentrations are respectively 1-10% and 0-10%, accounting for 0-40% by weight of the hydrophilic oil phase; use 0-50% by weight of glycerin and 50-100% of ethanol, ethylene glycol, propylene glycol or normal temperature liquid The polyethylene glycol solution accounts for 60-100% of the hydrophilic oil phase;

Preparation of ethanol phase (E): 100-1000mL of ethanol, ethylene glycol, propylene glycol or any mixture thereof;

2. Preparation of Blank Microspheres

Add the oil phase (O) dropwise to the hydrophilic oil phase (hO) and stir, sonicate or vortex for 0.5-5 minutes to form O/hO, then transfer to the ethanol phase to 100-1000mL and stir for 1-4 hours, centrifuge to collect the microspheres and Dry microspheres are obtained by lyophilization or evaporation of organic solvents.

2. Preparation of drug-loaded microspheres

1. To prepare drug granules, for small molecule drugs, it can be conventional grinding method, precipitation method, pulverization method or granulation method; for biomacromolecular drugs, it can be water phase-water phase emulsion method, spray drying method, phase separation method, supercritical method or the method of complexes formed by metal ions and biomacromolecules;

2. Preparation of oil phase (O) and hydrophilic oil phase (hO)

All follow the preparation method of blank oil phase (O) and hydrophilic oil phase (hO).

3. Mix the drug particles with the oil phase (O) to form a suspension;

4. Add the suspension in step 3 to the hydrophilic oil phase (hO) dropwise, and stir, vortex or sonicate for 0.5-5 minutes to form a double emulsion;

5. Add the double emulsion in step 4 dropwise to 100-1000mL ethanol phase (E), and stir for 1-4 hours, collect the microspheres by centrifugation and wash 3-5 times, and then lyophilize to obtain the microspheres.

The following examples overcome the shortcomings of low water-soluble drug encapsulation rate and serious burst release; Biomacromolecular drugs, such as proteins, peptides, vaccines, DNA, RNA, SiRNA, viruses or liposomes, are loaded into microspheres under certain conditions, which can maintain their activity for a long time. Greatly reduce the cost of preservation and improve the curative effect. The polysaccharide vitreous body prepared at the same time has small particle size and high activity, so when it is prepared into other dosage forms such as sustained-release microspheres, burst release and incomplete release can be reduced.

Embodiment one

Preparation of polylactic acid-polyglycolic acid (PLGA) microspheres loaded with small molecule drug particles

(1) According to the small molecule drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) and the weight percentage concentration is 2 %, 10%, 15%, 20% or 30% PLGA in dichloromethane solution weight ratio of 1:20, 1:30, 1:30, 1:45 or 1:4 equal proportion stirring, vortex or ultrasonic 0.5- Form a uniform suspension in 5 minutes, i.e. a solid in oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50%, 40%, 25%, 10% or 0% ;PLGA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 %, 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 800mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

Figure 1A shows the scanning electron microscope of microspheres prepared according to 15% by weight concentration of PLGA dichloromethane solution, and the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment two

Preparation of polylactic acid-polyglycolic acid (PLGA) microspheres loaded with biomacromolecular drug particles

(1) According to the concentration of 0.4mg, 2mg, 1.5mg, 1mg or 0mg of biomacromolecular drug particles (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) and the weight percentage concentration is The weight ratio of 2%, 10%, 15%, 20% or 30% PLGA in dichloromethane solution is 1:20, 1:30, 1:30, 1:45 or 1:4. Stir, vortex or sonicate 0.5 -5 minutes to form a uniform suspension i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components are respectively: 50%, 40%, 25%, 10% or 0 %; 50%, 60%, 75%, 90% or 100% for PLGA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 8%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 1000mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

Figure 1B is a scanning electron microscope of microspheres prepared according to 15% by weight concentration of PLGA dichloromethane solution, and the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment three

Preparation of polylactic acid (PLA) microspheres loaded with erythropoietin (EPO) biomacromolecular drug particles

(1) According to the erythropoietin (EPO) biomacromolecule drug particle 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA in dichloromethane solution weight ratio of 1:20, 1:30, 1:30, 1:45 or 1:4 etc. Proportional stirring, vortex or ultrasound for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40%, 25%, 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 800mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The scanning electron microscope image 1C of microspheres prepared according to 15% by weight concentration of PLGA in dichloromethane solution, the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment four

Preparation of polylactic acid-polyglycolic acid (PLGA) microspheres loaded with recombinant human granulocyte colony-stimulating factor (G-CSF) biomacromolecular drug particles

(1) According to the recombinant human granulocyte colony-stimulating factor (G-CSF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm respectively) , 2-10 μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA methylene chloride solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO): [The preparation of the hydrophilic oil phase (hO): formula 2: use polyethylene glycol (PEG) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounts for the weight percentage of hydrophilic oil phase respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40 or 50% by weight of glycerin and 90% of ethanol, 80% of ethylene glycol, 80% % ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase according to 99%, 95%, 90%, 80%, 60%] and stirring, vortex or Ultrasound for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to ethanol phase (E) [preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 10% ethylene glycol and 90% ethanol Ethanol or 400mL of 30% by weight 1,2 propylene glycol and 70% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

Scanning electron microscope image 1D of microspheres prepared according to 15% by weight concentration of PLGA dichloromethane solution, the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment five

Preparation of polyglycolic acid (PGA) microspheres loaded with cell-macrophage colony-stimulating factor (GM-CSF)

(1) According to the acetonitrile solution of granulocyte-macrophage colony-stimulating factor (GM-CSF) drug particles and PGA 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm respectively) , 2-6 μm, 2-10 μm or 0 μm) and the weight percent concentration of 2%, 10%, 15%, 20% or 30% PGA in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30. 1:45 or 1:4 equal ratio stirring, vortex or ultrasonic for 1-5 minutes to form a uniform suspension, that is, solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounted for the components of the microspheres) Percentages are: 50%, 40%, 25%, 10% or 0%, respectively; PGA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO): [The preparation of the hydrophilic oil phase (hO): formula 2: use polyethylene glycol (PEG) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounts for the weight percentage of hydrophilic oil phase respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40% or 50% by weight of glycerin and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase, according to 99%, 95%, 90%, 80%, 60%] and stirring, Vortex or ultrasonic for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 800mL weight percent 10% ethylene glycol and 90% ethanol or 1000mL of 30% by weight 1,2 propylene glycol and 70% ethanol], neutralized and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

According to the scanning electron microscope image 1E of the microspheres prepared according to the acetonitrile solution of 15% concentration of PGA by weight, the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PGA is similar to the above scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 20-100 μm, 60-150 μm, 70-180 μm Or 200-500μm.

Embodiment six

Preparation of polylactic acid (PLA) microspheres loaded with interferon (INF) biomacromolecular drug particles

(1) According to the interferon (INF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) And the weight percentage concentration is 2%, 10%, 15%, 20% or 30% PLA dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio stirring , vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounted for the percentage of microsphere components are: 50%, 40%, 25% , 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 2: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their percentage by weight concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the percentage by weight of accounting for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40% or 50% glycerin with 90% ethanol, 80% ethylene glycol, 80% Hydrophilic oil phase formulated with ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol, according to 99%, 95%, 90%, 80%, 60%] and stirring, vortex or Ultrasound for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 10% ethylene glycol and 90% ethanol Ethanol or 1000mL of 30% by weight of 1,2 propylene glycol and 70% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The scanning electron microscope image 1F of the microspheres prepared according to the 15% by weight concentration of PLA in dichloromethane solution, the particle size is about 50-120 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 20-100 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment seven

Preparation of polylactic acid-polyglycolic acid (PLGA) microspheres loaded with platelet growth factor (PDGF) biomacromolecular drug particles

(1) According to the platelet growth factor (PDGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio Stir, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding percentages of drug particles accounting for microsphere components: 50%, 40%, 25% %, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 2: use polyethylene glycol (PEG) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounts for the weight percentage of hydrophilic oil phase respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40% or 50% by weight of glycerin and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase, according to 99%, 95%, 90%, 80%, 60%] and stirring, Vortex or ultrasonic for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to ethanol phase (E) [preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 10% ethylene glycol and 90% ethanol Ethanol or 400mL of 30% by weight of 1,2 propylene glycol and 70% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 40-100 μm, 60- 150μm, 70-180μm or 200-500μm.

Embodiment eight

Preparation of polycaprolactone (PCL) microspheres loaded with growth hormone (GH) biomacromolecular drug particles

(1) According to growth hormone (GH) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) And the weight percentage concentration is that the dichloromethane solution weight ratio of 2%, 10%, 15%, 20% or 30% polycaprolactone (PCL) is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio stirring, vortex or ultrasonic 1-5 minutes to form a uniform suspension, that is, solid-in-oil (S/O) emulsion (the percentage of the corresponding drug particles accounting for the components of the microspheres is respectively: 50% , 40%, 25%, 10% or 0%; polycaprolactone (PCL) is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 2: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their percentage by weight concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the percentage by weight of accounting for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40% (w/w); and 10%, 20%, 25%, 40% or 50% by weight of glycerol with 90% ethanol, 80% Ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase according to 99%, 95%, 90%, 80%, 60%] And stir, vortex or ultrasonic for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): the weight percent 10% ethylene glycol and 90% ethanol of 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL or 1000mL of 30% by weight 1,2 propylene glycol and 70% ethanol], neutralized and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

According to 2%, 10%, 15%, 20% or 30% polycaprolactone (PCL), the appearance of microspheres is similar to the above-mentioned scanning electron microscope Fig. 1 is not shown on the figure, and its size is respectively: 1-20 μ m, 40-120 μm, 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment nine

Preparation of polylactic acid-polyethylene glycol (PLA-PEG) microspheres loaded with vascular endothelial growth factor (VEGF) biomacromolecular drug particles

(1) According to vascular endothelial cell growth factor (VEGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm respectively or 0 μm) and the weight percent concentration of 2%, 10%, 15%, 20% or 30% PLA-PEG in ethyl acetate solution weight ratio of 1:20, 1:30, 1:30, 1:45 or 1 : 4 Stir in equal proportions, vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLA-PEG is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-120μm , 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment ten

Preparation of polylactic-acid polyglycolic acid (PLA-PEG) microspheres loaded with coagulation factor IX genetic factor biomacromolecular drug particles

(1) According to coagulation factor IX genetic factor biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) And the weight percentage concentration is 2%, 10%, 15%, 20% or 30% PLGA-PEG ethyl acetate solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 etc. Proportional stirring, vortex or ultrasound for 1-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-120μm , 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment Eleven

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol (PLGA-PEG) microspheres loaded with bone morphogenic protein (BMP) biomacromolecular drug particles

(1) According to the bone morphogenic protein (BMP) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA-PEG ethyl acetate solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.1-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; PLGA-PEG is 50%, 60%, 75%, 90% or 100%));

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-120μm , 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment 12

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol (PLGA-PEG) microspheres loaded with nerve growth factor (NGF) biomacromolecular drug particles

(1) According to nerve growth factor (NGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA-PEG dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; PLGA-PEG is 50%, 60%, 75%, 90% or 100%));

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL weight percent 60% ethylene glycol and 40% ethanol or 800mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-120 μm, 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment Thirteen

Preparation of polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA) microspheres loaded with bone-derived growth factor (BDGF) biomacromolecular drug particles

(1) According to bone-derived growth factor (BDGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm respectively or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA methylene chloride solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; PLA-PEG-PLA is 50%, 60%, 75%, 90% or 100%));

(2) Add the emulsion obtained in step (1) dropwise to the hydrophilic oil phase (hO): [Preparation of the hydrophilic oil phase (hO): formula 1: use polyvinylpyrrolidone (PVP) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50- 130μm, 60-160μm, 70-180μm or 200-500μm.

Embodiment Fourteen

Preparation of polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA) microspheres loaded with transforming growth factor (TGF-β) biomacromolecular drug particles

(1) According to transforming growth factor (TGF-β) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is respectively 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA methylene chloride solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; PLA-PEG-PLA is 50%, 60%, 75%, 90% or 100%));

(2) Add the emulsion obtained in step (1) dropwise to the hydrophilic oil phase (hO): [Preparation of the hydrophilic oil phase (hO): formula 1: use poloxmer (poloxmer) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the weight percentage of hydrophilic oil phase is 0, 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL weight percent 60% ethylene glycol and 40% ethanol or 1000mL of 60% by weight 1,2 propylene glycol and 40% ethanol], neutralized and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50- 130μm, 60-160μm, 70-180μm or 200-500μm.

Embodiment 15

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA) microspheres loaded with vaccine biomacromolecular drug particles

(1) According to the concentration of 0.4mg, 2mg, 1.5mg, 1mg or 0mg of vaccine biomacromolecule drug particles (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and weight percentage The weight ratio of 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA in dichloromethane is 1:20, 1:30, 1:30, 1:45 or 1:4 and is stirred in equal proportions , vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounted for the percentage of microsphere components are: 50%, 40%, 25% , 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA-PEG-PLGA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [Preparation of the hydrophilic oil phase (hO): formula 1: mixed aqueous solution with polysorbate surfactant and sodium chloride salt, Their percentage by weight concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the percentage by weight of accounting for hydrophilic oily phase is 0, 5% , 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 800mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment sixteen

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA) microspheres loaded with antibody biomacromolecular drug particles

(1) According to the antibody (antibody) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and Weight percent concentration is 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA-PEG-PLGA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to ethanol phase (E) [preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 60% ethylene glycol by weight of 500mL and 40% ethanol Ethanol or 700mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50- 130μm, 60-160μm, 70-180μm or 200-500μm.

Embodiment 17

Preparation of polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLA-PEG) microspheres loaded with epidermal growth factor (EGF) biomacromolecular drug particles

(1) According to the epidermal growth factor (EGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1 : 4 Stir in equal proportions, vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL weight percent 60% ethylene glycol and 40% ethanol or 800mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment eighteen

Preparation of polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLA-PEG) microspheres loaded with fibroblast growth factor (FGF) biomacromolecular drug particles

(1) According to the fibroblast growth factor (FGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment nineteen

Preparation of polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLGA-PEG) microspheres loaded with bone-derived growth factor (BDGF) biomacromolecular drug particles

(1) According to bone-derived growth factor (BDGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm respectively or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLGA-PEG dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 1-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50 %, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLGA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 800mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% of PEG-PLGA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope, which is not shown in Figure 1, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment 20

Preparation of polyethylene glycol-polylactic acid-polyglycolic acid-polyethylene glycol (PEG-PLGA-PEG) microspheres loaded with coagulation factor VIII (VIII) biomacromolecular drug particles

(1) According to coagulation factor VIII (VIII) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLGA-PEG dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1 : 4 Stir in equal proportions, vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLGA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% of PEG-PLGA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope, which is not shown in Figure 1, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment 21

Preparation of hyaluronic acid microspheres loaded with protein biomacromolecular drug particles

(1) According to the protein biomacromolecule drug particle 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) and the weight percentage concentration The weight ratio of 2%, 10%, 15%, 20% or 30% hyaluronic acid in dichloromethane solution is 1:20, 1:30, 1:30, 1:45 or 1:4. Stir and vortex Or ultrasonic 0.5-5 minute forms the uniform suspension liquid that is solid in oil (S/O) emulsion (preparing the percentage that the corresponding medicine particle accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% % or 0%; hyaluronic acid is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% hyaluronic acid is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-130 μm , 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment 22

Preparation of hyaluronic acid microspheres loaded with nucleic acid biomacromolecular drug particles

(1) According to the nucleic acid biomacromolecule drug PEI or polyarginine particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% hyaluronic acid in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; hyaluronic acid is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% hyaluronic acid is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-130 μm , 60-160μm, 70-180μm or 200-500μm

Embodiment 23

Preparation of gelatin microspheres loaded with small molecule drug sodium alginate particles

(1) According to the small molecule drug sodium alginate particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and weight percentage Concentration is 2%, 10%, 15%, 20% or 30% gelatin dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio stirring, vortex or Ultrasound for 0.5-5 minutes to form a uniform suspension, that is, a solid in oil (S/O) emulsion (the percentages of the prepared corresponding drug particles accounting for the components of the microspheres are: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for gelatin);

(2) Add the emulsion obtained in step (1) dropwise to the hydrophilic oil phase (hO): [Preparation of the hydrophilic oil phase (hO): formula 1: mix the aqueous solution with ethyl cellulose surfactant and sodium chloride salt , and their weight percent concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percent of the hydrophilic oil phase is 0,5 %, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 1000mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

According to the preparation of microspheres according to 2%, 10%, 15%, 20% or 30% gelatin, the morphology is similar to the above scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-125 μm, 60 -150 μm, 70-180 μm or 200-500 μm.

Embodiment 24

Preparation of gelatin microspheres loaded with dextran biomacromolecular drug particles

(1) According to the 0.4mg, 2mg, 1.5mg, 1mg or 0mg of dextran biomacromolecular drug particles (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and weight The percentage concentration is 2%, 10%, 15%, 20% or 30% gelatin in dichloromethane solution, and the weight ratio is 1:20, 1:30, 1:30, 1:45 or 1:4. Stir and vortex Or ultrasonic 0.5-5 minute forms the uniform suspension liquid that is solid in oil (S/O) emulsion (preparing the percentage that the corresponding medicine particle accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% % or 0%; gelatin is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain dry microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% gelatin is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-150μm, 60 -150 μm, 70-180 μm or 200-500 μm.

Example 25

Preparation of polylactic acid polyglycolic acid (PLGA) microspheres loaded with nucleic acid drug particles

(1) According to the nucleic acid drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) and the weight percentage concentration is 2% , 10%, 15%, 20% or 30% PLGA in acetonitrile solution with a weight ratio of 1:20, 1:30, 1:30, 1:45 or 1:4 in equal proportions and stirred, vortexed or sonicated for 1-5 minutes to form Uniform suspension is solid in oil (S/O) emulsion (preparing the corresponding drug particles accounted for the percentage of microsphere components are respectively: 50%, 40%, 25%, 10% or 0%; PLGA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 50-150 μm, 60 -150 μm, 70-180 μm or 200-500 μm.

(1) According to the erythropoietin (EPO) biomacromolecule drug particle 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA in dichloromethane solution weight ratio of 1:20, 1:30, 1:30, 1:45 or 1:4 etc. Proportional stirring, vortex or ultrasound for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40%, 25%, 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 800mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The scanning electron microscope image 1C of microspheres prepared according to 15% by weight concentration of PLGA in dichloromethane solution, the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment four

Preparation of polylactic acid-polyglycolic acid (PLGA) microspheres loaded with recombinant human granulocyte colony-stimulating factor (G-CSF) biomacromolecular drug particles

(1) According to the recombinant human granulocyte colony-stimulating factor (G-CSF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm respectively) , 2-10 μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA methylene chloride solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO): [The preparation of the hydrophilic oil phase (hO): formula 2: use polyethylene glycol (PEG) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounts for the weight percentage of hydrophilic oil phase respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40 or 50% by weight of glycerin and 90% of ethanol, 80% of ethylene glycol, 80% % ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase according to 99%, 95%, 90%, 80%, 60%] and stirring, vortex or Ultrasound for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to ethanol phase (E) [preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 10% ethylene glycol and 90% ethanol Ethanol or 400mL of 30% by weight 1,2 propylene glycol and 70% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

Scanning electron microscope image 1D of microspheres prepared according to 15% by weight concentration of PLGA dichloromethane solution, the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment five

Preparation of polyglycolic acid (PGA) microspheres loaded with cell-macrophage colony-stimulating factor (GM-CSF)

(1) According to the acetonitrile solution of granulocyte-macrophage colony-stimulating factor (GM-CSF) drug particles and PGA 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm respectively) , 2-6 μm, 2-10 μm or 0 μm) and the weight percent concentration of 2%, 10%, 15%, 20% or 30% PGA in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30. 1:45 or 1:4 equal ratio stirring, vortex or ultrasonic for 1-5 minutes to form a uniform suspension, that is, solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounted for the components of the microspheres) Percentages are: 50%, 40%, 25%, 10% or 0%, respectively; PGA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO): [The preparation of the hydrophilic oil phase (hO): formula 2: use polyethylene glycol (PEG) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounts for the weight percentage of hydrophilic oil phase respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40% or 50% by weight of glycerin and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase, according to 99%, 95%, 90%, 80%, 60%] and stirring, Vortex or ultrasonic for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 800mL weight percent 10% ethylene glycol and 90% ethanol or 1000mL of 30% by weight 1,2 propylene glycol and 70% ethanol], neutralized and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

According to the scanning electron microscope image 1E of the microspheres prepared according to the acetonitrile solution of 15% concentration of PGA by weight, the particle size is about 50-150 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PGA is similar to the above scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 20-100 μm, 60-150 μm, 70-180 μm Or 200-500μm.

Embodiment six

Preparation of polylactic acid (PLA) microspheres loaded with interferon (INF) biomacromolecular drug particles

(1) According to the interferon (INF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) And the weight percentage concentration is 2%, 10%, 15%, 20% or 30% PLA dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio stirring , vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounted for the percentage of microsphere components are: 50%, 40%, 25% , 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 2: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their percentage by weight concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the percentage by weight of accounting for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40% or 50% glycerin with 90% ethanol, 80% ethylene glycol, 80% Hydrophilic oil phase formulated with ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol, according to 99%, 95%, 90%, 80%, 60%] and stirring, vortex or Ultrasound for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 10% ethylene glycol and 90% ethanol Ethanol or 1000mL of 30% by weight of 1,2 propylene glycol and 70% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The scanning electron microscope image 1F of the microspheres prepared according to the 15% by weight concentration of PLA in dichloromethane solution, the particle size is about 50-120 μm. The morphology of other microspheres prepared according to 2%, 10%, 20% or 30% PLA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 20-100 μm, 60-150 μm, 70- 180μm or 200-500μm.

Embodiment seven

Preparation of polylactic acid polyglycolic acid (PLGA) microspheres loaded with platelet growth factor (PDGF) biomacromolecular drug particles

(1) According to the platelet growth factor (PDGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio Stir, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding percentages of drug particles accounting for microsphere components: 50%, 40%, 25% %, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO): [The preparation of the hydrophilic oil phase (hO): formula 2: use polyethylene glycol (PEG) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounts for the weight percentage of hydrophilic oil phase respectively 1%, 5%, 10%, 20% or 40%; and 10%, 20%, 25%, 40% or 50% by weight of glycerin and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase, according to 99%, 95%, 90%, 80%, 60%] and stirring, Vortex or ultrasonic for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to ethanol phase (E) [preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 10% ethylene glycol and 90% ethanol Ethanol or 400mL of 30% by weight of 1,2 propylene glycol and 70% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 40-100 μm, 60- 150μm, 70-180μm or 200-500μm.

Embodiment eight

Preparation of polycaprolactone (PCL) microspheres loaded with growth hormone (GH) biomacromolecular drug particles

(1) According to growth hormone (GH) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) And the weight percentage concentration is that the dichloromethane solution weight ratio of 2%, 10%, 15%, 20% or 30% polycaprolactone (PCL) is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio stirring, vortex or ultrasonic 1-5 minutes to form a uniform suspension, that is, solid-in-oil (S/O) emulsion (the percentage of the corresponding drug particles accounting for the components of the microspheres is respectively: 50% , 40%, 25%, 10% or 0%; polycaprolactone (PCL) is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 2: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their percentage by weight concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the percentage by weight of accounting for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40% (w/w); and 10%, 20%, 25%, 40% or 50% by weight of glycerol with 90% ethanol, 80% Ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or 50% propylene glycol prepared hydrophilic oil phase according to 99%, 95%, 90%, 80%, 60%] And stir, vortex or ultrasonic for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): the weight percent 10% ethylene glycol and 90% ethanol of 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL or 1000mL of 30% by weight 1,2 propylene glycol and 70% ethanol], neutralized and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

According to 2%, 10%, 15%, 20% or 30% polycaprolactone (PCL), the appearance of microspheres is similar to the above-mentioned scanning electron microscope Fig. 1 is not shown on the figure, and its size is respectively: 1-20 μ m, 40-120 μm, 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment nine

Preparation of polylactic acid-polyethylene glycol (PLA-PEG) microspheres loaded with vascular endothelial growth factor (VEGF) biomacromolecular drug particles

(1) According to vascular endothelial cell growth factor (VEGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm respectively or 0 μm) and the weight percent concentration of 2%, 10%, 15%, 20% or 30% PLA-PEG in ethyl acetate solution weight ratio of 1:20, 1:30, 1:30, 1:45 or 1 : 4 Stir in equal proportions, vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLA-PEG is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-120μm , 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment ten

Preparation of polylactic acid-polyglycolic acid (PLA-PEG) microspheres loaded with coagulation factor IX genetic factor biomacromolecular drug particles

(1) According to coagulation factor IX genetic factor biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) And the weight percentage concentration is 2%, 10%, 15%, 20% or 30% PLGA-PEG ethyl acetate solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 etc. Proportional stirring, vortex or ultrasound for 1-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-120μm , 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment Eleven

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol (PLGA-PEG) microspheres loaded with bone morphogenic protein (BMP) biomacromolecular drug particles

(1) According to the bone morphogenic protein (BMP) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA-PEG ethyl acetate solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.1-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; PLGA-PEG is 50%, 60%, 75%, 90% or 100%));

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.1-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-120μm , 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment 12

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol (PLGA-PEG) microspheres loaded with nerve growth factor (NGF) biomacromolecular drug particles

(1) According to nerve growth factor (NGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLGA-PEG dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; PLGA-PEG is 50%, 60%, 75%, 90% or 100%));

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL weight percent 60% ethylene glycol and 40% ethanol or 800mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-120 μm, 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment Thirteen

Preparation of polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA) microspheres loaded with bone-derived growth factor (BDGF) biomacromolecular drug particles

(1) According to bone-derived growth factor (BDGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm respectively or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA methylene chloride solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; PLA-PEG-PLA is 50%, 60%, 75%, 90% or 100%));

(2) Add the emulsion obtained in step (1) dropwise to the hydrophilic oil phase (hO): [Preparation of the hydrophilic oil phase (hO): formula 1: use polyvinylpyrrolidone (PVP) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60% respectively] and stir, vortex or ultrasonic for 0.5-5 minutes to form double emulsion

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50- 130μm, 60-160μm, 70-180μm or 200-500μm.

Embodiment Fourteen

Preparation of polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA) microspheres loaded with transforming growth factor (TGF-β) biomacromolecular drug particles

(1) According to transforming growth factor (TGF-β) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is respectively 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA methylene chloride solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; PLA-PEG-PLA is 50%, 60%, 75%, 90% or 100%));

(2) Add the emulsion obtained in step (1) dropwise to the hydrophilic oil phase (hO): [Preparation of the hydrophilic oil phase (hO): formula 1: use poloxmer (poloxmer) surfactant and sodium chloride Salt mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the weight percentage of hydrophilic oil phase is 0, 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL weight percent 60% ethylene glycol and 40% ethanol or 1000mL of 60% by weight 1,2 propylene glycol and 40% ethanol], neutralized and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLA-PEG-PLA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50- 130μm, 60-160μm, 70-180μm or 200-500μm.

Embodiment 15

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA) microspheres loaded with vaccine biomacromolecular drug particles

(1) According to the concentration of 0.4mg, 2mg, 1.5mg, 1mg or 0mg of vaccine biomacromolecule drug particles (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and weight percentage The weight ratio of 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA in dichloromethane is 1:20, 1:30, 1:30, 1:45 or 1:4 and is stirred in equal proportions , vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounted for the percentage of microsphere components are: 50%, 40%, 25% , 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA-PEG-PLGA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [Preparation of the hydrophilic oil phase (hO): formula 1: mixed aqueous solution with polysorbate surfactant and sodium chloride salt, Their percentage by weight concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, and the percentage by weight of accounting for hydrophilic oily phase is 0, 5% , 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 800mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment sixteen

Preparation of polylactic acid-polyglycolic acid-polyethylene glycol-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA) microspheres loaded with antibody biomacromolecular drug particles

(1) According to the antibody (antibody) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and Weight percent concentration is 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA-PEG-PLGA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to ethanol phase (E) [preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 60% ethylene glycol by weight of 500mL and 40% ethanol Ethanol or 700mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA-PEG-PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50- 130μm, 60-160μm, 70-180μm or 200-500μm.

Embodiment 17

Preparation of polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLA-PEG) microspheres loaded with epidermal growth factor (EGF) biomacromolecular drug particles

(1) According to the epidermal growth factor (EGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1 : 4 Stir in equal proportions, vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL weight percent 60% ethylene glycol and 40% ethanol or 800mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment eighteen

Preparation of polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLA-PEG) microspheres loaded with fibroblast growth factor (FGF) biomacromolecular drug particles

(1) According to the fibroblast growth factor (FGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 0.5-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50 %, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% PEG-PLA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment nineteen

Preparation of polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLGA-PEG) microspheres loaded with bone-derived growth factor (BDGF) biomacromolecular drug particles

(1) According to bone-derived growth factor (BDGF) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm respectively or 0 μm) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLGA-PEG dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 Or 1: 4 equal ratio stirring, vortex or ultrasonic 1-5 minutes to form a uniform suspension, i.e. solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50 %, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLGA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 800mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% of PEG-PLGA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope, which is not shown in Figure 1, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment 20

Preparation of polyethylene glycol-polylactic acid-polyglycolic acid-polyethylene glycol (PEG-PLGA-PEG) microspheres loaded with coagulation factor VIII (VIII) biomacromolecular drug particles

(1) According to coagulation factor VIII (VIII) biomacromolecular drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% PEG-PLGA-PEG dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1 : 4 Stir in equal proportions, vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are respectively: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PEG-PLGA-PEG);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% of PEG-PLGA-PEG, the morphology of the microspheres is similar to the above-mentioned scanning electron microscope, which is not shown in Figure 1, and their sizes are: 1-20 μm, 50 - 130 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment 21

Preparation of hyaluronic acid microspheres loaded with protein biomacromolecular drug particles

(1) According to the protein biomacromolecule drug particle 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) and the weight percentage concentration The weight ratio of 2%, 10%, 15%, 20% or 30% hyaluronic acid in dichloromethane solution is 1:20, 1:30, 1:30, 1:45 or 1:4. Stir and vortex Or ultrasonic 0.5-5 minute forms the uniform suspension liquid that is solid in oil (S/O) emulsion (preparing the percentage that the corresponding medicine particle accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% % or 0%; hyaluronic acid is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% hyaluronic acid is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-130 μm , 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment 22

Preparation of hyaluronic acid microspheres loaded with nucleic acid biomacromolecular drug particles

(1) According to the nucleic acid biomacromolecule drug PEI or polyarginine particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% hyaluronic acid in dichloromethane solution weight ratio of 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 Stir in equal proportions, vortex or sonicate for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50%, 40% , 25%, 10% or 0%; hyaluronic acid is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% hyaluronic acid is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-130 μm , 60-160 μm, 70-180 μm or 200-500 μm.

Embodiment 23

Preparation of gelatin microspheres loaded with small molecule drug sodium alginate particles

(1) According to the small molecule drug sodium alginate particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and weight percentage Concentration is 2%, 10%, 15%, 20% or 30% gelatin dichloromethane solution weight ratio is 1: 20, 1: 30, 1: 30, 1: 45 or 1: 4 equal ratio stirring, vortex or Ultrasound for 0.5-5 minutes to form a uniform suspension, that is, a solid in oil (S/O) emulsion (the percentages of the prepared corresponding drug particles accounting for the components of the microspheres are: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for gelatin);

(2) Add the emulsion obtained in step (1) dropwise to the hydrophilic oil phase (hO): [Preparation of the hydrophilic oil phase (hO): formula 1: mix the aqueous solution with ethyl cellulose surfactant and sodium chloride salt , and their weight percent concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percent of the hydrophilic oil phase is 0,5 %, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) Add the double emulsion of step (2) dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL of 60% ethylene glycol and 40% ethanol Ethanol or 1000mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

According to the preparation of microspheres according to 2%, 10%, 15%, 20% or 30% gelatin, the morphology is similar to the above scanning electron microscope Figure 1, which is not shown on the figure, and their sizes are: 1-20 μm, 50-125 μm, 60 -150 μm, 70-180 μm or 200-500 μm.

Embodiment 24

Preparation of gelatin microspheres loaded with dextran biomacromolecular drug particles

(1) According to the 0.4mg, 2mg, 1.5mg, 1mg or 0mg of dextran biomacromolecular drug particles (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and weight The percentage concentration is 2%, 10%, 15%, 20% or 30% gelatin in dichloromethane solution, and the weight ratio is 1:20, 1:30, 1:30, 1:45 or 1:4. Stir and vortex Or ultrasonic 0.5-5 minute forms the uniform suspension liquid that is solid in oil (S/O) emulsion (preparing the percentage that the corresponding medicine particle accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% % or 0%; gelatin is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise to the ethanol phase (E) [preparation of the ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL of 60% ethylene glycol and 40% ethanol Ethanol or 400mL of 60% by weight of 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain dry microspheres.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% gelatin is similar to the above scanning electron microscope Figure 1 is not shown on the figure, but their sizes are: 1-20μm, 50-150μm, 60 -150 μm, 70-180 μm or 200-500 μm.

Example 25

Preparation of polylactic acid polyglycolic acid (PLGA) microspheres loaded with nucleic acid drug particles

(1) According to the nucleic acid drug particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm respectively) and the weight percentage concentration is 2% , 10%, 15%, 20% or 30% PLGA in acetonitrile solution with a weight ratio of 1:20, 1:30, 1:30, 1:45 or 1:4 in equal proportions and stirred, vortexed or sonicated for 1-5 minutes to form Uniform suspension is solid in oil (S/O) emulsion (preparing the corresponding drug particles accounted for the percentage of microsphere components are respectively: 50%, 40%, 25%, 10% or 0%; PLGA is 50%, 60%, 75%, 90% or 100%);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

The morphology of microspheres prepared according to 2%, 10%, 15%, 20% or 30% PLGA is similar to the above-mentioned scanning electron microscope Figure 1, which is not shown on the figure, but their sizes are: 1-20 μm, 50-150 μm, 60 -150 μm, 70-180 μm or 200-500 μm.

Embodiment 26

Preparation of Polyethylene Glycol-Polycaprolactone (PEG-PCL) Microspheres Loaded with Vaccine Drug Particles

(1) According to the vaccine drug particle 0.4mg, 2mg, 1.5mg, 1mg or 0mg (its particle size is respectively 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm) and the weight percentage concentration is 2% , 10%, 15%, 20% or 30% polyethylene glycol-polycaprolactone (PEG-PCL) in dichloromethane solution weight ratio of 1:20, 1:30, 1:30, 1:45 or Stir in 1:4 equal proportions, vortex or ultrasonic for 0.5-5 minutes to form a uniform suspension, that is, a solid-in-oil (S/O) emulsion (the percentages of the corresponding drug particles accounting for the components of the microspheres are: 50% , 40%, 25%, 10% or 0%; PEG-PCL is 50%, 60%, 75%, 90% or 100%));

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to 2%, 10%, 15%, 20% or 30% polyethylene glycol-polycaprolactone, the appearance of microspheres is similar to the above-mentioned scanning electron microscope. Figure 1 is not shown on the figure, but its dimensions are: 1 - 20 μm, 50-150 μm, 60-150 μm, 70-180 μm or 200-500 μm.

Embodiment 27

Preparation of Microspheres of Polylactic Acid and Polylactic-Polyglycolic Acid (PLA and PLGA) Blends Loaded with Protein Drug Particles

(1) According to protein drug (with EPO as model protein drug) particles 0.4mg, 2mg, 1.5mg, 1mg or 0mg (the particle size is 0.2-1μm, 1-5μm, 2-6μm, 2-10μm or 0μm ) and weight percent concentration of 2%, 10%, 15%, 20% or 30% polylactic acid and polylactic acid-polyglycolic acid (polylactic acid: polylactic acid-polyglycolic acid is 1:1, 2:1 by weight , 4:1, 4:1, or 2:1 mixed) dichloromethane solution weight ratio of 1:20, 1:30, 1:30, 1:45 or 1:4 equal proportion stirring, vortex or ultrasonic Form a uniform suspension in 0.5-5 minutes, that is, a solid in oil (S/O) emulsion (preparing the corresponding drug particles accounts for the percentage of microsphere components: 50%, 40%, 25%, 10% or 0%; 50%, 60%, 75%, 90% or 100% for PLGA and PLA);

(2) Step (1) is added dropwise to the hydrophilic oil phase (hO) by the emulsion: [The preparation of the hydrophilic oil phase (hO): formula 1: use polyvinyl alcohol (PVA) surfactant and sodium chloride salt Mixed aqueous solution, their weight percentage concentration is respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7%, or 10%, accounting for the weight percentage of hydrophilic oil phase is 0 , 5%, 10%, 25% or 40%; ethylene glycol is 100%, 95%, 90%, 75% or 60%] and stirred, vortexed or sonicated for 0.5-5 minutes to form double emulsion;

(3) the double emulsion of step (2) is added dropwise to ethanol phase (E) [the preparation of ethanol phase (E): 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL weight percent 60% ethylene glycol and 40% ethanol or 400mL of 60% by weight 1,2 propylene glycol and 40% ethanol], and stirred for 1-4 hours;

(4) Collect the microspheres obtained in the step (3) by centrifugation, wash with water for 3-5 times, and obtain dry microspheres after freeze-drying.

According to the preparation of 2%, 10%, 15%, 20% or 30% mixtures of polylactic acid and polylactic acid-polyglycolic acid (PLA and PLGA), the morphology of the microspheres is similar to that of the above scanning electron microscope. Figure 1 is not shown on the figure. , but their sizes are: 1-25 μm, 50-155 μm, 60-160 μm, 70-180 μm or 200-500 μm.

Claims (10)

1. the method for the hydrophilic oil of ethanol bag-hydrophilic oil bag oil oil bag microspheres with solid preparation is characterized in that, may further comprise the steps:

1. the organic solution that drug particles is joined slow release or controlled-release material is stirring in the oil phase, whirlpool or ultrasonic, makes it homodisperse and forms suspension;

Described drug particles, slow release or controlled-release material, their percentage by weight are that 0-50%, slow release or controlled release micro-material are 50-100%;

2. suspension being added in another hydrophilic organic solution is hydrophilic oil phase, and the organic solvent of insoluble slow release of this oil phase or controlled-release material stirs the ball that formed 1-500 μ m in 0.5-5 minute;

3. the completing steps microsphere suspension that contains is 2. transferred in the ethanol and to be solidified 1-4 hour;

4. completing steps sample lyophilizing is 3. got exsiccant microsphere.

2. the method for the hydrophilic oil of ethanol bag according to claim 1-hydrophilic oil bag oil oil bag microspheres with solid preparation is characterized in that described medicine comprises small-molecule drug or macromolecular drug; The size of its drug particles is at 0.2-10 μ m.

3. the method for the hydrophilic oil of ethanol bag according to claim 2-hydrophilic oil bag oil oil bag microspheres with solid preparation, it is characterized in that, described small-molecule drug refers to chemicals, and macromolecular drug is the biopharmaceutical macromolecular drug of protein macromolecule medicine, vaccine, antibody, nucleic acid or liposome medicament; Described nucleic acid is: antisense nucleotide, microRNA or gene.

4. the method for the hydrophilic oil of ethanol bag according to claim 3-hydrophilic oil bag oil oil bag microspheres with solid preparation, it is characterized in that described protein macromolecule medicine is: erythropoietin, recombinant human granulocyte colony stimulating factor, granulocyte-macrophage colony stimutaing factor, vaccine, interferon, growth hormone, insulin, epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin like growth factor, vascular endothelial cell growth factor, PDGF, endothelial cell growth factor (ECGF), nerve growth factor, bone-derived growth factor, bone morphogenetic protein(BMP), tissue polypeptide antigen, antibody, blood coagulation factor VIII or IX genetic factor.

5. the method for the hydrophilic oil of ethanol bag according to claim 1-hydrophilic oil bag oil oil bag microspheres with solid preparation is characterized in that described oil phase is: the organic solution of the material of slow release or controlled release.

6. the method for the hydrophilic oil of ethanol bag according to claim 5-hydrophilic oil bag oil-oil bag microspheres with solid preparation, it is characterized in that the organic solution of the material of described slow release or controlled release is: polylactic acid, polyglycolic acid, the polylactic acid poly hydroxyacetic acid, the combination in any of polylactic acid and polylactic acid-polyglycolic acid, collagen protein, polybutylcyanoacrylate, poly phosphazene, poly phosphate, Fibrinogen, fibrin, hyaluronic acid, gelatin, the Polyethylene Glycol polylactic acid, the polyglycolic acid Polyethylene Glycol, Polyethylene Glycol polylactic acid poly hydroxyacetic acid, Polyethylene Glycol polylactic acid poly hydroxyacetic acid Polyethylene Glycol, polylactic acid poly hydroxyacetic acid Polyethylene Glycol polylactic acid poly hydroxyacetic acid, polylactic acid poly ethylene glycol polylactic acid, Polyethylene Glycol polylactic acid poly ethylene glycol, poly-Acetic acid, hydroxy-, bimol. cyclic ester, the polyethylene glycol-caprolactone, polycaprolactone gathers-the polyethylene glycol-caprolactone, polycaprolactone, silicon is as glue, politef, polrvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, Merlon, or the dichloromethane of Carmomer, ethyl acetate, or the organic solution of acetonitrile;

The weight percent concentration of the organic solution of the material of described slow release or controlled release is: 2-30%.

7. the method for the hydrophilic oil of ethanol bag according to claim 1-hydrophilic oil bag oil-oil bag microspheres with solid preparation, it is characterized in that described hydrophilic oil phase is: surfactant, glycerol, ethanol, propylene glycol, ethylene glycol or liquid macrogol or their combination;

Described they be combined as: glycerol and ethanol add aqueous solution, the liquid macrogol that aqueous solution that aqueous solution that the aqueous solution of surfactant, aqueous solution, glycerol and propylene glycol that ethanol adds surfactant add surfactant, aqueous solution, glycerol and ethylene glycol that propylene glycol adds surfactant add surfactant, aqueous solution, glycerol and liquid macrogol that ethylene glycol adds surfactant add surfactant and add surfactant.

8. the method for the hydrophilic oil of ethanol bag according to claim 7-hydrophilic oil bag oil-oil bag microspheres with solid preparation, it is characterized in that, described their combination, its part by weight is: aqueous surfactant solution 0-30%, glycerol 0-50%, ethanol, propylene glycol, ethylene glycol or liquid macrogol 50-100%.

9. the method for the hydrophilic oil of ethanol bag according to claim 7-hydrophilic oil bag oil oil bag microspheres with solid preparation, it is characterized in that, described aqueous surfactant solution is: the mixed water solution of salt such as polyvinyl alcohol and sodium chloride, and their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of salt such as Polyethylene Glycol and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of salt such as polyvinylpyrrolidone and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of salt such as poloxamer and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of salt such as poly-sorbic alcohol and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; Or the mixed water solution of salt such as ethyl cellulose and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%.

10. the method for the hydrophilic oil of ethanol bag according to claim 1-hydrophilic oil bag oil-oil bag microspheres with solid preparation is characterized in that described microsphere, its particle diameter are 1-500 μ m.

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