CN1308034C - Method of preparing microsphere of ethoxyl copolymer PLGA in interferon poly acid - Google Patents
Method of preparing microsphere of ethoxyl copolymer PLGA in interferon poly acid Download PDFInfo
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- CN1308034C CN1308034C CNB2004100776220A CN200410077622A CN1308034C CN 1308034 C CN1308034 C CN 1308034C CN B2004100776220 A CNB2004100776220 A CN B2004100776220A CN 200410077622 A CN200410077622 A CN 200410077622A CN 1308034 C CN1308034 C CN 1308034C
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Abstract
The present invention discloses a preparation method for an interferon polylactic acid-glycolate copolymer (PLGA) microsphere, which is characterized in that the microsphere is prepared by a multiple emulsion-solvent volatilization method. A polylactic acidglycolate copolymer (PLGA) is a microsphere carrier, and recombinant human interferon-alpha is taken as a packaged object in a freeze-drying injection process. The manufactured interferon microsphere has round and normal form and uniform particle size distribution. The particle size distribution is below 30 microns, the drug bearing capacity can reach more than 8%, and the encapsulation efficiency is about 40%. The extrasomatic drug release property accords with the character of a long-acting preparation.
Description
Technical field
The present invention relates to a kind of preparation method of biodegradable control-release microsphere of biologically active drug, belong to the crossing research field of biological medical polymer material and biologically active drug controlled release preparation.
Background technology
In recent years, along with the high speed development of biotechnology, increasing polypeptide, pharmaceutical grade protein are developed out, and show the curative effect of many uniquenesses in clinical practice, for example insulin, Hepatitis B virus vaccine, interferon etc.But because polypeptide and protein medicaments biological half-life are short, poor stability easily by enzymes metabolism in the body, and is difficult to absorb in gastrointestinal tract, so need for a long time, drug administration by injection continually, has hindered that it is convenient clinically, extensively and has effectively used.
Human recombinant interferon-α is the product that adopts biological high-tech preparation, by the engineering bacteria that has the human interferon gene through efficiently expressing, and the off-white powder highly purified, that lyophilization is made, it has stronger immunoloregulation function, can suppress the effect of tumor proliferation, antiviral and enhancing human body immunity regulatory function.Be mainly used in treatment rheumatoid arthritis, tumor, viral disease.Because the oral inactivation of interferon, need drug administration by injection, and the half-life in blood is short, poor stability, easily by enzymes metabolism in the body, be eliminated soon after the injection or degrade,, need frequent, heavy dose of and long-time administration in order to reach curative effect, usually usage is intramuscular injection every day 50-200 ten thousand IU, and 3 months is a course of treatment.
Utilizing the biological degradation polyalcohol microparticulate systems to come transport protein matter medicine is a main method of current domestic and international research, report is arranged, with the polylactic acid-hydroxide acetic acid copolymer is human growth hormone's microball preparation of carrier, and its drug effect than injection every day is once injected 28 days better effects if continuously.Interferon is made polylactic acid microsphere, can reach the raising medicine stability, prolong action time and purpose easy to use.The report of interferon microball preparation was abroad promptly arranged at twentieth century in 90 years, and its carrier material has albumin, gelatin and polylactic acid-hydroxide acetic acid copolymer (PLGA) etc., and the PLGA microsphere is long and more satisfactory because of drug release time.
At present, scientist is when the development sustained release microsphere agents both at home and abroad, and using maximum biodegradated polymer materals is polylactic acid-hydroxide acetic acid copolymer, because of it has favorable biological degradability, the compatibility and absorbability, is pharmaceutic adjuvant by the FDA approval.And polylactic acid sustained-release micro-spheres product such as luteinising hormone-releasing hormo, leuprorelin official listing abroad.Because polylactic acid-hydroxide acetic acid copolymer is not also produced at home as medicinal materials, and human recombinant interferon-α has how tame manufacturer production in China, price is suitable, if interferon-' alpha ' is made the polylactic acid-hydroxide acetic acid copolymer sustained release microsphere agents, to improve its added value greatly, not only can promote the exploitation of biotech drug, but also can promote the industrialization process of pharmaceutic adjuvant.
Summary of the invention
The objective of the invention is to adopt polylactic acid-hydroxide acetic acid copolymer (PLGA) is carrier, interferon-' alpha ' is made microball preparation, reach the increase medicine stability, prolong drug action time, reduce frequency injection and consumption, improve the purpose of curative effect of medication and economic benefit, and the preparation method of interferon microsphere is provided.
Interferon polylactic acid-hydroxide acetic acid copolymer of the present invention (PLGA) method for preparing microsphere is to adopt emulsion-solvent evaporated method.Polylactic acid-hydroxide acetic acid copolymer is dissolved in the organic solvent; Lyophilizing injection recombinant human interferon alpha 2 is dissolved in the interior water of formation in the PBS solution that contains stabilizing agent; Interior water is injected in the organic facies, stirring and emulsifying becomes the W/O colostrum again; In the water PVA aqueous solution, be emulsified into the W/O/W emulsion outside then colostrum being added under stirring condition; Stirring at low speed in containing electrolytical solution makes the organic solvent volatilization fully again; Centrifugal, washing, collect microsphere, lyophilization promptly gets the microsphere of ethoxyl copolymer PLGA in interferon poly acid powder.
Interferon polylactic acid-hydroxide acetic acid copolymer (PLGA) method for preparing microsphere is characterized in that preparation method may further comprise the steps:
(1) preparation of polylactic acid-hydroxide acetic acid copolymer PLGA solution
Polylactic acid-glycolic guanidine-acetic acid copolymer p LGA is dissolved in forms organic facies in the organic solvent, concentration 20%～30%g/ml of PLGA;
(2) preparation of interferon PBS solution
Lyophilizing injection recombinant human interferon-alpha is dissolved in the interior water of formation in the PBS solution that contains a certain amount of stabilizing agent, and the concentration of interferon is 13.3%～20%g/ml, and the concentration of stabilizing agent is 11%～30%g/ml;
(3) preparation of colostrum
Interior water is injected into formation W/O colostrum in the organic facies, and emulsifying rate is 1000～9000rpm, and emulsification times is 1～3 minute, and the volume ratio of organic facies and interior water is 20: 3;
(4) preparation of emulsion
Colostrum is added under condition of stirring in the outer water aqueous solution of certain density PVA and NaCl, is emulsified into the W/O/W emulsion; The mixing speed that emulsion forms is 1600～2000rpm, and the concentration of PVA solution is 1～3%g/ml, and the concentration of NaCl solution is 0.5～1%g/ml, and emulsification times is 10～20 minutes, and colostrum is 2.3: 200 to 2.3: 300 with the volume ratio of outer water;
(5) formation of microsphere
Emulsion solution is transferred in the NaCl aqueous solution, and stirring at low speed is complete, centrifugal to the organic solvent volatilization, microsphere is collected in the washing back; The concentration of NaCl solution is 1%g/ml, and volume is 2-3 a times of emulsion, and mixing speed is 1000rpm, and centrifugal speed is 1200～1500rpm, and centrifugation time is 15-20 minute, and washing times is 3 times;
(6) lyophilization.
Good effect of the present invention is: interferon polylactic acid-hydroxide acetic acid copolymer (PLGA) microsphere that the present invention adopts emulsion-solvent evaporation method to prepare, microsphere stable preparation process, feasible, the form rounding of microsphere, smooth surface, good fluidity, even particle size distribution, mean diameter is 30 μ m, drug loading is more than 8%, and envelop rate is about 40%, and its external Release Performance meets the durative action preparation feature.Interferon-' alpha ' is made sustained release microsphere agents, but prolong drug action time is improved curative effect of medication and economic benefit.
Accompanying drawing and explanation
Fig. 1 adopts the optical microscope and the electron scanning micrograph of the interferon PLGA microsphere that the present invention prepares, and observes configuration of surface.
Fig. 2 is the DSC curve of the blank microsphere (1) of PLGA, interferon PLGA medicine carrying microballoons (2).
The specific embodiment
The invention will be further described for following examples, so that those skilled in the art further understands the present invention, but do not limit the present invention in any form.
The preparation of embodiment 1 interferon polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) microsphere
(1) preparation of polylactic acid-hydroxide acetic acid copolymer (PLGA) solution
400mg polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) is dissolved in forms organic facies (20%g/ml) in the 2ml dichloromethane.
(2) preparation of interferon PBS solution
40mg lyophilizing injection recombined human interferon-alpha is dissolved in form in the gelatin solution of 300 μ l1% (g/ml) concentration in water;
(3) preparation of colostrum
Interior water is injected in the organic facies, and stirring and emulsifying 1 minute is to form colostrum (W/O) under the 9000rpm condition;
(4) preparation of emulsion
Colostrum is added to 300ml contains in 1% (g/ml) PVA and 1% (g/ml) NaCl solution (outer water) under the 2000rpm condition of stirring, emulsifying 10 minutes is emulsified into emulsion (W/O/W);
(5) formation of microsphere
Emulsion solution is transferred in 3 times of amount 1% (g/ml) NaCl aqueous solutions, and the 1000rpm stirring at low speed is complete to the organic solvent volatilization, and centrifugal 15 minutes of 1500rpm collects microsphere with distilled water wash 3 times, washing back;
(6) made microsphere is carried out lyophilization in the following manner successively: in-40 ℃, dry 4h; Follow in-25 ℃ dry 10h; Follow in-10 ℃ dry 20h; At last in-4 ℃, dry 48h.
Made microsphere is carried out lyophilization, under above-mentioned lyophilization condition, obtain the Powdered interferon polylactic acid-glycolic of white loose guanidine-acetic acid copolymer microsphere.
The form rounding of gained interferon microsphere, smooth surface, good fluidity, even particle size distribution, mean diameter are 10.71 μ m, and drug loading is 6.96%, and envelop rate is 32.90%, and external Release Performance meets the durative action preparation feature.
The preparation of embodiment 2 interferon polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) microspheres
(1) preparation of polylactic acid-hydroxide acetic acid copolymer (PLGA) solution
600mg polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) is dissolved in forms organic facies (30%g/ml) in the 2ml dichloromethane.
(2) preparation of interferon PBS solution
60mg lyophilizing injection recombined human interferon-alpha is dissolved in the interior water of formation in the 300 μ l 30%PEG400 solution;
(3) preparation of colostrum
Interior water is injected in the organic facies, and stirring and emulsifying 3 minutes is to form colostrum (W/O) under the 1000rpm condition;
(4) preparation of emulsion
Colostrum is added to 200ml contains in 3% (g/ml) PVA and 0.5% (g/ml) NaCl solution (outer water) under the 1600rpm condition of stirring, emulsifying 20 minutes is emulsified into emulsion (W/O/W);
(5) formation of microsphere
Emulsion solution is transferred in 2 times of amount 1% (g/ml) NaCl aqueous solutions, and the 1000rpm stirring at low speed is complete to the organic solvent volatilization, and centrifugal 20 minutes of 1200rpm collects microsphere with distilled water wash 3 times, washing back;
(6) made microsphere is carried out lyophilization in the following manner successively: in-40 ℃, dry 4h; Follow in-25 ℃ dry 10h; Follow in-10 ℃ dry 20h; At last in-4 ℃, dry 48h.
Made microsphere is carried out lyophilization, under above-mentioned lyophilization condition, obtain the Powdered interferon polylactic acid-glycolic of white loose guanidine-acetic acid copolymer microsphere.
The form rounding of gained interferon microsphere, smooth surface, good fluidity, even particle size distribution, mean diameter are 12.36 μ m, and drug loading is 6.06%, and envelop rate is 36.80%, and external Release Performance meets the durative action preparation feature.
Claims (5)
1, a kind of preparation method of interferon polylactic acid-hydroxide acetic acid copolymer microsphere, carry out according to the following steps:
Polylactic acid-hydroxide acetic acid copolymer is dissolved in the organic solvent; Lyophilizing injection recombinant human interferon alpha 2 is dissolved in the interior water of formation in the PBS solution that contains stabilizing agent; Interior water is injected in the organic facies, stirring and emulsifying becomes the W/O colostrum again; In the water PVA aqueous solution, be emulsified into the W/O/W emulsion outside then colostrum being added under stirring condition; Stirring at low speed in containing electrolytical solution makes the organic solvent volatilization fully again; Centrifugal, washing, collect microsphere, lyophilization promptly gets the microsphere of ethoxyl copolymer PLGA in interferon poly acid powder.
2, interferon polylactic acid-hydroxide acetic acid copolymer microsphere preparation method as claimed in claim 1, it is characterized in that: preparation method may further comprise the steps:
(1) preparation of polylactic acid-hydroxide acetic acid copolymer PLGA solution
Polylactic acid-glycolic guanidine-acetic acid copolymer p LGA is dissolved in forms organic facies in the organic solvent, concentration 20%～30%g/ml of PLGA;
(2) preparation of interferon PBS solution
Lyophilizing injection recombinant human interferon-alpha is dissolved in the interior water of formation in the PBS solution that contains a certain amount of stabilizing agent, and the concentration of interferon is 13.3%～20%g/ml, and the concentration of stabilizing agent is 11%～30%g/ml;
(3) preparation of colostrum
Interior water is injected into formation W/O colostrum in the organic facies, and emulsifying rate is 1000～9000rpm, and emulsification times is 1～3 minute, and the volume ratio of organic facies and interior water is 20: 3;
(4) preparation of emulsion
Colostrum is added under condition of stirring in the outer water aqueous solution of certain density PVA and NaCl, is emulsified into the W/O/W emulsion; The mixing speed that emulsion forms is 1600～2000rpm, and the concentration of PVA solution is 1～3%g/ml, and the concentration of NaCl solution is 0.5～1%g/ml, and emulsification times is 10～20 minutes, and colostrum is 2.3: 200 to 2.3: 300 with the volume ratio of outer water;
(5) formation of microsphere
Emulsion solution is transferred in the NaCl aqueous solution, and stirring at low speed is complete, centrifugal to the organic solvent volatilization, microsphere is collected in the washing back; The concentration of NaCl solution is 1%g/ml, and volume is 2-3 a times of emulsion, and mixing speed is 1000rpm, and centrifugal speed is 1200～1500rpm, and centrifugation time is 15-20 minute, and washing times is 3 times;
(6) lyophilization.
3. interferon polylactic acid-hydroxide acetic acid copolymer microsphere preparation method as claimed in claim 1 or 2 is characterized in that: described organic solvent is a dichloromethane.
4. interferon polylactic acid-hydroxide acetic acid copolymer microsphere preparation method as claimed in claim 1 or 2 is characterized in that: the stabilizing agent of described interferon is gelatin or PEG400.
5. interferon polylactic acid-hydroxide acetic acid copolymer microsphere preparation method as claimed in claim 1 or 2 is characterized in that: made microsphere is carried out lyophilization in the following manner successively: in-40 ℃, and dry 4h; Follow in-25 ℃ dry 10h; Follow in-10 ℃ dry 20h; At last in-4 ℃, dry 48h.
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| CNB2004100776220A CN1308034C (en) | 2004-12-27 | 2004-12-27 | Method of preparing microsphere of ethoxyl copolymer PLGA in interferon poly acid |
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| CNB2004100776220A CN1308034C (en) | 2004-12-27 | 2004-12-27 | Method of preparing microsphere of ethoxyl copolymer PLGA in interferon poly acid |
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| CN100460441C (en) * | 2006-09-25 | 2009-02-11 | 南开大学 | Biodegradable star-shaped polylactide drug-loaded microspheres and preparation method thereof |
| CN100457187C (en) * | 2006-11-10 | 2009-02-04 | 中国人民解放军第二军医大学 | VEGF slowly releasing injection microsphere support and its prepn and use |
| UY33517A (en) * | 2010-07-19 | 2012-02-29 | Astrazeneca Ab | Pharmaceutical depot for 5-fluoro-2 - [[(1S) -1- (5-fluoro-2-pyridyl) ethyl] amino] -6 - [(5-isopropoxy-1H-pyrazol-3-yl) amino] pyridin -3-carbonitrile ?. |
| CN107281111A (en) * | 2016-04-05 | 2017-10-24 | 南方医科大学南方医院 | A kind of degradable polymer contains the preparation method of NBD polypeptide microballoons |
| CN107698795B (en) * | 2017-09-30 | 2020-10-27 | 西安交通大学 | A method for preparing porous polymer microspheres with controllable structure and its application |
| CN109172874A (en) * | 2018-09-18 | 2019-01-11 | 福建师范大学 | A method of preparing the polylactic acid microsphere of gelatin surface graft modification |
| CN114634634B (en) * | 2022-03-22 | 2024-08-09 | 陈凌卉 | Biological function composite porous polyester microsphere and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1140058A (en) * | 1996-03-29 | 1997-01-15 | 中国科学院成都有机化学研究所 | Slow-release microsphere powder injection of androgenic hormone, its preparing method and use |
| BR0300713A (en) * | 2003-03-24 | 2004-11-16 | Ct Ingenieria Genetica Biotech | Immunogenic compositions for prevention and treatment against endoparasites and ectoparasites |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1140058A (en) * | 1996-03-29 | 1997-01-15 | 中国科学院成都有机化学研究所 | Slow-release microsphere powder injection of androgenic hormone, its preparing method and use |
| BR0300713A (en) * | 2003-03-24 | 2004-11-16 | Ct Ingenieria Genetica Biotech | Immunogenic compositions for prevention and treatment against endoparasites and ectoparasites |
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