CN101721377B

CN101721377B - Method for preparing microspheres with hydrophilic oil in ethanol, oil in hydrophilic oil, solid in oil

Info

Publication number: CN101721377B
Application number: CN2010103003959A
Authority: CN
Inventors: 金拓; 袁伟恩; 吴飞; 任甜甜; 胡振华
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2010-01-18
Filing date: 2010-01-18
Publication date: 2013-04-17
Anticipated expiration: 2030-01-18
Also published as: CN101721377A

Abstract

The invention relates to a method for preparing microspheres with solid-in-oil-in-hydrophilic oil-in-ethanol, belonging to the field of the pharmaceutical technology and comprising the following steps: (1) adding and stirring or swirling medicament particles to the organic solution, i.e. the oil phase of sustained-release or controlled-release material; (2) adding mixed suspension to another hydrophilic organic solution, i.e. hydrophilic oil phase and stirring the mixed suspension to the hydrophilic organic solution, i.e. the hydrophilic oil phase for 0.1-5 min to form spheres of 1-500 Mum; (3) transferring the mixed suspension containing the microspheres to ethanol and solidifying the mixed suspension for 1-4h; (4) and drying and freezing the sample to obtain the dry microspheres. The diameter of the microspheres can be controlled and adjusted from 1 Mum to 500 Mum according to the need; the microspheres can not cause pollution to the environment; the surfaces of the microspheres are smooth and round; the microspheres are consistent without being adhered; the frozen-dried powder of the microspheres is white, fine and loose, can not collapse, can not be adhered and has good dispersity. The method can be used for preparing various sustained-release or controlled-release microspheres of various medicament or preparing the adjuvant of vaccines.

Description

The method of the hydrophilic oil of ethanol bag-hydrophilic oil bag oil-oil bag microspheres with solid preparation

Technical field

What the present invention relates to is the preparation method in a kind of pharmaceutical technology field, the method for the hydrophilic oil of especially a kind of ethanol bag-hydrophilic oil bag oil oil bag solid (S/O/hO/E) microsphere preparation.

Background technology

Pharmaceutical industry is from drug discovery, and to clinical application, last link is pharmaceutical preparation.Wherein some medicine needs long term administration to cure; Some needs the topicals such as targeting.Reach these purposes, crude drug must be prepared into corresponding dosage form.For example need long term administration but short medicine of in vivo half-life should be prepared into slow release formulation; For the treatment of some tumors, need some drug targetings in the disease photograph, such as targeting in tumor vascular embolism microball preparation etc.; Gene recombination technology be used for the treatment of protein expression and production 20 for many years, up to the present, existing more than 30 protein drug product drops into clinical use, nearly 200 examine with R﹠D process in, emerge and a collection ofly enter a collection of new large-scale medical companies such as (Amgen), gene technology (Genentech) such as peace.With respect to the fast development of protein macromolecule medicine itself, its dosage form technical progress is slow.On the one hand, the protein macromolecule drug oral does not absorb, Half-life in vivo short, needs drug administration by injection; On the other hand, the protein drug treatment cycle of many He Ermeng, cytokine class is long, and for a long time and continually injection becomes necessary, also affects the main cause of patient's compliance.The research and development of the dosage form of slow release protein drug are owing to cause the easily prominent shortcoming such as release of the standby microsphere of the not high S/O/W method of active loss such as W/O/W method, envelop rate and S/O/O legal system in preparation microgranule process.The protein microsphere that development preparation has an active protection can improve again envelop rate and the prominent method of releasing is imperative.Yet there are no up till now and utilize the hydrophilic oil of ethanol bag-hydrophilic oil bag oil oil bag solid (S/O/hO/E) method to prepare the report of microsphere.

Find by prior art documents, [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, International Journal ofPharmaceutics 331 (2007) 27-37], (Lee E.S., Kwon M.J., Lee H., and Kim J.J. has reported and has utilized new viscosity S/W/O/W method that protein stabilized is encapsulated in the PLGA microsphere Inpharm magazine, 2007,331:27-37).The people such as Lee E.S. have reported in the document and have utilized the S/W/O/W method to prepare microsphere.The document utilizes the lyophilizing of albumen cyclodextrin then to grind, and the albumen cyclodextrin that grinds is added to the dichloromethane solution emulsifying of full-bodied polysaccharide solution and PLGA, arrives at last aqueous phase sclerosis microsphere and just for protein medicaments, other drug does not appear in the newspapers.But the granule of protein loop dextrin contacts unavoidable protein undissolved with polysaccharide solution, after the dissolving protein solution be easy to contact with the dichloromethane of PLGA, have oil-water interfaces and cause assembling.Cause equally envelop rate also not high, have incomplete release.[Morita T., Sakamura Y., Horikiri Y., Suzuki T., Yoshino H., Protein encapsulation intobiodegradable microspheres by a novel S/O/W emulsion method using poly (ethyleneglycol) as a protein micronization adjuvant, Journal of Controlled Release 69 (2000) 435-444], (people such as Morita T. has reported in the document and has utilized new S/O/W emulsion process preparation to carry a protein microsphere.Just changed surfactant in the past report more be use PVA, use PEG instead at this piece document.But it is low still can not to overcome envelop rate, has the prominent shortcoming of releasing with incomplete release.

Summary of the invention

The object of the invention is to overcome deficiency of the prior art, the method for the hydrophilic oil of a kind of ethanol bag-hydrophilic oil bag oil-oil bag microspheres with solid preparation is provided.Make the smooth rounding of microparticle surfaces of its preparation, good evenness, granule regularizing is without adhesion; Envelop rate is high, prominently releases littlely, and drug loading is high.

The present invention is achieved by the following technical solutions, and the present invention is dispersed in drug particles in the organic solution with slow release or controlled-release material, and stirring, whirlpool or the formation suspension that makes it to be uniformly dispersed such as ultrasonic; Then suspension is added to outer oil phase, restir or whirlpool form microsphere, at last it are transferred to large aqueous phase and solidify 1-4 hour; Then centrifugal collection microsphere, lyophilizing is preserved.

The present invention includes following steps:

1. the organic solution that drug particles is joined slow release or controlled-release material is that stirring or whirlpool etc. make it Uniform Dispersion formation suspension in the oil phase (O);

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

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

3. the completing steps microsphere suspension that contains is 2. transferred in the ethanol (E) and solidified 1-4 hour.

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

Described drug particles refers to water miscible drug particles, or medicine is loaded into the granule for preparing in the adjuvant, or oil-soluble method by preparation is prepared into the granule that is insoluble to organic solvent;

Described medicine comprises small-molecule drug and macromolecular drug;

Described small-molecule drug refers to chemicals, and macromolecular drug mainly refers to biopharmaceutical macromolecular drug, especially finger protein macromolecular drug, vaccine, antibody, nucleic acid or liposome medicament.

Described protein macromolecule medicine is: erythropoietin (EPO), recombinant human granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimutaing 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 cell growth factor (VEGF), PDGF (PDGF), endothelial cell growth factor (ECGF) (ECGF), nerve growth factor (NGF), bone-derived growth factor (BDGF), bone morphogenetic protein(BMP) (BMP), tissue polypeptide antigen (TPA), antibody (antibody), blood coagulation factor VIII (VIII), or plasma thromboplastin component genetic factor;

Described nucleic acid is: antisense nucleotide (anti-RNA), microRNA (RNAi) or gene (DNA);

Described adjuvant refers to mainly is can the injection rank, especially little saccharide (sucrose, trehalose, glucose, maltose or lactose), and polyhydroxy compounds (mannitol, sorbitol, glycerol, 1,2-PD, erythritol, Polyethylene Glycol, polyvinyl alcohol, poly(ethylene oxide) or polypyrrole alkane ketone; ), polysaccharide compound (glucosan, sodium alginate, chitosan, starch, cellulose, PEI, poly-smart ammonia or cyclodextrin material), amino-acid compound (glycine, lysine, arginine, glutamic acid or histidine; ) or a kind of or combination in any of inorganic salts material (zinc salt, calcium salt, mantoquita, magnesium salt or molybdenum salt);

Described drug particles, the size of its drug particles are at 0.2-10 μ mm, take particle diameter as 1-5 μ mm as good.

Described drug particles accounts for microsphere total weight percent 0-50%;

The material of described slow release or controlled release is: polylactic acid (PLA), polylactic acid-polyglycolic acid (PLGA), the combination in any of PLA and PLGA, silicon is as glue, politef, polrvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, Merlon, Carmomer, hyaluronic acid, gelatin, collagen protein, polybutylcyanoacrylate, poly phosphazene, poly phosphate, Fibrinogen, fibrin, polyethylene glycol-polylactic acid (PLA-PEG), polyethylene glycol-hydroxyacetic acid (PLGA-PEG), mPEG-PLGA-polyglycolic acid (PLGA-PEG-PLGA), PLA-PEG-PLA (PLA-PEG-PLA), PGA, PEG-PCL (PEG-PCL), polycaprolactone gathers-ethylene glycol-polycaprolactone (PCL-PEG-PCL), or polycaprolactone (PCL);

The material of described slow release or controlled release, accounting for the microsphere total weight percent is 50-100%;

Described oil phase (O) is: the organic solution of the material of slow release or controlled release;

The organic solution of the material of described slow release or controlled release is: polylactic acid (PLA), polylactic acid-polyglycolic acid (PLGA), polyglycolic acid (PGA), the combination in any of polylactic acid (PLA) and polylactic acid-polyglycolic acid (PLGA), hyaluronic acid, gelatin, collagen protein, polybutylcyanoacrylate, poly phosphazene, poly phosphate, Fibrinogen, fibrin, polylactic acid-polyglycol (PLA-PEG), polylactic acid-mPEG-PLGA (PLGA-PEG), polylactic acid-mPEG-PLGA-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA), PLA-PEG-PLA (PLA-PEG-PLA), polyethylene glycol-polylactic acid-Polyethylene Glycol (PEG-PLA-PEG), polyethylene glycol-polylactic acid-mPEG-PLGA (PEG-PLGA-PEG), Merlon, Carmomer, hyaluronic acid, gelatin, collagen protein, PEG-PCL (PEG-PCL), polycaprolactone gathers-PEG-PCL (PCL-PEG-PCL), polycaprolactone, silicon is as glue, politef, polrvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, Merlon, or the dichloromethane of Carmomer, ethyl acetate, acetonitrile, heptane, chloroform, or acetone organic solution, with dichloromethane, ethyl acetate, the organic solution of acetonitrile or their combination in any is good;

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

Described hydrophilic oil phase (hO) is: surfactant, glycerol, ethanol, propylene glycol, ethylene glycol solution, liquid macrogol or their compositions;

Described their compositions is: glycerol and ethanol add aqueous solution or 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;

The water-soluble of described surfactant is: the mixed water solution of the salt such as polyvinyl alcohol (PVA) and sodium chloride, and their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of the salt such as Polyethylene Glycol (PEG) and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of the salt such as polyvinylpyrrolidone (PVP) and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of the salt such as poloxamer (poloxmer) and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of the 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 the salt such as ethyl cellulose (EC) and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%;

Described proper ratio is: the percentage by weight 0-50% of glycerol, the percentage by weight 0-30% of aqueous surfactant solution, the percentage by weight 50-100% of ethanol, propylene glycol, ethylene glycol or liquid macrogol; With the glycerol of percentage by weight 20-30%, the ethanol of 70-80%, propylene glycol, ethylene glycol or liquid macrogol 50-100% are good, the percentage by weight 5-15% of aqueous surfactant solution is good;

Described ethanol phase (E) is for can dissolve oil phase and hydrophilic oil phase ground ethanol, propylene glycol or their any mixed solution;

The particle diameter of described microsphere is 1-500 μ m, take 10-100 μ m as good;

The present invention has selected the mutually material of (E) and suitable controlled release or slow release of suitable oil phase (O), hydrophilic oil phase (hO) and ethanol, make water miscible drug particles or oil-soluble medicine be prepared into the insoluble granule of organic solvent by the method for preparation, avoid not high with the envelop rate of conventional W/O, W/O/W, S/O/W, release seriously with the prominent of S/O/O, and the shortcoming of the environmental pollution that causes; Adopt the method to prepare microsphere, the size of its particle diameter can be controlled according to different needs, and is free from environmental pollution; Can avoid the function influence to the treatment of medicine, especially those physicochemical properties are unsettled, to the medicine of oil-water interfaces sensitivity, such as biopharmaceutical macromolecular drug such as protein macromolecule medicine, DNA, RNA or siRNA medicine.The smooth surface rounding of microgranule, granule regularizing are without adhesion, and particle diameter can be regulated and control as required from 1 μ m to 500 μ m, and its freeze dried powder is that white is fine and smooth, loose, can not subside, adhesion, and redispersibility is good.Can apply to the preparation of various medicament slow-release microspheres and the adjuvant preparation of vaccine.

Description of drawings

Fig. 1 prepares the scanning electron microscope (SEM) photograph of microsphere

The specific embodiment

Below embodiments of the invention are elaborated: following examples are implemented under take technical solution of the present invention as prerequisite, have provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.

The implementation condition of following examples:

One, blank microsphere preparation

1. prepare oil phase (O) and hydrophilic oil phase (hO)

The preparation of oil phase (O): controlled release or slow-release material are dissolved in organic solvent, and to be prepared into weight percent concentration be 1-50%, forms oil phase (O).

The preparation of hydrophilic oil phase (hO): prescription 1: with salt mixed water solutions such as surfactant and sodium chloride, their weight percent concentration are respectively 1-10% and 0-10%, and the percentage by weight that accounts for hydrophilic oil phase is 0-40%; The Polyethylene Glycol of ethanol, ethylene glycol, propylene glycol or room temperature liquid state is 60-100%; Or fill a prescription 2: with salt mixed water solutions such as surfactant and sodium chloride, their weight percent concentration are respectively 1-10% and 0-10%, and the percentage by weight that accounts for hydrophilic oil phase is 0-40%; Be that the percentage ratio that the polyglycol solution of ethanol, ethylene glycol, propylene glycol or the room temperature liquid state of the glycerol of 0-50% and 50-100% accounts for hydrophilic oil phase is 60-100% with percentage by weight;

The preparation of ethanol phase (E): the ethanol of 100-1000mL, ethylene glycol, propylene glycol or their any mixture;

2. blank microsphere preparation

Oil phase (O) is added dropwise to also stirring of hydrophilic oil phase (hO), ultrasonic or whirlpool 0.5-5 minute formation O/hO, then change ethanol over to and be mutually 100-1000mL and stirring 1-4 hour, centrifugal collection microsphere and lyophilizing or volatilization are removed organic solvent and are obtained dry microsphere.

Two, the microsphere of preparation medicine carrying thing

1. preparation drug particles, to small-molecule drug can be conventional grinding method, the sedimentation method, comminuting method or comminution granulation; Can be the method for the complex that forms of aqueous phase-aqueous phase emulsion method, spray drying method, phase separation method, supercritical methanol technology or metal ion and biomacromolecule for biopharmaceutical macromolecular drug;

2. prepare oil phase (O) and hydrophilic oil phase (hO)

All according to blank oil phase (O) and hydrophilic oil phase (hO) preparation method.

3. drug particles and oil phase (O) mixing are formed suspension;

4. the suspension of step 3 is added dropwise to hydrophilic oil phase (hO), and stirring, whirlpool or ultrasonic 0.5-5 minute, emulsion formed;

5. the emulsion of step 4, be added drop-wise to 100-1000mL ethanol phase (E), and stirred 1-4 hour, centrifugal collection microsphere also washs 3-5 time, and then lyophilizing gets microsphere.

The entrapment efficiency that following examples have overcome good water solubility is not high, prominently release serious shortcoming; For biopharmaceutical macromolecular drug, oil-water interfaces, high shear force, interface and cross-linking agent etc. have been overcome, at the lower biopharmaceutical macromolecular drug of the condition of gentleness, be written in the microsphere such as protein, polypeptide, vaccine, DNA, RNA, SiRNA, virus or liposome, can keep for a long time active.Reduce greatly the expense of preserving and improve curative effect.The polysaccharide vitreous particle diameter of preparation is little simultaneously, and is active high, therefore is being prepared into other dosage form such as sustained-release micro-spheres, can reduce prominent releasing and incomplete release.

Embodiment one

Be loaded with the preparation of small-molecule drug granule polylactic acid-polyglycolic acid (PLGA) microsphere

(1) according to small-molecule drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with polyvinyl alcohol (PVA) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0%, 5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

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

(4) the centrifugal collection microsphere that step (3) is obtained, and wash with water 3-5 time, dry microsphere got after the lyophilizing.

Weight percent concentration PLGA dichloromethane solution according to 15% prepares scanning electron microscope Figure 1A of microsphere, the about 50-150 μ of particle diameter m.The pattern that other PLGA according to 2%, 10%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment two

Be loaded with the preparation of biopharmaceutical macromolecular drug granule polylactic acid-polyglycolic acid (PLGA) microsphere

(1) according to biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with polyvinyl alcohol (PVA) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 8% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): 1 of the percentage by weight 60% of the ethylene glycol of the percentage by weight 60% of 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL and 40% ethanol or 1000mL, 2 propylene glycol and 40% ethanol], in and stirred 1-4 hour;

(4) the centrifugal collection microsphere that step (3) is obtained, and wash with water 3-5 time, microsphere obtained after the lyophilizing.

Weight percent concentration PLGA dichloromethane solution according to 15% prepares scanning electron microscope Figure 1B of microsphere, the about 50-150 μ of particle diameter m.The pattern that other PLGA according to 2%, 10%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment three

Be loaded with the preparation of erythropoietin (EPO) biopharmaceutical macromolecular drug granule polylactic acid (PLA) microsphere

(1) according to erythropoietin (EPO) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with polyvinyl alcohol (PVA) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.1-5 minute;

Weight percent concentration PLGA dichloromethane solution according to 15% prepares the scanning electron microscope (SEM) photograph 1C of microsphere, the about 50-150 μ of particle diameter m.The pattern that other PLGA according to 2%, 10%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment four

Be loaded with the preparation of recombinant human granulocyte colony stimulating factor (G-CSF) biopharmaceutical macromolecular drug granule polylactic acid-polyglycolic acid (PLGA) microsphere

(1) according to recombinant human granulocyte colony stimulating factor (G-CSF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 2: with Polyethylene Glycol (PEG) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40%; With with percentage by weight be 10%, 20%, 25%, 40 or 50% glycerol and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or the hydrophilic oil phase of 50% propylene glycol preparation according to 99%, 95%, 90%, 80%, 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): the percentage by weight 30%1 of the ethylene glycol of the percentage by weight 10% of 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL and 90% ethanol or 400mL, 2 propylene glycol and 70% ethanol], in and stirred 1-4 hour;

Weight percent concentration PLGA dichloromethane solution according to 15% prepares the scanning electron microscope (SEM) photograph 1D of microsphere, the about 50-150 μ of particle diameter m.The pattern that other PLGA according to 2%, 10%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment five

Be loaded with the preparation of cell-M-CSF (GM-CSF) composition granule polyglycolic acid (PGA) microsphere

(1) according to the acetonitrile solution 0.4mg of granulocyte-macrophage colony stimutaing factor (GM-CSF) drug particles and PGA, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, the dichloromethane solution weight ratio of 20% or 30% PGA is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 1-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PGA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 2: with Polyethylene Glycol (PEG) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40%; With with percentage by weight be 10%, 20%, 25%, 40% or 50% glycerol and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or the hydrophilic oil phase of 50% propylene glycol preparation, according to 99%, 95%, 90%, 80%, 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): percentage by weight 10% ethylene glycol of 100ml ethanol, 200mL ethanol, 400mL ethanol, 800mL and the percentage by weight 30%1 of 90% ethanol or 1000mL, 2 propylene glycol and 70% ethanol], in and stirred 1-4 hour;

The acetonitrile solution of weight percent concentration PGA according to 15% prepares the scanning electron microscope (SEM) photograph 1E of microsphere, the about 50-150 μ of particle diameter m.Other according to 2%, 10%, 20% or the 30%PGA pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 20-100 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment six

Be loaded with the preparation of interferon (INF) biopharmaceutical macromolecular drug granule polylactic acid (PLA) microsphere

(1) according to interferon (INF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 2: with polyvinyl alcohol (PVA) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40%; With with percentage by weight be 10%, 20%, 25%, 40% or 50% glycerol and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or the hydrophilic oil phase of 50% propylene glycol preparation, according to 99%, 95%, 90%, 80%, 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): 1 of the percentage by weight 30% of the ethylene glycol of the percentage by weight 10% of 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL and 90% ethanol or 1000mL, 2 propylene glycol and 70% ethanol], in and stirred 1-4 hour;

The dichloromethane solution of weight percent concentration PLA according to 15% prepares the scanning electron microscope (SEM) photograph 1F of microsphere, the about 50-120 μ of particle diameter m.The pattern that other PLA according to 2%, 10%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 20-100 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment seven

Be loaded with the preparation of PDGF (PDGF) biopharmaceutical macromolecular drug granule polylactic acid-polyglycolic acid (PLGA) microsphere

(1) according to PDGF (PDGF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA is 50%, 60%, 75%, 90% or 100%);

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): 1 of the percentage by weight 30% of the ethylene glycol of the percentage by weight 10% of 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL and 90% ethanol or 400mL, 2 propylene glycol and 70% ethanol], in and stirred 1-4 hour;

According to 2%, 10%, 15%, 20% or the 30%PLGA pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., its size is respectively: 1-20 μ m, 40-100 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment eight

Be loaded with the preparation of growth hormone (GH) biopharmaceutical macromolecular drug granule polycaprolactone (PCL) microsphere

(1) according to growth hormone (GH) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, the dichloromethane solution weight ratio of 20% or 30% polycaprolactone (PCL) is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 1-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; Polycaprolactone (PCL) is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 2: with polyvinyl alcohol (PVA) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is respectively 1%, 5%, 10%, 20% or 40% (w/w); With with percentage by weight be 10%, 20%, 25%, 40% or 50% glycerol and 90% ethanol, 80% ethylene glycol, 80% ethylene glycol, 75% ethylene glycol, 60% propylene glycol or the hydrophilic oil phase of 50% propylene glycol preparation according to 99%, 95%, 90%, 80%, 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): percentage by weight 10% ethylene glycol of 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL and the percentage by weight 30%1 of 90% ethanol or 1000mL, 2 propylene glycol and 70% ethanol], in and stirred 1-4 hour;

Pattern according to 2%, 10%, 15%, 20% or 30% polycaprolactone (PCL) preparation microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., and its size is respectively: 1-20 μ m, 40-120 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment nine

Be loaded with the preparation of vascular endothelial cell growth factor (VEGF) biopharmaceutical macromolecular drug granule polylactic acid-polyglycol (PLA-PEG) microsphere

(1) according to vascular endothelial cell growth factor (VEGF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the ethyl acetate solution weight ratio of 30%PLA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLA-PEG is 50%, 60%, 75%, 90% or 100%);

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): percentage by weight 60% ethylene glycol of 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL and the percentage by weight 60%1 of 40% ethanol or 400mL, 2 propylene glycol and 40% ethanol], in and stirred 1-4 hour;

According to 2%, 10%, 15%, 20% or the 30%PLA-PEG pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 50-120 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment ten

Be loaded with the preparation of poly-breast-sour polyglycolic acid (PLA-PEG) microsphere of plasma thromboplastin component genetic factor biopharmaceutical macromolecular drug granule

(1) according to plasma thromboplastin component genetic factor biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the ethyl acetate solution weight ratio of 30%PLGA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 1-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA-PEG is 50%, 60%, 75%, 90% or 100%);

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): 1 of the percentage by weight 60% of the ethylene glycol of the percentage by weight 60% of 100ml ethanol, 200mL ethanol, 400mL ethanol, 200mL and 40% ethanol or 400mL, 2 propylene glycol and 40% ethanol], in and stirred 1-4 hour;

According to 2%, 10%, 15%, 20% or the 30%PLGA-PEG pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 50-120 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 11

Be loaded with the preparation of bone morphogenetic protein(BMP) (BMP) biopharmaceutical macromolecular drug granule polylactic acid-polyglycolic acid-PVOH (PLGA-PEG) microsphere

(1) according to bone morphogenetic protein(BMP) (BMP) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the ethyl acetate solution weight ratio of 30%PLGA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.1-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA-PEG is 50%, 60%, 75%, 90% or 100%);

Embodiment 12

Be loaded with the preparation of nerve growth factor (NGF) biopharmaceutical macromolecular drug granule polylactic acid-mPEG-PLGA (PLGA-PEG) microsphere

(1) according to nerve growth factor (NGF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA-PEG is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with polyvinyl alcohol (PVA) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

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

According to 2%, 10%, 15%, 20% or the 30%PLGA-PEG pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., its size is respectively: 1-20 μ m, 50-120 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 13

Be loaded with the preparation of bone-derived growth factor (BDGF) biopharmaceutical macromolecular drug granule PLA-PEG-PLA (PLA-PEG-PLA) microsphere

(1) according to bone-derived growth factor (BDGF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLA-PEG-PLA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLA-PEG-PLA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with polyvinylpyrrolidone (PVP) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

According to 2%, 10%, 15%, 20% or the 30%PLA-PEG-PLA pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., its size is respectively: 1-20 μ m, 50-130 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 14

Be loaded with the preparation of transforming growth factor (TGF-β) biopharmaceutical macromolecular drug granule PLA-PEG-PLA (PLA-PEG-PLA) microsphere

(1) according to transforming growth factor (TGF-β) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLA-PEG-PLA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLA-PEG-PLA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with poloxamer (poloxmer) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): percentage by weight 60% ethylene glycol of 100ml ethanol, 200mL ethanol, 400mL ethanol, 600mL and the percentage by weight 60%1 of 40% ethanol or 1000mL, 2 propylene glycol and 40% ethanol], in and stirred 1-4 hour;

Embodiment 15

Be loaded with the preparation of vaccine biopharmaceutical macromolecular drug granule polylactic acid-mPEG-PLGA-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA) microsphere

(1) according to vaccine biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA-PEG-PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA-PEG-PLGA is 50%, 60%, 75%, 90% or 100%);

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with poly-sorbic alcohol surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

The pattern that PLGA-PEG-PLGA according to 2%, 10%, 15%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., and its size is respectively: 1-20 μ m, 50-130 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 16

Be loaded with the preparation of antibody (antibody) biopharmaceutical macromolecular drug granule polylactic acid-mPEG-PLGA-polylactic acid-polyglycolic acid (PLGA-PEG-PLGA) microsphere

(1) according to antibody (antibody) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PLGA-PEG-PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA-PEG-PLGA is 50%, 60%, 75%, 90% or 100%);

(3) emulsion of step (2) is added drop-wise to ethanol phase (the E) [preparation of ethanol phase (E): 1 of the percentage by weight 60% of the ethylene glycol of the percentage by weight 60% of 100ml ethanol, 200mL ethanol, 400mL ethanol, 500mL and 40% ethanol or 700mL, 2 propylene glycol and 40% ethanol], in and stirred 1-4 hour;

According to 2%, 10%, 15%, 20% or the 30%PLGA-PEG-PLGA pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., its size is respectively: 1-20 μ m, 50-130 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 17

Be loaded with the preparation of epidermal growth factor (EGF) biopharmaceutical macromolecular drug granule polyethylene glycol-polylactic acid-Polyethylene Glycol (PEG-PLA-PEG) microsphere

(1) according to epidermal growth factor (EGF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PEG-PLA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PEG-PLA-PEG is 50%, 60%, 75%, 90% or 100%);

The pattern that PEG-PLA-PEG according to 2%, 10%, 15%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., and its size is respectively: 1-20 μ m, 50-130 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 18

Be loaded with the preparation of fibroblast growth factor (FGF) biopharmaceutical macromolecular drug granule polyethylene glycol-polylactic acid-Polyethylene Glycol (PEG-PLA-PEG) microsphere

(1) according to fibroblast growth factor (FGF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PEG-PLA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PEG-PLA-PEG is 50%, 60%, 75%, 90% or 100%);

Embodiment 19

Be loaded with the preparation of bone-derived growth factor (BDGF) biopharmaceutical macromolecular drug granule polyethylene glycol-polylactic acid-Polyethylene Glycol (PEG-PLGA-PEG) microsphere

(1) according to bone-derived growth factor (BDGF) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PEG-PLGA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 1-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PEG-PLGA-PEG is 50%, 60%, 75%, 90% or 100%);

The pattern that PEG-PLGA-PEG according to 2%, 10%, 15%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., and its size is respectively: 1-20 μ m, 50-130 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 20

Be loaded with the preparation of blood coagulation factor VIII (VIII) biopharmaceutical macromolecular drug granule polyethylene glycol-polylactic acid-mPEG-PLGA (PEG-PLGA-PEG) microsphere

(1) according to blood coagulation factor VIII (VIII) biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the dichloromethane solution weight ratio of 30%PEG-PLGA-PEG be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PEG-PLGA-PEG is 50%, 60%, 75%, 90% or 100%);

Embodiment 21

Be loaded with the preparation of protein biology macromolecular drug granule hyaluronic acid microsphere

(1) according to protein biology macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or 30% hyaluronic dichloromethane solution weight ratio is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; Hyaluronic acid is 50%, 60%, 75%, 90% or 100%);

The pattern that hyaluronic acid according to 2%, 10%, 15%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., and its size is respectively: 1-20 μ m, 50-130 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 22

Be loaded with the preparation of biological nucleic acid macromolecular drug granule hyaluronic acid microsphere

(1) according to biological nucleic acid macromolecular drug PEI or poly-smart ammonia granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or 30% hyaluronic dichloromethane solution weight ratio is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; Hyaluronic acid is 50%, 60%, 75%, 90% or 100%);

The pattern that hyaluronic acid according to 2%, 10%, 15%, 20% or 30% prepares microsphere is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., and its size is respectively: 1-20 μ m, 50-130 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m

Embodiment 23

Be loaded with the preparation of small-molecule drug sodium alginate Granular gelatin microsphere

(1) according to small-molecule drug sodium alginate granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, the dichloromethane solution weight ratio of 20% or 30% gelatin is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles 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 got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with ethyl cellulose surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute;

Be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG. according to the pattern for preparing microsphere according to 2%, 10%, 15%, 20% or 30% gelatin, its size is respectively: 1-20 μ m, 50-125 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 24

Be loaded with the preparation of glucosan biopharmaceutical macromolecular drug Granular gelatin microsphere

(1) according to glucosan biopharmaceutical macromolecular drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, the dichloromethane solution weight ratio of 20% or 30% gelatin is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; Gelatin is 50%, 60%, 75%, 90% or 100%);

(4) the centrifugal collection microsphere that step (3) is obtained, and wash with water 3-5 time, lyophilizing gets dry microsphere.

The pattern for preparing microsphere according to 2%, 10%, 15%, 20% or 30% gelatin is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 50-150 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 25

Be loaded with the preparation of nucleic acid drug granule polylactic acid poly hydroxyacetic acid (PLGA) microsphere

(1) according to nucleic acid drug granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, 20% or the acetonitrile solution weight ratio of 30%PLGA be 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 1-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA is 50%, 60%, 75%, 90% or 100%);

According to 2%, 10%, 15%, 20% or the 30%PLGA pattern for preparing microsphere be similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 50-150 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment four

Embodiment five

Embodiment six

Embodiment seven

Be loaded with the preparation of PDGF (PDGF) biopharmaceutical macromolecular drug granule polylactic acid poly hydroxyacetic acid (PLGA) microsphere

Embodiment eight

Embodiment nine

Embodiment ten

Be loaded with the preparation of plasma thromboplastin component genetic factor biopharmaceutical macromolecular drug granule polylactic acid-polyglycolic acid (PLA-PEG) microsphere

Embodiment 11

Embodiment 12

Embodiment 13

(2) step (1) is got emulsion droplets and be added to hydrophilic oil phase (hO): [preparation of hydrophilic oil phase (hO): prescription 1: with polyvinylpyrrolidone (PVP) surfactant and sodium chloride salt mixed water solution, their weight percent concentration are respectively 1%, 2%, 5%, 8% or 10% and 0%, 1%, 5%, 7% or 10%, and the percentage by weight that accounts for hydrophilic oil phase is 0,5%, 10%, 25% or 40%; Ethylene glycol is respectively 100%, 95%, 90%, 75% or 60%] and formed emulsion in stirring, whirlpool or ultrasonic 0.5-5 minute

Embodiment 14

Embodiment 15

Embodiment 16

Embodiment 17

Embodiment 18

Embodiment 19

Embodiment 20

Embodiment 21

Embodiment 22

Embodiment 23

Embodiment 24

Embodiment 25

Embodiment 26

Be loaded with the preparation of vaccine drug particles PEG-PCL (PEG-PCL) microsphere

(1) according to vaccine drug particles 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, the dichloromethane solution weight ratio of 20% or 30% PEG-PCL (PEG-PCL) is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PEG-PCL is 50%, 60%, 75%, 90% or 100%);

The pattern for preparing microsphere according to 2%, 10%, 15%, 20% or 30% PEG-PCL is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-20 μ m, 50-150 μ m, 60-150 μ m, 70-180 μ m or 200-500 μ m.

Embodiment 27

Be loaded with the preparation of microsphere of the mixture of the polylactic acid of protein drug granule and polylactic acid-polyglycolic acid (PLA and PLGA)

(1) according to protein drug (take EPO as the model protein medicine) granule 0.4mg, 2mg, 1.5mg, (its size is respectively 0.2-1 μ m for 1mg or 0mg, 1-5 μ m, 2-6 μ m, 2-10 μ m or 0 μ m) and weight percent concentration be 2%, 10%, 15%, (polylactic acid: polylactic acid-polyglycolic acid is 1: 1 according to weight ratio for 20% or 30% polylactic acid and polylactic acid-polyglycolic acid, 2: 1,4: 1,4: 1, or 2: 1 mixing) dichloromethane solution weight ratio is 1: 20,1: 30,1: 30,1: 45 or equal proportion stirring in 1: 4, formed in whirlpool or ultrasonic 0.5-5 minute even suspension namely oil bag solid (S/O) emulsion (preparing the percentage ratio that corresponding drug particles accounts for the component of microsphere is respectively: 50%, 40%, 25%, 10% or 0%; PLGA and PLA are 50%, 60%, 75%, 90% or 100%);

Pattern according to the preparation microsphere of the mixture of 2%, 10%, 15%, 20% or 30% polylactic acid and polylactic acid-polyglycolic acid (PLA and PLGA) is similar to above-mentioned scanning electron microscope Fig. 1 not shown on the FIG., but its size is respectively: 1-25 μ m, 50-155 μ m, 60-160 μ m, 70-180 μ m or 200-500 μ m.

Claims

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 Uniform Dispersion and forms suspension;

The percentage by weight of the percentage by weight of 0＜described drug particles≤50%, 50%≤described slow release or controlled-release material＜100%;

Described medicine is small-molecule drug or macromolecular drug; Described slow release or controlled-release material are polylactic acid-polyglycolic acid;

2. suspension being added in another hydrophilic organic solution is hydrophilic oil phase, and the organic solvent of the 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 dry 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 hydrophilic oil of ethanol bag according to claim 2-hydrophilic oil bag oil oil wraps the method for 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; Described nucleic acid is: antisense nucleotide, microRNA or gene.

4. the hydrophilic oil of ethanol bag according to claim 3-hydrophilic oil bag oil oil wraps the method for 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, 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, the bone formation egg certainly, tissue polypeptide antigen, platelet cofactor Ⅰ or IX genetic factor.

5. the hydrophilic oil of ethanol bag according to claim 1-hydrophilic oil bag oil oil wraps the method for microspheres with solid preparation, it is characterized in that described hydrophilic oil phase is: aqueous surfactant solution, 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 aqueous surfactant solution.

6. the hydrophilic oil of ethanol bag according to claim 5-hydrophilic oil bag oil oil wraps the method for 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%.

7. the hydrophilic oil of ethanol bag according to claim 6-hydrophilic oil bag oil oil wraps the method for microspheres with solid preparation, it is characterized in that, described aqueous surfactant solution is: the mixed water solution of polyvinyl alcohol and sodium chloride, and their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of Polyethylene Glycol and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of polyvinylpyrrolidone and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; The mixed water solution of poloxamer and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%; Or the mixed water solution of ethyl cellulose and sodium chloride, their weight percent concentration is respectively 1-10% and 0-10%.

8. 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.