JP7177436B2 - antibacterial agent composition - Google Patents

Description

The present invention relates to antimicrobial compositions. More specifically, biomaterials, detergents, cosmetics, paints, resins, wood preservatives, cement admixtures, water treatment agents, industrial water, pulp and paper, plastics, fibers, food additives, medical equipment, optical equipment, modules. , to antimicrobial compositions useful in electronic products and the like.

Since infectious diseases caused by bacteria such as O157 threaten human life, countermeasures against bacteria and the like are required worldwide. In recent years, in Japan, various antibacterial processed products have been marketed not only in the medical and sanitary fields but also in various fields due to consumers' preference for cleanliness. Antibacterial agents used in antibacterial processing can be classified into organic agents and inorganic agents. Regarding inorganic agents, Non-Patent Document 1 describes the classification and antibacterial mechanism of inorganic antibacterial agents, The antibacterial performance of metal ions of is disclosed. Regarding antibacterial agents using metal compounds, for example, Patent Document 1 discloses an antibacterial and fungicidal agent characterized by containing a water-sparingly water-soluble zinc-containing inorganic compound as an active ingredient. Patent Document 2 discloses that a sparingly soluble orthophosphoric acid double salt of two or more metals selected from the group consisting of magnesium, calcium, strontium, manganese, nickel, copper and zinc is added with 0.1-5. An antibacterial/antifungal double salt of phosphoric acid is disclosed, which is characterized by carrying 0% by weight of silver. In addition, regarding an antibacterial agent using a carbon material, Patent Document 3 discloses a dispersion of graphene oxide particles dispersed in a mixed solution of water and N-methyl-2-pyrrolidone, which is applied to the tooth surface and sustained. A dental coating composition used for imparting antibacterial properties, wherein the mixed solution of water and N-methyl-2-pyrrolidone has a concentration of N-methyl-2-pyrrolidone of 0.1 to 20% by mass. The compositions are disclosed, which are ranges. Patent Document 4 includes a substrate made of a porous scaffold and graphene oxide as a coating on at least part of the surface of the substrate, and the coating amount of the graphene oxide is based on the mass of the substrate. Bone or skin regeneration scaffolds are disclosed ranging from 0.1 to 2.5% by weight.

Japanese Patent Application Laid-Open No. 2007-314478 JP-A-05-294808 JP 2016-074636 A JP 2016-158680 A

Noriyuki Yamamoto, “Inorganic Materials” (Japan), 1999, Vol. 6, p468-473

As described above, although various antibacterial agents have been disclosed in the past, their antibacterial performance is not sufficient, and there has been a demand for an antibacterial agent that is superior in antibacterial performance to conventional antibacterial agents.

The present invention has been made in view of the above-mentioned current situation, and an object of the present invention is to provide an antibacterial agent composition having antibacterial properties superior to those of conventional antibacterial agents.

The inventor of the present invention conducted various studies on compounds exhibiting antibacterial properties, and found that by combining a carbon material with a specific structure and a zinc component, the antibacterial properties were superior to those of conventional antibacterial agents. The present invention has been arrived at by conceiving that the above problem can be solved.

That is, the present invention is an antibacterial agent composition containing a carbon material having a G band in the Raman spectrum and a zinc component.

The carbon material is preferably graphene oxide.

It is preferable that the mass ratio of the carbon material and the zinc component (carbon material/zinc) is 1/30 to 300/1.

The present invention is also an antimicrobial material comprising the above antimicrobial agent composition and a substrate.

The content of the zinc component is preferably 0.01 to 1% by mass with respect to 100% by mass of the antibacterial material.

Preferably, the substrate is a porous scaffold.

The antibacterial agent composition of the present invention has the above-described structure and is superior in antibacterial performance to conventional antibacterial agents. agents, industrial water, paper pulp, plastics, fibers, food additives, medical equipment, optical equipment, modules, electronic products and the like.

4 is a chart showing the Raman spectrum of the antibacterial material (2) of Example 6. FIG. 4 is a chart showing the Raman spectrum of the comparative antibacterial material (1) of Comparative Example 1. FIG. 4 is a chart showing the Raman spectrum of the comparative antibacterial material (2) of Comparative Example 2. FIG. 3 is a chart showing the Raman spectrum of the comparative antibacterial material (3) of Comparative Example 3. FIG. 10 is a chart showing XRD measurement results of the antibacterial material (2) of Example 6. FIG. 10 is a chart showing XRD measurement results of the antibacterial material (3) of Example 7. FIG. 4 is a chart showing XRD measurement results of comparative antibacterial material (1) of Comparative Example 1. FIG. 4 is a chart showing XRD measurement results of the comparative antibacterial material (2) of Comparative Example 2. FIG. 4 is a chart showing XRD measurement results of the comparative antibacterial material (3) of Comparative Example 3. FIG. 4 is a chart showing XRD measurement results of comparative antibacterial material (4) of Comparative Example 4. FIG. 10 is an SEM photograph (magnification: 10,000 times) of the antibacterial material (2) of Example 6. FIG. 4 is an SEM photograph (magnification: 5000 times) of the comparative antibacterial material (1) of Comparative Example 1. FIG. 4 is an SEM photograph (magnification: 5000 times) of the comparative antibacterial material (2) of Comparative Example 2. FIG. 3 is an SEM photograph (magnification: 5000 times) of the comparative antibacterial material (3) of Comparative Example 3. FIG. 10 is an SEM photograph (magnification: 20,000 times) of the antibacterial material (2) of Example 6. FIG. 3 is an SEM photograph (magnification: 20,000 times) of the comparative antibacterial material (3) of Comparative Example 3. FIG. A graph showing the results of the antibacterial evaluation (1) against S. mutans for the antibacterial materials (1) to (4) of Examples 5 to 8 and the comparative antibacterial materials (1) and (2) of Comparative Examples 1 and 2. be. 1 is a graph showing the results of antibacterial evaluation (2) against S. mutans for antibacterial material (2) of Example 6 and comparative antibacterial materials (1) to (3) of Comparative Examples 1 to 3. A. for the antibacterial material (2) of Example 6 and the comparative antibacterial materials (1) to (3) of Comparative Examples 1 to 3. It is a graph which shows the result of antibacterial evaluation (2) with respect to naeslundii. 1 is a graph showing cell proliferation evaluation results for antibacterial materials (1) to (4) of Examples 5 to 8 and comparative antibacterial materials (1) and (2) of Comparative Examples 1 and 2. FIG. 1 is a graph showing the cytotoxicity evaluation results of antibacterial materials (1) to (4) of Examples 5 to 8 and comparative antibacterial materials (1) and (2) of Comparative Examples 1 and 2. FIG. 2 is a graph showing cell proliferation evaluation results for antibacterial material (2) of Example 6 and comparative antibacterial materials (1) to (3) of Comparative Examples 1 to 3. FIG. 2 is a graph showing the results of cytotoxicity evaluation of antibacterial material (2) of Example 6 and comparative antibacterial materials (1) to (3) of Comparative Examples 1 to 3. FIG. 4 is a chart showing the Raman spectrum of the antibacterial material (5) of Example 10. FIG. 4 is a chart showing the Raman spectrum of the comparative antibacterial material (6) of Comparative Example 6. FIG. 4 is a chart showing the Raman spectrum of the comparative antibacterial material (7) of Comparative Example 7. FIG. 10 is a chart showing the Raman spectrum of the comparative antibacterial material (8) of Comparative Example 8. FIG. A graph showing the results of antibacterial evaluation (3) against S. mutans for the antibacterial material (5) of Example 10 and the comparative antibacterial materials (5), (6), and (8) of Comparative Examples 5, 6, and 8. be.

Preferred embodiments of the present invention will be specifically described below, but the present invention is not limited to the following description, and can be appropriately modified and applied without changing the gist of the present invention. In addition, the form which combined 2 or 3 or more of each preferable form of this invention described below also corresponds to the preferable form of this invention.

<Antibacterial agent composition>
The antibacterial agent composition of the present invention contains a carbon material having a G band in the Raman spectrum (hereinafter also referred to as the carbon material of the present invention) and a zinc component. By combining the specific carbon material and the zinc component, it is believed that the dispersibility of the zinc component is increased and the antibacterial performance is superior to that of conventional antibacterial agents containing the zinc component. Conventionally, various metals have been used as antibacterial ingredients, but the antibacterial agent composition of the present invention containing a zinc component can exhibit sufficient antibacterial performance even when the zinc concentration is low. Furthermore, since zinc has low cytotoxicity, it is also excellent in safety.

Here, the antibacterial agent means an agent having antibacterial properties. Antibacterial performance refers to the performance of sterilization (killing microorganisms), bacteriostasis (suppressing the growth of microorganisms), sterilization, disinfection, antibacterial, sterilization, antiseptic, antifungal, etc. The target microorganisms are , bacteria and fungi.
Specifically, target microorganisms include S. mutans, S. sobrinus, Lactobacillus, P. gingivalis, T. forsythia, P. intermedia, T. denticola, A. actinomycetemcomitans, A. naeslundii, E. corrodens, Spirochetes, Fusobacterium, and C. albicans.

In the antibacterial agent composition of the present invention, the mass ratio of the carbon material of the present invention to the zinc component (carbon material/zinc) is preferably 1/30 to 300/1. As a result, the antibacterial performance is further improved. The mass of the zinc component when calculating the above mass ratio shall be calculated in terms of zinc atoms. For example, when a zinc element-containing compound is included as a zinc component, it is calculated as the mass of zinc atoms in the zinc element-containing compound. The mass ratio is more preferably 1/15 to 150/1, still more preferably 1/5 to 100/1, and particularly preferably 1/1 to 50/1.

In the antibacterial agent composition of the present invention, the content ratio of the zinc component in terms of zinc atoms is preferably 0.0001 to 20% by mass with respect to 100% by mass of the antibacterial agent composition. More preferably 0.0002 to 10% by mass, still more preferably 0.001 to 5% by mass, and particularly preferably 0.005 to 1% by mass.

The antibacterial agent composition of the present invention preferably contains the carbon material of the present invention in an amount of 0.0001 to 10% by mass based on 100% by mass of the antibacterial agent composition. More preferably 0.0005 to 5% by mass, still more preferably 0.001 to 2% by mass, and particularly preferably 0.005 to 1% by mass.

(carbon material)
The carbon material contained in the antibacterial composition of the present invention may have a G band in the Raman spectrum. The G band is a peak appearing near the Raman shift of 1580 cm ⁻¹ in the Raman spectrum.
A Raman spectrum can be measured under the conditions described in Examples.
The carbon material has a G band in the Raman spectrum, meaning that it has a graphene skeleton. The carbon material having a graphene skeleton has carbon (C) bonded by sp ² bonds, and the carbons are arranged in a plane. Graphene oxide in which carbon is bonded to oxygen (O) is more preferable, and graphene oxide in which oxygen is bonded to carbon in graphene is more preferable.
In general, graphene refers to a sheet consisting of a single layer in which carbon atoms bonded by sp ² bonds are arranged in a plane, and a stack of many graphene sheets is called graphite, but the graphene skeleton in the present invention and graphene oxide in the present invention include not only a sheet consisting of only one layer but also those having a structure in which several layers to about 100 layers are laminated.
Graphene oxide having such a stacked structure can be obtained, for example, by treating graphite with a known oxidizing agent.
The carbon material may further have functional groups such as carboxyl groups, hydroxyl groups, sulfur-containing groups, etc., but the content of carbon, hydrogen, and oxygen as constituent elements with respect to all constituent elements is 97 mol%. It is preferably 99 mol % or more, more preferably 99 mol % or more, and further preferably contains only carbon, hydrogen, and oxygen as constituent elements.

When the carbon material of the present invention has carbon bonded to oxygen (O), the ratio of the number of carbon atoms to the number of oxygen atoms (C/O) is preferably 0.5-20. The ratio is more preferably 1 or more, and even more preferably 1.2 or more. The ratio is more preferably 10 or less, even more preferably 6 or less, still more preferably 4 or less, and particularly preferably 3 or less. The ratio of the number of carbon atoms to the number of oxygen atoms can be confirmed by the ratio of the total peak area of the O1s region and the total peak area of the C1s region obtained by XPS measurement.

The carbon material of the present invention preferably has a thickness of 0.4 to 10 nm. As a result, the adhesiveness is further improved by adhering along the fine irregularities on the surface of the antibacterially processed surface, and the antibacterial performance can be exhibited for a longer period of time. More preferably, the thickness is 0.4-5 nm, and still more preferably 0.4-3 nm.

The carbon material of the present invention preferably has a planar size of 20 nm or more. A form in which the carbon material is a film-like particle having a thickness of 0.4 to 10 nm and a size of 20 nm or more in the planar direction is also one of the preferred embodiments of the present invention. The size in the plane direction is more preferably 100 nm to 100 μm, still more preferably 1 μm to 30 μm.

When the carbon material of the present invention has carbon bonded to oxygen (O), the general method for producing it is to react graphite with a strong oxidizing agent in an acid solvent. The Hummers method with potassium manganate can be used. As other methods, the Brodie method using nitric acid and potassium chlorate, the Staudenmaier method using sulfuric acid, nitric acid and potassium chlorate as oxidizing agents, and the like can be used. When the oxidation method in the Hummers method is employed, a method of adding a permanganate to a mixed solution containing graphite and sulfuric acid may be used. The carbonaceous material thus obtained is usually purified by techniques such as filtration, decantation, centrifugation, liquid separation extraction, and water washing. Purification may be performed in air or in an atmosphere of an inert gas such as nitrogen, helium, argon or the like. Also, the reaction may be carried out under pressurized conditions, normal pressure conditions, or reduced pressure conditions.

(zinc component)
The zinc component contained in the antibacterial agent composition of the present invention is not particularly limited as long as it contains zinc element and can be ionized. Examples thereof include zinc metal, zinc element-containing compounds, and zinc ions. Among them, a zinc element-containing compound is preferable, and a zinc element-containing salt is more preferable.
The salt containing zinc element is not particularly limited as long as it is a compound in which a zinc ion and an anion are ionically bonded. , acetate ion, carbonate ion, hydrogen carbonate ion, phosphate ion, silicate ion, borate ion, halogen ion, hydroxide ion and the like. Preferred are nitrate ions, nitrite ions, sulfate ions, sulfite ions, hydrogen sulfite ions, and acetate ions. As the zinc-containing salt, zinc acetate, zinc sulfate, zinc nitrate, zinc phosphate and the like are preferable, and zinc sulfate and zinc acetate are more preferable.
Further, when the zinc element-containing compound is a salt, the salt may be in an ionized form in the composition. may be combined with other anions in the

(other ingredients)
The antibacterial agent composition of the present invention may contain components other than the above carbon material and zinc component. The above-mentioned other components are not particularly limited as long as they do not inhibit the antibacterial performance of the present invention, and examples thereof include solvents;
From the viewpoint of improving antibacterial properties, the antibacterial agent composition may contain metal salts, metal oxides, metal hydroxides, and the like other than the zinc component. As the metal in the metal salts, oxides, and metal hydroxides, transition metals such as copper, silver, and titanium are preferred.

Examples of the solvent include water; amides such as N-methyl-2-pyrrolidone, N,N'-dimethylformamide and N,N'-dimethylacetamide; polar solvents such as alcohols such as methanol, ethanol and isopropyl alcohol. preferable. By including a polar solvent in the antibacterial agent composition, the dispersibility of the carbon material is further improved, and the antibacterial treatment can be performed more uniformly.
In the case of forming an antibacterial material using the antibacterial agent composition and a porous base material as a base material described later, amides are preferable as the solvent. This allows the antibacterial agent composition to more sufficiently permeate the substrate. More preferred is N-methyl-2-pyrrolidone. A mixed solvent of water and the above solvent other than water can also be used.

Although the osmotic pressure adjusting substance is not particularly limited, examples thereof include alkali metal ions such as sodium ion and potassium ion, and halide ions such as chloride ion.
The pH buffer is not particularly limited, and examples thereof include phosphate buffers, acetate buffers, citric acid buffers, and the like.

<Antimicrobial material>
The present invention is also an antimicrobial material comprising the antimicrobial composition of the present invention and a substrate.
By using the antibacterial agent composition of the present invention supported on a substrate, the antibacterial performance can be exhibited more effectively.

The content of the zinc component in the antibacterial material is preferably 0.01 to 5% by mass with respect to 100% by mass of the antibacterial material. Thereby, antibacterial performance can be exhibited more fully. It is more preferably 0.01 to 1% by mass, still more preferably 0.02 to 1% by mass, and particularly preferably 0.02 to 0.5% by mass.

The antibacterial material preferably contains 0.01 to 5% by mass of the carbon material having a G band in the Raman spectrum with respect to 100% by mass of the antibacterial material. Thereby, antibacterial performance can be exhibited more fully. It is more preferably 0.01 to 1% by mass, still more preferably 0.02 to 1% by mass, and particularly preferably 0.02 to 0.5% by mass.

(Base material)
The substrate is not particularly limited as long as it can support the antibacterial agent composition, and examples thereof include paper, fiber substrates, wood, resin foams, porous substrates, film substrates, particulate substrates, and the like. be done.
Examples of the fiber base material include woven fabrics such as gauze, knitted fabrics, braids, laces, nets, and non-woven fabrics.
Examples of the resin foam include, but are not limited to, polyurethane foam, polystyrene foam, and the like.

The material constituting the base material is not particularly limited and may be appropriately selected depending on the application. Examples include collagen, gelatin, fibrin, chitosan, hyaluronic acid, polylactic acid; animal fibers such as silk and wool; polyester. , polypropylene; cellulosic fibers such as cotton, hemp, rayon, cupra, lyocell, polynosic, acetate and triacetate; polymer materials such as hydroxyapatite and inorganic materials such as calcium triphosphate.
As a material constituting the base material, only one of these may be used, or two or more of them may be used in combination.
At least one material selected from the group consisting of collagen, gelatin, fibrin, chitosan, hyaluronic acid, polylactic acid, hydroxyapatite, and calcium triphosphate is preferable as the material constituting the base material. Collagen is more preferred.

The substrate is preferably a fiber substrate or a porous substrate, and more preferably a porous substrate.
The porous substrate is not particularly limited as long as it has pores, but preferably has a porosity of 50 to 99%. The porosity is the following formula (1);
Porosity (%) = 100 x (1-ρ1/ρ2) (1)
(In the formula, ρ1 represents the density of the porous substrate, and ρ2 represents the true density of the material constituting the porous substrate.).
In addition, the porous structure is preferably a continuous pore structure, and the continuous pore structure is a structure in which at least some of the pores in the porous substrate are communicated, and isolated pores are separated from each other. preferably not.

The porous substrate is preferably a scaffold that functions as a scaffold for regeneration of cells, tissues, and the like. By using a scaffold as a base material, the antibacterial material of the present invention can be suitably used for tissue regeneration and the like. A form in which the base material is a porous scaffold is one of preferred embodiments of the present invention. By using a porous scaffold as a substrate for the antimicrobial material, the scaffold will be coated with the carbon material and the zinc component. By using such an antibacterial material for tissue regeneration and the like, infections and the like caused by bacteria and the like can be more effectively prevented.

The porous scaffold is preferably composed mainly of at least one material selected from the group consisting of collagen, gelatin, fibrin, chitosan, hyaluronic acid, polylactic acid, hydroxyapatite, and calcium triphosphate.
A collagen scaffold containing collagen as a main component is preferable as the porous scaffold. The porous scaffold may contain components other than the above materials, and may contain, for example, the above-described osmotic pressure adjusting substance, pH buffering agent, and the like.

As described above, the antibacterial material may contain an osmotic pressure adjusting substance, a pH buffering agent, and the like. etc.
The alkali metal component and the halogen component should just contain an alkali metal element and a halogen element, respectively.
Examples of the oxoacid include phosphoric acid, phosphorous acid, pyrophosphoric acid, carbonic acid, orthocarbonic acid, carboxylic acid, silicic acid, nitrous acid, nitric acid, arsenic acid, sulfurous acid, sulfuric acid, sulfonic acid, sulfinic acid, chromic acid, and dichromic acid. acid, permanganic acid, and the like. Preferred are phosphoric acid, phosphorous acid and pyrophosphoric acid, and more preferred is phosphoric acid.

A form in which the antibacterial material contains an alkali metal component or a phosphoric acid component is one of preferred embodiments of the present invention.
The content of the alkali metal element in the antibacterial material is preferably 0.005 to 2% by mass with respect to 100% by mass of the antibacterial material. More preferably, it is 0.02 to 1% by mass.
The content of phosphorus element in the antibacterial material is preferably 0.005 to 2% by mass with respect to 100% by mass of the antibacterial material. More preferably, it is 0.03 to 1% by mass.

When the antibacterial material contains an alkali metal component or a phosphoric acid component, the form thereof in the antibacterial material is not particularly limited, and they may be ionized or form a salt.
When the antibacterial material contains an alkali metal component or a phosphoric acid component, it is preferable that the zinc component, the alkali metal component, and the phosphoric acid component form a double salt.
The content of these elements can be measured by X-ray fluorescence (XRF) analysis described in Examples.

The structure of the double salt is not particularly limited, but the following formula (2);
_MxHy ( _{ZnPO4 ) z.mH2O} ( ₂ )
(In the formula, M represents an alkali metal element. x represents a number greater than 0. y represents a number of 0 or more. z represents a number of 1 to 10. However, x, y and z satisfies x+y=z, and m represents a number from 0 to 16.).
It should be noted that each composition ratio in the composition formula of a compound or the like is generally represented by the smallest integer ratio, but the structure represented by the composition ratio represented by the smallest integer ratio is the unit structure in the crystal structure. In the present invention, the compound having such a crystal structure is represented by the minimum integer ratio in the above formula (1). Therefore, the above formula (1) does not necessarily represent the actual unit structure of the double salt. x is preferably 1 to 10, more preferably 1 to 6.
y is preferably 0 to 5, more preferably 0 to 3, and most preferably 0.
z is preferably 1 to 8, more preferably 1 to 6.
m is preferably 0 to 12, more preferably 0 to 8.
The combination of x, y, z, m is preferably (x, y, z, m) = (3, 0, 3, 6), (1, 0, 1, 1), more preferably ( x,y,z,m)=(3,0,3,6).

More preferably, the double salt is represented by the following formula (3);
M6( _ZnPO4 ) _{6.8H2O ( 3} )
(In the formula, M represents an alkali metal element). The double salt represented by the above formula (3) does not contain water molecules as hydration water in the crystal, but the structure represented by the above formula (3) is the minimum unit structure (unit cell). Therefore, the composition ratio in the above formula (3) is not the minimum integer ratio, but the actual composition ratio of the unit cell.
Na ₆ (ZnPO ₄ ) _{6.8H 2} O is most preferable as the double salt.

<Method for producing antibacterial material>
The method for producing the antibacterial material of the present invention is not limited as long as the antibacterial agent composition containing the carbon material and the zinc component can be supported on the substrate. and/or injecting an antibacterial agent composition containing a carbon material, a zinc component, and a solvent into the substrate.
The solvent is as described above.
In the injection step, the injection method is not particularly limited as long as the antibacterial agent composition containing the carbon material, the zinc component, and the solvent can be injected into the substrate. can be injected.

In the production of the antibacterial material, the step of washing and drying the obtained antibacterial material may be performed after the dipping step and/or the injection step. Washing and drying can be carried out according to commonly used methods.

The antibacterial agent composition and antibacterial material of the present invention are composed of the above-described structures and are superior in antibacterial performance to conventional antibacterial agents containing elemental zinc. Detergents such as detergents, softeners, household cleaners, dishwashing agents, and hard surface cleaners; cosmetics such as shampoos, rinses, lotions, milky lotions, creams, sunscreens, foundations, eye makeup products, and antiperspirants Cosmetic applications such as agents; Industrial applications such as paints, wood preservatives, cement admixtures, industrial water (papermaking process water in the papermaking process, cooling water and washing water for various industries); masks, eye patches, bandages, plasters, sanitary products such as gauze; medical instruments; textile products such as clothing; food additives; In particular, the antibacterial material of the present invention can be suitably used for biomaterial applications.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited only to these examples. Unless otherwise specified, "part" means "mass part" and "%" means "mass %".

Various physical properties were measured by the following methods.
<Measurement of Raman spectrum>
Raman spectroscopic analysis was performed using the following equipment and conditions.
Measuring device: Microscopic Raman (JASCO NRS-3100)
Measurement conditions: Use of 532 nm laser, 20x objective lens, CCD capture time of 1 second, integration of 32 times (resolution = 4 cm ^-1 )

<Measurement of X-ray fluorescence (XRF)>
The XRF measurement was performed using a fluorescent X-ray analyzer (ZSX Primus2, manufactured by Rigaku Corporation) and an Rh (4 kW) X-ray tube, using a calibration curve method.

<Measurement of X-ray diffraction (XRD)>
The XRD measurement was performed under the following conditions using a fully automatic horizontal X-ray diffractometer (SMART LAB manufactured by Rigaku Corporation).
CuKα1 line: 0.15406 nm
Scan range: 10°-90°
X-ray output setting: 45kV-200mA
Step size: 0.020°
Scanning speed: 0.5°min ^-1 -4°min ^-1
The XRD measurement was performed in a state in which an inert atmosphere was maintained by loading the sample onto an airtight sample table in a glove box.

<SEM Observation>
SEM observation was performed using a scanning electron microscope (manufactured by JEOL Ltd., JSM-7600F) with accelerated electrons of 5.0 keV, and a secondary electron image was observed.

<Antibacterial evaluation (1)>
Each sample was placed in a 48-well plate, seeded with a Streptococcus mutans (ATCC35668) (5.5×10 ⁶ CFU/well) suspension, and anaerobically cultured at 37° C. for 24 hours, and then the turbidity of the medium was measured. . A smaller turbidity indicates a higher antimicrobial activity.

<Antibacterial evaluation (2)>
Each sample was placed in a 48-well plate and seeded with a suspension of Streptococcus mutans (ATCC35668) (6.0×10 ⁶ CFU/well) and Actinomyces naeslundii (ATCC 27039) (6.0×10 ⁶ CFU/well). After anaerobic culture at 37° C. for 24 hours, the turbidity of the medium was measured. A smaller turbidity indicates a higher antimicrobial activity. As a control, a similar test was conducted without using a sample.

<Antibacterial evaluation (3)>
Each sample was placed in a 48-well plate, seeded with a Streptococcus mutans (ATCC35668) (5.5×10 ⁶ CFU/well) suspension, and anaerobically cultured at 37° C. for 24 hours, and then the turbidity of the medium was measured. . A smaller turbidity indicates a higher antimicrobial activity.

<Cell proliferation evaluation>
Each sample was placed in a 48-well plate, seeded with MC3T3-E1 cells (1.0×10 ⁴ cells/0.5 ml, well), and cultured at 37° C. and a CO ₂ concentration of 5%. On the 1st, 3rd, 5th and 7th days from the start of culture, cell proliferation was evaluated based on enzyme activity derived from living cells using WST-8 (Cell Counting Kit-8, manufactured by Dojindo Laboratories). In the cell proliferation evaluation, the higher the absorbance, the higher the enzymatic activity derived from living cells.

<Cytotoxicity evaluation>
Each sample was placed in a 48-well plate, seeded with MC3T3-E1 cells (1.0×10 ⁴ cells/0.5 ml, well), and cultured at 37° C. and a CO ₂ concentration of 5%. On days 1, 3, 5, and 7 from the start of culture, Cytotoxicity LDH Assay Kit-WST (manufactured by Dojindo Laboratories) was used to detect cytotoxicity based on lactate dehydrogenase (LDH) activity derived from dead cells. evaluated. In the cytotoxicity evaluation, higher absorbance indicates higher LDH activity.

<Preparation Example 1> Preparation of Graphene Oxide Dispersion (1) A graphene oxide dispersion was synthesized by the following steps. 15 g of graphite (Z-25 manufactured by Ito Graphite Co., Ltd.) and 640 g of sulfuric acid (manufactured by FUJIFILM Wako Pure Chemical Co., Ltd.) are placed in a reaction vessel in advance, and potassium permanganate (FUJIFILM Wako Pure Chemical Co., Ltd.) is added while adjusting the temperature to 30 ° C. product) was added. After charging, the temperature was raised to 35° C. for 30 minutes and the reaction was allowed to proceed for 2 hours. After the reaction, 1070 ml of water and 42 ml of 30% hydrogen peroxide solution (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to the reaction solution to terminate the reaction. The resulting reaction solution was purified by static sedimentation by repeatedly removing the supernatant and redispersing with ion-exchanged water. After the purification, a peeling operation was performed using a homogenizer to prepare a graphene oxide dispersion (1).

<Example 1> Preparation of antibacterial agent composition (1) Using graphene oxide dispersion (1) obtained in Preparation Example 1 and zinc acetate as a reagent, graphene oxide (hereinafter also referred to as GO) and zinc acetate were prepared. (Hereinafter, also referred to as Zn), a dispersion was prepared using N-methylpyrrolidone so that the concentrations of 0.01% by mass and 0.001% by mass, respectively, were used as an antibacterial agent composition (1). .

<Example 2> Preparation of antibacterial agent composition (2) A dispersion was prepared in the same manner as in Example 1, except that the concentration of zinc acetate was 0.01% by mass. did.

<Example 3> Preparation of antibacterial agent composition (3) A dispersion was prepared in the same manner as in Example 1, except that the concentration of zinc acetate was 0.1% by mass. did.

<Example 4> Preparation of antibacterial agent composition (4) A dispersion was prepared in the same manner as in Example 1, except that the concentration of zinc acetate was changed to 1% by mass, and used as antibacterial agent composition (4).

<Examples 5 to 8> Preparation of antibacterial materials (1) to (4) A scaffold (Teldermis (registered trademark), Olympus Termo Biomaterial) (hereinafter also referred to as Ter) molded to 6 x 6 x 3 mm was prepared. , respectively, immersed in 5 mL of the antibacterial agent compositions (1) to (4) obtained in Examples 1 to 4, washed with ethanol, and dried to obtain antibacterial materials (1) to (4).

<Comparative Example 1> Preparation of Comparative Antibacterial Material (1) Comparative antibacterial material (1) was obtained in the same manner as in Examples 5 to 8, except that N-methylpyrrolidone was used instead of the antibacterial agent composition.

<Comparative Example 2> Preparation of Comparative Antibacterial Material (2) In the same manner as in Examples 5 to 8, except that a 0.01% by mass N-methylpyrrolidone dispersion of graphene oxide was used instead of the antibacterial agent composition. A comparative antimicrobial material (2) was obtained.

<Comparative Example 3> Preparation of comparative antibacterial material (3) Comparative materials were prepared in the same manner as in Examples 5 to 8, except that a 0.01% by mass zinc acetate N-methylpyrrolidone solution was used instead of the antibacterial agent composition. An antimicrobial material (3) was obtained.

<Comparative Example 4> Preparation of Comparative Antibacterial Material (4) Comparative materials were prepared in the same manner as in Examples 5 to 8, except that a 0.1 mass% zinc acetate N-methylpyrrolidone solution was used instead of the antibacterial agent composition. An antimicrobial material (4) was obtained.

The antibacterial material (2) obtained in Example 6 and the comparative antibacterial materials (1) to (3) obtained in Comparative Examples 1 to 3 were subjected to Raman spectrum measurement. The results are shown in Figures 1-4.
From FIG. 1, it became clear that the antibacterial material (2) of Example 6 has a G band and contains a carbon material having a G band.

The antibacterial materials (2) and (3) obtained in Examples 6 and 7 and the comparative antibacterial materials (1) to (4) obtained in Comparative Examples 1 to 4 were subjected to XRF measurement. Table 1 shows the results.
From Table 1, it is clear that antibacterial materials (2) and (3) carry zinc.

XRD measurement was performed on the antibacterial materials (2) and (3) obtained in Examples 6 and 7 and the comparative antibacterial materials (1) to (4) obtained in Comparative Examples 1 to 4. The results are shown in Figures 5-10.
In FIGS. 5, ₆ , 9, and 10, peaks derived from the structure represented by Na ₆ (ZnPO ₄ ) 6.8H ₂ O were observed, antibacterial materials (2) and (3) and comparative antibacterial material (3). , (4) contained a double salt represented by Na ₆ (ZnPO ₄ ) _{6.8H 2} O.

The antibacterial material (2) obtained in Example 6 and the comparative antibacterial materials (1) to (3) obtained in Comparative Examples 1 to 3 were observed by SEM. SEM photographs are shown in Figures 11-16.
From the comparison between FIG. 15 and FIG. 16, it is clear that the inclusion of GO and Zn results in the presence of more fine particles than in the case of Zn alone, indicating that Zn has a high dispersibility.

For the antibacterial materials (1) to (4) obtained in Examples 5 to 8 and the comparative antibacterial materials (1) and (2) obtained in Comparative Examples 1 and 2, evaluation of antibacterial properties against S. mutans (1) did The results are shown in FIG.
The antibacterial material (2) obtained in Example 6 and the comparative antibacterial materials (1) to (3) obtained in Comparative Examples 1 to 3 were evaluated for antibacterial properties (2) against S. mutans. The results are shown in FIG.
Regarding the antibacterial material (2) obtained in Example 6 and the comparative antibacterial materials (1) to (3) obtained in Comparative Examples 1 to 3, A. naeslundii antibacterial evaluation (2) was performed. The results are shown in FIG.
From the results of FIGS. 17 to 19, the antibacterial material of the present invention containing a carbon material having a G band in the Raman spectrum and a zinc component is a comparative antibacterial material (1) containing neither a carbon material nor a zinc component. It became clear that the antibacterial performance is superior to the comparative antibacterial materials (2) and (3) containing only the antibacterial agent. In addition, in FIG. 17, the results of the antibacterial materials (3) and (4) of Examples 7 and 8 were below the detection limit.

Antibacterial materials (1) to (4) obtained in Examples 5 to 8 and comparative antibacterial materials (1) to (3) obtained in Comparative Examples 1 to 3 were evaluated for cell proliferation and cytotoxicity. . The results are shown in Figures 20-23.
From the results of FIGS. 20 to 23, it is clear that the antibacterial materials (1) to (4) of the present invention have little effect on cell proliferation and little cytotoxicity even when the zinc concentration is increased.

<Example 9> Preparation of antibacterial agent composition (5) Using the graphene oxide dispersion (1) obtained in Preparation Example 1 and zinc sulfate as a reagent, the concentrations of graphene oxide and zinc sulfate were each 0.01% by mass. A dispersion was prepared using water so as to obtain an antibacterial agent composition (5).

<Example 10> Preparation of antibacterial material (5) A piece of gauze cut to 10 x 10 mm was immersed in 0.1 mL of the antibacterial agent composition (5) obtained in Example 9, dried, and prepared into antibacterial material (5). got

<Comparative Example 5> Preparation of Comparative Antibacterial Material (5) Comparative antibacterial material (5) was prepared in the same manner as in Example 10, except that an aqueous dispersion of 0.001% by mass of graphene oxide was used instead of the antibacterial agent composition. ).

<Comparative Example 6> Preparation of Comparative Antibacterial Material (6) Comparative antibacterial material (6) was prepared in the same manner as in Example 10, except that an aqueous dispersion of 0.01% by mass of graphene oxide was used instead of the antibacterial agent composition. ).

<Comparative Example 7> Preparation of Comparative Antibacterial Material (7) Comparative antibacterial material (7) was prepared in the same manner as in Example 10, except that a 0.01% by mass aqueous solution of zinc sulfate was used instead of the antibacterial agent composition. Obtained.

Unprocessed gauze as the antibacterial material (5) obtained in Example 10, the comparative antibacterial materials (6) and (7) obtained in Comparative Examples 6 and 7, and the comparative antibacterial material (8) (Comparative Example 8) , a Raman spectrum measurement was performed. The results are shown in Figures 24-27.
FIG. 24 reveals that the antibacterial material (5) of Example 10 has a G band and contains a carbon material having a G band.

The antibacterial material (5) obtained in Example 10, the comparative antibacterial materials (6) and (7) obtained in Comparative Examples 6 and 7, and the comparative antibacterial material (8) were subjected to XRF measurement. Table 2 shows the results.
From Table 2, it became clear that the antibacterial material (5) carried zinc.

Antibacterial property evaluation against S. mutans (3 ) was performed. The results are shown in FIG.
From the results of FIG. 28, the antibacterial material (5) of the present invention containing a carbon material having a G band in the Raman spectrum and a zinc component has better antibacterial performance than the comparative antibacterial materials (5), (6), and (8). proved to be superior.

Claims (5)

An antimicrobial composition comprising graphene oxide and a zinc component. 2. The antibacterial agent composition according to claim 1 , wherein the mass ratio of the graphene oxide and the zinc component ( graphene oxide /zinc) is 1/30 to 300/1. An antibacterial material comprising the antibacterial agent composition according to claim 1 or 2 and a substrate. 4. The antibacterial material according to claim 3 , wherein the content of the zinc component is 0.01 to 1% by mass with respect to 100% by mass of the antibacterial material. 5. An antimicrobial material according to claim 3 or 4 , characterized in that said substrate is a porous scaffold.

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