CN101036785A - Chitosan nanoparticles oral preparations of hepatitis vaccine - Google Patents

Abstract

The invention relates to the technical field of medicine. At present, hepatitis vaccines are commonly used for hepatitis vaccination, and the existing dosage forms are injections, which have the problems of high production cost, poor tolerance, and the need for professional technicians to administer the vaccine. The invention takes hepatitis vaccine as a model and chitosan as a carrier material, adopts ion cross-linking-high pressure homogenization process, makes it into chitosan nanoparticle preparation, and further uses lectin to modify the surface of chitosan nanoparticle , to prepare nanoparticle formulations suitable for oral vaccination. The vaccine nanoparticle preparation involved in the invention has strong operability in the preparation process and low cost, and can provide a new oral vaccination route for the prevention of hepatitis B.

Description

Chitosan Nanoparticle Oral Formulation of Hepatitis Vaccine

technical field

The invention relates to the technical field of medicine, in particular to an oral chitosan nanoparticle preparation of hepatitis vaccine.

Background technique

Viral hepatitis is a common and frequently-occurring disease that is distributed all over the world. Due to the obvious regional differences in health conditions and population immunity in my country, the number of potential sources of infection is huge, it is difficult to prevent and control, and it has a wide range of hazards. The annual economic loss is huge. The cure rate for viral hepatitis is unsatisfactory, although a few medicines have certain curative effect, but side effect is big, cost is high. For viral hepatitis, prevention is the main focus. Hepatitis vaccine is currently a commonly used preventive method. However, vaccination is usually administered by injection, which has problems such as high production cost, poor tolerance, and the need for professional and technical personnel to administer it. Therefore, the development of a A new oral formulation of a vaccine that is easy to use is of great significance for the prevention of hepatitis.

The nanoparticle carrier prepared by polymer materials has a protective effect on the loaded protein drugs, can resist the influence of gastrointestinal enzymes, pH and other factors, and can realize oral administration. Chitosan is the second most abundant hydrophilic polysaccharide in nature. Its good biocompatibility, non-toxic and biodegradable properties endow it with broad prospects for medical applications. However, due to the inability to realize the positioning and uptake of nanoparticles, it is easy to cause a decrease in bioavailability.

At present, there is no relevant report on the oral preparation of chitosan nanoparticles for hepatitis vaccine.

Contents of the invention

The object of the present invention is to provide a chitosan nanoparticle preparation of a hepatitis vaccine that can realize effective positioning of the vaccine, improve the bioavailability of the vaccine, and be administered orally, and a preparation method thereof.

A chitosan nanoparticle oral preparation of a hepatitis vaccine provided by the invention is a nanoparticle prepared by using chitosan as a carrier to carry a hepatitis vaccine. The surface of the nanoparticle is activated by an amino group and then modified with a lectin. The particle diameter is 200nm-1.5μm, and the concentration of the hepatitis vaccine is 0.1μg/ml-1g/ml.

The particle size of the aforementioned nanoparticles is preferably 200 nm to 500 nm.

The above-mentioned lectin is Ulex europaeus lectin, Wheat germ agglutinin or Tomato lectin.

The aforementioned hepatitis vaccine is a hepatitis B vaccine.

The present invention also provides the preparation method of above-mentioned hepatitis vaccine chitosan nanoparticle oral preparation, and this preparation method comprises the following steps:

1, prepare chitosan solution: a certain amount of chitosan is dissolved in vinyl acetic acid, obtain chitosan solution, concentration is 0.1%～10% (w/v);

II. Preparation of sodium polyphosphate solution: dissolving sodium polyphosphate in distilled water to prepare a cross-linking solvent (0.1% to 5%, w/v);

III. Prepare drug-loaded chitosan nanoparticles: get an appropriate amount of hepatitis vaccine solution (hepatitis vaccine is dissolved in phosphate buffer) and disperse in the sodium polyphosphate solution prepared in II or disperse in the chitosan solution prepared in I Among them, the concentration of hepatitis vaccine is: 0.1μg/ml～1g/ml; Slowly add the II solution to the chitosan solution containing hepatitis vaccine under continuous stirring at 600 rpm, or add the polysaccharide containing hepatitis vaccine The sodium polyphosphate solution is added dropwise in the I liquid, and reacted for 10 minutes to obtain the chitosan nanoparticles of the hepatitis vaccine;

IV. High-pressure homogenization: Put the chitosan nanoparticles of the hepatitis vaccine obtained in III into a high-pressure milk homogenizer, and homogenize under a pressure of 100-1000 Bar to obtain chitosan of the hepatitis vaccine with a narrow particle size distribution range Nanoparticles;

V, the activation of the amino groups on the surface of chitosan: add the glutaraldehyde of 25% (v/v) to the nanoparticle colloidal liquid prepared in IV, mix and shake for 6 hours to activate the surface amino groups of the chitosan nanoparticles, Discard excess glutaraldehyde in the supernatant after high-speed centrifugation;

VI. Preparation of lectin solution: Take an appropriate amount of lectin and dissolve it in phosphate buffer solution with pH 7.4, the concentration is: 0.5μg/ml～2g/ml;

VII. Add the lectin solution in VI to the chitosan nanoparticles in V, vortex and mix well, incubate at room temperature for 24 hours, discard the unbound lectin in the supernatant after high-speed centrifugation, and obtain the lectin Modified chitosan nanoparticles.

As measured by NicompTM 380 ZLS Zeta Potential and Particle Size Analyzer, the particle size of the above-mentioned nanoparticles is between 200nm and 1.5μm.

In the hepatitis vaccine nanoparticle preparation prepared by the present invention, the hepatitis B vaccine is encapsulated in the chitosan carrier material, and the nanoparticle carrier prepared by the macromolecular material of chitosan has a protective effect on the protein medicine contained therein, and can It can resist the influence of gastrointestinal enzymes, pH and other factors, and can realize oral administration. The present invention also covalently combines the lectin with intestinal specific adhesion with the active amino groups on the surface of chitosan nanoparticles, and the exogenous lectin can specifically combine with the fucose on the microfold cells of the intestinal tract , prepared into nanoparticles with its anchoring materials, which can pass through morphological barriers such as intestinal mucosa and epithelial cells, and deliver the loaded vaccine to intestinal microfold cells. The lectin-modified chitosan nano-particles prepared by the method of the invention have relatively round appearance and narrow particle size distribution. This method has the characteristics of rapidity, simplicity, low cost, and strong operability, and is superior to other methods.

In addition, the above-mentioned nanoparticle preparation can be further made into tablets or capsules by using techniques known in the art for oral vaccination. Under the protection and delivery of chitosan nanoparticles, the activity of hepatitis B vaccine can be maintained, and the lectin bound to chitosan nanoparticles can further locate and transport hepatitis B vaccine into the intestinal mucosa, effectively triggering the immune response, The realization of oral vaccination has broad application prospects.

Detailed ways

Now, the present invention will be further described in conjunction with the embodiments, but the implementation of the present invention is not limited thereto.

Example 1:

Materials and Reagents:

Chitosan Shanghai Ruji Biotechnology Development Co., Ltd. has a deacetylation degree of 90%, and an average molecular weight of 40-80kDa;

Sodium tripolyphosphate Sinopharm Chemical Reagent Co., Ltd. Batch number: F20050127 Chemically pure hepatitis B vaccine stock solution Beijing Tiantan Biological Products Co., Ltd.

Glacial acetic acid Sinopharm Chemical Reagent Co., Ltd. Analytical grade

25% Glutaraldehyde Shanghai Pharmaceutical (Group) Shanghai Chemical Reagent Company 0.25g/10ml

Lectins Sigma Reagents

Weigh chitosan 200mg, be dissolved in 100mL 0.5% vinyl acetic acid, obtain 0.2% chitosan solution. A 0.2% sodium polyphosphate solution was prepared with distilled water, and the hepatitis B vaccine stock solution was added thereto, and the concentration of the hepatitis B vaccine was 200 μg/ml. Under continuous stirring at a speed of 600 rpm, the sodium polyphosphate solution containing the hepatitis B vaccine was slowly added dropwise to the chitosan solution, and reacted for 10 minutes to obtain the chitosan nanoparticles of the hepatitis B vaccine. The above-mentioned nanoparticle suspension was subjected to a high-pressure milk homogenizer and homogenized for one cycle under a pressure of 660 Bar to obtain hepatitis B vaccine chitosan nanoparticles with a narrow particle size distribution range.

Add 1 mL of 25% (v/v) glutaraldehyde to the nanoparticle colloid (containing 20 mg of nanoparticles) after high-pressure homogenization, shake and mix for 6 hours, activate the amino groups on the surface of chitosan nanoparticles, and then centrifuge at a high speed (10000rpm, 30 minutes) and discard excess glutaraldehyde in the supernatant (to prevent it from cross-linking hepatitis B vaccine and lectins). Take 200 μg of Ulex europaeus lectin and dissolve it in 1000 μL of phosphate buffer (pH 7.4). After incubation for 24 hours, high-speed centrifugation (10,000 rpm, 30 minutes) discarded unbound lectin in the supernatant, and collected the centrifuged colloid in the lower layer to obtain lectin-modified chitosan nanoparticles. Measured by NicompTM 380 ZLS Zeta potential and particle size analyzer, the particle size is 293.2nm.

Example 2

The dosage of chitosan is 300mg, the concentration of sodium polyphosphate solution is 0.5%, and the dosage of glutaraldehyde is 1.2mL. All the other are the same as Example 1, and the particle diameter is 496.3nm.

Example 3

The amount of chitosan was 100 mg, the concentration of sodium polyphosphate solution was 0.2%, the amount of glutaraldehyde was 0.5 mL, and the rest were the same as in Example 1, and the particle diameter was 330.5 nm.

Example 4

Chitosan consumption is 100mg, and the concentration of sodium polyphosphate solution is 0.1%, and glutaraldehyde consumption is 0.5mL, and used lectin is 200 μ g wheat germ agglutinin (Wheat germ agglutinin), all the other are the same as embodiment 1, particle diameter is 276.8nm.

Example 5

The preparation method is the same as in Example 1, the concentration of the sodium polyphosphate solution is 0.1%, the dosage of glutaraldehyde is 0.5 mL, and the lectin used is 200 μg of tomato lectin. The stock solution of hepatitis B vaccine was added into chitosan solution, and then cross-linked with sodium polyphosphate solution, the rest were the same as in Example 1, and the particle diameter was 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.