CN105194662B - Sustained release microsphere agents and preparation method thereof containing recombination hepatitis B surface antigen - Google Patents
Sustained release microsphere agents and preparation method thereof containing recombination hepatitis B surface antigen Download PDFInfo
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- CN105194662B CN105194662B CN201510588930.8A CN201510588930A CN105194662B CN 105194662 B CN105194662 B CN 105194662B CN 201510588930 A CN201510588930 A CN 201510588930A CN 105194662 B CN105194662 B CN 105194662B
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Abstract
The invention discloses a kind of sustained release microsphere agents and preparation method thereof containing recombination hepatitis B surface antigen; the sustained release microsphere agents are to form kernel with stability protection agent and recombination hepatitis B surface antigen particle, and making outsourcing sealing by high molecular material polylactic acid poly hydroxyacetic acid block copolymer is composed；The stabilizer is trehalose, human serum albumins.Preparation method is：(1）Stabilizer and recombined human hepatitis B surface antigen are mixed into obtain inner aqueous phase；（2）Polylactic acid poly hydroxyacetic acid block copolymer is dissolved in organic solvent, oil phase is made, taken inner aqueous phase that oil phase stirring is added and homogenize；（3）Outer aqueous phase is added in W/O colostric fluids, stirring forms W/O/W double emulsions after homogenizing；It is stirred for 4~6 hours, centrifuge washing, collects, vacuum freeze drying.The present invention, which can overcome the problems, such as that the immunogenicity of hepatitis B surface antigen is destroyed during prepared by microballoon and discharge, to be caused antigen to be denaturalized and then influences immune effect.
Description
Technical field
The present invention relates to a kind of biological agent, especially a kind of sustained release microsphere agents containing recombination hepatitis B surface antigen and its
Preparation method.
Background technology
Hepatitis B is a kind of pandemic infection disease seriously threatening human health, it is by hepatitis type B virus
（HBV）Infection caused by disease.Currently, HB vaccination be prevent and control HBV propagate most effective measure it
One.Now most-often used hepatitis B vaccine is the hepatitis B surface antigen obtained by gene recombination technology（HBsAg）Purified, inactivation
And aluminium adjuvant hepatitis B vaccine made of aluminium adjuvant absorption is added.However, in order to reach lasting and effective immune response effect, aluminium
Adjuvant hepatitis B vaccine need at 0,1,2 month or 0,1,6 month immune programme injected three times, due to immune programme complexity,
Quite a few people is caused to be difficult to complete immune programme, this phenomenon is even more serious in developing country.In addition, aluminium adjuvant hepatitis B
Vaccine only selective stimulating body generates humoral immune response, and cannot effectively induce generation cellullar immunologic response, cannot be effective
Existing HBV viruses in scavenger-cell.Thus, clinic needs to research and develop novel potent hepatitis B vaccine, simplifies immune programme, makes wider
General crowd is effectively protected.
Polyglycolic-polylactic acid block copolymer (PLGA) is a kind of macromolecule polymer material, and antigen is encapsulated in
Single injection sustained-release micro-spheres vaccine preparation is made in the slow release that antigen may be implemented in PLGA microballoons.But it is prepared in microballoon
It is related to the emulsion process that freeze-drying and antigen disperse in organic solvent in the process, the two processes are easy to cause antigen
Denaturation, immunogenicity decline.
Invention content
It is an object of the invention to provide a kind of sustained release microsphere agents of the recombination hepatitis B surface antigen of single injection, with gram
Taking the immunogenicity of hepatitis B surface antigen and being destroyed during prepared by microballoon and discharge causes antigen to be denaturalized and then influences immune
The problem of effect.
It is a further object of the present invention to provide a kind of preparation methods of the sustained release microsphere agents containing recombination hepatitis B surface antigen.
The invention is realized in this way：Sustained release microsphere agents containing recombination hepatitis B surface antigen, the sustained release microsphere agents are
Kernel is formed with stability protection agent and recombination hepatitis B surface antigen particle, it is embedding by high molecular material polyglycolic-polylactic acid
Section copolymer is made outsourcing sealing and is composed；The stabilizer is trehalose, human serum albumins.
In the present invention, recombination hepatitis B surface antigen and the mass ratio of the stability protection agent are preferably 1:（1~20）.Compared with
Preferably 1:（1~10）, consider the influence factors, more preferably 1: 5 such as production cost.
In the present invention, the recombined human hepatitis B surface antigen is that yeast hepatitis B surface antibody or Chinese hamster ovary are thin
The hepatitis B surface antibody of intracrine.
The preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen of the present invention, includes the following steps：
(1）Stabilizer and recombined human hepatitis B surface antigen are mixed into obtain inner aqueous phase, the stabilizer is trehalose, human serum
Albumin；
（2）Polyglycolic-polylactic acid block copolymer is dissolved in organic solvent, oil phase is made, takes the made interior water of step
Above-mentioned oil phase stirring is added to homogenize to form W/O colostric fluids；
（3）W/O colostric fluids are added in outer aqueous phase poly-vinyl alcohol solution, stirring forms W/O/W double emulsions after homogenizing；It stirs again
It mixes 4~6 hours, centrifuge washing, collects, vacuum freeze drying.
In the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen of the present invention, recombination hepatitis B surface antigen
Mass ratio with the stability protection agent is preferably 1:（1~20）, it is more preferably 1:（1~10）, consider the shadows such as production cost
The factor of sound, more preferably 1: 5.
In the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen of the present invention, the（2）Stirring turns in step
Speed is 8500~20000 revs/min；It is preferred with 15000 revs/min；Mixing time is 30~300 seconds；The（3）It is stirred in step homogenized
Rotating speed is 8500~15000 revs/min；It is preferred 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 × 103~5.5 × 104, wherein
Polylactic acid and polyglycolic acid（PLA:PLG）Mass ratio be（50:50）~（85:15）.
It is used organic in the preparation method of the sustained release microsphere agents containing recombination hepatitis B surface antigen described in the present invention
Solvent is the mixed liquor of dichloromethane or dichloromethane and acetone, wherein when the mixed liquor that organic solvent is dichloromethane and acetone
When, the volume ratio of dichloromethane and acetone is 75:25.
In the preparation method of the present invention, a concentration of the 75 of polyglycolic-polylactic acid block copolymer in the oil phase
~250 mg/ml.A concentration of the 0.5%~5% of the outer aqueous phase decentralized medium polyvinyl alcohol, it can be added a concentration of 0~
10% inorganic salts, to increase the encapsulation rate of antigen.In the present invention inner aqueous phase, the pH value of outer aqueous phase should be located at 5.0~7.4 it
Between, inner aqueous phase, outer aqueous phase pH value are identical, and preferably 6.0 with the formation of sharp finished product.
In the present invention, the centrifugation rate for receiving product microballoon is 8500 revs/min, and the time is about 20 minutes.
The present invention selects stability of the suitable stabilizer protection hepatitis B surface antigen in microballoon preparation process；Then make
With Biodegradable polymer material polyglycolic-polylactic acid block copolymer（PLGA）For carrier material encapsulate stabilizer and
Recombination hepatitis B surface antigen, the polyglycolic-polylactic acid block copolymer sustained release that recombination hepatitis B surface antigen is prepared are micro-
Ball, the immunogenicity that hepatitis B surface antigen during prepared by microballoon and discharge can be effectively ensured are not destroyed.
The present invention by add stability protection agent made from sustained-release micro-spheres, surface is smooth, appearance uniform, regular particles without
Adhesion, average grain diameter is at 1.0-10.0 μm；Drugloading rate, encapsulation rate are high.It is slow as the single injection for preventing spreading
Microspheres vaccine preparation is released, Antigen Stability is good, and immunocompetence is high, and the sustained release phase is more than 60 days or more, is suitable for non-vein and is administered to
Medicine is used for healthy population, improves crowd's HBsAb levels, and can be with inducing cellular immune, the intracellular hepatitis B for removing infection
Virus.Therefore, this preparation acts not only as preventing spreading, and can reach treatment chronic hepatitis B and hepatitis B
The effect of carrier.In addition, lactic acid-glycolic acid block copolymer microballoon is biodegradable, good biocompatibility.
Description of the drawings
Fig. 1 is the sustained release microsphere agents release in vitro containing recombination hepatitis B surface antigen prepared by the embodiment of the present invention 8
The electron scanning micrograph of metamorphosis.
Fig. 2 is the sustained release microsphere agents containing recombination hepatitis B surface antigen prepared by the embodiment of the present invention 7 and embodiment 8
Drug release patterns in vitro.
Fig. 3 is that anti-HBsAg IgG antibody level changes with time in rat blood serum.
Fig. 4 is that IL-2 and IFN-γ are horizontal in the rat body of different preparations induction generation.
Specific implementation mode
Technical scheme of the present invention is further described below by embodiment.
The surface characteristics of microballoon is observed using scanning electron microscope.
The assay method of encapsulation rate：20 mg HBsAg microballoons accurately are weighed in 2ml centrifuge tubes, and 1ml acetonitriles are added,
14000 r·min﹣ 120 min are centrifuged, supernatant is abandoned, after precipitation is dried in vacuo 2 h, with 1ml sodium radio-phosphate,P-32 solutions（0.02
mol·L﹣ 1, pH7.4）Again disperse, centrifuge again, and retain supernatant；It repeats the above process, merges supernatant twice,
HBsAg contents are measured with Lowry methods.Calculate the encapsulation rate and drugloading rate of microballoon.The encapsulation rate of microballoon=（It is encapsulated in microballoon
Encapsulating and non-encapsulated HBsAg total amounts in HBsAg contents/microballoon）×100%.The drugloading rate of microballoon=（Contained HBsAg in microballoon
Weight/microballoon total weight）×100%.The measurement of burst release rate：Accurately 20mg HBsAg microballoons are weighed in 2ml centrifuge tubes,
1ml sodium phosphates are added（0.02mol·L﹣ 1pH7.4）Buffer solution is placed in 37 DEG C of constant-temperature tables, rate 160rmin-1, for 24 hours after
Centrifuging and taking supernatant calculates the burst release rate of microballoon with its HBsAg content of Lowry methods kit measurement.The burst release rate of microballoon=（20mg
Microballoon for 24 hours in amount/20mg microballoons of discharged HBsAg HBsAg total amount）×100%.
Microballoon release in vitro:The HBsAg-HSA microballoons prepared by 20mg are accurately weighed respectively with HBsAg microballoons in 2ml's
In centrifuge tube, 1ml sodium phosphates are added thereto（0.02mol·L﹣ 1pH7.4）Buffer solution is placed in 36.5 DEG C of constant-temperature tables, rate
120 revs/min, according to pre-set time interval centrifuging and taking supernatant, then fresh sodium phosphate is added in precipitation
（0.02mol·L﹣ 1pH7.4）Buffer solution, which is replaced in shaking table, sways release.With total egg in BCA method kit measurement supernatants
Bai Hanliang, double antibody sandwich ELISA survey HBsAg antigen actives in supernatant, and the stability of the HBsAg of release in vitro uses body
The liquid chromatography detection of product Exclusion High Performance.
Double antibody sandwich ELISA measures HBsAg antigen actives：（1）Coating：With the carbonate buffer of 0.05M pH9.6
Liquid 1:2000 dilution rabbit AntiHBsAg antibodies, 100 holes μ l/, 4 DEG C overnight；（2）Washing：Abandoning coating buffer, PBST washs 3 times, and every time 3
Minute, it pats dry；（3）Closing：1%BSA-PBST, 200 holes μ l/ are added per hole, 37 DEG C of wet box incubate 2 hours.Washing is same as above；（4）
Sample-adding：HBsAg standard samples are added（Concentration range 5.0 ng/ml ~ 700 ng/ml) and sample to be tested, 100 holes μ l/, 37 DEG C are wet
Box incubates 1 hour.Washing is same as above；（5）Enzyme labeling antibody：With l%BSA-PBST-4%PEG6000 by enzyme mark rabbit AntiHBsAg antibody
1:4000 dilutions, 100 holes μ l/, 37 DEG C of wet box incubate 1 hour.Washing is same as above；（6）Add substrate：It is molten that TMB soluble substrates are added
100 holes μ l/ of liquid, room temperature are protected from light 15min；（7）It terminates：100 holes μ l/ terminate liquids are added dropwise；（8）Detection：Microplate reader measures
Absorbance value under 450nm and 630nm.The difference of OD450 and OD630 is sample itself absorbance.It is built using standard solution
Day-mark directrix curve calculates HBsAg antigen actives in sample to be tested.Size exclusion-high performance liquid chromatography method detects HBsAg：（1）Instrument
Device is Japanese Shimadzu high performance liquid chromatograph, the model SEC-300 of chromatographic column（5 μm, 150 × 7.8 mm）, Detection wavelength is
280 nm, mobile phase are the PBS containing 0.1% sodium azide（1 pH7.4 of 0.02mol L ﹣）Buffer solution, flow velocity：0.3 ml/min,
Sample size：20μl.
Humoral immune response of the HBsAg sustained-release micro-spheres in rat Immune inducing in vivo：Select 8 week old female sd inbred rats 28, body
Weight 200g or so, gives Free water and food, is randomly divided into recombinant hepatitis B vaccine in the entire experiment process
（alum-HBsAg）Group, blank microballoon group, HBsAg sustained-release micro-spheres group, HBsAg-TS sustained-release micro-spheres group and HBsAg-HSA sustained releases are micro-
Five groups of ball group.Experiment is using being subcutaneously injected, and alum-HBsAg groups are administered at twice injected at 0,1 month respectively, per injection agent
Amount is 10 μ g/；HBsAg sustained-release micro-spheres group, HBsAg-TS sustained-release micro-spheres group and HBsAg-HSA sustained-release micro-spheres groups are primary
Administration, based on HBsAg 20 μ of dosage g/, a blank microballoon group shot, injectable microsphere amount and HBsAg sustained-release micro-spheres groups
Injection volume is identical.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 upon administration leads to
It crosses the intraocular corner of the eyes to take in blood 0.5ml to centrifuge tube, serum is collected by centrifugation.Hepatitis B surface antibody in rat blood serum（HBsAb）Measurement：
Antibody quantitative determination is carried out using the HBsAb Quantikine ELISA kits of Bio-Swamp Immunoassay R＆D Center.
Cellullar immunologic response of the HBsAg sustained-release micro-spheres in rat Immune inducing in vivo：Complete above-mentioned five groups of female sd inbred rats the 18th
After week blood sampling, gives the urethane of this five groups of rat injection optimal doses to anaesthetize it, perfusion operation is carried out to it after anesthesia,
Spleen is won in centrifuge tube, quick freeze preserves to be measured in -80 DEG C of refrigerators.Rats Spleen is weighed, and is added by the tissue per 10mg
150 μ l sodium phosphates（0.02mol·L﹣ 1pH7.4）Corresponding body is added according to the weight of different Rats Spleens in the ratio of buffer solution
Long-pending sodium phosphate（0.02mol·L﹣ 1pH7.4）Buffer solution shreds tissue under ice bath state, and uses Ultrasonic Cell Disruptor will
The historrhexis shredded, 12000 revs/min of the progress centrifugation 10 minutes in 4 DEG C of centrifuges, gained supernatant is white for cell factor
The measurement of interleukin IL-2 and interferon IFN-γ.The measurement of rat interleukin I L-2 and interferon IFN-γ level：Using Bio-
The IL-2 and IFN-γ b Quantikine ELISA kits of Swamp Immunoassay R＆D Center is carried out.
Embodiment 1
It is stability protection agent to select trehalose and human serum albumins, takes multigroup 0.5 mg/ml HBsAg solution, respectively
Trehalose is added and its concentration is made to respectively reach 5 mg/ml, 10 mg/ml；Human serum albumins is added（HSA）And make its concentration
0.5 mg/ml, 1.0 mg/ml, 2.5 mg/ml, 5.0 mg/ml are respectively reached, then vacuum freeze drying 12h, with double antibody
Sandwich ELISA surveys HBsAg antigen active retention rates, the results are shown in Table 1.Vacuum freeze drying 12h is represented with freeze-drying in table 1.
Embodiment 2
It is stability protection agent to select trehalose and human serum albumins, takes a concentration of 0.5 mg/ml of more parts of 0.2 ml
HBsAg solution, is separately added into trehalose, human serum albumins wherein, and acquisition trehalose concentration is 5.0 mg/ml, human serum
Albumin concentration be 1.0 mg/ml, 2.5 mg/ml, 5.0 mg/ml, 10.0 mg/ml HBsAg solution, then with dichloromethane
Mixing, 15000 revs/min emulsify 150 seconds, and supernatant is taken to be pressed from both sides with double antibody after dichloromethane volatilization is clean after 5000 revs/min of centrifugations
Heart ELISA method surveys HBsAg antigen active retention rates.Test data is shown in Table 1.Aforementioned emulsifying step is indicated in table 1 by emulsification.
1 stability protection agent of table is freeze-dried to HBsAg solution, after emulsification antigen active retention rate influence
Embodiment 3
With human serum albumins（HAS）As the stability protection agent for preparing HBsAg sustained-release micro-spheres, HBsAg and human serum
Albumin in mass ratio 1:5 ratio mixings, by solvent of the sodium radio-phosphate,P-32 solution that pH value is 5.0 be made mixed phosphate sodium solution as
Inner aqueous phase.In the mixed solution, a concentration of 1 mg/ml of HBsAg, a concentration of 5 mg/ml of human serum albumins.Taking 100 μ l, this is molten
Liquid is added to 2 ml and contains 150 mg 23kDa polyglycolic-polylactic acid block copolymers（Wherein polylactic acid and poly- hydroxyl second
The mass ratio of acid（PLA:PLG）It is 50:50）Dichloromethane and acetone（65:35）Mixed solution in, existed with high-shear homogenizer
By its homogeneous 300 seconds under 20000 revs/min of rotating speed, stable W/O emulsion is formed, which is slowly noted with pipettor
Enter the sodium phosphate for containing NaCl and 2.5% PVA to 20ml（pH5.0）In buffer solution, high-shear homogenizer is homogeneous at 13000 revs/min
Emulsification in 15 minutes forms W/O/W micro emulsions, the aqueous solution that 20 ml contain NaCl is added into the W/O/W micro emulsions of formation, in digital display
Stirring at low speed 4 hours in constant temperature blender with magnetic force keep dichloromethane volatilization clean, obtain solidified microsphere.It is centrifuged after microballoon solidification
It washs three times, collection HBsAg-HSA microballoons, 80 DEG C of freeze overnights of ﹣, 5.0 × 10﹣ 3Pa vacuum under pressure is freeze-dried 12 h and obtains
To HBsAg-HSA sustained-release micro-spheres.The grain size of thus obtained microsphere is 4.87 microns, and encapsulation rate is 78.5%, drugloading rate 0.41%.
Embodiment 4
With human serum albumins（HAS）As the stability protection agent for preparing HBsAg sustained-release micro-spheres, HBsAg and human serum
Albumin in mass ratio 1:Mixed phosphate sodium solution is made by solvent of the sodium radio-phosphate,P-32 solution that pH value is 7.0 and makees for 10 ratio mixings
For inner aqueous phase.In the mixed solution, a concentration of 1 mg/ml of HBsAg, a concentration of 10 mg/ml of human serum albumins.Take 400 μ l
Sodium radio-phosphate,P-32 solution containing HSA and HBsAg is added to 2 ml and contains 500 mg 23kDa polyglycolic-polylactic acid block copolymerizations
Object（Wherein polylactic acid and polyglycolic acid（PLA:PLG）Mass ratio be 85:15）Dichloromethane solution in, use high speed homogenization
Machine homogeneous 200 seconds under 8500 revs/min of rotating speed, by its homogeneous W/O emulsion for forming stabilization, by the emulsion liquid relief
Device is slowly injected into the sodium phosphate that 20ml contains 5% NaCl and 2.5% PVA（pH7.0）In buffer solution, existed with high-shear homogenizer
15000 revs/min of emulsifications in homogeneous 10 minutes form W/O/W microemulsions, and 20 ml, which are added, into the W/O/W microemulsions of formation contains
The aqueous solution of NaCl, stirring at low speed 5 hours in digital display constant temperature blender with magnetic force keep dichloromethane volatilization clean, are cured
Microballoon.Microballoon solidification after centrifuge washing three times, collect HBsAg-HSA microballoons, 80 DEG C of freeze overnights of ﹣, 5.0 × 10﹣ 3Under Pa pressure
12 h of vacuum freeze drying obtains HBsAg-HSA sustained-release micro-spheres.The grain size of thus obtained microsphere is 9.67 microns, encapsulation rate 50.5%,
Drugloading rate is 0.22%.
Embodiment 5
With human serum albumins（HAS）As the stability protection agent for preparing HBsAg sustained-release micro-spheres, HBsAg and human serum
Albumin in mass ratio 1:5 ratio mixings, by solvent of the sodium radio-phosphate,P-32 solution that pH value is 6.0 be made mixed phosphate sodium solution as
Inner aqueous phase（pH6.0）.In the mixed solution, a concentration of 1 mg/ml of HBsAg, a concentration of 5 mg/ml of human serum albumins.Take 200
The sodium radio-phosphate,P-32 solution that μ l should contain HSA and HBsAg is added to 2 ml and contains 500 mg 23kDa polyglycolic-polylactic acid blocks
Copolymer (wherein polylactic acid and polyglycolic acid（PLA:PLG）Mass ratio be 75:25) in dichloromethane solution, with high speed
Homogenizer homogeneous 290 seconds under 10000 revs/min of rotating speed, by its homogeneous W/O colostric fluid for forming stabilization, by the emulsion
It is slowly injected into the sodium phosphate that 20ml contains 5% NaCl and 2.5% PVA with pipettor（pH6.0）In buffer solution, high-shear homogenizer
20 minutes homogeneous at 12000 revs/min, emulsification forms W/O/W double emulsions, and 20 ml, which are added, into the W/O/W micro emulsions of formation contains
The aqueous solution of NaCl, stirring at low speed 5 hours in digital display constant temperature blender with magnetic force keep dichloromethane volatilization clean, are cured
Microballoon.Microballoon solidification after centrifuge washing three times, collect HBsAg-HSA microballoons, 80 DEG C of freeze overnights of ﹣, 5.0 × 10﹣ 3Under Pa pressure
12 h of vacuum freeze drying obtains HBsAg-HSA sustained-release micro-spheres.The grain size of thus obtained microsphere is 7.67 microns, encapsulation rate 60.7%,
Drugloading rate is 0.72%.
Embodiment 6
Using HSA as the stability protection agent for preparing HBsAg sustained-release micro-spheres, HBsAg and human serum albumins are in mass ratio
1:5 ratio mixings obtain mixed phosphate sodium solution by solvent of the sodium radio-phosphate,P-32 solution that pH is 6.0 as inner aqueous phase, the mixed solution
In, a concentration of 1 mg/ml of HBsAg, a concentration of 5 mg/ml of human serum albumins.Take 400 PBSs of the μ l containing HSA and HBsAg molten
The solution is added to 3 ml and contains 200 mg 23kDa polyglycolic-polylactic acid block copolymers by liquid（Wherein polylactic acid
With polyglycolic acid（PLA:PLG）Mass ratio be 85:15）Dichloromethane solution in, with high-shear homogenizer 20000
Homogeneous 100 seconds under the rotating speed of rpm/min its homogeneous W/O emulsion for forming stabilization is slowly noted the emulsion with pipettor
Enter the sodium phosphate for containing 5% NaCl and 2.5% PVA to 20ml（pH6.0）In buffer solution, high-shear homogenizer is even at 15000 revs/min
Matter emulsification in 15 minutes forms W/O/W micro emulsions, the aqueous solution that 20 ml contain NaCl is added into the W/O/W micro emulsions of formation, in number
Stirring at low speed 5 hours in aobvious constant temperature blender with magnetic force, keeps dichloromethane volatilization clean, obtains solidified microsphere.Microballoon solidification after from
The heart washs three times, collection HAS-HBsAg microballoons, 80 DEG C of freeze overnights of ﹣, and 5.0 × 10﹣ 3Pa vacuum under pressure is freeze-dried 12 h
Obtain HBsAg-HSA sustained-release micro-spheres.The grain size of thus obtained microsphere is 7.67 microns, encapsulation rate 50.5%, drugloading rate 0.97%.
Embodiment 7
Take the sodium phosphate of 200 μ l 1 mg/ml containing HBsAg（pH6.0）The solution is added to 2 ml and contains 200 by solution
Mg molecular weight is in the dichloromethane solution of 23kDa, 43kDa, 87kDa polyglycolic-polylactic acid block copolymer, with height
Fast homogenizer homogeneous 300 seconds under 17500 revs/min of rotating speed, by its homogeneous W/O emulsion for forming stabilization, by the emulsion
It is slowly injected into the sodium phosphate for containing NaCl and 2.5% PVA to 20 ml with pipettor（pH6.0）In buffer solution, 13000 revs/min even
Homogeneous 15 min of 15 minutes homogenizers of matter emulsifies to form W/O/W micro emulsions, and 20 ml, which are added, into the W/O/W micro emulsions of formation contains
The aqueous solution of NaCl, the stirring at low speed 4h in digital display constant temperature blender with magnetic force keep dichloromethane volatilization clean, are cured
23kDa, 43kDa, 87kDa PLGA microballoon, are denoted as A1, B1, C1 respectively.Centrifuge washing three times, collects HBsAg after microballoon solidification
Microballoon, 80 DEG C of freeze overnights of ﹣, 5.0 × 10﹣ 3Pa vacuum under pressure is freeze-dried 12 h and obtains dry HBsAg microballoons.Gained
Between the grain size of microballoon is 4.0 microns ~ 7.87 microns, for encapsulation rate between 76.5% ~ 86.1%, drugloading rate is 0.39% ~ 0.45%
（Table 2）.
Embodiment 8
Using HSA as the stability protection agent for preparing HBsAg sustained-release micro-spheres, with HBsAg mixings in proportion, obtained with pH6.0
Mixed phosphate sodium（pH6.0）As solvent mixed phosphate sodium solution is made as inner aqueous phase in solution, in the mixed solution, HBsAg
A concentration of 1 mg/ml, a concentration of 5 mg/ml of human serum albumins.200 solution of the μ l containing HSA and HBsAg are taken, which is added
Enter to 2 ml to contain 200 mg 23kDa, the dichloromethane of 43kDa, 87kDa polyglycolic-polylactic acid block copolymer molten
In liquid, with high-shear homogenizer under the rotating speed of 17500 rpm/min homogeneous 200 seconds, its homogeneous is formed into stable W/O emulsus
The emulsion is slowly injected into the sodium phosphate that 20ml contains NaCl and 2.5% PVA by liquid with pipettor（pH6.0）In buffer solution,
High-shear homogenizer forms W/O/W micro emulsions in 13000 revs/min of emulsifications in homogeneous 15 minutes, and 20 are added into the W/O/W micro emulsions of formation
Ml contains the aqueous solution of NaCl, stirring at low speed 4 hours in digital display constant temperature blender with magnetic force, keeps dichloromethane volatilization clean, obtains
To solidified microsphere.Microballoon solidification after centrifuge washing three times, collect HSA-HBsAg microballoons, 80 DEG C of freeze overnights of ﹣, 5.0 × 10﹣ 3 Pa
Vacuum under pressure is freeze-dried 12 h and obtains the HBsAg-HSA sustained-release micro-spheres that PLGA molecular weight is 23kDa, 43kDa, 87kDa, point
A2, B2, C2 are not denoted as it.The form of thus obtained microsphere and the metamorphosis during release are as shown in Figure 1.The grain size of thus obtained microsphere is
Between 4.67 microns ~ 6.87 microns, for encapsulation rate between 76.5% ~ 89.5%, drugloading rate is 0.40% ~ 0.47%（Table 2）.
The property of 2 HBsAg-HSA microballoons of table and HBsAg microballoons
By total with BCA methods kit measurement HBsAg-HSA microballoons and HBsAg in HBsAg microballoon release in vitro supernatants
Content, and HBsAg antigen actives in release supernatant are surveyed with double antibody sandwich ELISA, and microballoon is calculated in proportion
The curve of burst size such as Fig. 2.It can be seen that in the presence of no HSA is as stabilizer, the HBsAg activity discharged is largely lost
It loses, activity preservation rate only about 60%；When have HSA as stabilizer in the presence of, the antigen active of the HBsAg discharged retains
Rate is significantly improved, and antigen active retention rate is increased to 80.0% ~ 90.0%.
The release profiles of microballoon show two stages as seen from Figure 2：（1）The burst release behavior of starting stage, largely
HBsAg release in for 24 hours；（2）After burst release, the release behavior of microballoon shows a kind of lasting release conditions.Through
Cross 60 days extracorporeal releasing experiments, the HBsAg total amounts that microballoon is discharged are up to as many as 50%.
Embodiment 9
Using trehalose as the stability protection agent for preparing HBsAg sustained-release micro-spheres, and HBsAg mixings in proportion, with pH value
Sodium radio-phosphate,P-32 solution for 6.0 is that mixed phosphate sodium solution is made as inner aqueous phase in solvent（pH6.0）, in the mixed solution, HBsAg
A concentration of 1 mg/ml, trehalose concentration are 5 mg/ml.Solution of the 200 μ l containing trehalose and HBsAg is taken, which is added
Contain the dichloromethane solution of 200 mg 23kDa, 43kDa, 87kDa polyglycolic-polylactic acid block copolymer to 2 ml
In, with high-shear homogenizer under the rotating speed of 17500 rpm/min homogeneous 200 seconds, by its it is homogeneous form stable W/O emulsion,
The emulsion is slowly injected into the sodium phosphate that 20ml contains NaCl and 2.5% PVA with pipettor（pH6.0）It is high in buffer solution
Fast homogenizer forms W/O/W micro emulsions in 13000 revs/min of emulsifications in homogeneous 15 minutes, and 20 ml are added into the W/O/W micro emulsions of formation
Aqueous solution containing NaCl, stirring at low speed 4 hours in digital display constant temperature blender with magnetic force keep dichloromethane volatilization clean, obtain
Solidified microsphere.Three times, collection contains HBsAg (HBsAg-TS) of the trehalose as stabilizer to centrifuge washing after microballoon solidification
PLGA microballoons, 80 DEG C of freeze overnights of ﹣, 5.0 × 10﹣ 312 h of Pa vacuum under pressure freeze-drying obtain PLGA molecular weight and are
The HBsAg-TS sustained-release micro-spheres of 23kDa, 43kDa, 87kDa, are denoted as A3, B3, C3.The grain size of thus obtained microsphere be 4.30 microns ~
Between 6.50 microns, for encapsulation rate between 50.2% ~ 60.5%, drugloading rate is 0.47% ~ 0.65%.
Embodiment 10
8 week old female sd inbred rats 28, weight 200g or so are selected to give Free water and food in the entire experiment process
Object is randomly divided into recombinant hepatitis B vaccine（alum-HBsAg）Without stabilizer in group, blank microballoon group, embodiment 7
HBsAg PLGA sustained-release micro-spheres groups in A1, in embodiment 9 in the HBsAg-TS PLGA sustained-release micro-spheres groups of addition trehalose
Totally five groups of A2 in sero-abluminous HBsAg-HAS PLGA sustained-release micro-spheres groups is added in A3 and embodiment 8.Lead to before administration
It crosses the intraocular corner of the eyes to take in blood 0.5ml to centrifuge tube, serum is collected by centrifugation, it is to be measured to be denoted as negative control sera.
Experiment is using being subcutaneously injected, and alum-HBsAg groups are administered at twice injected at 0,1 month respectively, per injection dosage
Only for 10 μ g/；HBsAg PLGA sustained-release micro-spheres group, HBsAg-TS PLGA sustained-release micro-spheres groups and HBsAg-HAS PLGA sustained releases are micro-
Ball group is single administration, based on HBsAg 20 μ g/ of dosage only, a blank microballoon group shot, injectable microsphere amount with
HBsAg sustained-release micro-spheres group injection volumes are identical.
Microballoon is added in the suspending agent for being 0.9% containing NaCl concentration, test medicine is made, is injected into leg skin after rat
Under.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 upon administration passes through the intraocular corner of the eyes
It takes in blood 0.5ml to centrifuge tube, serum is collected by centrifugation, it is to be measured to be denoted as positive control serum.
By the blood of taking-up, natural coagulation 30 minutes, 3500rpm/min are centrifuged in careful collection in 20 minutes at room temperature
Clearly, 20 DEG C of freezen protectives of ﹣ are to be measured, as precipitated during preservation, should centrifuge again.
After completing the blood sampling in the 18th week of above-mentioned five groups of female sd inbred rats, the urethane pair of this five groups of rat injection optimal doses is given
It is anaesthetized, and perfusion operation is carried out to it after anesthesia, wins spleen in centrifuge tube, quick freeze is protected in -80 DEG C of refrigerators
It deposits to be measured.
Specific anti-HBsAg in serum is measured by rat hepatitis B surface antibody (HBsAb) enzyme-linked immunoassay kit
IgG antibody level carries out the research of rat humoral immune reaction.The results are shown in Figure 3, and blank PLGA microballoons are almost without induction
To the antibody response of HBsAg specificity.HBsAg-HSA PLGA microball preparations, HBsAg-TS PLGA sustained-release micro-spheres groups and HBsAg
PLGA microball preparations are through inducing the anti-HBsAg IgG antibody persistent levels generated to increase after single-dose.And by double injection
Alum-HBsAg preparations induction anti-HBsAg IgG antibody level showed as ascendant trend at first 8 weeks, then show later by
The trend gradually reduced.
By figure we have observed that from 0 week to 4 week, HBsAg-HSA PLGA microball preparations, HBsAg-TS PLGA sustained-release micro-spheres groups
It is almost the same with the anti-HBsAg IgG antibody level of HBsAg PLGA microball preparations and the induced generation of alum-HBsAg preparations
(P>0.05).However alum-HBsAg preparations are after 4th week booster shots, the anti-HBsAg IgG antibody water at the 5th week
It is flat to increase, hence it is evident that be higher than HBsAg-HSA PLGA sustained-release micro-spheres group, HBsAg-TS PLGA sustained-release micro-spheres groups and HBsAg PLGA
Microball preparation（P <0.05）.The HBsAg-HSA PLGA microball preparations induction that single dose is injected after the 7th week generates anti-
HBsAg IgG antibody levels are apparently higher than HBsAg PLGA microball preparations and HBsAg-TS PLGA sustained-release micro-spheres groups induce production
Raw anti-HBsAg IgG antibody is horizontal, and surmounts the anti-HBsAg IgG of the induced generation of alum-HBsAg preparations after 12 weeks
Antibody level（P <0.05）.Show that HBsAg-HSA PLGA microball preparations can the more effectively immune response of induction body fluid.
Pass through Rat Interleukin 2（IL-2）Enzyme-linked immunoassay kit measures IL-2 levels in spleen and carries out rat
The research of cell immune response.The IL-2 levels that blank PLGA microballoons induction that the results are shown in Figure 4 generates are nearly close to 0；And
Alum-HBsAg, HBsAg-HAS PLGA sustained-release micro-spheres group, HBsAg-TS PLGA sustained-release micro-spheres groups and HBsAg PLGA sustained releases are micro-
Ball group induces the IL-2 for generating different level；The IL-2 levels that three kinds of microball preparation inductions generate are apparently higher than alum-HBsAg
Induce the IL-2 generated horizontal（P <0.05）, and it is horizontal by the IL-2 that the induction of HBsAg-HSA PLGA sustained-release micro-spheres groups generates
The IL-2 for being higher than HBsAg PLGA sustained-release micro-spheres groups and the induced generation of HBsAg-TS PLGA sustained-release micro-spheres groups is horizontal（P <
0.05）.The IL-2 levels that the induction of HBsAg-HSA PLGA sustained-release micro-spheres group generates as seen from Figure 4 are HBsAg respectively
PLGA sustained-release micro-spheres group, HBsAg-TS PLGA sustained-release micro-spheres group, the IL-2 of the induced generation of alum-HBsAg preparations are horizontal
1.3,1.2 and 3.2 times.
Pass through rat gamma interferon（IFN-γ）Enzyme-linked immunoassay kit measures IFN-γ level in spleen and carries out greatly
The research of mouse cell immune response.The IFN-γ level that the induction of the results are shown in Figure 4 blank PLGA microballoons generates nearly close to
0；And alum-HBsAg, HBsAg-HSA PLGA sustained-release micro-spheres group and HBsAg PLGA sustained-release micro-spheres group induce and generate different water
Flat IFN-γ；The IFN-γ level that three kinds of microball preparation inductions generate is apparently higher than the IFN- of the induced generations of alum-HBsAg
γ is horizontal（P <0.05）, and the IFN-γ level generated by the induction of HBsAg-HSA PLGA sustained-release micro-spheres groups is higher than HBsAg
The IFN-γ of the induced generation of PLGA sustained-release micro-spheres groups is horizontal（P <0.05）.HBsAg-HSA PLGA are sustained as seen from the figure
The IFN-γ level that the induction of microballoon group generates is alum-HBsAg, HBsAg-TS PLGA sustained-release micro-spheres group and HBsAg respectively
4.3,1.2 and 1.2 times of the IFN-γ level of the induced generation of PLGA sustained-release micro-spheres groups.Therefore, with alum-HBsAg, HBsAg
In comparison, HBsAg-HSA PLGA sustained-release micro-spheres groups can be more for PLGA sustained-release micro-spheres group and HBsAg-TS PLGA sustained-release micro-spheres group
Good inducing cellular immune response, generates higher levels of IFN-γ.
Therefore, alum-HBsAg can induce very weak cellullar immunologic response, and HBsAg PLGA microball preparations are induction of relatively strong
Cellullar immunologic response, HBsAg-HSA PLGA sustained-release micro-spheres group microball preparations can induce stronger cellullar immunologic response.
Claims (6)
1. a kind of sustained release microsphere agents containing recombination hepatitis B surface antigen, which is characterized in that the sustained release microsphere agents are with stabilization
Property protective agent and recombination hepatitis B surface antigen particle form kernel, by high molecular material polyglycolic-polylactic acid block copolymerization
Object is made outsourcing sealing and is composed, wherein the molecular weight of the polyglycolic-polylactic acid block copolymer is 4.0 × 103~
5.5×104, the mass ratio of polylactic acid and polyglycolic acid is 50: 50 ~ 85: 15, the average grain diameter of prepared microballoon 1.0 ~
10.0μm；The stability protection agent is human serum albumins, the matter of recombination hepatitis B surface antigen and the stability protection agent
Amount is than being 1: (5 ~ 10)；The sustained release microsphere agents are made by following methods：
（1）Stability protection agent and recombination hepatitis B surface antigen are mixed into obtain inner aqueous phase；
（2）Polyglycolic-polylactic acid block copolymer is dissolved in organic solvent, oil phase is made, the made inner aqueous phase of step is taken to add
Enter above-mentioned oil phase stirring to homogenize to form W/O colostric fluids；
（3）W/O colostric fluids are added in outer aqueous phase poly-vinyl alcohol solution, stirring forms W/O/W double emulsions after homogenizing；It is stirred for 4
~6 hours, centrifuge washing was collected, vacuum freeze drying.
2. the sustained release microsphere agents according to claim 1 containing recombination hepatitis B surface antigen, which is characterized in that recombination hepatitis B
The mass ratio of surface antigen and the stability protection agent human serum albumins is 1: 5.
3. the sustained release microsphere agents according to claim 1 or 2 containing recombination hepatitis B surface antigen, which is characterized in that described
Recombined human hepatitis B surface antigen is the hepatitis B surface that yeast hepatitis B surface antibody or Chinese hamster ovary cell are secreted
Antigen.
4. the preparation method of the sustained release microsphere agents described in claim 1 containing recombination hepatitis B surface antigen, which is characterized in that packet
Include following steps：
（1）Stability protection agent and recombination hepatitis B surface antigen are mixed into obtain inner aqueous phase, the stability protection agent is human serum
Albumin；Recombination hepatitis B surface antigen and the mass ratio of the stability protection agent are 1:（5~10）；
（2）Polyglycolic-polylactic acid block copolymer is dissolved in organic solvent, oil phase is made, the made inner aqueous phase of step is taken to add
Enter above-mentioned oil phase stirring to homogenize to form W/O colostric fluids, wherein the molecular weight of the polyglycolic-polylactic acid block copolymer exists
4.0×103~5.5×104, the mass ratio of polylactic acid and polyglycolic acid is 50: 50 ~ 85: 15；
（3）W/O colostric fluids are added in outer aqueous phase poly-vinyl alcohol solution, stirring forms W/O/W double emulsions after homogenizing；It is stirred for 4
~6 hours, centrifuge washing was collected, and vacuum freeze drying, the average grain diameter of prepared microballoon is at 1.0 ~ 10.0 μm.
5. the preparation method of the sustained release microsphere agents according to claim 4 containing recombination hepatitis B surface antigen, feature exist
In the mass ratio of recombination hepatitis B surface antigen and the stability protection agent human serum albumins is 1: 5.
6. the preparation method of the sustained release microsphere agents according to claim 4 or 5 containing recombination hepatitis B surface antigen, feature
It is,
The（2）Speed of agitator is 8500~20000 revs/min in step；Mixing time is 30~300 seconds；
The（3）It is 8500~15000 revs/min that homogenized rotating speed is stirred in step；Mixing time is 10~20 minutes.
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| CN1769453A (en) * | 2005-09-23 | 2006-05-10 | 张永亮 | GRF(1-32)and SS-HbsAg fusion gene combination and its constructed expression system and pharmaceutical preparation |
| CN101507712A (en) * | 2009-03-20 | 2009-08-19 | 河北师范大学 | Sustained-release micro-spheres preparation containing recombined erythropoietin and preparation method and use thereof |
| CN102327611A (en) * | 2011-09-14 | 2012-01-25 | 西安交通大学 | Vaccine taking HBeAg egg white and/or HBeAg epitope polypeptide as immunogen |
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| CN101601860A (en) * | 2008-06-13 | 2009-12-16 | 中国科学院过程工程研究所 | Vaccine |
| CN103432570B (en) * | 2013-09-09 | 2015-10-07 | 深圳翰宇药业股份有限公司 | A kind of thymalfasin sustained-release micro-spheres and preparation method thereof |
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| CN101507712A (en) * | 2009-03-20 | 2009-08-19 | 河北师范大学 | Sustained-release micro-spheres preparation containing recombined erythropoietin and preparation method and use thereof |
| CN102327611A (en) * | 2011-09-14 | 2012-01-25 | 西安交通大学 | Vaccine taking HBeAg egg white and/or HBeAg epitope polypeptide as immunogen |
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