CN1370601A - Prepn of single-component slowly released genetically engineered hepatitis B vaccine microballoon - Google Patents
Prepn of single-component slowly released genetically engineered hepatitis B vaccine microballoon Download PDFInfo
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- CN1370601A CN1370601A CN 01107158 CN01107158A CN1370601A CN 1370601 A CN1370601 A CN 1370601A CN 01107158 CN01107158 CN 01107158 CN 01107158 A CN01107158 A CN 01107158A CN 1370601 A CN1370601 A CN 1370601A
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Abstract
The present invention applies biological degradable poly-DL-lactide-b-polyethylene glycol (PELA) as vaccine carrier material. By means of double-emulsion system solvent extraction process, microballoon with regular and smooth appearance and controllable size is prepared mechanically. The microballoon has antigen coating amount over 3 % and coating efficiency over 80 %. The present invention can be used in preparing long-acting delayed releasing hepatitis B vaccine.
Description
The present invention relates to a kind of medicine and pharmaceutical carrier, can be used for preparing target controlling and releasing vaccine or medicine, belong to biological medical polymer material and targeted drug controlled release system field.
Hepatitis B (Hepatitis B, HB) be a kind of global infectious disease that has a strong impact on human health, up to the present, the whole world has 1/3rd population (about 2,000,000,000) to infect hepatitis B virus (HBV) approximately, there are 3.5 hundred million people to be chronic carrier among them approximately, wherein have 1/4th people finally to die from liver cirrhosis and the primary hepatocarcinoma that hepatitis B virus infection causes again.China is the unique hepatitis B in Asia popular district occurred frequently, has 200,000,000 populations to carry hepatitis B surface antigen (HBsAg) approximately.Up to now, still there is not effective medicine, so prophylactic immunization is particularly important.Safe, efficient, cheap vaccine is to control the main means that HBV infects at present.1986, obtained reconstituted hepatitis B vaccine by genetic engineering, advantage such as this vaccine has good immune effect (reaching 85%-95%), and side effect is littler, can obtain in a large number, and price is low.But gene recombinaton Hepatitis B virus vaccine at present commonly used all be with 0,1, the mode of June or 0,1,2, December inoculates.Inoculation expense height, often, required time is long, cause inconvenient operation, child and old man to be difficult for accepting, and easily causes leaking problems such as kind of a rate height, whole immune effect difference.Vaccine is subjected to the effect of acid, alkali and enzyme in vivo, and the albumen higher structure is vulnerable to destroy, and degraded takes place or get rid of externally, therefore often needs escalated dose and increases inoculation times.Simultaneously, some crowd is lower to the reconstituted hepatitis B vaccine reactivity as people of old, fat, smoking and hypoimmunity etc.
The objective of the invention is in order to overcome the above problems, Development of New Generation is more convenient reliable, the long-acting controlled release bacterin preparation that immunogenicity is stronger.Oral sustained release, controlled release preparation are the important directions of domestic and international medical industry development in modern age, because the construction cycle is short, less input, and economic risk is low, and are had an optimistic view of by pharmaceutical industry because of product technology content increases advantages such as added value significantly improves.The microsphere technology has been widely used in the research and development of medicine, polypeptide and protein vaccine.Vaccine microsphere sustained release system based on biodegradable polymers can not only improve bioavailability of medicament, reduce the toxic and side effects of medicine, simplify administering mode, and the release of medicine can be by the degraded control of polymer, slowly discharge or pulse release, after drug release finished, carrier material need not take out.The single agent injection or the oral biodegradable recombinant hepatitis b vaccine microsphere dosage form of the present invention preparation, will overcome common bacterin preparation needs the multi-agent injection can gather the shortcoming of imitating, and has great importance.The expense of finishing single-component slowly released genetically engineered hepatitis B vaccine microballoon preparation inoculation only finish present use reconstituted hepatitis B vaccine inoculation expense 2/3rds, can alleviate the financial burden of people's immunity inoculation widely, and by single agent injection or oral way inoculation, the easier acceptance of child and old man, this can improve general kind of rate of Hepatitis B virus vaccine greatly, thereby can reduce the hepatitis B sickness rate, finally be that the China and even world radical cure hepatitis B lays the foundation.
The superiority of genetically engineered drug and vaccine biodegradable polymers sustained-release micro-spheres is fairly obvious, carrier material to this class targeting sustained release system, once adopted polystyrene and albumin, liposome etc. both at home and abroad as carrier, the former can not biodegradation, and its biodegradation period of the latter is short; Obviously, to be used to prepare slow release target controlling and releasing preparation be inappropriate to these materials.In recent years, set about research abroad with biodegradable polymers immunological adjuvant material, as polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA) etc. prepare targeted drug sustained release system, have obtained some gratifying results.1984, and Allen (Am.J.Nat.1984,170, the probability of polylactic acid microgranule as the angtigen presentation system used in studies confirm that 483-489); Eldridge (Molecular Immunology, 1991,28,287-292) and O ' Hagan (Immunology, 1991,73,239) studies confirm that oral microsphere with specified particle diameter and distribution and surface nature, can enter human body by digestive tract, reticuloendothelial system is reached good targeting, have slow release antigen and adjuvant effect simultaneously, can stimulate body to produce stronger immunological effect in a long time; Singh (Vaccine, after studies confirm that 1997,15 (5), 475) wrapped up hepatitis B virus surface antigen (HBsAg) with PLGA, detect the antigen that discharges in the microsphere with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, find that antigen does not almost change; The research of Chang (J.Pharm.Sci, 1996,65,129) adopts the emulsion solvent evaporation method to prepare tetanus toxin (TT) PLGA microsphere, and the TT envelop rate surpasses 90%, and drug loading is 0.48-0.9%; Tabata (Biometerials, 1988,9,356-362) find that with the microsphere of narrow diameter distribution, surface hydrophobicity macrophage is that the microsphere amount of engulfing of 1.0-2.0 micron is big to particle diameter after injecting.But, studies show that the biodegradable polymers carrier that is used to prepare microsphere at present still comes with some shortcomings, as albumen parcel amount low (about about 1%), the oral back continuous action time is short, and immune effect strengthens not obvious etc.In order to improve polymer and antigenic affinity, the present invention introduces hydrophilic Polyethylene Glycol in the hydrophobic polylactic acid chain segment, prepared amphiphilic poly--D, L-lactic acid-polyethyleneglycol block copolymer (PELA).This material Sichuan Province's industrial hygiene institute carries out biomedicine and estimates, and proves that it has nontoxic, safety and excellent biological compatibility.Deng Xianmo (J.Control.Release, 1999,58,123-131) and Li Xiaohong (J.Int.Pharm.1999,178,245-255) the microglobulin parcel amount with PELA preparation can reach 3～5%, parcel efficient is about 80%.
The microsphere with polylactic acid and copolymer thereof of domestic and foreign literature report mainly has the following disadvantages:
(1) polylactic acid and the copolymer thereof as drug carrier material is hydrophobic material, and be not strong to hydrophilic vaccine or medicine affinity, causes parcel amount and parcel efficient low;
(2) process conditions instability;
(3) vaccine of biologically active or medicine easy loss of activity after wrapping up;
(4) microsphere preparation technology instability is difficult to amplify.
The object of the present invention is to provide a kind of is carrier material with amphiphatic poly-DL-lactic acid-polyethyleneglycol block copolymer (PELA), particle diameter is below 5 microns, smooth surface does not have adhesion, the vaccine amount of carrying is more than 3%, not only can the induction ratio aluminium adjuvant Hepatitis B virus vaccine persistent period longer, the immune effect that antibody horizontal is higher, and can induce the preparation method of the slow release Hepatitis B virus vaccine microsphere of lymph T cell proliferation.
Technology contents of the present invention:
(1) synthetic having abandoned with the stannous octoate of polylactic acid-polyglycol block copolymer (PELA) carrier material is the traditional method of Preparation of Catalyst PELA, and adopt nontoxic non-tin catalyst, prepare medical PELA by the normal pressure solution polymerization process, the production domesticization of carrier material can reduce the production cost of microball preparation greatly;
(2) preparation of microsphere adopts the emulsion solvent extraction process to prepare parcel amount 3～5%, and parcel efficient about 80%, animal immune reinforced effects are obviously and have a good targeting recombinant hepatitis b vaccine slow releasing preparation.
Microsphere host material of the present invention is selected viscosity-average molecular weight 40,000 for use, and Polyethylene Glycol (PEG) block is the PELA copolymer of 10% (W/W).Compare with other present biodegradable polymers carrier, it has more advantages: can improve protein vaccine parcel amount and parcel efficient in the microsphere, improve proteic specific activity of parcel and preparation process protein active yield etc. in the microsphere.The method also is suitable for other lactic acid analog copolymer.But to the present invention, the lactic acid analog copolymer molecular weight of selection should be 8 * 10
3～9 * 10
4In the scope.
Water disperse medium of the present invention, the employing degree of hydrolysis is 80%～95% polyvinyl alcohol (PVA) aqueous solution, and the weight average molecular weight of PVA is 10,000～100,000, and its concentration range is 1%～50%.
Organic solvent as host material of the present invention is an acetone, methanol and dichloromethane or their mixed solvent.
Adopt the electronics constant speed stirrer as function input equipment of the present invention, its mixing speed is 200～2000 rev/mins.
As early stage of the present invention emulsification times be 1～30 minute.
Adopt the present invention to make and has good biocompatibility, biodegradable, vaccine construct is kept perfectly, and by single agent injection or orally just can obtain good immune effect, and can also treat the slow release polymer microsphere of hepatitis B to a certain extent.This microspherulite diameter is below 5 microns, and smooth surface does not have adhesion (Fig. 1), and particle size distribution meets requirement oral or the injection target administration at 0.5～5 micron (Fig. 2).
Good effect of the present invention is: the present invention has guaranteed antigenic activity in the carrier owing to adopt the carrier of amphiphatic polylactic acid-polyether block copolymer (PELA) as recombinant hepatitis b vaccine.Adopt to improve the two emulsion system solvent evaporation methods of property and prepared parcel amount 3～5%, parcel efficient about 80%, animal immune reinforced effects are obviously and have a good targeting recombinant hepatitis b vaccine sustained release microsphere agents.It is more convenient that microsphere of the present invention certainly will become administration of new generation, and the prophylactic immunization expense is lower, the easier acceptance of child and old man, the pharmaceutical preparation that immunity is stronger.It will play huge impetus to the development of China's pharmaceuticals industry, will promote the research and development of China's chemical synthetic drug, hormone, vaccine, polypeptide and Gene Handling release dosage form.
Be embodiments of the invention below:
Embodiment 1: particle diameter is less than the preparation of 5 microns copolymer microsphere.Accurately weighing PELA10g is dissolved in the 100ml dichloromethane.Measure PVA aqueous solution (12%) 150ml, the three-necked bottle of the 500ml that packs into.Start the electronics constant speed and stir 150 rev/mins of rotating speeds.Consoluet PELA solution slowly is added dropwise to the water disperse medium.Behind reinforced the finishing, rotating speed increases to 800 rev/mins, keeps this mixing speed 25 minutes, with speed drop to 200 rev/min, continues to stir 8 hours then.Treat that solvent evaporates finishes, with the emulsion centrifugalize, 7000 rev/mins of centrifugal speeds, 10 minutes time, go the supernatant, precipitate with distilled water wash.Repeat four times, collect white precipitate, lyophilization, productive rate 95%.
Embodiment 2: the parcel amount is 3%, and particle diameter is less than the preparation of 5 microns recombinant hepatitis b vaccine sustained-release micro-spheres.Accurately measure 4ml Hepatitis B virus vaccine aqueous solution (3.0mg/ml).Accurately weighing PELA0.8g is dissolved in the 8ml dichloromethane.Copolymer organic solution is put into the ultra-sonic dispersion device, under the ultra-sonic dispersion condition, slowly add the Hepatitis B virus vaccine aqueous solution, ultra-sonic dispersion obtained water-in-oil emulsion in 20 minutes.Measure PVA aqueous solution (12%) 15ml, the three-necked bottle of the 100ml that packs into.Start the electronics constant speed and stir 150 rev/mins of rotating speeds.By embodiment 1 water-in-oil emulsion is added dropwise to the water disperse medium, rotating speed increases to 800 rev/mins, keeps this mixing speed 25 minutes, then with speed drop to 200 rev/min, continues to stir 8 hours, presses embodiment 1 carrying out washing treatment then.Lyophilization, productive rate 95%.
Accompanying drawing and explanation: Fig. 1. adopt the electron scanning micrograph of the PELA microsphere of the present invention's preparation.Observe configuration of surface with scanning electron microscope (SEM U.S. AMRAY).Fig. 2. adopt the particle size distribution figure of the PELA microsphere of the present invention's preparation.Measure size and distribution with laser particle size scatterometer (Tianjin, island SALD-2009).
Claims (10)
1, a kind of preparation method of single-component slowly released genetically engineered hepatitis B vaccine microballoon is characterized in that microsphere substrate is novel poly-DL-lactic acid-polyethyleneglycol block copolymer (PELA), and the viscosity-average molecular weight of PELA is 8 * 10
3～9 * 10
4, recombinant hepatitis b vaccine is the parcel object, and the parcel amount can reach more than 3%, and parcel efficient is about 80%, and microspherulite diameter is distributed in below 5 microns, and microsphere can be used for target administration and sustained release to biology as the carrier that carries vaccine.
2, according to the method for preparing microsphere of claim 1, its feature prepares according to the following steps:
(1) the PELA copolymer is dissolved in and forms organic solution in the organic solvent;
(2) under agitation Hepatitis B virus vaccine is added organic solution;
(3) under agitation will go up solution joins in the water disperse medium;
(4) treat that under agitation organic solvent is evaporated completely;
(5) water flush away water disperse medium under centrifugal condition;
(6) lyophilization obtains recombinant hepatitis b vaccine sustained-release micro-spheres powder.
3, according to the microsphere of claim 1, it is characterized in that the percentage by weight of Polyethylene Glycol (PEG) in microsphere substrate is 5%～50%, the molecular weight of PEG is 4 * 10
2～2 * 10
4
4,, it is characterized in that organic solvent is an acetone, methanol and dichloromethane or their mixed solvent according to the method for claim 2.
5, according to the method for claim 2, the concentration that it is characterized in that PELA organic solution is 1%～50% (W/W).
6, according to the method for claim 2, it is characterized in that the water disperse medium is polyvinyl alcohol (PVA) aqueous solution, the degree of hydrolysis of PVA is 80～95%, and weight average molecular weight is 10,000～100,000, and concentration is 1%～50%.
7,, it is characterized in that mixing speed is 200～2000RPM according to the method for claim 2.
8,, it is characterized in that not only obtaining good immune effect, and can reach the effect of treatment hepatitis B to a certain extent by single agent injection or oral this Hepatitis B virus vaccine sustained-release micro-spheres according to the microsphere of claim 1 preparation.
9, according to the microsphere of claim 1 preparation, it is characterized in that this microsphere dosage form has the toxic and side effects that can reduce aluminium adjuvant, simplify administering mode, improve the bioavailability of vaccine.
10, according to the microsphere of claim 1 preparation, it is characterized in that this Hepatitis B virus vaccine microsphere system can be used for the preparation of sustained release dosage forms such as polypeptide drugs, other vaccine and genomic medicine.
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| CN 01107158 CN1370601A (en) | 2001-02-22 | 2001-02-22 | Prepn of single-component slowly released genetically engineered hepatitis B vaccine microballoon |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105646911A (en) * | 2014-09-23 | 2016-06-08 | 中南大学 | Liver-targeting drug-loaded microspheres with pH and reduction responsiveness, and preparation method and application thereof |
| CN109069440A (en) * | 2016-03-02 | 2018-12-21 | 得克萨斯州大学系统董事会 | The nano vaccine of activation STING for immunization therapy |
-
2001
- 2001-02-22 CN CN 01107158 patent/CN1370601A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105646911A (en) * | 2014-09-23 | 2016-06-08 | 中南大学 | Liver-targeting drug-loaded microspheres with pH and reduction responsiveness, and preparation method and application thereof |
| CN109069440A (en) * | 2016-03-02 | 2018-12-21 | 得克萨斯州大学系统董事会 | The nano vaccine of activation STING for immunization therapy |
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