Preparation method of recombined human blood-vessel endothelia inhibin sustained-released microsphere

Abstract

The invention discloses a method for preparing a recombinant human endostatin sustained release microsphere. In the method, recombinant human endostatin is solved in buffer solution containing protein stabilizer to obtain an inner water phase; lacto-glycolic acid copolymer is solved in mixed organic solvent to obtain an inner oil phase; the mixed solution of the inner water phase and the inner oil phase is dispersed fast to obtain primary emulsion; the obtained primary emulsion is arranged in a stirring vessel which is filled with plant oil or mineral oil containing emulsifier to be stirred into water-in-oil type compound emulsion; the water-in-oil type compound emulsion is stirred continuously to volatilize the mixed organic solvent in the inner oil phase and then is filtered and separated by a microporous filtering film after the microsphere is solidified; the residual plant oil or mineral oil on the surface of the microsphere is washed by the solvent; and the microsphere finished product is obtained after vacuum drying. The microsphere prepared by the method has high drug loading quantity, high entrapment rate, proper burst release quantity and long sustained release period.

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 the growth of blood vessels", and in 1971 proposed the theory of "starve tumor therapy". Today, under the impetus of molecular biology technology, the anti-cancer protein - Endo has been developed using genetic engineering technology. Chinese patent CN 1237072C Luo Yongzhang et al. added 9 amino acids to the N-terminus 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 b...

Examples

Embodiment 1

[0041] 400 mg of Endostar was accurately weighed and dissolved in 1 ml of PBS buffer at pH 7.4 (containing 3% gelatin) to form an inner aqueous phase. 400 mg of PLGA (Mw=20000, 75:25) was dissolved in 8 ml of a mixed solvent of dichloromethane and acetonitrile (1:1) to obtain an inner oil phase. Add the above-mentioned inner water phase to the inner oil phase, disperse and emulsify at high speed at 4000 rpm to form W / O colostrum, then pour this colostrum into soybean oil containing 0.3% lecithin and 0.1% sucrose ester, and mechanically stir to evaporate the solvent (500rpm) for 4 hours, use a 0.8 μm organic microporous membrane to filter to obtain microspheres, wash three times with petroleum ether, and finally freeze-dry to obtain the finished microspheres. The finished microspheres were dissolved in dichloromethane, the drug was extracted with a PBS solution of pH=7.4, and the protein concentration in the extracted solution was measured by HPLC to obtain the encapsulation ef...

Embodiment 2

[0044] Accurately weigh 2000mg Endostar and 200mg Chondroitin Sulfate and dissolve it in 10ml Tris (10Mm PH7.4), adjust the pH of the solution to 6.5-8.3, and measure when its zeta potential is between 0 and -48mv to form a chondroitin sulfate-Endostar complex. thing. Take 0.5 ml of the complex solution as the inner water phase. 400 mg of PLGA (Mw=20000, 50:50) was dissolved in 8 ml of a mixed solvent of dichloromethane, acetonitrile and ethyl acetate (0.5:1:0.5) to obtain an inner oil phase. Add the above-mentioned inner water phase to the inner oil phase, disperse and emulsify at high speed at 4000 rpm to form W / O colostrum, then pour this colostrum into liquid paraffin containing 0.8% Span-80 and 0.35% sucrose ester, stir mechanically The solvent was evaporated (500 rpm) for 4 hours, and the microspheres were obtained by filtration with a 0.8 μm organic microporous membrane, washed three times with petroleum ether, and finally freeze-dried to obtain the finished microspher...

Embodiment 3

[0047] Accurately weigh 400 mg of Endostar and dissolve it in 1 ml of sodium acetate solution with pH=5.5 (containing 5% bovine serum albumin) to form an inner aqueous phase. PLGA (A: Mw=12000, 50:50), (B: Mw=20000, 50:50), (C: Mw=40000, 50:50), (D: Mw=80000, 100:0) , (E: Mw=20000, 75:25), (F: Mw=130000, 75:25) 400 mg were dissolved in 8 ml of a mixed solvent of dichloromethane, acetonitrile and ethyl acetate (0.5:1:0.5) to become Internal oil phase. Add the above-mentioned inner water phase to the above-mentioned inner oil phase respectively, disperse and emulsify at high speed at 4000 rpm to form W / O colostrum, then pour this colostrum into soybean oil containing 0.5% lecithin and 0.2% sucrose ester, and stir mechanically. The solvent was evaporated (500 rpm) for 4 hours, and the microspheres were obtained by filtration with a 0.8 μm organic microporous membrane, washed three times with petroleum ether, and finally freeze-dried to obtain the finished microspheres. The fini...

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 the growth of blood vessels", and in 1971 proposed the theory of "starve tumor therapy". Today, under the impetus of molecular biology technology, the anti-cancer protein - Endo has been developed using genetic engineering technology. Chinese patent CN 1237072C Luo Yongzhang et al. added 9 amino acids to the N-terminus 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 b...

Examples

Embodiment 1

[0041] 400 mg of Endostar was accurately weighed and dissolved in 1 ml of PBS buffer at pH 7.4 (containing 3% gelatin) to form an inner aqueous phase. 400 mg of PLGA (Mw=20000, 75:25) was dissolved in 8 ml of a mixed solvent of dichloromethane and acetonitrile (1:1) to obtain an inner oil phase. Add the above-mentioned inner water phase to the inner oil phase, disperse and emulsify at high speed at 4000 rpm to form W / O colostrum, then pour this colostrum into soybean oil containing 0.3% lecithin and 0.1% sucrose ester, and mechanically stir to evaporate the solvent (500rpm) for 4 hours, use a 0.8 μm organic microporous membrane to filter to obtain microspheres, wash three times with petroleum ether, and finally freeze-dry to obtain the finished microspheres. The finished microspheres were dissolved in dichloromethane, the drug was extracted with a PBS solution of pH=7.4, and the protein concentration in the extracted solution was measured by HPLC to obtain the encapsulation ef...

Embodiment 2

[0044] Accurately weigh 2000mg Endostar and 200mg Chondroitin Sulfate and dissolve it in 10ml Tris (10Mm PH7.4), adjust the pH of the solution to 6.5-8.3, and measure when its zeta potential is between 0 and -48mv to form a chondroitin sulfate-Endostar complex. thing. Take 0.5 ml of the complex solution as the inner water phase. 400 mg of PLGA (Mw=20000, 50:50) was dissolved in 8 ml of a mixed solvent of dichloromethane, acetonitrile and ethyl acetate (0.5:1:0.5) to obtain an inner oil phase. Add the above-mentioned inner water phase to the inner oil phase, disperse and emulsify at high speed at 4000 rpm to form W / O colostrum, then pour this colostrum into liquid paraffin containing 0.8% Span-80 and 0.35% sucrose ester, stir mechanically The solvent was evaporated (500 rpm) for 4 hours, and the microspheres were obtained by filtration with a 0.8 μm organic microporous membrane, washed three times with petroleum ether, and finally freeze-dried to obtain the finished microspher...

Embodiment 3

[0047] Accurately weigh 400 mg of Endostar and dissolve it in 1 ml of sodium acetate solution with pH=5.5 (containing 5% bovine serum albumin) to form an inner aqueous phase. PLGA (A: Mw=12000, 50:50), (B: Mw=20000, 50:50), (C: Mw=40000, 50:50), (D: Mw=80000, 100:0) , (E: Mw=20000, 75:25), (F: Mw=130000, 75:25) 400 mg were dissolved in 8 ml of a mixed solvent of dichloromethane, acetonitrile and ethyl acetate (0.5:1:0.5) to become Internal oil phase. Add the above-mentioned inner water phase to the above-mentioned inner oil phase respectively, disperse and emulsify at high speed at 4000 rpm to form W / O colostrum, then pour this colostrum into soybean oil containing 0.5% lecithin and 0.2% sucrose ester, and stir mechanically. The solvent was evaporated (500 rpm) for 4 hours, and the microspheres were obtained by filtration with a 0.8 μm organic microporous membrane, washed three times with petroleum ether, and finally freeze-dried to obtain the finished microspheres. The fini...

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