CN101653598B - Thymic peptide alpha1PLGA slow release microsphere preparation and method for preparing same - Google Patents

Thymic peptide alpha1PLGA slow release microsphere preparation and method for preparing same Download PDF

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CN101653598B
CN101653598B CN200810045838.7A CN200810045838A CN101653598B CN 101653598 B CN101653598 B CN 101653598B CN 200810045838 A CN200810045838 A CN 200810045838A CN 101653598 B CN101653598 B CN 101653598B
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plga
standby
colostrum
pva
rev
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CN101653598A (en
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叶兵
刘金花
黄梅
鲍锐
武勇
谢毅
毛华
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CHENGDU DIAO JIUHONG PHARMACEUTICAL FACTORY
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CHENGDU DIAO JIUHONG PHARMACEUTICAL FACTORY
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Abstract

The invention relates to a thymic peptide alpha1PLGA slow release microsphere preparation, a preparation method thereof and application thereof. The thymic peptide alpha1 microspheres prepared by the technical scheme have the advantages of greatly improving drug-loading rate, simultaneously keeping extremely high entrapping rate of the thymic peptide alpha1, and having unique substantive characteristics and obvious improvement.

Description

A kind of Thymosin alpha 1pLGA sustained release microsphere agents and preparation method thereof
Technical field
The present invention relates to a kind of pharmaceutical preparation for the treatment of with polypeptide, be specifically related to PLGA sustained release microsphere agents of Thymosin alpha 1 and its production and use, belong to field of pharmaceutical preparations.
Background technology
Thymosin alpha 1 (thymosin α 1 is called for short T α 1) is a kind of polypeptide with immunologic function, is used for clinically immunostimulant or treatment viral hepatitis, and its aminoacid sequence is
AC-Ser-A sp-Alα-Alα-Vαl-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-lys-Glu-Lys-Lys-Glu-Vαl-Vαl-Glu-Glu-Alα-Glu-Asn_-OH
Molecular formula: C 129h 215n 33o 55-
Molecular weight: 3108.37
Existing T α 1preparation, because short treatment time of half-life is long, needs frequent drug administration, has the shortcomings such as the poor and bioavailability of patient compliance is low.Microball preparation passes through thymosin T α 1be wrapped in spheroid slow, controlled release thymosin T α 1, make patient by T α 1 microball preparation of injection, reach longer therapeutic purposes.PLGA (poly lactic coglycolic acid) is Biodegradable material, has good biocompatibility and safety, and vivo degradation is carbon dioxide and water, is usually used in preparing injection microsphere preparation.PLGA can be by lactic acid and hydroxyacetic acid polymerization, or with lactide and Acetic acid, hydroxy-, bimol. cyclic ester through organotin catalytic polymerization.
At present about Thymosin alpha 1the report of microsphere is more, as: (preparation of Thymosin alpha 1 microsphere and the evaluation thereof such as Zhu Yan, pharmaceutical services and research, 2005, vol (5) 3:248～251) 1mg T α 1 is dissolved in the pure water of 50 μ L as interior water, CH with the concentration PLGA (50:50, specificity viscosity is 19mL/g) that is 200mg/mL 2cl 2solution 500 μ L mix, ultrasonic emulsification (400ms/ time under condition of ice bath, 10 times) make colostrum, colostrum is added drop-wise in finite concentration (3%, w/v) PVA1788 aqueous solution (30mL), high-speed stirred (1000r/min) 2min obtains emulsion, and it is used to about 150mL distilled water diluting immediately, stirring at low speed 4h, centrifugal collection microsphere, distilled water wash 3 times, lyophilization 24h.The envelop rate for preparing thus microsphere is (85.2 ± 2.2) %, (87.6 ± 3.5) %, (86.6 ± 2.7) %, and theoretical drug loading is 1%.(the research of Thymosin alpha 1 release injectable microsphere such as Zhu Yan, Acta Pharmaceutica Sinica, vol (42) 2:211～215) report and get a certain amount of T α 1 lyophilized powder, with 10mmol/L pH7.4 phosphate buffer 1,00 μ L dissolves lyophilized powder as interior water, with the CH of PLGA 2cl 2solution (mass concentration is 200mg/mL) mixes, ultrasonic emulsification (400ms/ time under condition of ice bath, 10 times) after make colostrum, colostrum is added drop-wise to fast in 3% (w/v) PVA aqueous solution 30mL that contains 5%NaCl or 10% glucose to 1200rmin -1stir 2min and obtain emulsion, and it is diluted with the about 100mL of distilled water immediately, stirring at low speed (400r/min) 4h, centrifugal collection microsphere, distilled water wash 3 times, lyophilization 24h makes microsphere.ZL02136181.9 discloses the microsphere being prepared by the Thymosin alpha 1 of 3～5mg and 10ml5～20%PLGA (25:75, molecular weight is 3000) dichloromethane solution, envelop rate 20% left and right.The microsphere that it is 0.5:100 to 5:100 that ZL200610118413.5 discloses by Thymosin alpha 1 and PLGA (25:75～75:25, molecular weight 3000～40000) weight ratio, microballoons room temperature particle diameter <100 μ m.Specifically disclosing microsphere envelop rate is 87.8%, 90.2%, the microball preparation of theoretical drug loading ball 1% and preparation technology thereof.
Relation with regard to envelop rate and drug loading is illustrated: on the one hand, under same preparation condition, 100 unit volume PLGA more can be by the Thymosin alpha of 1 unit volume than 50 unit volume PLGA 1seal completely the Thymosin alpha dropping into when making preparation and producing 1more be written into medicine, improve Thymosin alpha 1envelop rate, realize pharmaceutical preparation economization produce.On the other hand, the input of high dose PLGA must cause the decline of microsphere drug loading, by 100 unit volume PLGA, seals the Thymosin alpha obtaining 1the drug loading of microsphere must be less than 50 unit volume PLGA and seal and obtain microsphere drug loading (weight/microsphere weight of drug loading=microsphere Chinese medicine).In view of the price of PLGA higher, be about 12-25 dollar/g, the use of relatively large PLGA must improve pharmaceutical production cost, the production equipment capacity of simultaneously having relatively high expectations and apparatus of load rate, and need to use a large amount of power, consume mass energy, the direction that is not suitable for energy-saving and emission-reduction and builds intensive society.Zhu Yan (research [C] of thymosin alphal long-acting injection microsphere, The 2nd Army Medical College academic dissertation) report: along with the increase of medicine and PLGA ratio, the corresponding raising of drug loading of microsphere, the envelop rate of microsphere reduces.Microball preparation is a kind of very complicated dosage form, and in pharmaceutical industry microspheres in the urgent need to address, these those long problems that disappear of drug loading and envelop rate, realize microball preparation and the preparation technology thereof with high drug load and high envelop rate.
Summary of the invention
The invention provides a kind of Thymosin alpha 1pLGA sustained-release micro-spheres, wherein Thymosin alpha 1with the content ratio of PLGA be 0.5～10:100, the range of viscosities of PLGA is 0.4～1.2dl/g.Further, Thymosin alpha 1with the content ratio of PLGA be 1～9:100, the range of viscosities of PLGA is 0.5～0.94dl/g.
Further Thymosin alpha 1with the content ratio of PLGA be 1.5～8:100, the range of viscosities of PLGA is 0.55～0.75dl/g.
Described PLGA, the PLGA that preferably ratio of polylactic acid and hydroxyacetic acid is 50:50.
In addition, the invention also discloses one and prepare Thymosin alpha 1the method of PLGA sustained-release micro-spheres, comprises the following steps composition:
(1), by the Ta1 of recipe quantity, be dissolved in the buffer solution of pH6.5～8.0; Obtain water, standby;
(2) PLGA of recipe quantity is dissolved in to dichloromethane, obtains oil phase, standby;
(3) water is injected to oil phase, emulsifying 5-20 minute, obtains colostrum (W/O), standby;
(4) colostrum is dropped in the aqueous solution lower than colostrum temperature containing PVA and NaCl or glucose, stir, then add in uniform temp, the aqueous solution containing identical PVA and NaCl or concentration of glucose, stir 1～12 hour;
(5) filter, wash, lyophilization, obtains microsphere.
The present invention further discloses one and prepare Thymosin alpha 1the method of PLGA sustained-release micro-spheres, comprising:
(1) Ta1 of recipe quantity is dissolved in the phosphate buffer of pH7.4, making the concentration of Ta1 is 25～100mg/ml, obtains water, standby;
(2) PLGA of recipe quantity is dissolved in dichloromethane, making the concentration of PLGA is 50～500mg/ml, obtains oil phase, standby;
(3) under 10-25 ℃ of condition, mulser rotating speed is adjusted to 1500-8000 rev/min, water is injected to oil phase, emulsifying, obtains water-in-oil type colostrum, standby;
(4) will be containing 1～10%PVA, and the aqueous solution of 1～10%NaCl or glucose is chilled to below the temperature lower than colostrum, under the condition of speed of agitator 300-2000 rev/min, colostrum is added wherein, stir, add again uniform temp, in the aqueous solution of the PVA of same concentrations and NaCl or glucose, stir 1～12 hour; Stop stirring, cross 100-200 mesh sieve, filter, washing, drains, and lyophilization, obtains microsphere.
Low envelop rate causes Thymosin alpha 1a large amount of losses and waste; Low drug loading causes effective unit weight PLGA energy load Thymosin alpha 1amount considerably less, and patient generally need to inject a certain amount of Thymosin alpha in order to reach therapeutic purposes 1, this just means this microsphere of a large amount of injection of needs, causes misery huge while injecting and the health care expenditures of great number; The microsphere of low drug loading certainly will consume a large amount of PLGA simultaneously, and production difficulty, energy resource consumption and manufacturing cost are significantly raise.Inventor is in research practice, and in order to solve, envelop rate raises and drug loading declines, or the problem that drug loading rising envelop rate declines has expended a large amount of manpower and materials and time.
The Thymosin alpha being prepared by technical scheme of the present invention 1microsphere has still kept high Thymosin alpha in the drug loading that significantly raises 1envelop rate, has outstanding substantive distinguishing features and significant progressive.
With concrete example, describe below, but do not limit the invention.
figure of description
The release curve of microsphere prepared by Fig. 1: embodiment 1
The release curve of microsphere prepared by Fig. 2: embodiment 2
The release curve of microsphere prepared by Fig. 3: embodiment 3
The release curve of microsphere prepared by Fig. 4: embodiment 4
The release curve of microsphere prepared by Fig. 5: embodiment 5
The release curve of microsphere prepared by Fig. 6: embodiment 6
The release curve of microsphere prepared by Fig. 7: embodiment 7
The release curve of microsphere prepared by Fig. 8: embodiment 8
The release curve of microsphere prepared by Fig. 9: embodiment 9
The specific embodiment
Embodiment 1
·Ta1 1.0g
·PLGA(50/50)(0.95～1.2dl/g) 26.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 400ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 2000-2800 rev/min, water is injected to oil phase, emulsifying 5-10 minute, obtains colostrum (O/W), standby;
Will be containing 3%PVA, the aqueous solution 800ml of 5%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 500-1000 rev/min, add colostrum, stir 2 minutes, then add the 3%PVA that contains of uniform temp, the aqueous solution 200ml of 5%NaCl, obtains emulsion (W/O/W), under 1200～1800 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 2
·Ta1 1.0g
·PLGA(50/50)(0.40～0.56dl/g) 32.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 100ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 2500-3200 rev/min, water is injected to oil phase, emulsifying 5-10 minute, obtains colostrum (O/W), standby;
Will be containing 2%PVA, the aqueous solution 1600ml of 5%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 500-1000 rev/min, add colostrum, stir 2 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 400ml of 3%NaCl, obtains emulsion (W/O/W), under 500～1000 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 3
·Ta1 1.0g
·PLGA(65/35)(0.55～0.75dl/g) 30.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity, join in the phosphate buffer of 20mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 200ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 3200-3800 rev/min, water is injected to oil phase, emulsifying 5-10 minute, obtains colostrum (O/W), standby;
Will be containing 2%PVA, the aqueous solution 2500ml of 2%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 400ml of 2%NaCl, obtains emulsion (W/O/W), under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 4
·Ta1 1.0g
·PLGA(85/15)(0.55～0.75dl/g) 38.5g
Mannitol 1.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity and mannitol, join in the phosphate buffer of 35mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 400ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 3800-4300 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
Will be containing 2%PVA, the aqueous solution 3500ml of 2%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 1500ml of 2%NaCl, obtains emulsion (W/O/W), under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 5
·Ta1 1.0g
·PLGA(50/50)(0.76～0.94dl/g) 32.0g
·PEG-6000 1.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity and PEG-6000, join in the phosphate buffer of 40mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 280ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 4000-4500 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
Will be containing 2%PVA, the aqueous solution 4000ml of 2%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 2000ml of 2%NaCl, obtains emulsion (W/O/W).Under 600～1200 revs/min of conditions, stir 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 6
·Ta1 1.0g
·PLGA(25/75)(0.76～0.94dl/g) 11.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity and mannitol, join in the phosphate buffer of 30mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 280ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 4000-4500 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
Will be containing 2%PVA, the aqueous solution 3000ml of 1.5%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 1000ml of 1.5%NaCl, obtains emulsion (W/O/W).Under 600～1200 revs/min of conditions, stir 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 7
·Ta1 1.0g
·PLGA(50/50)(0.76～0.94dl/g) 28.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 450ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 4000-4500 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum O/W), standby;
Will be containing 3%PVA, the aqueous solution 6000ml of 5%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 2000ml of 2%NaCl, obtains emulsion (W/O/W), under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 8
·Ta1 1.0g
·PLGA(75/25)(0.76～0.94dl/g) 30.0g
Mannitol 1.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity, join in the phosphate buffer of 35mlpH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 500ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 4000-4500 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
Will be containing 2%PVA, the aqueous solution 6000ml of 8% glucose is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 2000ml of 8% glucose, obtains emulsion (W/O/W), under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Embodiment 9
·Ta1 1.0g
·PLA(0.26～0.54dl/g) 59.0g
Preparation technology
Under normal temperature condition, by the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4, it is dissolved completely, obtain water, standby;
The PLA of recipe quantity, under normal temperature condition, is joined in 200ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 3500-4000 rev/min, water is injected to oil phase, emulsifying 5-10 minute, obtains colostrum (O/W), standby;
Will be containing 3%PVA, the aqueous solution 3500ml of 5%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 3%PVA that contains of uniform temp, the aqueous solution 1500ml of 5%NaCl, emulsion (W/O/W), under 600～1200 revs/min of conditions, stirred and crossed 100 mesh sieves in 3 hours, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
Below according to described method, carry out respectively the mensuration of microsphere drug loading and envelop rate, release, organic solvent residual.
Ta1 content assaying method in microsphere
The about 20mg of accurately weighed microsphere, adds 0.5mlCH 2cl 2, vortex makes it to dissolve, and adds the phosphate buffer of 2.5mlpH7.4, vortex makes it to dissolve for 3 minutes, and centrifugal 7 minutes of 3000r/min is standing, get supernatant standby, lower floor's liquid adds the phosphate buffer of 2.5mlpH7.4, vortex 3 minutes, centrifugal 7 minutes of 3000r/min, standing, get supernatant standby, twice gained supernatant merged, be test sample, get test sample and carry out HPLC mensuration, calculate Ta1 content.
The mensuration of microsphere envelop rate
The Ta1 content of practical measurement is multiplied by 100% again divided by Ta1 inventory, obtains microsphere envelop rate.
The mensuration of microsphere drug loading
The Ta1 content of practical measurement is multiplied by 100% again divided by the amount that participates in the actual microsphere of measuring, obtains microsphere envelop rate.
The assay method of release
Test specimen: the sample of preparing according to embodiment of the present invention method.
Test reagent: containing the phosphate buffer of 0.2% Tween 80 PH7.4
Test apparatus: water-bath constant temperature oscillator, centrifuge.
Experimental condition: temperature: 37 ℃ ± 0.5 ℃
Test method: precision takes the about 12mg of laboratory sample and be placed in the tool plug centrifuge tube of 10ml, adds 5ml release medium (containing the phosphate buffer of 0.2% Tween 80 PH7.4), is placed in water-bath constant temperature oscillator and keeps certain temperature to sample on time.
Sampling method: centrifugal, abandon or adopt supernatant, dry 48h, by the content of Ta1 content assaying method mensuration remaining solid.(0.5h directly measures T α in aqueous solution 1content)
Sample time 0.5h, 1d, 5d, 7d, 10d, 15d
Organic solvent residual: with reference to official method
Test data table (one)
The data such as the actual drug loading of microsphere prepared by each embodiment, envelop rate
Embodiment Actual drug loading (%) Theoretical drug loading (%) Envelop rate (%) (μ m) for mean diameter
Embodiment 1 3.34 3.70 90.3 36.40
Embodiment 2 2.81 3.03 92.8 34.56
Embodiment 3 3.07 3.23 95.2 58.64
Embodiment 4 2.33 2.47 94.5 55.05
Embodiment 5 2.75 2.94 93.6 55.53
Embodiment 6 7.28 8.33 87.4 52.68
Embodiment 7 3.31 3.45 96.0 57.58
Embodiment 8 2.96 3.13 94.7 67.36
Embodiment 9 1.53 1.67 91.7 75.08
The release curve of the microsphere of embodiment 1～9 preparation is shown in Fig. 1-9.

Claims (1)

1. a Thymosin alpha 1pLGA sustained-release micro-spheres, it is characterized in that: it comprises one of following mode thus obtained microsphere:
(1) Thymosin alpha 1 1.0g
·PLGA 26.0g
In described PLGA, the ratio of polylactic acid and hydroxyacetic acid is that 50/50, PLGA viscosity is 0.95～1.2dl/g
Preparation technology
Under normal temperature condition, by the Thymosin alpha 1 of recipe quantity, join in the phosphate buffer of 30ml pH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 400ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 2000-2800 rev/min, water is injected to oil phase, emulsifying 5-10 minute, obtains colostrum O/W, standby;
Will be containing 3%PVA, the aqueous solution 800ml of 5%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 500-1000 rev/min, add colostrum, stir 2 minutes, then add the 3%PVA that contains of uniform temp, the aqueous solution 200ml of 5%NaCl, obtains emulsion W/O/W, under 1200～1800 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere;
·
(2) Thymosin alpha 1 1.0g
·PLGA 30.0g
In described PLGA, the ratio of polylactic acid and hydroxyacetic acid is that 65/35, PLGA viscosity is 0.55～0.75dl/g
Preparation technology
Under normal temperature condition, by the Thymosin alpha 1 of recipe quantity, join in the phosphate buffer of 20ml pH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 200ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 3200-3800 rev/min, water is injected to oil phase, emulsifying 5-10 minute, obtains colostrum O/W, standby;
Will be containing 2%PVA, the aqueous solution 2500ml of 2%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 400ml of 2%NaCl, obtains emulsion W/O/W, under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere;
(3) Thymosin alpha 1 1.0g
·PLGA 38.5g
Mannitol 1.0g
In described PLGA, the ratio of polylactic acid and hydroxyacetic acid is that 85/15, PLGA viscosity is 0.55～0.75dl/g preparation technology
Under normal temperature condition, by the Thymosin alpha 1 of recipe quantity and mannitol, join in the phosphate buffer of 35ml pH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 400ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 3800-4300 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum O/W, standby;
Will be containing 2%PVA, the aqueous solution 3500ml of 2%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 1500ml of 2%NaCl, obtains emulsion W/O/W, under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere;
(4) Thymosin alpha 1 1.0g
·PLGA 32.0g
·PEG-6000 1.0g
In described PLGA, the ratio of polylactic acid and hydroxyacetic acid is that 50/50, PLGA viscosity is 0.76～0.94dl/g
Preparation technology
Under normal temperature condition, by the Thymosin alpha 1 of recipe quantity and PEG-6000, join in the phosphate buffer of 40ml pH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 280ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 4000-4500 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum O/W, standby;
Will be containing 2%PVA, the aqueous solution 4000ml of 2%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 2000ml of 2%NaCl, obtains emulsion W/O/W; Under 600～1200 revs/min of conditions, stir 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere;
(5) Thymosin alpha 1 1.0g
·PLGA 28.0g
In described PLGA, the ratio of polylactic acid and hydroxyacetic acid is that 50/50, PLGA viscosity is 0.76～0.94dl/g
Preparation technology
Under normal temperature condition, by the Thymosin alpha 1 of recipe quantity, join in the phosphate buffer of 30ml pH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 450ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 4000-4500 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum O/W, standby;
Will be containing 3%PVA, the aqueous solution 6000ml of 5%NaCl is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 2000ml of 2%NaCl, obtains emulsion W/O/W, under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere;
(6) Thymosin alpha 1 1.0g
·PLGA 30.0g
Mannitol 1.0g
In described PLGA, the ratio of polylactic acid and hydroxyacetic acid is that 75/25, PLGA viscosity is 0.76～0.94dl/g
Preparation technology
Under normal temperature condition, by the Thymosin alpha 1 of recipe quantity, join in the phosphate buffer of 35ml pH7.4, it is dissolved completely, obtain water, standby;
The PLGA of recipe quantity, under normal temperature condition, is joined in 500ml dichloromethane, make it to dissolve completely, obtain oil phase, standby;
Under 10-25 ℃ of condition, mulser rotating speed is adjusted to 4000-4500 rev/min, water is injected to oil phase, emulsifying 5-10 minute, colostrum O/W, standby;
Will be containing 2%PVA, the aqueous solution 6000ml of 8% glucose is chilled to below 10 ℃, and blender rotating speed is adjusted to 600-1200 rev/min, add colostrum, stir 5 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 2000ml of 8% glucose, obtains emulsion W/O/W, under 600～1200 revs/min of conditions, stirs 3 hours, cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times, drain, lyophilization, obtains microsphere.
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CN102895192B (en) * 2012-09-26 2014-08-27 上海交通大学 Method for producing microsphere through oil-in-nanoparticle suspension-water-in-oil mode

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CN1965810A (en) * 2006-11-17 2007-05-23 中国人民解放军第二军医大学 Sustained release microsphere formulation of thymosin alpha-1, preparation process and use thereof

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