JP6358933B2 - Particles containing polysaccharide-peptidoglycan complex - Google Patents

Description

  The present invention relates to a polysaccharide-peptidoglycan complex-containing particle and use thereof.

  Polysaccharide-peptidoglycan complex (PS-PG) is a component present in the cell walls of gram-positive bacteria such as lactic acid bacteria and gram-negative bacteria such as Escherichia coli, and has recently been effective in preventing inflammatory bowel disease and enteritis-associated cancer. (Patent Document 1, Non-Patent Document 1). PS-PG is a cell wall component of Gram-negative bacteria and has a distinct difference from lipopolysaccharide (LPS), which is an endotoxin, in terms of its structure and properties. PS-PG is known to have two types, PS-PG1 and PS-PG2, having different molecular weights. Among them, PS-PG1 has a molecular weight of about 1 million kDa or more, and PS-PG2 has a molecular weight of about 300,000 kDa. The ratio of these contents in Lactobacillus casei YIT9029 is PS-PG1: PS-PG2 = about 1: 8 (Non-patent Document 2).

JP 2011-193730 A

Immunology, Vol.128, 1 Suppl, e170-e180 (2009) "Lactobacillus casei Shirota strain-Relationship with intestinal flora and health-" pp. 26-33 (Yakult Central Research Institute, issued January 1, 1999)

However, PS-PG is significantly different from lipopolysaccharide, which is an endotoxin, and the innate immune stimulating ability expressed in live bacteria having PS-PG does not occur at all in PS-PG separated and solubilized from the cell wall. Therefore, in PS-PG, it was not possible to study changes in the characteristics of PS-PG, the relationship between structure and physiological activity, etc. without using live bacteria.
Accordingly, an object of the present invention is to provide a model cell wall system that retains the physiological function of PS-PG.

  Thus, as a result of various studies to construct a PS-PG model system that generates physiological activity, the present inventors obtained PS-PG-containing particles by using a particulate carrier and supporting PS-PG on the surface thereof. As a result, it was found that the particles had an IL-12 production promoting effect and an immunostimulatory ability, thereby completing the present invention.

That is, the present invention provides the following [1] to [13].
[1] PS-PG-containing particles obtained by supporting a polysaccharide-peptidoglycan complex (PS-PG) on the surface of a particulate carrier.
[2] PS-PG-containing particles according to [1], wherein PS-PG is derived from bacteria.
[3] PS-PG-containing particles according to [1] or [2], wherein PS-PG is derived from lactic acid bacteria.
[4] The polysaccharide-peptidoglycan complex-containing particle according to any one of [1] to [3], wherein the polysaccharide-peptidoglycan complex is derived from a lactic acid bacterium belonging to the genus Lactobacillus.
[5] The PS-PG-containing particles according to any one of [1] to [4], wherein PS-PG is derived from Lactobacillus casei and / or Lactobacillus johnsonii.
[6] The PS-PG-containing particle according to any one of [1] to [5], wherein the particulate carrier is a nanoparticle.
[7] The PS-PG-containing particles according to any one of [1] to [6], wherein the particulate carrier is latex nanoparticles or silica nanoparticles.
[8] The PS-PG-containing particles according to any one of [1] to [7], wherein the average particle diameter of the particulate carrier is 20 nm to 3100 nm.
[9] The polysaccharide-peptidoglycan complex-containing particle according to any one of [1] to [8], wherein the average particle size of the particulate carrier is 300 nm to 2000 nm.
[10] The PS-PG-containing particle according to any one of [1] to [9], wherein the amount of PS-PG bound to one particulate carrier is 0.1 to 50 amol.
[11] A medicament containing the PS-PG-containing particles according to any one of [1] to [10].
[12] An immunostimulant comprising the PS-PG-containing particles according to any one of [1] to [10] as an active ingredient.
[13] An interleukin 12 production promoter comprising the PS-PG-containing particles according to any one of [1] to [10] as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the artificial particle which has the bioactivity of PS-PG which did not show bioactivity unless it was the state of a living microbe can be provided. By using the PS-PG-containing particles of the present invention, it becomes possible to study the mechanism by which PS-PG produces immunostimulatory ability without using viable bacteria. The PS-PG-containing particles of the present invention are also useful as pharmaceuticals such as immunostimulants and IL-12 production promoters, foods having these functions, and the like.

It is a figure which shows the relationship between the average particle diameter of the particulate support | carrier of Lactobacillus casei origin PS-PG containing particle | grains, and IL-12 production induction ability.

  The PS-PG-containing particles of the present invention are formed by supporting PS-PG on the surface of a particulate carrier.

PS-PG includes those derived from bacterial cell walls and artificially produced. Of these, those derived from bacterial cell walls are preferred in terms of physiological activity. Here, the bacteria include gram-positive bacteria such as lactic acid bacteria, bifidobacteria and Clostridium, and gram-negative bacteria such as Escherichia coli and bacteroides, but lactic acid bacteria and bifidobacteria are preferable from the viewpoint of handleability, safety and physiological activity. Lactic acid bacteria are more preferred. As the lactic acid bacteria, lactic acid bacteria belonging to the genus Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Pediococcus, etc. are preferred, and more specifically, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus Acidophilus, Lactobacillus salivaius, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus euglety, Lactobacillus delbruecki Subspices. Bulgaricus, Streptococcus thermophilus, Lactococcus lactis Subspecies. Lactis, Lactococcus lactis Subspecies. Cremoris, Leuconostoc mesenteroides, Pediococcus pentosaceus and the like. Of these, lactic acid bacteria belonging to the genus Lactobacillus are preferred, Lactobacillus casei and Lactobacillus johnsonii are more preferred, and Lactobacillus casei is particularly preferred. As Lactobacillus casei, Lactobacillus casei YIT 9029 (FERM BP-1366) is preferable, and as Lactobacillus johnsonii, Lactobacillus johnsonii YIT 0219 ^T (JCM 2012 ^T ) is preferable.

To separate PS-PG from these bacteria, for example, the description in Patent Document 1 and Non-Patent Document 2 may be followed.
PS-PG is not particularly limited as long as it contains PS-PG1 and / or PS-PG2 (derived from a bacterial cell wall, artificially produced, etc.). It includes those that have been further subjected to separation / purification such as separation by chromatography and density gradient centrifugation.

  As the particulate carrier, nanoparticles are preferable from the viewpoint of exerting physiological activity. The specific average particle diameter is preferably 1 nm to 3100 nm, more preferably 20 nm to 3100 nm, further preferably 100 nm to 3100 nm, further preferably 300 nm to 2000 nm, further preferably 500 nm to 2000 nm, and particularly preferably 1000 nm.

  The shape of the particulate carrier is not particularly limited, but a spherical or substantially spherical shape is preferable from the viewpoint of the amount of PS-PG bound, the loading rate, and physiological activity.

Examples of particulate carriers include polystyrene beads such as styrene / divinylbenzene copolymer beads, latex beads, dendrimers, silica beads, titanium oxide beads, inorganic nano beads such as alumina beads, carbon nano materials, gold nanoparticles, and silver nanoparticles. Metal nanoparticles (including nanorods), semiconductor nanoparticles such as cadmium selenide, self-assembled aggregate particles such as liposomes and micelles, etc., but stable nanoparticles with small particle diameters can be obtained From the viewpoint, latex nanoparticles and silica nanoparticles are preferable. As the latex nanoparticles, polystyrene latex nanoparticles are preferable.
Moreover, since these particulate carrier surfaces carry PS-PG, they are amino groups, aminooxy groups, hydrazide groups, azide groups, carbonyl groups, α, β-unsaturated carbonyl groups, formyl groups, carboxyl groups, sulfones. It is preferably modified with an acid group, a thiol group, a disulfide group, an alkynyl group or the like. Examples of these modifying groups include aliphatic and aromatic modifying groups such as aliphatic amino groups and aromatic amino groups. Further, it may be modified with a ligand that forms a coordinate bond with a metal ion such as histidine oligomer, bipyridine, dipicolylamine, iminodiacetic acid and the like.

  The support form of PS-PG on the surface of the particulate carrier may be any of adsorption, ionic bond, covalent bond, metal bond, van der Waals bond, etc., but stability of PS-PG-containing particles, physiological activity, etc. From the viewpoint, a covalent bond is preferable. More preferably, PS-PG and the formyl group or amino group modified on the surface of the particulate carrier are covalently bonded through Schiff base formation and reductive amination.

  The amount of PS-PG bound to the particulate carrier is not particularly limited, but the amount of PS-PG bound to the surface of one particulate carrier (number of moles) is preferably 0.1 to 250 amol, 0.1 to 50 amol is more preferable, 1 to 50 amol is more preferable, and 10 to 50 amol is particularly preferable. Here, as shown in the examples described later, the amount of PS-PG bound to one particulate carrier surface is X (pg), and the weight average molecular weight of PS-PG is Y (g / mol). It can be calculated by X / Y (amol).

  The support rate of PS-PG on the particulate carrier is not particularly limited, but is preferably 0.1 to 100%, more preferably 0.1 to 20%, more preferably 1 to 20%, based on the number of moles of amino groups on the surface of the particulate carrier. -20% is more preferable, and 10-20% is particularly preferable. Here, the loading ratio is calculated by (X / Y) / Z × 100 (%), where Z (amol) is the number of moles of amino groups on the surface of one particulate carrier, as shown in the Examples below. Can do.

  Examples of the method for supporting PS-PG on the surface of the particulate carrier include formation methods such as adsorption, ionic bond, covalent bond, metal bond and van der Waals bond as described above. It is preferable to use a reductive amination reaction in which the sugar chain of PS-PG is reacted on the surface under reducing conditions. More specifically, the amino group contained in the amino acid of PS-PG is protected by acetylation or the like, and in the presence of a reducing agent such as sodium cyanoborohydride, the amino group or aliphatic amino group modified particulate carrier is used. It is preferable to react.

The PS-PG-containing particles of the present invention have a strong IL-12 production action in macrophages and are useful as an immunostimulant, as shown in Examples below. Conventionally, PS-PG did not show physiological activity in a state separated from cells, but the PS-PG-containing particles of the present invention show physiological activity despite being artificial particles. -It is also useful as a research reagent for the mechanism of action of physiological activity of PG.
Therefore, the PS-PG-containing particles of the present invention can be used in the fields of pharmaceuticals, foods, cosmetics, reagents and the like as IL-12 production promoters, immunostimulators and the like.

  The pharmaceutical of the present invention can be used either orally or parenterally, but oral administration is desirable. Regarding administration, PS-PG-containing particles as an active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for the administration method and administered in the form of a conventional pharmaceutical preparation.

There is no strict limitation on the dosage when using PS-PG-containing particles which are the active ingredients of the present invention. Since the effect obtained varies depending on various use modes such as the subject and applicable disease, it is desirable to set the dose as appropriate, but the preferred dose is 1 × 10 ⁷ particles per time as PS-PG-containing particles. The above is preferable, 1 × 10 ⁷ to 1 × 10 ¹³ is more preferable, 1 × 10 ⁹ to 1 × 10 ¹³ is further preferable, and 1 × 10 ¹¹ to 1 × 10 ¹³ is particularly preferable.

  Examples of the preparation in the case of a pharmaceutical product include solid agents such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, and freeze-dried agents. These preparations can be prepared by conventional means on the preparation. Examples of the non-toxic pharmaceutical carrier include starch, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water, and physiological saline. It is done. If necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, an isotonic agent, and an excipient can be appropriately added.

Moreover, the immunostimulant and IL-12 production promoter of the present invention can be used not only as a pharmaceutical preparation as described above but also as a food or drink. In this case, the PS-PG-containing particles of the present invention may be contained in food or drink as they are or with various nutritional components added. This food and drink can be used as a health food or food material useful for the prevention and treatment of immunity improvement, diseases caused by insufficient production of IL-12, such as infections, tumors, and allergies. The container may be marked with the effect described above. Specifically, when the immunostimulant or IL-12 production promoter of the present invention is blended in a food or drink, an additive that can be used as a food or drink is appropriately used, and a form suitable for food using conventional means, For example, it may be formed into granules, granules, tablets, capsules, pastes, etc., and various foods such as processed meat products such as ham and sausage, processed fishery products such as kamaboko and chikuwa, bread, confectionery and butter In addition, it may be used by adding to milk powder, fermented food or drink, or may be used by adding to beverages such as water, fruit juice, milk, soft drinks and tea drinks. The food and drink includes animal feed.
These drugs, when used in foods, etc., the concentration of the present invention PS-PG-containing particles is preferably not less than 1 × 10 ⁷ cells / g, 1 × 10 ⁷ cells / g to 1 × 10 ¹³ cells / g Gayori Preferably, 1 × 10 ⁹ pieces / g to 1 × 10 ¹³ pieces / g are more preferable, and 1 × 10 ¹¹ pieces / g to 1 × 10 ¹³ pieces / g are particularly preferable.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to this.

Example 1
(1) Separation of PS-PG
The cultured cells of Lactobacillus casei YIT 9029 (FERM BP-1366) were heated at 90 ° C. for 30 minutes and freeze-dried. 500 mg of this freeze-dried microbial cell freeze-dried product was suspended in 30 mL of 5 mM Tris-maleate buffer (pH 6.4), Mutanolysin (SIGMA) was added and reacted at 37 ° C. for 24 hours. The supernatant obtained by centrifuging the reaction solution was dialyzed at 4 ° C. for 20 hours using 10 mM phosphate buffer (pH 6.0) /0.25 M NaCl as an external solution. The dialysis internal solution was freeze-dried, and the freeze-dried product was dissolved in distilled water for injection (Fuso Yakuhin) to a final concentration of 5 mg / mL. This was applied to Sephacryl S-200 HR (GE Healthcare, column size: 5 × 60 cm), and gel filtration was performed (flow rate: 1 mL / min, temperature: 4 ° C.). About the obtained fraction, PS-PG fraction was detected by the phenol-sulfuric acid method. The PS-PG fraction was collected and dialyzed at 4 ° C. for 20 hours using distilled water for injection as an external solution. This was freeze-dried to obtain 73 mg of purified PS-PG.

(2) Protection of the amino group of PS-PG 3 mg of PS-PG was added to a 1.5 mL microtube and dissolved in 500 μL of milli-Q water. To the prepared PS-PG aqueous solution, 50 μL of 50 mM phosphate buffer (pH 8.5) and 20 μL of acetic anhydride were added so that deprotonation of the amino group occurred, and acetylation reaction was started. The reaction solution was allowed to stand at room temperature and vortexed every 15 minutes. Further, 20 μL of acetic anhydride was added every hour after the start of the reaction in order to block all amino groups. This operation was continued for 5 hours. After 5 hours, the reaction solution was put into a dialysis membrane (manufactured by Funakoshi (Spectropore), molecular weight cut off 3500), and dialyzed against distilled water for 1 day. The solution slightly turbid by dialysis was transferred to a 200 mL eggplant-shaped flask and lyophilized. The yield of acetylated PS-PG after lyophilization was 1.6 mg (53% recovery).

(3) Preparation of sugar chain stock solution for reaction i) Preparation of dextran stock solution To a 0.5 mL microtube, 5 mg of dextran was added and dissolved in 50 μL of milli-Q water (0.1 mg / μL). In order to change the modification rate of dextran, a stock solution series in which 0.1 mg / μL prepared previously was diluted 10-fold (maximum 10 ^25- fold dilution: 1 × 10 ⁻¹ to 1 × 10 ⁻²⁶ mg / μL) Was prepared. In the preparation method, 10 μL of the stock solution was taken, added to a 0.5 mL microtube, 90 μL of milli-Q water was added and mixed to prepare a diluted solution.

ii) Preparation of PS-PG stock solution 0.8 mg of acetylated PS-PG was added to a 0.5 mL microtube and dissolved in 40 μL of milli-Q water (0.02 mg / μL). In order to change the modification rate of PS-PG, a stock solution series in which 0.02 mg / μL prepared previously was diluted 10-fold (maximum 10 ^10- fold dilution: 2 × 10 ⁻² to 2 × 10 ⁻¹² mg / μL) was prepared. In the preparation method, 10 μL of the stock solution was taken, added to a 0.5 mL microtube, 90 μL of milli-Q water was added and mixed to prepare a diluted solution.

  The sugar chain-modified nanobeads synthesized in Example 1 are summarized in Tables 1 and 2, and a method for synthesizing these sugar chain-modified nanobeads will be described in detail below.

iii) Modification of sugar chain to nanobeads As nanobeads, aliphatic amino group-modified latex nanobeads (particle size: 24 ± 3 nm: Molecular Probe) (20LA-NH ₂ ), aliphatic amino group-modified latex nanobeads (particle size: 110 ± 5 nm) : Molecular Probe) (100LA-NH ₂ ), aliphatic amino group modified latex beads (particle size 3.1 μm: Molecular Probe) (NP ₃₀₀₀ ), amino group modified silica nano beads (particle size 100 nm: Micromod Partikeltechnologie GmbH) (100SI-NH ₂ ) and amino group-modified silica nano beads (particle size 500 nm: Micromod Partikeltechnologie GmbH) (500SI-NH ₂ ) were used. Dextran having a molecular weight of 40,000 (Wako Pure Chemical Industries) was used.
A. Modification of dextran to silica nanobeads In a 0.5 mL microtube, 20 μL of 50 mM phosphate buffer (pH 8.5), 20 μL of silica nanobeads (100SI-NH ₂ or 500SI-NH ₂ ), 100 mM sodium cyanoborohydride aqueous solution 30 μL was added and 20 μL of dextran stock solution was added. The solution was vortexed and then heated at 45 ° C. for 1 day. The reaction solution was centrifuged (9,000 rpm, 5 min, 25 ° C.) to precipitate silica nanobeads. The supernatant was removed so as not to suck the beads, and 70 μL of milli-Q water was added to the microtube to wash the reaction solution from the nanobeads, and vortexed. After centrifugation again (9,000 rpm, 5 min, 25 ° C.), the supernatant was removed, 70 μL of milli-Q water was added, and the mixture was vortexed. This operation was repeated twice.
Here, the coated beads 100 SI-NH ₂ in the dextran used ^{1 × 10 -1 ~1 × 10 -26} mg / μL, and 100SD-1 to 100SD-26, 1 × 10 dextran in 500SI-NH ₂ ⁻¹ to 1 × 10 ⁻²⁶ mg / μL coated beads were designated as 500SD-1 to 500SD-26.
A. Modification of PS-PG to silica nanobeads In a 0.5 mL microtube, 20 μL of 50 mM phosphate buffer (pH 8.5), 20 μL of silica nanobeads (100SI-NH ₂ or 500SI-NH ₂ ), 100 mM cyanoborohydride 30 μL of aqueous sodium solution was added, and 20 μL of PS-PG stock solution was added. The solution was vortexed and then heated at 45 ° C. for 3 days. The reaction solution was centrifuged (9,000 rpm, 5 min, 25 ° C.) to precipitate silica nanobeads. The supernatant was removed so as not to suck the beads, and 70 μL of milli-Q water was added to the microtube to wash the reaction solution from the nanobeads, and vortexed. After centrifugation again (9,000 rpm, 5 min, 25 ° C.), the supernatant was removed, 70 μL of milli-Q water was added, and the mixture was vortexed. This operation was repeated 4 times.
Here, the beads were coated with ^{PS-PG 2 × 10 -2 ~2} × 10 -12 mg / μL in 100 SI-NH _2, and 100SP-2 to 100SP-12, the 500SI-NH ₂ PS-PG The beads coated with 2 × 10 ^{−2 to} 2 × 10 ⁻¹² mg / μL were designated as 500SP-2 to 500SP-12.

C. Modification of dextran to latex nanobeads To a 0.5 mL microtube, 20 μL of 50 mM phosphate buffer (pH 8.5), 20 μL of latex nanobeads, 30 μL of 100 mM sodium cyanoborohydride aqueous solution were added, and 20 μL of dextran stock solution was added. The solution was vortexed and then heated at 45 ° C. for 1 day. The reaction solution was centrifuged (10,000 rpm, 15 min, 25 ° C.) to precipitate latex nanobeads. The supernatant was removed so as not to suck the beads, and 70 μL of milli-Q water was added to the microtube to wash the reaction solution from the nanobeads, and vortexed. After centrifugation again (10,000 rpm, 15 min, 25 ° C.), the supernatant was removed, 70 μL of milli-Q water was added, and the mixture was vortexed. This operation was repeated twice.
Here, beads coated with ₂₀ LA-NH ₂ using 1 × 10 ⁻¹ to 1 × 10 ⁻²⁶ mg / μL of dextran are designated as 20LD-1 to 20LD-26, and dextran is added to 100LA-NH ₂ with 1 × 10 ⁻¹ to 1 × 10 ⁻²⁶ mg / μL of beads were used as 100LD-1 to 100LD-26.

D. Modification of PS-PG to latex nanobeads To a 0.5 mL microtube, 20 μL of 50 mM phosphate buffer (pH 8.5), 20 μL of latex nanobeads, 30 μL of 100 mM sodium cyanoborohydride aqueous solution were added, and 20 μL of PS-PG stock solution was added. Was added. The solution was vortexed and then heated at 45 ° C. for 3 days. The reaction solution was centrifuged (10,000 rpm, 15 min, 25 ° C.) to precipitate latex nanobeads. The supernatant was removed so as not to suck the beads, and 70 μL of milli-Q water was added to the microtube to wash the reaction solution from the nanobeads, and vortexed. After centrifugation again (10,000 rpm, 15 min, 25 ° C.), the supernatant was removed, 70 μL of milli-Q water was added, and the mixture was vortexed. This operation was repeated 4 times.
Here, beads coated with ₂₀ LA-NH ₂ using 2 × 10 ⁻² to 2 × 10 ⁻¹² mg / μL of PS-PG are designated as 20LP-2 to 20LP-12, and 100LA-NH ₂ is treated with PS-PG. The beads coated with 2 × 10 ^{−2 to} 2 × 10 ⁻¹² mg / μL were designated as 100LP-2 to 100LP-12.
E. Determination of PS-PG binding amount to nanobead surface i) Material
About NP ₃₀₀₀ Aliphatic amino group-modified latex nanobeads (Molecular Probe)
Particle diameter = 3.1 μm (data published by Molecular Probe)
Amino-occupied area of particle surface = 21Å ² = 0.21 × 10 ⁻⁶ μm ² (Data published by Molecular Probe)
Particle surface area = 4 × (× (3.1 / 2) ² = 30.2 μm ²
Amino groups on the particle surface ^{= 30.2μm 2 /0.21×10 -6 μm 2 =} 143,692,380
Number of moles of amino groups on the particle surface = 143,692,380 / 6.02 × 10 ²³ mol ⁻¹ = 24.3 × 10 ^-17 mol = 243 amol

ii) Synthesis of NP ₃₀₀₀ -8 PSPG In a microtube, 150 μL of PBS (50 mM, pH 8.5), 75 μL of an acetylated PS-PG aqueous solution (2.0 mg / 75 μL: 8 equivalents of acetyl to the amino group on the bead surface) PS-PG (in moles)), 25 μL of NP ₃₀₀₀ aqueous solution (3.75 × 10 ⁷ , 0.230 mg), and 75 μL of sodium cyanoborohydride aqueous solution (100 mM) were added. The microtube was allowed to stand at room temperature and stirred by vortex every 30 minutes. This was repeated 8 times and then incubated at 45 ° C. for 72 hours with stirring at 250 rpm.
The reaction solution was centrifuged (11,000 rpm, 15 min, 4 ° C.) to precipitate the NP ₃₀₀₀ component, and the supernatant was removed. The washing operation using 200 μL of ultrapure water was repeated 7 times to obtain PS-PG-bonded NP ₃₀₀₀ (NP ₃₀₀₀ -8PSPG) as a pellet. In the same way as above, except that the number of moles of acetylated PS-PG added to the amino group on the bead surface (1 equivalent, 2 equivalents, 4 equivalents, 6 equivalents) was changed, NP _{3000 -PSPG, NP 3000 -2PSPG, NP} 3000 -4PSPG, to give the NP ₃₀₀₀ -6PSPG.

iii) Each PS-PG bond NP ₃₀₀₀ obtained as a single PS-PG amount calculation method pellets bound to NP ₃₀₀₀ of lyophilized to measure the weight of each PS-PG bond NP ₃₀₀₀ with a precision electronic balance . The weight increased before and after each reaction was attributed to PS-PG bound to nanobeads, and the amount of PS-PG bound to one NP ₃₀₀₀ was calculated. The following is an example of the calculation process of NP ₃₀₀₀ -8PSPG.
The weight of NP ₃₀₀₀ (3.75 × 10 ⁷ pieces) before the reaction was 0.230 mg. In the case of NP ₃₀₀₀ -8PSPG, the weight after reaction and purification was 0.284 mg. From this, it was found that 54 μg of PS-PG was bound to 3.75 × 10 ⁷ NP ₃₀₀₀ . Further, the weight of PS-PG bound to one NP ₃₀₀₀ is calculated as 1.44 pg (54 μg / 3.75 × 10 ⁷ ), and the weight average molecular weight of PS-PG is 30,000 (g / mol). The number of moles of PS-PG bound to one NP ₃₀₀₀ was calculated to be 48 amol (1.44 pg / 30,000 g · mol ⁻¹ ). From the fact that the number of moles of amino groups on the surface of NP ₃₀₀₀ is 243 amol, it can be seen that in the case of NP ₃₀₀₀ -8PSPG, 19.8% of the amino groups presented on the surface of NP ₃₀₀₀ were used for binding to PS-PG. It was.

iv) Quantification of the amount of PS-PG bound to NP ₃₀₀₀ Table 3 summarizes the amount of PS-PG bound to the surface of one nanobead (number of moles). As the amount of PS-PG added increased, the amount of PS-PG bound to one nanobead increased. The amount of PS-PG added and the amount of PS-PG binding were not in direct proportion, and the amount of binding was the same at 1 mol equivalent of PS-PG and 2 mol equivalent. At 4 molar equivalents, a binding amount approximately twice that of 2 molar equivalents was obtained. With 6 molar equivalents, a binding amount of about 14 times the 2 molar equivalents was obtained, and with 8 molar equivalents, a binding amount of about 43 times the 2 molar equivalents was obtained.

Test example 1
(Evaluation of cytokine production using mouse-derived macrophage cells)
(1) Cell culture and passage J774.1 cells (mouse macrophage-like cell line) cultured in a 10 cm dish are peeled off from the bottom of the dish by pipetting and trypsin treatment, and the cell suspension is centrifuged (1000 rpm, 3 min), the sample was collected in a centrifuge tube. After removing the supernatant by aspiration, a fresh medium (10% inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.05 mM 2-mercaptoethanol-containing RPMI-1640 medium) was added thereto, and pipetting was performed. J774.1 cells were suspended in the medium. The obtained J774.1 cell suspension was poured into a new 10 cm dish (2.0 × 10 ⁵ cells / mL, 8 mL) and cultured in a 37 ° C., 5% CO ₂ incubator. These operations were repeated every 3 days to perform subculture.

(2) Cytotoxicity test J774.1 cell suspension was dispensed into a 96-well plate for cell culture at 100 μL / well (1.0 × 10 ⁵ cells / well), and 2 in a 37 ° C., 5% CO ₂ incubator. Incubated for hours. Thereafter, various PS-PG bound nano-beads were suspended in use media and control (20LD-1,100LD-1,500SD-1,20LA -NH 2, 100LA-NH 2, 500SI-NH 2) was added in 100 [mu] L, The cells were cultured for 24 hours in a 37 ° C., 5% CO ₂ incubator. Thereafter, WST-1 assay solution to each well (the 9/1 mixed aqueous solution of WST-1/1-methoxy PMS ) was added 10 [mu] L, 37 ° C., was 2 hours color reaction in a 5% CO ₂ incubator. Thereafter, absorbance at 450 nm and 620 nm was measured with a microplate reader.

(3) IL-12 production induction experiment The J774.1 cell suspension was dispensed into a 96-well plate for cell culture at 100 μL / well (1.0 × 10 ⁵ cells / well), 37 ° C., 5% CO ₂ incubator. Incubated for 2 hours. Thereafter, various PS-PG binding nanobeads and controls were suspended in spent medium (20LD-1,100LD-1,500SD-1,20LA -NH 2, 100LA-NH 2, 500SI-NH 2) was added in 100μL The cells were cultured at 37 ° C. in a 5% CO ₂ incubator for 24 hours. Thereafter, the culture supernatant was filtered with a 0.22 μm filter, and the obtained filtrate was stored at −30 ° C. Purified rat anti-mouse IL-12 p40 / p70 was diluted with Na ₂ CO ₃ buffer (pH 9.6), 50 μL was added to a 96-well plate for ELISA, and incubated at 4 ° C. overnight. After washing each well with PBS (pH 7.4) containing 0.05% Tween 20, 100 μL of Na ₂ CO ₃ buffer containing 1% BSA was added and incubated at 37 ° C. for 90 minutes. After washing each well with PBS, 50 μL of the sample and recombinant mouse IL-12 p70 diluted to a predetermined concentration with PBS containing 0.03% NaN ₃ were added and reacted at room temperature for 90 minutes. After washing each well with PBS, 50 μL of biotin rat anti-mouse IL-12 p40 / p70 diluted with PBS containing 1% BSA was added and reacted at room temperature for 90 minutes. After washing each well with PBS, 50 μL of peroxidase-labeled streptavidin diluted 20000 times with PBS containing 1% BSA was added and reacted at room temperature in the dark for 30 minutes. After washing each well with PBS, 50 μL of TMB (3,3 ′, 5,5′-tetramethylbenzidine) substrate solution was added and reacted at room temperature in the dark for 20 minutes. The coloring reaction was stopped by adding 50 μL of 1 MH ₂ SO ₄ to each well, and the absorbance at 450 nm and 620 nm was measured with a microplate reader.

(4) Cytotoxicity It was found that modification of the nanobead surface with PS-PG reduces the size-dependent toxicity of nanobeads to J774.1 cells.

(5) IL-12 production induction effect i) When an IL-12 production induction experiment was conducted using PS-PG modified latex nanobeads (20LP-2) using 20LP-2, a particle concentration of 1.0 × 10 ¹³ particles / In mL, the expression level of IL-12 reached 28,878 pg / mL.

ii) When an experiment of inducing IL-12 production using 100LP-2 was performed on the PS-PG modified latex nanobeads (100LP-2) , the particle concentration was 1.0 × 10 ^{1 to} 1.0 × 10 ⁹ particles / mL. In this case, the expression level of IL-12 was changed in the concentration range of 251 pg / mL to 512 pg / mL as the concentration increased. However, when the particle concentration reached 1.0 × 10 ¹¹ particles / mL, IL-12 production increased rapidly to 5,026 pg / mL.

iii) When the IL-12 production induction experiment was conducted using 500SP-2 for the PS-PG modified silica nanobeads (500SP-2) , the particle concentration was 1.0 × 10 ^{1 to} 1.0 × 10 ⁷ particles / mL. Then, the expression level of IL-12 was changed in the concentration range of 257 pg / mL to 751 pg / mL as the concentration increased, but when the particle concentration reached 1.0 × 10 ⁹ particles / mL, the production amount of IL-12 Increased rapidly to 892 pg / mL.

iv) When an IL-12 production induction experiment was conducted using NP ₃₀₀₀ -8PSPG for PS-PG modified latex nanobeads (NP ₃₀₀₀ -8PSPG) , the particle concentration was 1.0 × 10 ⁷ particles / mL, and IL-12 The production increased rapidly to 921 pg / mL. Threshold values (1.0 × 10 ¹³ particles / mL for nanobeads of about 20 nm, 1.0 × 10 ¹¹ particles / mL for nanobeads of about 100 nm, and 1.0 × 10 ⁹ particles / mL for nanobeads of about 500 nm) Considering that there was a particle concentration at which the amount of IL-12 production increased rapidly), it became clear that the particle concentration serving as the threshold decreased as the particle diameter increased.
In addition, IL-12 production induction experiments were performed using solubilized PS-PG in an amount corresponding to the NP ₃₀₀₀ -8PSPG (added in a state where the solubilized PS-PG separated from the cell wall was not coated on the beads). However, IL-12 production was not induced in the concentration range corresponding to the particle concentration of NP ₃₀₀₀ -8PSPG, and there was no significant difference in the expression level of IL-12.

v) Regarding the dextran modified latex nanobeads (20LD-1) using 20LD-1, the production of IL-12 is not observed when the particle concentration is in the range of 1.0 × 10 ^{1 to} 1.0 × 10 ¹³ particles / mL. There was no significant difference in the expression level of IL-12.

Example 2
(Correlation between particle size of PS-PG modified nanobeads and IL-12 production)
(I) Synthesis of PS-PG-modified latex nanobeads In a 0.5 μL microtube, 20.0 μL of 50 mM phosphate buffer (pH 8.5), NP (diameter: 200, 300, 1000, 2000, 3000 nm) Latex nanobeads) Suspension 20 μL, 100 mM sodium cyanoborohydride aqueous solution 30.0 μL, Lactobacillus casei adjusted with MilliQ water to 8 molar equivalents with respect to amino groups present on the surface of each NP 20 μL of the derived PS-PG solution was added. The solution was then vortexed and heated at 45 ° C. for 72 hours. Thereafter, the reaction solution was centrifuged (11,000 rpm, 15 min, 4 ° C.) to precipitate nanobeads. The supernatant of the nanobead reaction solution was removed, Milli-Q water was added, and the mixture was vortexed. Again, after centrifugation (11,000 rpm, 15 min, 4 ° C.), the supernatant was removed, milliQ water was added to the nanobeads, and the mixture was vortexed. This operation was repeated 7 times.

(Ii) Determination of IL-12 concentration by ELISA J774.1 cells (mouse macrophage-like cell line) were dispensed at 1 × 10 ⁵ cells / well in a 96-well plate (100 μL), and ₂ at 37 ° C. with 5% CO ₂ . Incubated for hours. Thereafter, 100 μL each of PS-PG modified latex bead solution of each size adjusted (1.0 × 10 ⁹ cells / mL) was added to each well and incubated at 37 ° C. and 5% CO ₂ for 24 hours. After 24 hours, the culture supernatant was collected by filtration through a 0.45 μm filter and stored at −20 ° C. as a sample for ELISA. 50 μL of purified rat anti-mouse IL-12 p40 / p70 (diluted to 10 μg / mL with Na ₂ CO ₃ buffer at pH 9.6) was added to the 96-well plate for ELISA, and the plate was incubated overnight at 4 ° C. Turned into. Thereafter, the plate was washed 5 times with 0.05% Tween20-PBS, 100 μL of 1% BSA-Na ₂ CO ₃ buffer was added and incubated at 4 ° C. for 24 hours. Each well was washed 4 times with PBS, and the sample and 1% BSA-PBS (4000 pg / mL, 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 50 μL of recombinant mouse IL-12 p70 diluted to 0 pg / mL) was added and reacted at room temperature for 90 minutes. Each well was washed 4 times with PBS, 50 μL of biotin rat anti-mouse IL12 p40 / p70 (1.0 μg / mL) diluted with 1% BSA-PBS was added and reacted at room temperature for 90 minutes. After washing 4 times with PBS, 50 μL of a peroxidase-labeled streptavidin solution diluted 20000 times with 1% BSA-PBS was added and allowed to react for 30 minutes at room temperature in the dark. The plate was washed 4 times with PBS, 50 μL of TMB (3,3 ′, 5,5′-tetramethylbenzidine) substrate solution was added, and the mixture was reacted at room temperature for 20 minutes in the dark. The absorbance at 620 nm was measured with a microplate reader. . Thereafter, 50 μL of 1 MH ₂ SO ₄ was added to stop the reaction, and absorbance at 450 nm and 620 nm was measured with a microplate reader.

(Iii) Results FIG. 1 shows the relationship between the particle size (200 nm to 3000 nm) of the PS-PG modified latex nanobead carrier and the IL-12 producing action.
As is apparent from FIG. 1, it can be seen that the ability to induce IL-12 production of PS-PG-containing particles having an average particle diameter of the particulate carrier of 300 nm to 2000 nm is particularly excellent.

Example 3
(IL-12 production inducing ability of PS-PG-containing particles derived from Lactobacillus johnsonii )
(I) Synthesis of Lactobacillus johnsonii -derived PS-PG-modified latex nanobeads Lactobacillus johnsonii YIT 0219 ^T (JCM 2012 ^T ) -derived PS-PG solution was prepared from Lactobacillus casei-derived PS of Example 1. -Performed by the same method as the method for preparing the PG solution.
In a 0.5 μL microtube, 20.0 μL of 50 mM phosphate buffer (pH 8.5), 20 μL of a 1 μm diameter NP (latex nanobead) suspension, 30.0 μL of 100 mM sodium cyanoborohydride aqueous solution, present on the NP surface 20 μL of a Lactobacillus johnsonii- derived PS-PG solution whose concentration was adjusted with milli-Q water so as to be 8 molar equivalents relative to the amino group to be added. The solution was then vortexed and heated at 45 ° C. for 72 hours. Thereafter, the reaction solution was centrifuged (11,000 rpm, 15 min, 4 ° C.) to precipitate nanobeads. The supernatant of the nanobead reaction solution was removed, Milli-Q water was added, and the mixture was vortexed. Again, after centrifugation (11,000 rpm, 15 min, 4 ° C.), the supernatant was removed, milliQ water was added to the nanobeads, and the mixture was vortexed. This operation was repeated 7 times.
(Ii) Determination of IL-12 concentration by ELISA J774.1 cells were dispensed into 96-well plates at 1 × 10 ⁵ cells / well (100 μL) and incubated at 37 ° C. and 5% CO ₂ for 2 hours. Thereafter, 100 μL of each size-adjusted PS-PG modified latex bead solution was added to each well and incubated at 37 ° C., 5% CO ₂ for 24 hours. After 24 hours, the culture supernatant was collected by filtration through a 0.45 μm filter and stored at −20 ° C. as a sample for ELISA. 50 μL of purified rat anti-mouse IL-12 p40 / p70 (diluted to 10 μg / mL with Na ₂ CO ₃ buffer at pH 9.6) was added to the 96-well plate for ELISA, and the plate was incubated overnight at 4 ° C. Turned into. Thereafter, the plate was washed 5 times with 0.05% Tween20-PBS, 100 μL of 1% BSA-Na ₂ CO ₃ buffer was added and incubated at 4 ° C. for 24 hours. Each well was washed 4 times with PBS, and the sample and 1% BSA-PBS (4000 pg / mL, 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 50 μL of recombinant mouse IL-12 p70 diluted to 0 pg / mL) was added and reacted at room temperature for 90 minutes. Each well was washed 4 times with PBS, 50 μL of biotin rat anti-mouse IL12 p40 / p70 (1.0 μg / mL) diluted with 1% BSA-PBS was added and reacted at room temperature for 90 minutes. After washing 4 times with PBS, 50 μL of a peroxidase-labeled streptavidin solution diluted 20000 times with 1% BSA-PBS was added and allowed to react for 30 minutes at room temperature in the dark. The plate was washed 4 times with PBS, 50 μL of TMB (3,3 ′, 5,5′-tetramethylbenzidine) substrate solution was added, and the mixture was reacted at room temperature for 20 minutes in the dark. The absorbance at 620 nm was measured with a microplate reader. . Thereafter, 50 μL of 1 MH ₂ SO ₄ was added to stop the reaction, and absorbance at 450 nm and 620 nm was measured with a microplate reader.

(Iii) Results
The ability to induce IL-12 production for J774.1 cells of PS-PG modified nanoparticles derived from Lactobacillus johnsonii reached 870,000 pq / mL at a particle concentration of 1 × 10 ⁸ particles / mL. L. Since it is known that Johnson-derived PS-PG does not show IL-12 production-inducing ability at the bacterial cell level, this experimental result shows that even PS-PG derived from lactic acid bacteria that do not show IL-12 production induction This suggests that the ability to induce IL-12 production is exhibited by granulating (supporting on the surface of the particulate carrier) as in the invention.

Claims (13)

  A polysaccharide-peptidoglycan complex-containing particle obtained by supporting a polysaccharide-peptidoglycan complex on the surface of a particulate carrier.   The polysaccharide-peptidoglycan complex-containing particle according to claim 1, wherein the polysaccharide-peptidoglycan complex is derived from bacteria.   The polysaccharide-peptidoglycan complex-containing particles according to claim 1 or 2, wherein the polysaccharide-peptidoglycan complex is derived from lactic acid bacteria.   The polysaccharide-peptidoglycan complex-containing particle according to any one of claims 1 to 3, wherein the polysaccharide-peptidoglycan complex is derived from a lactic acid bacterium belonging to the genus Lactobacillus.   The polysaccharide-peptidoglycan complex-containing particle according to any one of claims 1 to 4, wherein the polysaccharide-peptidoglycan complex is derived from Lactobacillus casei and / or Lactobacillus johnsonii.   The polysaccharide-peptidoglycan complex-containing particle according to any one of claims 1 to 5, wherein the particulate carrier is a nanoparticle.   The polysaccharide-peptidoglycan complex-containing particles according to any one of claims 1 to 6, wherein the particulate carrier is latex nanoparticles or silica nanoparticles.   The polysaccharide-peptidoglycan complex-containing particle according to any one of claims 1 to 7, wherein the average particle diameter of the particulate carrier is 20 nm to 3100 nm.   The polysaccharide-peptidoglycan complex-containing particles according to any one of claims 1 to 8, wherein the average particle diameter of the particulate carrier is 300 nm to 2000 nm.   The polysaccharide-peptidoglycan complex-containing particle according to any one of claims 1 to 9, wherein the binding amount of the polysaccharide-peptidoglycan complex to one particulate carrier is 0.1 to 50 amol.   The pharmaceutical containing the polysaccharide-peptidoglycan complex containing particle | grains in any one of Claims 1-10.   The immunostimulant which uses the polysaccharide-peptidoglycan complex containing particle | grains in any one of Claims 1-10 as an active ingredient.   The interleukin 12 production promoter which uses the polysaccharide-peptidoglycan complex containing particle | grains in any one of Claims 1-10 as an active ingredient.