CN105194662A - Sustained-release microsphere preparation containing recombinant hepatitis b virus surface antigen and preparation method of preparation - Google Patents
Sustained-release microsphere preparation containing recombinant hepatitis b virus surface antigen and preparation method of preparation Download PDFInfo
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Abstract
The invention discloses a sustained-release microsphere preparation containing a recombinant hepatitis b virus surface antigen and a preparation method of the preparation. A kernel is composed of a stability protective agent and recombinant hepatitis b virus surface antigen particles, a high polymer material polylactic acid-polyglycolic acid block copolymer serves as an outer encapsulated layer, and the kernel and the outer encapsulated layer are combined to form the sustained-release microsphere preparation, wherein the stabilizer adopts mycose and human serum albumin. The preparation method comprises the steps that 1, the stabilizer and the recombinant hepatitis b virus surface antigen are mixed to obtain an inner water phase; 2, the polylactic acid-polyglycolic acid block copolymer is dissolved in organic solvent to be prepared into an oil phase, the inner water phase is added to the oil phase to be stirred and homogenized; 3, W/O initial emulsion is added to an outer water phase, and after stirring and homogenization are conducted, W/O/W multiple emulsion is formed; stirring is conducted for 4-6 h, centrifugal washing, collecting and vacuum freeze drying are conducted, and the preparation is obtained. According to tShe sustained-release microsphere preparation containing the recombinant hepatitis b virus surface antigen and the preparation method of the preparation, the problems that due to the fact that the immunogenicity of the hepatitis b virus surface antigen is destroyed in the preparation and releasing processes of microspheres, the antigen is denaturalized, and the immune effect is affected are solved.
Description
Technical field
The present invention relates to a kind of biological preparation, especially a kind of sustained release microsphere agents containing recombination hepatitis B surface antigen and preparation method thereof.
Background technology
Hepatitis B is a kind of pandemic infection disease of serious threat human health, and it is the disease caused by the infection of hepatitis B virus (HBV).At present, HB vaccination is one of most effective measures of propagating of prevention and corntrol HBV.The Hepatitis B virus vaccine the most often used is now that the hepatitis B surface antigen (HBsAg) obtained by gene recombination technology is purified, deactivation and add the aluminium adjuvant Hepatitis B virus vaccine of aluminium adjuvant absorption.But, the effective immunne response effect in order to reach lasting, aluminium adjuvant Hepatitis B virus vaccine need 0,1,2 months or 0, within 1,6 months, carry out the immune programme for children of three injections, because immune programme for children is complicated, cause quite a few people to be difficult to immune programme for children, this phenomenon is even more serious in developing country.In addition, aluminium adjuvant Hepatitis B virus vaccine only selective stimulating body produces humoral immunoresponse(HI), and effectively can not induce generation cellullar immunologic response, and the HBV that can not effectively exist in scavenger cell is viral.Thus, clinical needs researches and develops novel potent Hepatitis B virus vaccine, simplifies immune programme for children, makes crowd widely be subject to effective protection.
Polyglycolic-polylactic acid block copolymer (PLGA) is a kind of macromolecule polymer material, antigen is encapsulated in the slow releasing that can realize antigen in PLGA microsphere, makes single injection sustained-release micro-spheres bacterin preparation.But relate to the emulsion process that lyophilization and antigen disperse in organic solvent in microsphere preparation process, these two processes all easily cause antigen degeneration, immunogenicity declines.
Summary of the invention
Object of the present invention is just to provide a kind of sustained release microsphere agents of recombination hepatitis B surface antigen of single injection, to overcome immunogenicity destroyed problem causing antigen degeneration and then affect immune effect in microsphere preparation and dispose procedure of hepatitis B surface antigen.
Another object of the present invention is to provide a kind of preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen.
The present invention is achieved in that the sustained release microsphere agents containing recombination hepatitis B surface antigen, this sustained release microsphere agents is with stability protection agent and recombination hepatitis B surface antigen microgranule composition kernel, makes outsourcing sealing combine by macromolecular material polyglycolic-polylactic acid block copolymer; Described stabilizing agent is trehalose, human serum albumin.
In the present invention, the mass ratio of recombination hepatitis B surface antigen and described stability protection agent is preferably 1: (1 ~ 20).Be more preferably 1: (1 ~ 10), consider the influence factors such as production cost, be more preferably 1: 5.
In the present invention, described recombined human hepatitis B surface antigen is the hbs antigen of yeast hbs antigen or Chinese hamster ovary cell secretion.
The preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen of the present invention, comprises the following steps:
(1) stabilizing agent and recombined human hepatitis B surface antigen are mixed to get interior aqueous phase, described stabilizing agent is trehalose, human serum albumin;
(2) polyglycolic-polylactic acid block copolymer is dissolved in organic solvent makes oil phase, get the made interior aqueous phase of step and add above-mentioned oil phase and stir homogenize and form W/O colostric fluid;
(3) W/O colostric fluid is added in outer aqueous phase poly-vinyl alcohol solution, after stirring homogenize, form W/O/W double emulsion; Stir 4 ~ 6 hours again, centrifuge washing, collect, vacuum lyophilization.
In the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen of the present invention; the mass ratio of recombination hepatitis B surface antigen and described stability protection agent is preferably 1: (1 ~ 20); be more preferably 1: (1 ~ 10), consider the influence factors such as production cost, be more preferably 1: 5.
In the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen of the present invention, in (2) step, speed of agitator is 8500 ~ 20000 revs/min; Be good with 15000 revs/min; Mixing time is 30 ~ 300 seconds; The rotating speed stirring homogenize in (3) step is 8500 ~ 15000 revs/min; Be good with 12000 revs/min; Mixing time is 10 ~ 20 minutes.
The polyglycolic-polylactic acid block copolymer amount that the present invention selects is 4.0 × 10
3~ 5.5 × 10
4, wherein the mass ratio of polylactic acid and polyglycolic acid (PLA:PLG) is (50:50) ~ (85:15).
In the preparation method of the said sustained release microsphere agents containing recombination hepatitis B surface antigen of the present invention, the mixed liquor that the organic solvent used is dichloromethane or dichloromethane and acetone, wherein when organic solvent is the mixed liquor of dichloromethane and acetone, the volume ratio of dichloromethane and acetone is 75:25.
In preparation method of the present invention, in described oil phase, the concentration of polyglycolic-polylactic acid block copolymer is 75 ~ 250mg/ml.The concentration of described outer Aqueous dispersions medium polyvinyl alcohol is 0.5% ~ 5%, can add the inorganic salt that concentration is 0 ~ 10%, to increase the envelop rate of antigen.In in the present invention, the pH value of aqueous phase, outer aqueous phase should between 5.0 ~ 7.4, and interior aqueous phase, outer aqueous pH values are identical, and preferably 6.0 in order to the formation of finished product.
In the present invention, the centrifugation rate receiving long-pending microsphere is 8500 revs/min, and the time is approximately 20 minutes.
The present invention selects the suitable stability of stabilizing agent protection hepatitis B surface antigen in microsphere preparation process; Then use Biodegradable polymer material polyglycolic-polylactic acid block copolymer (PLGA) for carrier material encapsulating stabilizing agent and recombination hepatitis B surface antigen, prepare the polyglycolic-polylactic acid block copolymer sustained-release micro-spheres of recombination hepatitis B surface antigen, effectively can ensure that the immunogenicity of hepatitis B surface antigen in microsphere preparation and dispose procedure is not destroyed.
The present invention is by adding the obtained sustained-release micro-spheres of stability protection agent, smooth surface, appearance uniform, and regular particles is without adhesion, and mean diameter is at 1.0-10.0 μm; Drug loading, envelop rate are high.As the single injection sustained-release micro-spheres bacterin preparation of prevention spreading, Antigen Stability is good, immunocompetence is high, slow-release period was more than more than 60 days, be suitable for non-vein drug administration by injection, for healthy population, improve crowd HBsAb level, again can inducing cellular immune, for remove infection cell in hepatitis B virus.Therefore, this preparation not only as prevention spreading, can to reach again the effect for the treatment of chronic viral hepatitis B and hepatitis B virus carriers.In addition, PLGA microsphere is biodegradable, good biocompatibility.
Accompanying drawing explanation
The electron scanning micrograph of the metamorphosis of the sustained release microsphere agents release in vitro containing recombination hepatitis B surface antigen of Fig. 1 prepared by the embodiment of the present invention 8.
The drug release patterns in vitro of the sustained release microsphere agents containing recombination hepatitis B surface antigen of Fig. 2 prepared by the embodiment of the present invention 7 and embodiment 8.
Fig. 3 is that in rat blood serum anti-HBsAg IgG antibody level is over time.
Fig. 4 be different preparation induction produce rat body in IL-2 and IFN-γ level.
Detailed description of the invention
Below by embodiment, technical scheme of the present invention is further described.
The surface character of microsphere adopts sem observation.
The assay method of envelop rate: accurately take 20mgHBsAg microsphere in 2ml centrifuge tube, add 1ml acetonitrile, 14000rmin
﹣ 1centrifugal 20min, abandons supernatant, after precipitating vacuum drying 2h, with 1ml sodium radio-phosphate,P-32 solution (0.02molL
﹣ 1, pH7.4) again disperse, recentrifuge, and retain supernatant; Repeat said process, merge the supernatant of twice, measure HBsAg content by Lowry method.Calculate envelop rate and the drug loading of microsphere.Envelop rate=(in the HBsAg content/microsphere encapsulated in microsphere encapsulating and non-encapsulated HBsAg total amount) × 100% of microsphere.Drug loading=(gross weight of the weight/microsphere of HBsAg contained by microsphere) × 100% of microsphere.The prominent mensuration releasing rate: accurately take 20mgHBsAg microsphere in 2ml centrifuge tube, add 1ml sodium phosphate (0.02molL
﹣ 1pH7.4) buffer, is placed in 37 DEG C of constant-temperature tables, speed 160rmin
-1, centrifuging and taking supernatant after 24h, with its HBsAg content of Lowry method kit measurement, calculates the prominent of microsphere and releases rate.Microsphere prominent releases rate=(20mg microsphere 24h discharge the total amount of HBsAg in the amount/20mg microsphere of HBsAg) × 100%.
Microsphere release in vitro: accurately take HBsAg-HSA microsphere prepared by 20mg and HBsAg microsphere respectively in the centrifuge tube of 2ml, add 1ml sodium phosphate (0.02molL wherein
﹣ 1pH7.4) buffer, is placed in 36.5 DEG C of constant-temperature tables, speed 120 revs/min, according to pre-set interval centrifuging and taking supernatant, then in precipitation, adds fresh sodium phosphate (0.02molL
﹣ 1pH7.4) buffer is again placed in shaking table and sways release.With total protein content in BCA method kit measurement supernatant, double antibody sandwich ELISA surveys HBsAg antigen active in supernatant, and the stability of the HBsAg of release in vitro adopts size exclusion-high performance liquid chromatography method to detect.
Double antibody sandwich ELISA measures HBsAg antigen active: (1) bag quilt: dilute rabbit AntiHBsAg antibody, 100 μ l/ holes with the carbonate buffer solution 1:2000 of 0.05MpH9.6,4 DEG C are spent the night; (2) wash: abandon coating buffer, PBST washs 3 times, each 3 minutes, pats dry; (3) close: every hole adds 1%BSA-PBST, 200 μ l/ holes, 37 DEG C of wet box incubations 2 hours.Wash the same; (4) application of sample: add HBsAg standard sample (concentration range 5.0ng/ml ~ 700ng/ml) and testing sample, 100 μ l/ holes, 37 DEG C of wet box incubations 1 hour.Wash the same; (5) enzyme labelled antibody is added: diluted by enzyme mark rabbit AntiHBsAg antibody 1:4000 with l%BSA-PBST-4%PEG6000,100 μ l/ holes, 37 DEG C of wet box incubations 1 hour.Wash the same; (6) substrate is added: add TMB soluble substrate solution 100 μ l/ hole, room temperature lucifuge reaction 15min; (7) stop: drip 100 μ l/ hole stop buffers; (8) detect: microplate reader measures the absorbance under 450nm and 630nm.The difference of OD450 and OD630 is the absorbance of sample own.Use standard solution Criterion curve, calculate HBsAg antigen active in testing sample.Size exclusion-high performance liquid chromatography method detects HBsAg:(1) instrument is Japanese Shimadzu high performance liquid chromatograph, the model of chromatographic column is SEC-300(5 μm, 150 × 7.8mm), determined wavelength is 280nm, mobile phase is the PBS(0.02molL ﹣ 1pH7.4 containing 0.1% Hydrazoic acid,sodium salt) buffer, flow velocity: 0.3ml/min, sample size: 20 μ l.
HBsAg sustained-release micro-spheres is in the humoral immunoresponse(HI) of rat Immune inducing in vivo: select female sd inbred rats 28 in 8 week age, about body weight 200g, give and Free water and food in whole experimentation, be divided into recombinant hepatitis B vaccine (alum-HBsAg) group, blank microsphere group, HBsAg sustained-release micro-spheres group, HBsAg-TS sustained-release micro-spheres group and HBsAg-HSA sustained-release micro-spheres group five groups randomly.Experiment adopts subcutaneous injection, and twice administration of alum-HBsAg component is respectively injection in 0,1 month, and per injection dosage is 10 μ g/; HBsAg sustained-release micro-spheres group, HBsAg-TS sustained-release micro-spheres group and HBsAg-HSA sustained-release micro-spheres group are single administration, and by HBsAg 20 μ g/ only, the shot of blank microsphere group, injectable microsphere amount is identical with HBsAg sustained-release micro-spheres group injection volume for dosage.Within upon administration the 1st week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, 15 weeks, 18 weeks, get blood 0.5ml in centrifuge tube by the ophthalmic corner of the eyes, collected by centrifugation serum.The mensuration of hepatitis B surface antibody (HBsAb) in rat blood serum: antibody quantitative assay adopts the HBsAb Quantikine ELISA kits of Bio-SwampImmunoassayR & DCenter to carry out.
HBsAg sustained-release micro-spheres is at the cellullar immunologic response of rat Immune inducing in vivo: after completing the blood sampling in the 18th week of above-mentioned five groups of female sd inbred rats, to the urethane of these five groups of rat injection optimal doses, it is anaesthetized, after anesthesia, perfusion operation is carried out to it, win spleen in centrifuge tube, quick freeze is preserved to be measured in-80 DEG C of refrigerators.Rats Spleen is weighed, and adds 150 μ l sodium phosphate (0.02molL by every 10mg tissue
﹣ 1pH7.4) ratio of buffer, adds the sodium phosphate (0.02molL of respective volume according to the weight of different Rats Spleen
﹣ 1pH7.4) buffer, under ice bath state, tissue is shredded, and use the historrhexis that Ultrasonic Cell Disruptor will shred, carry out in 4 DEG C of centrifuges 12000 revs/min centrifugal 10 minutes, gained supernatant be used for cytokine interleukin element IL-2 and interferon IFN-γ mensuration.The mensuration of rat interleukin I L-2 and interferon IFN-γ level: adopt IL-2 and the IFN-γ b Quantikine ELISA kits of Bio-SwampImmunoassayR & DCenter to carry out.
Embodiment 1
Select trehalose and human serum albumin to be stability protection agent, get many group 0.5mg/mlHBsAg solution, add trehalose respectively and make its concentration reach 5mg/ml, 10mg/ml respectively; Add human serum albumin (HSA) and make its concentration reach 0.5mg/ml, 1.0mg/ml, 2.5mg/ml, 5.0mg/ml respectively, then vacuum lyophilization 12h, surveying HBsAg antigen active retention rate with double antibody sandwich ELISA, the results are shown in Table 1.Vacuum lyophilization 12h is represented with lyophilizing in table 1.
Embodiment 2
Trehalose and human serum albumin is selected to be stability protection agent; getting many parts of 0.2ml concentration is 0.5mg/mlHBsAg solution; add trehalose, human serum albumin respectively wherein; the HBsAg solution that acquisition trehalose concentration is 5.0mg/ml, human serum albumin's concentration is 1.0mg/ml, 2.5mg/ml, 5.0mg/ml, 10.0mg/ml; mix with dichloromethane again; 15000 revs/min of emulsifying 150 seconds; 5000 revs/min centrifugal after get supernatant; after dichloromethane volatilization is clean, survey HBsAg antigen active retention rate with double antibody sandwich ELISA.Test data is in table 1.Aforementioned emulsifying step is represented by emulsifying in Table 1.
The agent of table 1 stability protection is on the impact of HBsAg solution antigen active retention rate after lyophilizing, emulsifying
Embodiment 3
Stability protection agent using human serum albumin (HAS) as preparation HBsAg sustained-release micro-spheres, HBsAg and human serum albumin in mass ratio 1:5 ratio mix, and are that the sodium radio-phosphate,P-32 solution of 5.0 obtains mixed phosphate sodium solution as interior aqueous phase for solvent using pH value.In this mixed solution, HBsAg concentration is 1mg/ml, and human serum albumin's concentration is 5mg/ml.Get 100 these solution of μ l to join 2ml and contain in the dichloromethane of 150mg23kDa polyglycolic-polylactic acid block copolymer (wherein the mass ratio (PLA:PLG) of polylactic acid and polyglycolic acid is 50:50) and the mixed solution of acetone (65:35), with high-shear homogenizer under the rotating speed of 20000 revs/min by its homogeneous 300 seconds, form stable W/O emulsion, this emulsion pipettor being slowly injected into 20ml contains in sodium phosphate (pH5.0) buffer of NaCl and 2.5%PVA, high-shear homogenizer forms W/O/W microemulsion 13000 revs/min of emulsifyings in homogeneous 15 minutes, the aqueous solution that 20ml contains NaCl is added in the W/O/W microemulsion formed, stirring at low speed 4 hours in digital display constant temperature blender with magnetic force, dichloromethane is volatilized clean, obtain solidified microsphere.After microsphere solidification, centrifuge washing three times, collects HBsAg-HSA microsphere, ﹣ 80 DEG C of freeze overnight, 5.0 × 10
﹣ 3pa vacuum under pressure lyophilization 12h obtains HBsAg-HSA sustained-release micro-spheres.The particle diameter of thus obtained microsphere is 4.87 microns, and envelop rate is 78.5%, and drug loading is 0.41%.
Embodiment 4
Stability protection agent using human serum albumin (HAS) as preparation HBsAg sustained-release micro-spheres, HBsAg and human serum albumin in mass ratio 1:10 ratio mix, and are that the sodium radio-phosphate,P-32 solution of 7.0 obtains mixed phosphate sodium solution as interior aqueous phase for solvent using pH value.In this mixed solution, HBsAg concentration is 1mg/ml, and human serum albumin's concentration is 10mg/ml.Get 400 μ l to join 2ml containing the sodium radio-phosphate,P-32 solution of HSA and HBsAg and contain in the dichloromethane solution of 500mg23kDa polyglycolic-polylactic acid block copolymer (wherein the mass ratio of polylactic acid and polyglycolic acid (PLA:PLG) is 85:15), with high-shear homogenizer homogenizing 200 seconds under the rotating speed of 8500 revs/min, by W/O emulsion stable for its homogeneous formation, this emulsion pipettor being slowly injected into 20ml contains in sodium phosphate (pH7.0) buffer of 5%NaCl and 2.5%PVA, W/O/W microemulsion is formed 15000 revs/min of emulsifyings in homogeneous 10 minutes with high-shear homogenizer, the aqueous solution that 20ml contains NaCl is added in the W/O/W microemulsion formed, stirring at low speed 5 hours in digital display constant temperature blender with magnetic force, dichloromethane is volatilized clean, obtain solidified microsphere.After microsphere solidification, centrifuge washing three times, collects HBsAg-HSA microsphere, ﹣ 80 DEG C of freeze overnight, 5.0 × 10
﹣ 3pa vacuum under pressure lyophilization 12h obtains HBsAg-HSA sustained-release micro-spheres.The particle diameter of thus obtained microsphere is 9.67 microns, and envelop rate is 50.5%, and drug loading is 0.22%.
Embodiment 5
Stability protection agent using human serum albumin (HAS) as preparation HBsAg sustained-release micro-spheres; HBsAg and human serum albumin in mass ratio 1:5 ratio mix, and are that the sodium radio-phosphate,P-32 solution of 6.0 obtains mixed phosphate sodium solution as interior aqueous phase (pH6.0) for solvent using pH value.In this mixed solution, HBsAg concentration is 1mg/ml, and human serum albumin's concentration is 5mg/ml.Get sodium radio-phosphate,P-32 solution that 200 μ l should contain HSA and HBsAg to join 2ml and contain in the dichloromethane solution of 500mg23kDa polyglycolic-polylactic acid block copolymer (wherein the mass ratio of polylactic acid and polyglycolic acid (PLA:PLG) is 75:25), with high-shear homogenizer 10000 revs/min rotating speed under homogenizing 290 seconds, by W/O colostric fluid stable for its homogeneous formation, this emulsion pipettor being slowly injected into 20ml contains in sodium phosphate (pH6.0) buffer of 5%NaCl and 2.5%PVA, high-shear homogenizer is homogeneous 20 minutes at 12000 revs/min, emulsifying forms W/O/W double emulsion, the aqueous solution that 20ml contains NaCl is added in the W/O/W microemulsion formed, stirring at low speed 5 hours in digital display constant temperature blender with magnetic force, dichloromethane is volatilized clean, obtain solidified microsphere.After microsphere solidification, centrifuge washing three times, collects HBsAg-HSA microsphere, ﹣ 80 DEG C of freeze overnight, 5.0 × 10
﹣ 3pa vacuum under pressure lyophilization 12h obtains HBsAg-HSA sustained-release micro-spheres.The particle diameter of thus obtained microsphere is 7.67 microns, and envelop rate is 60.7%, and drug loading is 0.72%.
Embodiment 6
Using the stability protection agent of HSA as preparation HBsAg sustained-release micro-spheres; HBsAg and human serum albumin in mass ratio 1:5 ratio mix; using pH be 6.0 sodium radio-phosphate,P-32 solution obtain mixed phosphate sodium solution as interior aqueous phase for solvent; in this mixed solution; HBsAg concentration is 1mg/ml, and human serum albumin's concentration is 5mg/ml.Get the PBS solution of 400 μ l containing HSA and HBsAg, this solution being joined 3ml contains in the dichloromethane solution of 200mg23kDa polyglycolic-polylactic acid block copolymer (wherein the mass ratio of polylactic acid and polyglycolic acid (PLA:PLG) is 85:15), with high-shear homogenizer homogenizing 100 seconds under the rotating speed of 20000rpm/min, by W/O emulsion stable for its homogeneous formation, this emulsion pipettor being slowly injected into 20ml contains in sodium phosphate (pH6.0) buffer of 5%NaCl and 2.5%PVA, high-shear homogenizer forms W/O/W microemulsion 15000 revs/min of emulsifyings in homogeneous 15 minutes, the aqueous solution that 20ml contains NaCl is added in the W/O/W microemulsion formed, stirring at low speed 5 hours in digital display constant temperature blender with magnetic force, dichloromethane is volatilized clean, obtain solidified microsphere.After microsphere solidification, centrifuge washing three times, collects HAS-HBsAg microsphere, ﹣ 80 DEG C of freeze overnight, 5.0 × 10
﹣ 3pa vacuum under pressure lyophilization 12h obtains HBsAg-HSA sustained-release micro-spheres.The particle diameter of thus obtained microsphere is 7.67 microns, and envelop rate is 50.5%, and drug loading is 0.97%.
Embodiment 7
Get sodium phosphate (pH6.0) solution of 200 μ l containing HBsAg1mg/ml, this solution being joined 2ml, to contain 200mg molecular weight be 23kDa, 43kDa, in the dichloromethane solution of 87kDa polyglycolic-polylactic acid block copolymer, with high-shear homogenizer homogenizing 300 seconds under the rotating speed of 17500 revs/min, by W/O emulsion stable for its homogeneous formation, this emulsion pipettor being slowly injected into 20ml contains in sodium phosphate (pH6.0) buffer of NaCl and 2.5%PVA, 13000 revs/min of homogeneous 15min emulsifyings of homogeneous 15 minutes homogenizers form W/O/W microemulsion, the aqueous solution that 20ml contains NaCl is added in the W/O/W microemulsion formed, stirring at low speed 4h in digital display constant temperature blender with magnetic force, dichloromethane is volatilized clean, obtain solidifying 23kDa, 43kDa, 87kDaPLGA microsphere, be designated as A1 respectively, B1, C1.After microsphere solidification, centrifuge washing three times, collects HBsAg microsphere, ﹣ 80 DEG C of freeze overnight, 5.0 × 10
﹣ 3pa vacuum under pressure lyophilization 12h obtains dry HBsAg microsphere.The particle diameter of thus obtained microsphere is between 4.0 microns ~ 7.87 microns, and envelop rate is between 76.5% ~ 86.1%, and drug loading is 0.39% ~ 0.45%(table 2).
Embodiment 8
Using the stability protection agent of HSA as preparation HBsAg sustained-release micro-spheres; mix in proportion with HBsAg; obtain mixed phosphate sodium (pH6.0) solution using pH6.0 and obtain mixed phosphate sodium solution as interior aqueous phase as solvent; in this mixed solution; HBsAg concentration is 1mg/ml, and human serum albumin's concentration is 5mg/ml.Get the solution of 200 μ l containing HSA and HBsAg, this solution is joined 2ml and contains 200mg23kDa, 43kDa, in the dichloromethane solution of 87kDa polyglycolic-polylactic acid block copolymer, with high-shear homogenizer homogenizing 200 seconds under the rotating speed of 17500rpm/min, by W/O emulsion stable for its homogeneous formation, this emulsion pipettor being slowly injected into 20ml contains in sodium phosphate (pH6.0) buffer of NaCl and 2.5%PVA, high-shear homogenizer forms W/O/W microemulsion 13000 revs/min of emulsifyings in homogeneous 15 minutes, the aqueous solution that 20ml contains NaCl is added in the W/O/W microemulsion formed, stirring at low speed 4 hours in digital display constant temperature blender with magnetic force, dichloromethane is volatilized clean, obtain solidified microsphere.After microsphere solidification, centrifuge washing three times, collects HSA-HBsAg microsphere, ﹣ 80 DEG C of freeze overnight, 5.0 × 10
﹣ 3pa vacuum under pressure lyophilization 12h obtains the HBsAg-HSA sustained-release micro-spheres that PLGA molecular weight is 23kDa, 43kDa, 87kDa, is designated as A2, B2, C2 respectively.Metamorphosis in the form of thus obtained microsphere and dispose procedure as shown in Figure 1.The particle diameter of thus obtained microsphere is between 4.67 microns ~ 6.87 microns, and envelop rate is between 76.5% ~ 89.5%, and drug loading is 0.40% ~ 0.47%(table 2).
The character of table 2HBsAg-HSA microsphere and HBsAg microsphere
By with HBsAg total content in BCA method kit measurement HBsAg-HSA microsphere and HBsAg microsphere release in vitro supernatant, and survey HBsAg antigen active in release supernatant with double antibody sandwich ELISA, and the curve calculating microsphere burst size is in proportion as Fig. 2.Can find out when not having HSA as stabilizing agent, the active a large amount of forfeiture of the HBsAg discharged, activity preservation rate approximately only has 60%; When there being HSA to exist as stabilizing agent, the antigen active retention rate of the HBsAg discharged is significantly improved, and antigen active retention rate brings up to 80.0% ~ 90.0%.
The release profiles of microsphere presents two stages as seen from Figure 2: the prominent of (1) starting stage releases behavior, and a large amount of HBsAg discharges in 24h; (2) prominent release after, the release behavior of microsphere presents a kind of lasting release conditions.Through the extracorporeal releasing experiment of 60 days, the HBsAg total amount that microsphere discharges reached 50% more than.
Embodiment 9
Using the stability protection agent of trehalose as preparation HBsAg sustained-release micro-spheres; mix in proportion with HBsAg; using pH value be 6.0 sodium radio-phosphate,P-32 solution obtain mixed phosphate sodium solution as interior aqueous phase (pH6.0) for solvent; in this mixed solution; HBsAg concentration is 1mg/ml, and trehalose concentration is 5mg/ml.Get the solution that 200 μ l contain trehalose and HBsAg, this solution is joined 2ml and contains 200mg23kDa, 43kDa, in the dichloromethane solution of 87kDa polyglycolic-polylactic acid block copolymer, with high-shear homogenizer homogenizing 200 seconds under the rotating speed of 17500rpm/min, by W/O emulsion stable for its homogeneous formation, this emulsion pipettor being slowly injected into 20ml contains in sodium phosphate (pH6.0) buffer of NaCl and 2.5%PVA, high-shear homogenizer forms W/O/W microemulsion 13000 revs/min of emulsifyings in homogeneous 15 minutes, the aqueous solution that 20ml contains NaCl is added in the W/O/W microemulsion formed, stirring at low speed 4 hours in digital display constant temperature blender with magnetic force, dichloromethane is volatilized clean, obtain solidified microsphere.Centrifuge washing three times after microsphere solidification, collects containing HBsAg (HBsAg-TS) the PLGA microsphere of trehalose as stabilizing agent, ﹣ 80 DEG C of freeze overnight, 5.0 × 10
﹣ 3pa vacuum under pressure lyophilization 12h obtains the HBsAg-TS sustained-release micro-spheres that PLGA molecular weight is 23kDa, 43kDa, 87kDa, is designated as A3, B3, C3.The particle diameter of thus obtained microsphere is between 4.30 microns ~ 6.50 microns, and envelop rate is between 50.2% ~ 60.5%, and drug loading is 0.47% ~ 0.65%.
Embodiment 10
Select female sd inbred rats 28 in 8 week age, about body weight 200g, give and Free water and food in whole experimentation, be divided into randomly in recombinant hepatitis B vaccine (alum-HBsAg) group, blank microsphere group, embodiment 7 without the A2 totally five groups added in the A3 added in the A1 in the HBsAgPLGA sustained-release micro-spheres group of stabilizing agent, embodiment 9 in the HBsAg-TSPLGA sustained-release micro-spheres group of trehalose and embodiment 8 in sero-abluminous HBsAg-HASPLGA sustained-release micro-spheres group.Get blood 0.5ml in centrifuge tube by the ophthalmic corner of the eyes before administration, collected by centrifugation serum, is designated as negative control sera to be measured.
Experiment adopts subcutaneous injection, and twice administration of alum-HBsAg component is respectively injection in 0,1 month, and per injection dosage is 10 μ g/; HBsAgPLGA sustained-release micro-spheres group, HBsAg-TSPLGA sustained-release micro-spheres group and HBsAg-HASPLGA sustained-release micro-spheres group are single administration, dosage by HBsAg 20 μ g/ only, the shot of blank microsphere group, injectable microsphere amount is identical with HBsAg sustained-release micro-spheres group injection volume.
Being added by microsphere containing NaCl concentration is make test medicine in the suspending agent of 0.9%, and after being expelled to rat, leg is subcutaneous.Within upon administration the 1st week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, 15 weeks, 18 weeks, get blood 0.5ml in centrifuge tube by the ophthalmic corner of the eyes, collected by centrifugation serum, is designated as positive control serum to be measured.
By blood natural coagulation 30 minutes under room temperature of taking out, 3500rpm/min carefully collects supernatant in centrifugal 20 minutes, and ﹣ 20 DEG C of freezen protective are to be measured, as there is precipitation in preservation process, answers recentrifuge.
After completing the blood sampling in the 18th week of above-mentioned five groups of female sd inbred rats, anaesthetize to the urethane of these five groups of rat injection optimal doses to it, carry out perfusion operation to it after anesthesia, win spleen in centrifuge tube, quick freeze is preserved to be measured in-80 DEG C of refrigerators.
Measure specificity anti-HBsAg IgG antibody level in serum by rat hepatitis B surface antibody (HBsAb) enzyme-linked immunoassay kit and carry out the research of rat humoral immune reaction.Result as shown in Figure 3, almost do not induce the specific antibody response of HBsAg by blank PLGA microsphere.HBsAg-HSAPLGA microball preparation, HBsAg-TSPLGA sustained-release micro-spheres group and HBsAgPLGA microball preparation induce the anti-HBsAg IgG antibody persistent levels of generation to raise after single-dose.And the anti-HBsAg IgG antibody level of being induced by the alum-HBsAg preparation of double injection showed as ascendant trend at first 8 weeks, then show the trend reduced gradually afterwards.
By figure, we find out from 0 week to 4 weeks, HBsAg-HSAPLGA microball preparation, HBsAg-TSPLGA sustained-release micro-spheres group and HBsAgPLGA microball preparation and alum-HBsAg preparation induce the anti-HBsAg IgG antibody level almost identical (P>0.05) of generation.But alum-HBsAg preparation is after 4th week booster injection, the 5th week time, anti-HBsAg IgG antibody level raises, apparently higher than HBsAg-HSAPLGA sustained-release micro-spheres group, HBsAg-TSPLGA sustained-release micro-spheres group and HBsAgPLGA microball preparation (P<0.05).The anti-HBsAg IgG antibody level that the HBsAg-HSAPLGA microball preparation induction of single dose injection after the 7th week produces apparently higher than HBsAgPLGA microball preparation and HBsAg-TSPLGA sustained-release micro-spheres group induce the anti-HBsAg IgG antibody level of generation, and surmounted after 12 weeks alum-HBsAg preparation induce the anti-HBsAg IgG antibody level (P<0.05) of generation.Display HBsAg-HSAPLGA microball preparation can the response of more effectively elicit humoral immune.
By Rat Interleukin 2(IL-2) enzyme-linked immunoassay kit measures IL-2 level in spleen and carries out the immunoreactive research of rat cell.Result as shown in Figure 4 blank PLGA microsphere induces the IL-2 level produced almost close to 0; And alum-HBsAg, HBsAg-HASPLGA sustained-release micro-spheres group, HBsAg-TSPLGA sustained-release micro-spheres group and HBsAgPLGA sustained-release micro-spheres group all induce the IL-2 producing varying level; The IL-2 level that the induction of three kinds of microball preparations produces induces the IL-2 level (P<0.05) of generation apparently higher than alum-HBsAg institute, and by HBsAg-HSAPLGA sustained-release micro-spheres group induce the IL-2 level of generation be higher than HBsAgPLGA sustained-release micro-spheres group and HBsAg-TSPLGA sustained-release micro-spheres group induce the IL-2 level (P<0.05) of generation.The IL-2 level that the induction of HBsAg-HSAPLGA sustained-release micro-spheres group produces as seen from Figure 4 is HBsAgPLGA sustained-release micro-spheres group respectively, HBsAg-TSPLGA sustained-release micro-spheres group, alum-HBsAg preparation induce 1.3,1.2 and 3.2 times of the IL-2 level of generation.
Measure IFN-γ level in spleen by rat gamma interferon (IFN-γ) enzyme-linked immunoassay kit and carry out the immunoreactive research of rat cell.Result as shown in Figure 4 blank PLGA microsphere induces the IFN-γ level produced almost close to 0; And alum-HBsAg, HBsAg-HSAPLGA sustained-release micro-spheres group and HBsAgPLGA sustained-release micro-spheres group all induce the IFN-γ producing varying level; The IFN-γ level that the induction of three kinds of microball preparations produces induces the IFN-γ level (P<0.05) of generation apparently higher than alum-HBsAg institute, and by HBsAg-HSAPLGA sustained-release micro-spheres group induce the IFN-γ level of generation be higher than HBsAgPLGA sustained-release micro-spheres group induce the IFN-γ level (P<0.05) of generation.The IFN-γ level that the induction of HBsAg-HSAPLGA sustained-release micro-spheres group produces as seen from the figure be respectively alum-HBsAg, HBsAg-TSPLGA sustained-release micro-spheres group and HBsAgPLGA sustained-release micro-spheres group induce 4.3,1.2 and 1.2 times of the IFN-γ level of generation.Therefore, with alum-HBsAg, HBsAgPLGA sustained-release micro-spheres group and HBsAg-TSPLGA sustained-release micro-spheres group Comparatively speaking, HBsAg-HSAPLGA sustained-release micro-spheres group can better inducing cellular immune response, produces higher levels of IFN-γ.
Therefore, alum-HBsAg can induce very weak cellullar immunologic response, and HBsAgPLGA microball preparation is induction of stronger cellullar immunologic response, and HBsAg-HSAPLGA sustained-release micro-spheres group microball preparation can induce stronger cellullar immunologic response.
Claims (10)
1. the sustained release microsphere agents containing recombination hepatitis B surface antigen, it is characterized in that, this sustained release microsphere agents is with stability protection agent and recombination hepatitis B surface antigen microgranule composition kernel, makes outsourcing sealing combine by macromolecular material polyglycolic-polylactic acid block copolymer; Described stabilizing agent is trehalose, human serum albumin.
2. the sustained release microsphere agents containing restructuring liver surface antigen according to claim 1, it is characterized in that, the mass ratio of recombination hepatitis B surface antigen and described stability protection agent is 1: (1 ~ 20).
3. the sustained release microsphere agents containing restructuring liver surface antigen according to claim 2, it is characterized in that, the mass ratio of recombination hepatitis B surface antigen and described stability protection agent is 1: (1 ~ 10).
4. the sustained release microsphere agents containing recombination hepatitis B surface antigen according to claim 3, it is characterized in that, the mass ratio of recombination hepatitis B surface antigen and described stability protection agent human serum albumin is 1: 5.
5. the sustained release microsphere agents containing recombination hepatitis B surface antigen according to Claims 1-4, is characterized in that, described recombined human hepatitis B surface antigen is the hbs antigen of yeast hbs antigen or Chinese hamster ovary cell secretion.
6. the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen according to claim 1, is characterized in that, comprise the following steps:
(1) stabilizing agent and recombination hepatitis B surface antigen are mixed to get interior aqueous phase, described stabilizing agent is trehalose, human serum albumin;
(2) polyglycolic-polylactic acid block copolymer is dissolved in organic solvent makes oil phase, get the made interior aqueous phase of step and add above-mentioned oil phase and stir homogenize and form W/O colostric fluid;
(3) W/O colostric fluid is added in outer aqueous phase poly-vinyl alcohol solution, after stirring homogenize, form W/O/W double emulsion; Stir 4 ~ 6 hours again, centrifuge washing, collect, vacuum lyophilization.
7. the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen according to claim 6, it is characterized in that, the mass ratio of recombination hepatitis B surface antigen and described stability protection agent is 1: (1 ~ 20).
8. the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen according to claim 7, it is characterized in that, the mass ratio of recombination hepatitis B surface antigen and described stability protection agent is 1: (1 ~ 10).
9. the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen according to claim 8, it is characterized in that, the mass ratio of recombination hepatitis B surface antigen and described stability protection agent human serum albumin is 1: 5.
10., according to the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen described in claim 6 to 9, it is characterized in that,
In (2) step, speed of agitator is 8500 ~ 20000 revs/min; Mixing time is 30 ~ 300 seconds;
The rotating speed stirring homogenize in (3) step is 8500 ~ 15000 revs/min; Mixing time is 10 ~ 20 minutes.
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