Method for preparing sustained-release microspheres containing micronized recombinant human vascular endothelial inhibin

Abstract

The invention relates to a method for preparing sustained-release microspheres with recombinant human vascular endothelial inhibin, in particular to a high-entrapment-efficiency method for preparing sustained-release microspheres with recombinant human vascular endothelial inhibin. The method comprises the following steps: (1) dissolving copolymer of lactic acid and hydroxyacetic acid in an organic solvent to obtain an inner oil phase; (2) adding recombinant human vascular endothelial inhibin micropowder to the inner oil phase; (3) dispersing the recombinant human vascular endothelial inhibinmicropowder and the inner oil phase at a high speed between 4,000 and 50,000 revolutions to obtain solid-liquid suspension; (4) putting the suspension in a stir container holding emulsifier-containing vegetable oil or mineral oil and stirring at a temperature between 0 and 60 DEG C at a speed between 100 and 2,000 revolutions per minute to form oil-in-oil coated-fixed type compound emulsion; and (5) continuing stirring a mixed organic solvent in the volatile inner oil phase, using microporous membrane for filtration and separation after microspheres are cured, using the solvent to wash the vegetable oil or mineral oil remaining on the surfaces of the microspheres, drying under vacuum and then obtaining finished microsphere products.

Description

technical field

[0001] The invention relates to a preparation method of recombinant human endostatin sustained-release microspheres, in particular to a preparation method of recombinant human endostatin sustained-release microspheres with high encapsulation efficiency. Background technique

[0002] In the 1960s, Dr. Folkman of Harvard Medical School put forward the hypothesis that "tumor growth depends on blood vessel growth", and in 1971 he proposed the theory of "starving tumor therapy". Today, under the impetus of molecular biology technology, the anti-cancer protein-endu has been developed by using genetic engineering technology. Chinese patent CN 1237072C Luo Yongzhang et al. added 9 amino acids to the N-terminal of natural Endostatin, which not only improves the stability of Endostatin, prolongs the half-life, but also increases the biological activity, and the renaturation rate of the protein is also higher than that of the general production method. The drug has bee...

Examples

Embodiment 1

[0049] Accurately weigh 240 mg of recombinant human endostatin Endostar micropowder (containing 200 mg of recombinant human endostatin Endostar), dissolve 400 mg of PLGA (Mw=48000, 50:50) in 10 ml of acetonitrile, add the micropowder into PLGA dichloromethane In the mixed organic solution with acetonitrile, dichloromethane:acetonitrile=1:4 (volume ratio). 4000rpm high-speed dispersion and emulsification to form S / O colostrum. Pour the above S / O colostrum into soybean oil containing 0.3% lecithin and 0.1% sucrose ester, mechanically stir the volatile solvent (1000rpm) for 4 hours, use a 0.8 μm organic microporous membrane to filter to obtain microspheres, and use petroleum Washed three times with ether, and finally freeze-dried to obtain the finished microspheres. The finished microspheres were dissolved in dichloromethane, and the drug was extracted with a PBS solution with pH=7.4. The protein concentration in the extracted solution was determined by HPLC to obtain the drug l...

Embodiment 2

[0052] Accurately weigh 120 mg of recombinant human endostatin Endostar micropowder (containing 100 mg of recombinant human endostatin Endostar), dissolve 400 mg of PLGA (Mw=10000, 50:50) in 10 ml of acetonitrile, and add the micropowder into the acetonitrile solution of PLGA , 4000rpm high-speed dispersion and emulsification to form S / O colostrum. Pour the above S / O colostrum into the medicinal light liquid paraffin containing 0.5% Span 80, mechanically stir the volatile solvent (500rpm) for 4 hours, use a 0.8 μm organic microporous membrane to filter to obtain microspheres, and use petroleum Washed three times with ether, and finally freeze-dried to obtain the finished microspheres. The finished microspheres were dissolved in dichloromethane, and the drug was extracted with a PBS solution with pH=7.4. The protein concentration in the extracted solution was determined by HPLC to obtain the drug loading and encapsulation efficiency of the microspheres. The drug loading is 28....

Embodiment 3

[0055] Accurately weigh the same batch of micronized Endostar protein used in Example 1, redissolve with buffer, and dilute to 1500ng / ml, 1200ng / ml, 1000ng / ml, 800ng / ml, 600ng / ml, 400ng / ml, 200ng / ml, 100ng / ml, 50ng / ml. The immunological activity was measured with a commercially available ELISA (enzyme-linked immunoassay) kit. Method: Make a standard curve of unmicronized protein, and then make a standard curve of reconstituted micronized protein, compare the difference in absorbance value (OD) between the two curves at 100ng / ml-1000ng / ml, and obtain the immunological activity retention Rate. The calculation method of activity retention rate is: activity retention rate=100%-(non-micronized protein OD-micronized protein OD) / (non-micronized protein OD-blank value 0.0392)*100%. Table 1 shows that the protein activity retention rate after micronization is greater than 90%.

[0056] Table 1

[0057]

[0058]

technical field

[0001] The invention relates to a preparation method of recombinant human endostatin sustained-release microspheres, in particular to a preparation method of recombinant human endostatin sustained-release microspheres with high encapsulation efficiency. Background technique

[0002] In the 1960s, Dr. Folkman of Harvard Medical School put forward the hypothesis that "tumor growth depends on blood vessel growth", and in 1971 he proposed the theory of "starving tumor therapy". Today, under the impetus of molecular biology technology, the anti-cancer protein-endu has been developed by using genetic engineering technology. Chinese patent CN 1237072C Luo Yongzhang et al. added 9 amino acids to the N-terminal of natural Endostatin, which not only improves the stability of Endostatin, prolongs the half-life, but also increases the biological activity, and the renaturation rate of the protein is also higher than that of the general production method. The drug has bee...

Examples

Embodiment 1

[0049] Accurately weigh 240 mg of recombinant human endostatin Endostar micropowder (containing 200 mg of recombinant human endostatin Endostar), dissolve 400 mg of PLGA (Mw=48000, 50:50) in 10 ml of acetonitrile, add the micropowder into PLGA dichloromethane In the mixed organic solution with acetonitrile, dichloromethane:acetonitrile=1:4 (volume ratio). 4000rpm high-speed dispersion and emulsification to form S / O colostrum. Pour the above S / O colostrum into soybean oil containing 0.3% lecithin and 0.1% sucrose ester, mechanically stir the volatile solvent (1000rpm) for 4 hours, use a 0.8 μm organic microporous membrane to filter to obtain microspheres, and use petroleum Washed three times with ether, and finally freeze-dried to obtain the finished microspheres. The finished microspheres were dissolved in dichloromethane, and the drug was extracted with a PBS solution with pH=7.4. The protein concentration in the extracted solution was determined by HPLC to obtain the drug l...

Embodiment 2

[0052] Accurately weigh 120 mg of recombinant human endostatin Endostar micropowder (containing 100 mg of recombinant human endostatin Endostar), dissolve 400 mg of PLGA (Mw=10000, 50:50) in 10 ml of acetonitrile, and add the micropowder into the acetonitrile solution of PLGA , 4000rpm high-speed dispersion and emulsification to form S / O colostrum. Pour the above S / O colostrum into the medicinal light liquid paraffin containing 0.5% Span 80, mechanically stir the volatile solvent (500rpm) for 4 hours, use a 0.8 μm organic microporous membrane to filter to obtain microspheres, and use petroleum Washed three times with ether, and finally freeze-dried to obtain the finished microspheres. The finished microspheres were dissolved in dichloromethane, and the drug was extracted with a PBS solution with pH=7.4. The protein concentration in the extracted solution was determined by HPLC to obtain the drug loading and encapsulation efficiency of the microspheres. The drug loading is 28....

Embodiment 3

[0055] Accurately weigh the same batch of micronized Endostar protein used in Example 1, redissolve with buffer, and dilute to 1500ng / ml, 1200ng / ml, 1000ng / ml, 800ng / ml, 600ng / ml, 400ng / ml, 200ng / ml, 100ng / ml, 50ng / ml. The immunological activity was measured with a commercially available ELISA (enzyme-linked immunoassay) kit. Method: Make a standard curve of unmicronized protein, and then make a standard curve of reconstituted micronized protein, compare the difference in absorbance value (OD) between the two curves at 100ng / ml-1000ng / ml, and obtain the immunological activity retention Rate. The calculation method of activity retention rate is: activity retention rate=100%-(non-micronized protein OD-micronized protein OD) / (non-micronized protein OD-blank value 0.0392)*100%. Table 1 shows that the protein activity retention rate after micronization is greater than 90%.

[0056] Table 1

[0057]

[0058]

Images