KR100664466B1 - Microparticle for delivering oral vaccines and the method of manufacturing thereof - Google Patents

Abstract

The present invention relates to microparticles for vaccine delivery using extracellular polysaccharide, and more specifically, oral vaccine delivery microparticles prepared using GFP expressing Salmonella typhimurium cells as an antigen model and using extracellular polysaccharides containing them, and their preparation. It is about a method.

Drug delivery systems, microparticles, liposomes, polysaccharides, β-1,3-glucan, curdlan-sulfate, O-palmitoyl curdlan-sulfate, O-palmitoyl scleroglucan-sulfate

Description

Microparticles for delivering oral vaccines and manufacturing method thereof

1 is a TEM image of OPP and OPCS-coated liposomes ((a) uncoated liposomes, (b) OPP-coated liposomes, (c) OPCS-coated liposomes).

Figure 2 is a TEM image of OPCS coated liposomes ((a) lipid: OPCS = 9: 1, (b) lipid: OPCS = 6: 4].

3 is a graph showing the size distribution of OPCS coated liposomes [each lipid to OPCS ratio is as follows: (a) 10: 0, (b) 9: 1, (c) 8: 2, (d 7: 3 and (e) 6: 4].

Figure 4 is a graph showing the zeta potential of the OPCS-coated liposomes by OPCS concentration.

5 is a graph showing the average size change of the OPCS-coated liposomes in SGF (Stimulated Gastric Fluid).

6 is a graph showing the degree of release of 5-carboxyflourescien from OPCS coated liposomes.

7 is a cross-sectional view of the S. typhimurium chromosome.

Figure 8 is a photo cultured by performing a green fluorescent staining of GFP ⁺ S. typhimurium .

9 shows multifocal micrographs of microparticles loaded with GFP ⁺ S. typhimurium

10 is a graph showing the size distribution of GFP ⁺ S. typhimurium [(A) liposome loaded with ST only, (B) liposome coated with OPCS, (C) liposome coated with OPP, (D) pullulan acetate Prepared liposomes, (E) uncoated ST].

FIG. 11 is a graph showing the results of observing GFP ⁺ bacteria reaching Peyer's patch and mesenteric lymph nodes after oral administration of microparticles carrying cell particles.

12 is a graph showing the results of measuring antibody titers in serum after oral administration of microparticles.

Figure 13 is a graph showing the result of measuring the sIgA in feces after oral administration of the microparticles.

14 is a graph showing the results of measuring the number of living bacteria in Peyer's patch and spleen after oral administration of virulent S. typhimurium after immunization with microparticles.

The present invention relates to microparticles for vaccine delivery using extracellular polysaccharide, and more specifically, oral vaccine delivery microparticles prepared using GFP expressing Salmonella typhimurium cells as an antigen model and using extracellular polysaccharides containing them, and their preparation. It is about a method.

The mucosal surface of the human body becomes the main entry site for pathogens. Mucosal immunity provides the first immunological defense mechanism. To induce antibodies on mucosal surfaces, antigens must be administered directly to mucosal surfaces. In this case, oral vaccination has several advantages. It is a physiological route that expresses antigens and induces an IgA dominant antibody response that induces an IgG dominant antibody response that does not always reach the mucosal surface, which is the predominant site for most infectious pathogens. Oral vaccination also provides most common methods of inducing mucosal immunity.

Oral vaccination of antigens must overcome several immunological tests. Oral administration of vaccines generally requires large amounts of antigen. Many digestive enzymes reduce the vaccine administered. Oral administration of the antigen leads to antigen specific peripheral immune tolerance known as oral tolerance. In order to solve this disadvantage, a capsule for collecting antigen and a method of safely transporting it have been studied.

Microparticle delivery systems of oral vaccines have received considerable attention as they provide a number of significant benefits through systemic delivery. Polymer delivery systems can be engineered to enhance the efficacy of vaccines administered to the mucosa in many ways: for example, to defend against antigen through degradation, to aggregate by increasing the absorption of a portion of mucosal tissue, It may also extend the retention time in the body or target the site of absorption of the antigen (Pyer's patches in the digestive tract).

Therefore, the present inventors have found a derivative of β-1,3-glucan having anti-acid, mucoadhesive and immuno-promoting polymers, and have anti-acid, immunity and anti-cancer activity. And through liposome coating technology to prepare oral vaccine transport microparticles to complete the present invention.

Accordingly, it is an object of the present invention to provide an oral vaccine delivery microparticles prepared by coating an extracellular polysaccharide containing Salmonella typhimurium cells expressing GFP as an antigenic model and a method for producing the same.

The present invention relates to oral vaccine transport microparticles prepared by coating GFP-expressing Salmonella typhimurium cells as an antigen model and coating extracellular polysaccharides containing them, and preferably, the microparticles are prepared in the form of liposomes. .

Many microbial polysaccharides are widely accepted as products of biotechnology, and other polysaccharides are in various stages of development. Aureobasidium pullulans synthesizes α-D-glucan. These pullulans have high hydrophilicity and are used to form oil-resistant, water-soluble films. Several bacteria, including Agrobacterium and Rhizobium species, each produce several extracellular polysaccharides. One of them is curdlan, a relatively low molecular weight 1,3-β-D-glucan that produces a neutral gel.

In the present invention, hydrophobization of 1,3-β-D-glucan to coat the curdlan on liposomes, and coating liposomes using derivatives of hydrophobic 1,3-β-D-glucan for oral vaccine delivery Prepare microparticles.

Derivatives of 1,3-β-D- glucan according to the invention is larger them up-sulfated (Curdlan-sulfate), O-palmitoyl grow them up-sulfated (O -palmitoyl-curdlan sulfate), O-palmitoyl's Klee a glucan -, and the like palmitoyl pullulan (O -palmitoyl pullulan, OPP) - sulfate (O -palmitoyl scleroglucan-sulfate), O.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Curdlan-sulfate Synthesis of β-1,3-Glucan

Curdlan (purchased from Wako, Osaka, Japan), β-1,3-glucan with anti-acid and anticancer activity (Takashi Yoshida, et al ., Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV- 1 and HIV-2, Carbohydrate Research, (1995) 276: 425-436) was synthesized sulfate bound derivatives to enhance the immune activity.

0.5 g of Curdlan was dispersed in 40 ml of pyridine, 6.0 g of SO ₃ -pyridine complex was added thereto, stirred at 85 ° C. for 2 hours, and then transferred to an ice bath for cooling. 50 ml of 10% NaOH solution was added to the mixed solution with stirring. Acetone was added until phase separation occurred, followed by three washing steps, followed by dialysis to complete the synthesis.

The FT-IR spectra confirmed the synthesis of curdlan-sulfate, and showed S = O binding absorption peaks near 1259 cm ⁻¹ and COS binding absorption peaks near 619 cm ⁻¹ to confirm the synthesis.

[Example 2] O-palmitoyl grow them up-sulfated (O -palmitoyl-curdlan sulfate, OPCS) synthesis

The method proposed by Sunamoto (Macromolecules 25 , 5665-5670, 1992) group was synthesized with some modifications. First, 0.5 g curdlan-sulfate was dissolved in 11 ml dimethylformamide, and then 0.24 ml dimethylformamide solution containing 1 ml pyridine and 0.05 g palmitoyl chloride was added. The mixture was reacted with stirring at 60 ° C. for 8 hours, cooled at room temperature for 1 hour, then leached with ethanol and diethyl ether, washed and dried in vacuo.

The FT-IR spectra confirmed the synthesis of O -palmitoyl curdlan-sulfate, showing a stronger binding absorption peak of CH ₂ CH ₃ near 2917 cm ^-1 , which was weak in curdlan-sulfate. It became.

[Example 3] O-palmitate in weekends's Klee glucan-sulfated (O -palmitoyl scleroglucan-sulfate) synthesis

The method proposed by Sunamoto (Macromolecules 25 , 5665-5670, 1992) group was synthesized with some modifications. First, a 0.24 ml dimethylformamide solution containing 1 ml pyridine and 0.05 g palmitoyl chloride was added to a solution of 0.5 g scleroglucan-sulfate dissolved in 11 ml dimethylformamide. The mixture was reacted with stirring at 60 ° C. for 8 hours, cooled at room temperature for 1 hour, then leached with ethanol and diethyl ether, washed and dried in vacuo.

The FT-IR spectra confirmed the synthesis of O -palmitoyl scleroglucan-sulfate, showing a stronger binding absorption peak of CH ₂ CH ₃ near 2922 cm ^-1 , which is weak in scleroglucan-sulfate. Synthesis was confirmed.

[Example 4] O - palmitoyl pullulan (O -palmitoyl pullulan, OPP) synthesis

0.5 g of pullulan was dispersed in 40 ml of pyridine, and 6.0 g of SO ₃ -pyridine complex was added thereto, stirred at 85 ° C. for 2 hours, and then transferred to an ice bath for cooling. 50 ml of 10% NaOH solution was added to the mixed solution with stirring. Acetone was added until phase separation occurred, followed by three washing steps, followed by dialysis to complete the synthesis.

FT-IR spectra confirmed the synthesis of O -palmitoyl flurane, and showed a CH ₂ CH ₃ binding absorption peak near 2927 cm ^-1 and a CO binding absorption peak near 1153 cm ^-1 Confirmed.

Example 5 Preparation of Liposomes Coated with O -palmitoyl Pullulan ( O- palmitoyl pullulan, OPP)

Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (cholesterol, CH) in eggs are dissolved in 5 ml of a chloroform / methanol mixture at a molar ratio of 7: 1: 2, followed by a rotary evaporator. By evaporating the solvent to prepare a thin film (thin film) to prepare a vesicle (vesicles) by hydrolyzing the PBS buffer. In the prepared liposome solution, the OPP prepared in Example 4 was dissolved in 2 ml of PBS buffer so as to be liposome phospholipid and 7: 3 (lipid: OPP molar), added to the liposome suspension, and stirred for 1 hour to coat the OPP. Prepared liposomes.

The liposomes coated with O -palmitoyl flurane were observed by transmission electron microscopy (TEM), and the particle size was measured by dynamic light scattering (DLS). Is shown in Figure 1 (b).

① Particle morphology: The size of particles was 100-200 nm and the shape was spherical. In addition, it was observed that a thin film was formed on the surface of the liposome due to the added OPP.

[Example 6] O - palmitoyl grow them up the coated liposomes prepared sulfate (O-palmitoyl curdlan sulfate, OPCS )

Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (cholesterol, CH) in eggs were dissolved in 5 ml of a chloroform / methanol mixture at a molar ratio of 7: 1: 2, followed by a rotary evaporator. The thin film was prepared by evaporation of the solvent, followed by hydrolysis of the PBS buffer to prepare vesicles. In the prepared liposome solution, the OPCS prepared in Example 2 was dissolved in 2 ml of PBS buffer so as to be liposome phospholipid and 7: 3 (lipid: OPCS molar), added to the liposome suspension, and stirred for 1 hour to coat the OPCS. Prepared liposomes.

The prepared O -palmitoyl curdlan sulfate-coated liposomes were observed through a transmission electron microscope, and the particle size was measured using a dynamic light scattering method (DLS). c).

① Particle morphology: The size of particles was 100-200 nm and the shape was spherical. In addition, it was observed that a thin film was formed on the surface of the liposome due to the added OPCS.

[Test Example 1] Check the properties and stability of the OPCS-coated liposomes

The type of the OPCS-coated liposome prepared by the method of Example 6 was observed through a transmission electron microscope, the particle size was measured by dynamic light scattering, and the introduction of the OPCS through the measurement of zeta potential. The results were evaluated and their stability in gastric juice was investigated. The results are as follows:

① Particle shape: The size of particles was 100-200nm, and the shape was spherical when observed through transmission electron microscope.

2 is a TEM photograph of the change of the liposome surface according to the content of the OPCS by varying the mixing ratio of lipid and OPCS, the coating portion of the surface of the liposome was reliably observed as the amount of the added OPCS increases.

② Particle size: In FIG. 3, the particle size of the OPCS-coated liposomes prepared by varying the mixing ratio of lipids and OPCS was measured by using DLS, whereas the particle size of the liposomes not coated with OPCS was 120 nm on average. The particle size of the liposome added with OPCS increases with increasing amount of OPCS. However, when the ratio of lipid to OPCS exceeds 7: 3, the particle size change was small.

③ Zeta potential measurement: Figure 4 shows the zeta potential value of the OPCS-coated liposome prepared by varying the mixing ratio of lipid and OPCS, the zeta potential value was decreased with the addition amount of OPCS. This is because the charge of the OPCS has a negative charge and is introduced into the liposome to lower the zeta potential value. Therefore, these results support the results observed with a transmission electron microscope, confirming that the OPCS coated liposomes.

④ Stability measurement in SGF (Stimulated Gastric Fluid): The OPCS-coated liposomes were prepared by varying the amount of OPCS added, dispersed in SGF, and then evaluated for stability by measuring the change in particle size after 5 hours. 5 is shown. As a result, the change in particle size was small as the amount of OPCS added increased. This means that the acidity was added to the liposomes by OPCS, and the extent was improved according to the amount added to the OPCS.

⑤ Permeability measurement: A CF loaded OPCS coated liposome was prepared by loading 5-carboxyflourescien (CF) into liposomes and then reacting with the addition of OPCS (lipid: OPCS = 7: 3) and preparing PCS buffer The release trend of CF was measured at, and the results are shown in FIG. 6. As a result, CF was released more than 90% after 5 hours from the liposome without the OPCS while the OPCS-coated liposome released 40% less CF after 5 hours. This means that the addition of OPCS further increased the stability of the liposomes.

Test Example 2 Intestinal Target Delivery of Synthetic Microparticles / Liposomes

1) Preparation of Green Fluorescence Protein (GFP) positive S. typhimurium (ST)

First, the cloned CAT (Chloramphenicol Acetyl Transferase; CAT) gene was inserted into EcoRI and Spe1 in a multiple cloning site located downstream of the EGFP gene of pEGFP (clonetech, USA) to prepare a pEGFP-CAT construct. The CAT gene is composed of pKD3 as a template for SEQ ID NO: 1 (5'- GGA ATT CGT GTA GGC TGG AGC TGC TTC-3 ') and SEQ ID NO: 2 (5'- GAC TAG TCC TAC CTG TGA CGG AAG ATC -3'). The primers were amplified by PCR reaction. At this time, the PCR reaction was initially denatured at 94 ° C. for 5 minutes, denatured at 94 ° C. for 30 seconds, annealed at 58 ° C. for 30 seconds, and extended for 1 minute at 72 ° C. in 30 cycles, and finally, at 7 ° C. at 7 ° C. The reaction was carried out for a minute.

A fusion construct was inserted into the S. typhimurium chromosome using the linear DNA transformation method (Detsanko and Wanner, 2000) and transformed S. typhimurium containing plasmid (XXX) by electrophoresis. The strain carrying the lacZP :: EGFP :: CAT construct (SHJ2033) was finally isolated from SEQ ID NO: 3 (5'- GGC CGA TTC ATT AAT GCA GC -3 ') and SEQ ID NO: 4 (5'- GCA TGT GTC AGA GGT TTT Amplification was carried out by PCR using a primer of CGC C-3 ′), confirmed by DNA sequencing, and a cross-sectional view of the S. typhimurium chromosome is shown in FIG. 7. In this case, the PCR reaction was initially denatured at 94 ° C. for 5 minutes, then denatured at 94 ° C. for 30 seconds, annealed at 58 ° C. for 30 seconds, and extended for 1 minute at 72 ° C. in 30 cycles. The reaction was carried out for a minute.

2) Delivery test of microparticles containing antigen to mucosal organs

To carry an effective antigen, it must pass through the strong acid in the stomach to reach the Peyer's patch in the small intestine and the mesenteric lymph nodes. In order to confirm this, after fixing GFP ⁺ ST (2% paraformaldehyde) (see FIG. 8), it was collected in liposomes, and then the liposomes were prepared by O -palmitoylflurane (OPP) of Example 4, respectively. Prepared by the method of Example 5), O -palmitoylkerlan sulfate (OPCS) of Example 2 (prepared by the method of Example 6) and liposomes prepared by pullulan acetate (PA) were subjected to multifocal microscopy ( observation with a confocal microscope, and the results are shown in FIG. 9.

The procedure for preparing the microparticles using the furan acetate is as follows: 30 mg of the furan acetate is dissolved in 5 ml of dichloromethane (DCM), and a predetermined amount of GFP ⁺ ST is added thereto, followed by PVA 10% ( w / v) poured slowly into an aqueous solution and then sufficiently evaporated DCM while stirring to prepare microparticles.

As a result, the microparticles generally had a diameter of 1-3 μm, and as shown in FIG. 10, the largest particle size was shown in the case of pullulan acetate.

After 24 hours after oral administration of the microparticles, GFP ⁺ bacteria were observed in Peyer's patch and mesenteric lymph nodes using a fluorescence microscope or a multifocal microscope. The results are shown in FIG. 11. The transport of OPCS-coated microparticles was significantly increased in microparticles including GFP ⁺ ST delivered to Peyer's patch or mesenteric lymph nodes. For this reason, it was found that when OPCS is used to make microparticles, antigens can stably pass through the highly acidic environment and effectively transport to mucosal organs.

Test Example 2 Investigation of Immune Response after Administration of Cell Support Microparticles

1) Immunization using immunogen-microparticles / liposomes

Each immunogen-microparticle / liposome was used at a volume of 0.25 ml. Mice were divided into experimental groups according to the composition of the antigen and the route of administration, and each group was 10-12 mice. For oral administration, thin vinyl tubes or curved needles for oral experiments were used. Seven days after the final administration, spleen and blood were collected from mice and used for the experiment. As experimental animals, normal BALB / c female mice 6 to 8 weeks old were used. During the experiment, all mice were fed in an animal breeding facility maintained at constant temperature and humidity and sterile, with no more than 6 animals per cage having a size of 18 cm x 28 cm, and supplied with tap water and commercial animal feed.

2) antibody test

Antigen-specific antibodies IgG, IgM, IgA and all sIgA were detected in immune serum, splenocyte culture and saliva. In other words, the antigen and anti-mouse IgA were dissolved in carbonate buffer at a concentration of 5 μl / ml, and then 50 μl of the mixture was placed in a 96-well culture plate. Was reacted overnight at 4 ° C. Then, the antibody attached to the antigen is reacted with anti-mouse IgA (1: 1000 dilution) attached with peroxidase, and then reacted with the enzyme pigment substrate OPD (orthophenylenediamine) and stopped with 5N sulfate solution. The resulting pigment was read using an ELISA reader (Molecular Devices Inc.) at 490 nm wavelength.

<Result>

a) Serum antibody production capacity: To measure antibody response to oral immunity, various types of microparticles were administered twice a week for 10 ¹¹ microparticles orally and then serum was obtained one week after the final administration. The antibody titer in serum was measured using an ELISA test, and the results are shown in FIG. 12. IgG and IgA antibodies against ST antigens were measured in all immune animals. Liposomes containing OPCS-coated STs were significantly increased in IgG and IgA titers compared to liposomes packed with STs or those containing OPP-coated STs or ST alone.

b) Secretory antibody (sIgA) production capacity: The most characteristic of mucin immunity is secreted IgA. The secreted IgA can prevent epithelial infection and remove the antigen that has passed through the epithelial cell. In order to measure the mucosal immune response to oral immunity, the microparticles were orally administered in the same manner as described above, and one stool was obtained one week after the final administration. The sIgA in the stool was measured by using an ELISA test. It is shown in 13. Liposomes, including OPCS-coated STs, were also significantly increased as compared to the administration groups of other microparticles.

Test Example 3 Investigation of Resistance to Salmonella Infection After Immunization

In order to measure the effect as a vaccine, oral administration of various microparticles of 10 ¹¹ twice a week, followed by 5 × 10 ⁵ live Salmonella (SHJ2033) bacteria orally and 7 days later, Peyer's patch and spleen The number of was measured and the result is shown in FIG. The proliferation of ST was significantly inhibited in Peyer's patches, mesenteric lymph nodes and spleen of animals treated with OPCS-packed microparticles. As a result, it was found that the OPCS-coated ribosomes prepared in Example 2 were very effective as a vaccine delivery system.

As described above, in the present invention, by using extracellular polysaccharides produced in Agrobacterium, oral vaccine transport microparticles having excellent anti-acid, mucoadhesive and immune enhancing properties could be prepared.                     

▣ Reference

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Claims (8)

In oral vaccine delivery microparticles prepared using extracellular polysaccharide containing an antigen, The antigen is GFP expressing Salmonella typhimurium cells, The extracellular polysaccharides include 1,3-β-D-glucan produced in Agrobacterium; or Kerr them up-sulfated (Curdlan-sulfate), O-palmitoyl grow them up-sulfated (O -palmitoyl-curdlan sulfate), O-palmitoyl's Klee a glucan-sulfated (O -palmitoyl scleroglucan-sulfate) and O-palmitoyl Microparticles for oral vaccine delivery, characterized in that the derivative is selected from the group consisting of pullulan ( O -palmitoyl pullulan, OPP). According to claim 1, wherein the microparticles are microparticles, characterized in that prepared in the form of liposomes. delete delete delete An oral vaccine formulation comprising the microparticles of claim 1 or 2. delete delete

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