CN101036785A - Chitosan nanoparticles oral preparations of hepatitis vaccine - Google Patents
Chitosan nanoparticles oral preparations of hepatitis vaccine Download PDFInfo
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- CN101036785A CN101036785A CN 200710040264 CN200710040264A CN101036785A CN 101036785 A CN101036785 A CN 101036785A CN 200710040264 CN200710040264 CN 200710040264 CN 200710040264 A CN200710040264 A CN 200710040264A CN 101036785 A CN101036785 A CN 101036785A
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Abstract
The invention relates to medicine technique domain. At present, the hepatitis vaccine is in common use of protective inoculation of hepatitis. The current formulation is injection, with the shortcomings of high cost, poor tolerance and need specialized technical personnel administrating. The chitosan nanoparticles preparation is formed by ionic crosslinking-high pressure homogeneization technique through hepatitis vaccine model and chitosan as carrier material, furthermore the chitosan nanoparticles are surface modified by lectin to obtain nanoparticles preparation suitable for oral inoculation. The said nanoparticles preparation vaccine provides a new route of oral inoculation to prevent hepatitis B, with high operability and low cost.
Description
Technical field
The present invention relates to medical technical field, relate in particular to a kind of oral chitosan nanoparticle preparation of hepatitis vaccine.
Background technology
Viral hepatitis is a kind of commonly encountered diseases, frequently-occurring disease, is distributed in all over the world, because China's sanitary condition and community immunity situation regional difference are obvious, potential source of infection enormous amount is difficult to control and harm is wide, and annual economic loss is very big.Cure rate for viral hepatitis is undesirable, though there is the minority medicine to have certain curative effect, side effect is big, expense is high.To viral hepatitis to put prevention first, hepatitis vaccine is the preventive means of generally using at present, but the injection system administration is adopted in vaccination usually, there is the manufacturing cost height, problems such as toleration difference and need professional and technical personnel administration, therefore develop the oral novel formulation of a kind of vaccine easy to use, have crucial meaning for the prevention of hepatitis.
Nanoparticulate carriers by the macromolecular material preparation has protective effect to the wrap protein medicaments that carries, and can resist the influence of factors such as gastrointestinal enzyme, pH, can realize oral administration.Chitosan is the nature second largest abundant hydrophilic polysaccharide of originating, and its excellent biological compatibility, nontoxic and biodegradable characteristic are given its wide medical applications prospect.But the location picked-up effect owing to realizing nanoparticle causes the reduction of bioavailability easily.
The relevant report of not seeing the hepatitis vaccine chitosan nanoparticles oral preparations at present.
Summary of the invention
Bioavailability, the route of administration that the object of the present invention is to provide a kind of effective location that can realize vaccine, improves vaccine is the chitosan nanoparticle preparation of oral hepatitis vaccine, and preparation method thereof.
The chitosan nanoparticles oral preparations of a kind of hepatitis vaccine provided by the invention, be as carrier with chitosan, bag carries the nanoparticle that hepatitis vaccine makes, this nanoparticle surface after amino activation with Phytoagglutinin modified, the nanoparticle particle diameter is 200nm～1.5 μ m, and wherein the concentration of hepatitis vaccine is 0.1 μ g/ml～1g/ml.
Above-mentioned nanoparticle particle diameter is preferably 200nm～500nm.
Above-mentioned agglutinin is UEA (Ulex europaeus lectin), wheat germ agglutinin (Wheat germ agglutinin) or Tomato lectin (Tomato lectin).
Above-mentioned hepatitis vaccine is a hepatitis B vaccine.
The present invention also provides the preparation method of above-mentioned hepatitis vaccine chitosan nanoparticles oral preparations, and this preparation method may further comprise the steps:
I, preparation chitosan solution: certain amount of chitosan is dissolved in the alkene acetic acid, obtains chitosan solution, concentration is 0.1%～10% (w/v);
II, preparation polyphosphoric acids sodium solution: with the dissolved in distilled water sodium polyphosphate, prepare crosslinked solvent (0.1%～5%, w/v);
III, preparation medicine carrying chitosan nano: get an amount of hepatitis vaccine solution (hepatitis vaccine is dissolved in the phosphate buffer) and be scattered in the sodium polyphosphate liquid that makes among the II or be scattered in the chitosan solution of preparing among the I, hepatitis vaccine concentration is: 0.1 μ g/ml～1g/ml; With II solution, slowly drip in the chitosan solution that contains hepatitis vaccine under continuing to stir with 600 rev/mins, the sodium polyphosphate drips of solution that maybe will contain hepatitis vaccine adds in the I liquid, reacts 10 minutes, promptly gets the chitosan nano of hepatitis vaccine;
IV, high pressure homogenize: the chitosan nano of the hepatitis vaccine of gained among the III is put into the high pressure dispersing emulsification machine, under 100～1000Bar pressure, carry out homogenize, obtain the narrower hepatitis vaccine chitosan nano of particle size distribution range;
The activation of V, chitosan surface amino groups: in IV, make the glutaraldehyde that adds 25% (v/v) in the nanoparticle colloidal solution, mixed jolting 6 hours, make the surface amino groups activation of chitosan nano, unnecessary glutaraldehyde in the abandoning supernatant behind the high speed centrifugation;
The preparation of VI, agglutinin solution: it is an amount of to get agglutinin, is dissolved in the phosphate buffer of pH7.4, and concentration is: 0.5 μ g/ml～2g/ml;
VII, the agglutinin solution among the VI is added in the chitosan nano among the V, the whirlpool mixing was at room temperature hatched 24 hours, and unconjugated agglutinin in the abandoning supernatant promptly gets Phytoagglutinin modified chitosan nano behind the high speed centrifugation.
Measure through NicompTM 380 ZLS Zeta potentials and particle size analyzer, above-mentioned nanoparticle particle diameter is between 200nm～1.5 μ m.
The hepatitis vaccine nano particle preparations of the present invention's preparation; the Hepatitis B virus vaccine bag is stated from the chitin carrier material; nanoparticulate carriers by this macromolecular material preparation of chitosan has protective effect to the wrap protein medicaments that carries; the influence of factors such as gastrointestinal enzyme, pH can be resisted, oral administration can be realized.The present invention also carries out covalent bond to have agglutinin that the intestinal specificity sticks effect with the active amino on chitosan nano surface, special combination can take place with the fucose on the intestinal microfold cell in ectogenous agglutinine, material preparation with its grappling becomes nanoparticle, can pass through morphology barriers such as intestinal mucosa and epithelial cell, will wrap a year vaccine and be transported to the intestinal microfold cell.With the Phytoagglutinin modified chitosan nano that the inventive method makes, mode of appearance is rounding comparatively, narrower particle size distribution.That this method has is fast and convenient, cost is low, characteristics such as workable, is better than additive method.
In addition, can adopt techniques well known that above-mentioned nano particle preparations is further made tablet or capsule, use for oral vaccination.Hepatitis B virus vaccine is under the protection and delivery of chitosan nano; its activity can be maintained, and the agglutinin of bonding can further the location be transported to Hepatitis B virus vaccine in the intestinal mucosa on the chitosan nano, efficiently triggers immune response; realize oral vaccination, have broad application prospects.
The specific embodiment
Now in conjunction with the embodiments, the invention will be further described, but enforcement of the present invention is not limited in this.
Embodiment 1:
Material and reagent:
Chitosan Shanghai Ru Ji biotechnology Development Co., Ltd deacetylation is 90%, and mean molecule is 40-80kDa;
Sodium tripolyphosphate Chemical Reagent Co., Ltd., Sinopharm Group lot number: F20050127 chemical pure Hepatitis B virus vaccine stock solution Beijing Tiantan Biological Products Co.ltd
Glacial acetic acid Chemical Reagent Co., Ltd., Sinopharm Group analytical pure
25% glutaraldehyde Shanghai medicine (group) 0.25g/10ml of Shanghai chemical reagents corporation
Agglutinin Sigma reagent company
Take by weighing chitosan 200mg, be dissolved in the alkene acetic acid of 100mL 0.5%, obtain 0.2% chitosan solution.With the polyphosphoric acids sodium solution of distilled water preparation 0.2%, and to wherein adding Hepatitis B virus vaccine stock solution, the concentration of Hepatitis B virus vaccine is 200 μ g/ml.Under continuing to stir with 600 rev/mins of speed, the polyphosphoric acids sodium solution that will contain Hepatitis B virus vaccine slowly drips in chitosan solution, reacts 10 minutes, promptly gets the chitosan nano of Hepatitis B virus vaccine.Above-mentioned nanoparticle suspension through the high pressure dispersing emulsification machine, is carried out 1 circulation homogenize under 660Bar pressure, obtain the narrower Hepatitis B virus vaccine chitosan nano of particle size distribution range.
The glutaraldehyde 1mL that adds 25% (v/v) in the nanoparticle colloidal solution after the high pressure homogenize (containing nanoparticle 20mg), jolting mixed 6 hours, the amino on activation chitosan nano surface, unnecessary glutaraldehyde (preventing its crosslinked Hepatitis B virus vaccine and agglutinin) in high speed centrifugation (10000rpm, 30 minutes) and the abandoning supernatant then.Get UEA (Ulex europaeus lectin) 200 μ g, be dissolved in the 1000 μ L phosphate buffers (pH 7.4), this agglutinin solution is joined in the chitosan nano after the homogenize, the whirlpool mixing, under room temperature condition, hatched unconjugated agglutinin in the abandoning supernatant of high speed centrifugation (10000rpm, 30 minutes) back 24 hours, collect the centrifugal colloidal solution of lower floor, promptly get Phytoagglutinin modified chitosan nano.Measure through NicompTM 380 ZLS Zeta potentials and particle size analyzer, particle diameter is 293.2nm.
Embodiment 2
Chitosan dosage is 300mg, and the concentration of polyphosphoric acids sodium solution is 0.5%, and the glutaraldehyde consumption is 1.2mL.All the other are with embodiment 1, and particle diameter is 496.3nm.
Embodiment 3
Chitosan dosage is 100mg, and the concentration of polyphosphoric acids sodium solution is 0.2%, and the glutaraldehyde consumption is 0.5mL, and all the other are with embodiment 1, and particle diameter is 330.5nm.
Embodiment 4
Chitosan dosage is 100mg, and the concentration of polyphosphoric acids sodium solution is 0.1%, and the glutaraldehyde consumption is 0.5mL, and used agglutinin is 200 μ g wheat germ agglutinins (Wheat germ agglutinin), and all the other are with embodiment 1, and particle diameter is 276.8nm.
Embodiment 5
Preparation method is with embodiment 1, and the concentration of polyphosphoric acids sodium solution is 0.1%, and the glutaraldehyde consumption is 0.5mL, and used agglutinin is 200 μ g Tomato lectins (Tomato lectin).Hepatitis B virus vaccine stock solution adds in the chitosan solution, carries out crosslinkedly then with the polyphosphoric acids sodium solution, and all the other are with embodiment 1, and particle diameter is 201.4nm.
Claims (7)
1, a kind of chitosan nanoparticles oral preparations of hepatitis vaccine, it is characterized in that said preparation is as carrier with chitosan, bag carries the nanoparticle that hepatitis vaccine makes, this nanoparticle surface after amino activation with Phytoagglutinin modified, the nanoparticle particle diameter is 200nm～1.5 μ m, and wherein the concentration of hepatitis vaccine is 0.1 μ g/ml～1g/ml.
2, the chitosan nanoparticles oral preparations of a kind of hepatitis vaccine according to claim 1 is characterized in that the nanoparticle particle diameter is 200nm～500nm.
3, the chitosan nanoparticles oral preparations of a kind of hepatitis vaccine according to claim 1 and 2 is characterized in that agglutinin wherein is UEA, wheat germ agglutinin or Tomato lectin.
4, the chitosan nanoparticles oral preparations of a kind of hepatitis vaccine according to claim 1 and 2 is characterized in that hepatitis vaccine wherein is a hepatitis B vaccine.
5, a kind of preparation method of hepatitis vaccine chitosan nanoparticles oral preparations as claimed in claim 1 is characterized in that this preparation method may further comprise the steps:
I, preparation chitosan solution: certain amount of chitosan is dissolved in the alkene acetic acid, obtains chitosan solution, concentration is 0.1%～10%w/v;
II, preparation polyphosphoric acids sodium solution: with the dissolved in distilled water sodium polyphosphate, prepare crosslinked solvent, concentration is 0.1%～5%w/v;
III, preparation medicine carrying chitosan nano: get an amount of hepatitis vaccine solution and be scattered in the chitosan solution of preparing among the sodium polyphosphate liquid that makes among the II or the I, hepatitis vaccine concentration is: 0.1 μ g/ml～1g/ml; With II solution, slowly drip in the chitosan solution that contains hepatitis vaccine under continuing to stir with 600 rev/mins, the sodium polyphosphate drips of solution that maybe will contain hepatitis vaccine adds in the I liquid, reacts 10 minutes, promptly gets the chitosan nano of hepatitis vaccine;
IV, high pressure homogenize: the chitosan nano of the hepatitis vaccine of gained among the III is put into the high pressure dispersing emulsification machine, under 100～1000Bar pressure, carry out homogenize, obtain the narrower hepatitis vaccine chitosan nano of particle size distribution range;
The activation of V, chitosan surface amino groups: in IV, make in the nanoparticle colloidal solution glutaraldehyde that adds 25%v/v, mix jolting 6 hours, make the surface amino groups activation of chitosan nano, unnecessary glutaraldehyde in the abandoning supernatant behind the high speed centrifugation;
The preparation of VI, agglutinin solution: it is an amount of to get agglutinin, is dissolved in the phosphate buffer of pH7.4, and concentration is: 0.5 μ g/ml～2g/ml;
VII, the agglutinin solution among the VI is added in the chitosan nano among the V, the whirlpool mixing was at room temperature hatched 24 hours, and unconjugated agglutinin in the abandoning supernatant promptly gets Phytoagglutinin modified chitosan nano behind the high speed centrifugation.
6, the preparation method of the chitosan nanoparticles oral preparations of a kind of hepatitis vaccine according to claim 5 is characterized in that agglutinin wherein is UEA, wheat germ agglutinin or Tomato lectin.
7, according to the preparation method of the chitosan nanoparticles oral preparations of claim 5 or 6 described a kind of hepatitis vaccines, it is characterized in that hepatitis vaccine wherein is a hepatitis B vaccine.
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| CN 200710040264 CN101036785A (en) | 2007-04-29 | 2007-04-29 | Chitosan nanoparticles oral preparations of hepatitis vaccine |
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| CN 200710040264 CN101036785A (en) | 2007-04-29 | 2007-04-29 | Chitosan nanoparticles oral preparations of hepatitis vaccine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102284065A (en) * | 2011-08-16 | 2011-12-21 | 黑龙江大学 | Preparation method of chitosan swine-infuenza DNA (deoxyribonucleic acid) vaccine nanoparticles |
| CN102614527A (en) * | 2012-04-14 | 2012-08-01 | 南开大学 | Antiacid nano oral deoxyribonucleic acid (DNA) anti-tumor vaccine with potential of hydrogen (pH) sensitive characteristic and preparation method |
| CN102772785A (en) * | 2012-08-14 | 2012-11-14 | 中国人民解放军第四军医大学 | Composite medicine and ointment for hemostasis and preparation methods of composite medicine and ointment |
| CN107198771A (en) * | 2017-05-08 | 2017-09-26 | 广东渔跃生物技术有限公司 | The method that microcarrier suspension culture cell produces pseudorabies gE gene delection viral vaccines |
| CN107261136A (en) * | 2017-07-31 | 2017-10-20 | 中国医学科学院医学生物学研究所 | The application of sodium polyphosphate and the vaccine dose containing sodium polyphosphate |
| RU2713138C1 (en) * | 2019-09-18 | 2020-02-03 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Саратовский национальный исследовательский государственный университет имени Н.Г. Чернышевского" | Method of producing chitosan aspartate nanoparticles |
-
2007
- 2007-04-29 CN CN 200710040264 patent/CN101036785A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102284065A (en) * | 2011-08-16 | 2011-12-21 | 黑龙江大学 | Preparation method of chitosan swine-infuenza DNA (deoxyribonucleic acid) vaccine nanoparticles |
| CN102614527A (en) * | 2012-04-14 | 2012-08-01 | 南开大学 | Antiacid nano oral deoxyribonucleic acid (DNA) anti-tumor vaccine with potential of hydrogen (pH) sensitive characteristic and preparation method |
| CN102772785A (en) * | 2012-08-14 | 2012-11-14 | 中国人民解放军第四军医大学 | Composite medicine and ointment for hemostasis and preparation methods of composite medicine and ointment |
| CN107198771A (en) * | 2017-05-08 | 2017-09-26 | 广东渔跃生物技术有限公司 | The method that microcarrier suspension culture cell produces pseudorabies gE gene delection viral vaccines |
| CN107261136A (en) * | 2017-07-31 | 2017-10-20 | 中国医学科学院医学生物学研究所 | The application of sodium polyphosphate and the vaccine dose containing sodium polyphosphate |
| CN107261136B (en) * | 2017-07-31 | 2020-07-28 | 中国医学科学院医学生物学研究所 | Application of sodium polyphosphate and vaccine containing sodium polyphosphate |
| RU2713138C1 (en) * | 2019-09-18 | 2020-02-03 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Саратовский национальный исследовательский государственный университет имени Н.Г. Чернышевского" | Method of producing chitosan aspartate nanoparticles |
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Open date: 20070919 |