阅读说明：本技术 水分散性的微粒 (Water-dispersible microparticles ) 是由 白马弘文 于 2019-09-20 设计创作，主要内容包括：本发明的技术问题在于提供一种以抑制生物被膜的形成和/或除去形成的生物被膜的制剂的形式,可以在水中分散,并且在住宅用水区域等环境下其效果持续,并且安全性高,低环境负荷的材料。本发明涉及一种水分散性的微粒,其包含多元羧酸衍生物和药剂构成的复合物。(The present invention has been made in view of the above problems, and it is an object of the present invention to provide a material which can be dispersed in water in the form of a preparation for inhibiting biofilm formation and/or removing biofilm formed, and which has a sustained effect in an environment such as a residential water area, and which is highly safe and has a low environmental load. The present invention relates to a water-dispersible microparticle comprising a complex of a polycarboxylic acid derivative and a drug.)

1. A water-dispersible microparticle comprising a complex of a polycarboxylic acid derivative and a pharmaceutical agent.

2. The microparticle of claim 1, wherein the microparticle is an association of the complex.

3. The microparticle of claim 1 or 2, wherein the agent comprises an antimicrobial peptide.

4. The microparticle of any one of claims 1 to 3, wherein the agent comprises epsilon-poly-L-lysine.

5. The fine particles according to any one of claims 1 to 4, wherein the polycarboxylic acid derivative is a compound obtained by condensing a polycarboxylic acid with a propargyl amino acid of the following general formula (1),

[ chemical formula 5]

Wherein R represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms, wherein a plurality of hydrogen atoms in the hydrocarbon group may be substituted with a nitrogen atom, an oxygen atom or a sulfur atom, and n represents an integer of 1 to 3.

6. The microparticle according to any one of claims 1 to 5, wherein the polycarboxylic acid is at least one selected from the group consisting of polyglutamic acid, polyacrylic acid, alginic acid and salts thereof.

7. The microparticles according to any one of claims 1 to 6, wherein the average particle diameter is 0.05 to 0.7 μm.

8. The microparticle of any one of claims 1 to 7, wherein the molar ratio of the pharmaceutical agent to the propargylamino group of the polycarboxylic acid derivative is from 0.1 to 0.75.

9. An antimicrobial agent and/or biofilm remover comprising the microparticle according to any one of claims 1 to 8.

Technical Field

The present invention relates to a biofilm removing material produced by microorganisms, and more particularly, to a biofilm removing composition which can effectively inhibit and remove a biofilm even in an environment where a biofilm is easily formed or in a state where a biofilm is formed.

Background

Biofilms are structures produced by microorganisms and adhered to surfaces of materials, are known as so-called slime (sticky matter), and are involved in generation of pathogens, corrosion of materials, and the like in all environments such as medical treatment, life, and industry, and cause many problems.

For example, in medical environments such as hospitals and nursing care, the generation of pathogenic bacteria (chemical-resistant bacteria) that have acquired resistance to drugs such as antibacterial agents and antibiotics has become a problem. Chemical-resistant bacteria form a biofilm to create an environment that can easily resist chemicals and external pressure. In addition, periodontal biofilms occurring in the oral cavity and the like of a care recipient can cause serious diseases such as aspiration pneumonia. Further, in residential water areas around kitchens, bathrooms, toilets and the like, unpleasant odor, food poisoning, material corrosion and the like due to biofilm are also brought about.

In the case of a biofilm, microorganisms, which have adhered to the surface of a material, form and grow in the process of proliferation of the microorganisms, and are released to the outside as colonies containing the microorganisms and pathogenic substances. Once formed, biofilms exhibit resistance to environmental stress, antimicrobial agents, and the like, and it is difficult to remove microorganisms in biofilms.

As a method for removing a biofilm, a cleaning treatment with an oxidizing bactericide such as hypochlorous acid, ozone, or chlorine dioxide may be performed. However, since the oxidizing bactericide is highly corrosive, the application range is limited from the viewpoint of environment and safety.

In order to solve the above problems, non-oxidative biofilm bactericides have been known as alternatives to highly corrosive oxidative sterilization methods. For example, it is known that Isopropylmethylphenol (IPMP) in patent document 1 permeates periodontal pathogenic biofilm and exhibits bactericidal properties. Since IPMP is water-insoluble, when it is used as a liquid, it is necessary to use a solubilizing agent such as an organic solvent or a surfactant in combination, and there are cases where it cannot be applied to a formulation using water as a base.

Patent document 2 discloses a biofilm remover using epsilon-poly-L-lysine (epsilon-PL). In this invention, it is described that when an aqueous solution containing ε -PL is allowed to act on a biofilm, the biofilm is removed. It is known that epsilon-PL is an antimicrobial polyamino acid having high safety, has high solubility in water, and is excellent in formulation properties as an aqueous solution. However, when ε -PL is used as a coating agent for inhibiting biofilm formation, it is difficult to retain it on the material or biofilm surface because it readily dissolves out in water. In particular, depending on the use environment such as a residential water area where bacteria and biofilm are likely to propagate, the biofilm inhibiting and removing effects of e-PL may be lost.

As described above, in order to suppress the formation of biofilm, it is necessary to maintain the antibacterial effect within the range of an effective concentration at a site that becomes a foothold of microorganisms on the surface of the material. Patent document 3 describes that micelles containing an amphiphilic block copolymer and an antibacterial agent, as a drug delivery system for suppressing the formation of biofilm in the oral cavity, have a binding property with teeth and remain on the surface, thereby improving the persistence.

Non-patent document 1 describes a biofilm-resistant coating of silver nanoparticles. The biofilm-resistant effect of silver nanoparticles is considered to be that nano-sized silver particles permeate a biofilm to destroy bacteria and biofilm in the biofilm. However, silver nanoparticles have excellent antibacterial properties and biofilm resistance, but the particles are easily aggregated and their activity is significantly reduced when mixed with proteins, salts, and the like (non-patent document 2). Further, silver nanoparticles are considered to be highly safe, but have poor degradability, and may cause silver precipitation, environmental pollution, and the like.

In view of the prior art, as a preparation for effectively suppressing the removal of a biofilm, a nanomaterial such as a polymer micelle or a metal nanoparticle is exemplified, but there are many technical problems such as water dispersibility, residue on the surface of the material, durability in an environment such as a residential water area, safety, and biodegradability.

Documents of the prior art

Patent document

Patent document 1 Japanese patent No. 5573111

Patent document 2 Japanese patent No. 5982088

Patent document 3 Japanese patent No. 5771143

Non-patent document

Non-patent document 1, nanometer Research Letters, 2014, 9, 373.

Non-patent document 2 journal of the society for fungi and mildew prevention of Japan (J.Antibactt.Antifunging.Agents), 2018, 46, 7, 277-284.

Disclosure of Invention

Problems to be solved by the invention

The invention provides a material which can be dispersed in water as a preparation for inhibiting the formation and/or removal of a biofilm, has a sustained effect even in an environment such as a residential water area, and has high safety and low environmental load.

Technical scheme for solving problems

As a result of intensive studies, the inventors of the present invention have found that the above-mentioned problems can be solved by the means shown below, and have completed the present invention.

That is, the present invention has the following configuration.

(1) A water-dispersible microparticle comprising a complex of a polycarboxylic acid derivative and a pharmaceutical agent.

(2) The fine particles according to (1), wherein the fine particles are an association of the complex.

(3) The microparticle according to (1) or (2), wherein the drug contains an antimicrobial peptide.

(4) The microparticle according to any one of (1) to (3), wherein the drug contains epsilon-poly-L-lysine.

(5) The fine particles according to any one of (1) to (4), wherein the polycarboxylic acid derivative is a compound obtained by condensing a polycarboxylic acid with a propargyl amino acid of the following general formula (1).

[ chemical formula 1]

(wherein R represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; in the hydrocarbon group, a plurality of hydrogen atoms may be substituted with a nitrogen atom, an oxygen atom or a sulfur atom; and n represents an integer of 1 to 3).

(6) The microparticle according to any one of (1) to (5), wherein the polycarboxylic acid is at least one selected from the group consisting of polyglutamic acid, polyacrylic acid, alginic acid and salts thereof.

(7) The fine particles according to any one of (1) to (6), wherein the average particle diameter is from 0.05 to 0.7. mu.m.

(8) The fine particles according to any one of (1) to (7), wherein the molar ratio of the drug to the propargylamino group of the polycarboxylic acid derivative is 0.1 to 0.75.

(9) An antimicrobial agent and/or biofilm remover, which comprises the fine particles according to any one of (1) to (8).

ADVANTAGEOUS EFFECTS OF INVENTION

The fine particles of the present invention are excellent in water dispersibility and biofilm removability, can be used as a coating agent for inhibiting biofilm formation, and can continuously inhibit biofilm formation even in an aqueous environment in which bacteria easily proliferate. Further, since a raw material derived from a natural product is used, the load on the environment is small.

Drawings

Fig. 1 is a schematic diagram showing a process of forming fine particles.

Fig. 2 is an optical micrograph showing a dispersed state of fine particles in water.

FIG. 3 is an IR spectrum of the fine particles of the present invention.

Fig. 4 is a graph showing the relationship between the drug deposition amount and the biofilm inhibition rate.

Fig. 5 is a graph showing the relationship between the contact time of the agent with the biofilm and the biofilm removal rate.

Fig. 6 is a graph showing the relationship between the contact time of the agent with the biofilm and the biofilm inhibition rate.

Fig. 7 is a graph showing the relationship between the contact time of the agent with the biofilm and the number of viable bacteria in the biofilm.

Detailed Description

The present invention is a water-dispersible polymer microparticle containing a complex of a polycarboxylic acid derivative and a drug. The polycarboxylic acid derivative is a graft polymer obtained by introducing a substituent having an interaction with the above-mentioned chemical into a polycarboxylic acid. Then, the polycarboxylic acid derivative forms a strong bond such as a hydrogen bond or a covalent bond with the drug, whereby fine particles having excellent dispersibility can be formed. In addition, the microparticles can be retained on the material and the surface of the biofilm for a long period of time, and the permeability into the biofilm is improved, so that the retention of the contained drug and the persistence of the effect can be expected.

In the present invention, the above-mentioned agent is preferably an agent having an antimicrobial, microbicidal, disinfectant or microbe-removing action against microorganisms associated with biofilm production. Specifically, there may be mentioned organic antimicrobial (bacteria) agents such as antimicrobial (bacteria) peptides, quaternary ammonium salts, biguanide compounds, thiazoline compounds, imidazole compounds, glyceride compounds, carbamate compounds, sulfonamide compounds, pyridine compounds, phenol compounds and halogen compounds. Among them, antimicrobial (bacterial) peptides are preferable from the viewpoint of low environmental load and high safety. Preferred examples of the antimicrobial (bacterial) peptide include epsilon-poly-L-lysine (epsilon-PL), protamine, lysozyme, nisin, defensin, colistin, antimicrobial peptide (Indolicidin), melittin (melittin), and the like, and epsilon-PL is more preferred from the viewpoint of availability and versatility of raw materials. The epsilon-PL is an amino acid homopolymer in which the amino group at the epsilon position of L-lysine, which is one of essential amino acids, is linear with about 25 to 35 lysine bonds forming a peptide bond with a carboxyl group. Polylysine as described above is available, for example, from Toronto Research Chemicals.

In the present invention, the polycarboxylic acid derivative is a derivative obtained by modifying a carboxylic acid having 2 or more carboxyl groups in the molecule with at least 1 or more propargyl amino acids. Examples of the carboxylic acid having 2 or more carboxyl groups in the molecule include low-molecular carboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, citric acid, and tartaric acid; high molecular synthetic polymers such as polyacrylic acid and polymethacrylic acid; natural polysaccharides such as pectin, alginic acid, hyaluronic acid, and carboxymethyl cellulose; poly-gamma-glutamic acid, poly-alpha-glutamic acid, polyaspartic acid and other polypeptides. Among them, poly-gamma-glutamic acid, polyacrylic acid, alginic acid, and salts thereof are preferable because they have a plurality of crosslinking points in the molecule and are highly safe.

In the present invention, a propargyl amino acid is an amino acid derivative represented by the general formula (1). Wherein R is a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms. In the above hydrocarbon group, a plurality of hydrogen atoms may be replaced by nitrogen atoms, oxygen atoms or sulfur atoms. In the formula, R may be the same or different. In the formula, n represents an integer of 1 to 3.

[ chemical formula 2]

The amino acid constituting the propargyl amino acid of the general formula (1) may be used arbitrarily without limitation, and commercially available products are available, but from the viewpoint of safety, amino acids constituting biological proteins are preferably used. Examples of the amino acids constituting the biological protein include asparagine, aspartic acid, alanine, arginine, cysteine/cystine, glutamine, glutamic acid, glycine, proline, serine, tyrosine, isoleucine, leucine, valine, histidine, lysine (lysine), methionine, phenylalanine, threonine (threonine), and tryptophan, and L-form is preferably used. Among these, glycine, which is a material that is easily purchased, highly safe, and has the simplest structure, is preferably used.

The propargyl amino acid of the general formula (1) can be obtained by condensing the amino acid with propargyl alcohol, a propargyl halide or the like as shown in the following reaction formula (2). More specifically, the propargyl amino acid can be easily produced by mixing and dissolving the amino acid with propargyl alcohol or the like, and adding a condensing agent such as thionyl chloride to the resulting mixture to carry out a reaction. In the formula, X represents a hydroxyl group or a halogen group. In the formula, R represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms. In the above hydrocarbon group, a plurality of hydrogen atoms may be replaced by nitrogen atoms, oxygen atoms or sulfur atoms. In the formula, R may be the same or different. In the formula, n represents an integer of 1 to 3.

[ chemical formula 3]

In the present invention, the polycarboxylic acid derivative can be obtained by condensing a propargyl amino acid of the general formula (1) with a carboxyl group of the above polycarboxylic acid as shown in the following reaction formula (3). The method for condensing the polycarboxylic acid with the propargyl amino acid can be carried out by reacting the polycarboxylic acid with the propargyl amino acid at room temperature (1 to 30 ℃) in the presence of a dehydration condensing agent. More specifically, the compound can be produced by dissolving a polycarboxylic acid in a solvent, adding the propargyl amino acid and the condensing agent prepared above, and if necessary, adding a reaction accelerator to the resulting mixture to carry out a reaction. In this case, the propargyl amino acid as the carboxylic acid active group is preferably 0.25 to 1.0 equivalent to the carboxylic acid unit constituting the polycarboxylic acid. When the molar ratio of the propargyl amino acid to the carboxylic acid unit is too small, the reaction may be insufficient, and the modification rate may be lowered. On the other hand, when the molar ratio of the propargyl amino acid to the carboxylic acid unit is too large, the amounts of unreacted raw materials and by-products may increase.

[ chemical formula 4]

In the present invention, the dehydration-condensation agent is N, N '-Dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC), O- (benzotriazol-1-yl) -N, N' -tetramethylonium Hexafluorophosphate (HBTU), or the like. The dehydration condensation agent is preferably added in an amount of 0.25 to 1.0 equivalent to the carboxylic acid unit constituting the polycarboxylic acid.

In the present invention, the reaction solvent for the condensation reaction is not particularly limited as long as it is a solvent for dissolving the polycarboxylic acid, and examples thereof include water, Dimethylformamide (DMF), Dimethylsulfoxide (DMSO), and Tetrahydrofuran (THF).

In the present invention, when the condensation reaction is promoted, it is preferable to add a base, a catalyst and an active ester to the reaction mixture as a reaction promoter. As the base, triethylamine (Et) is exemplified₃And amines such as N), N-diisopropylethylamine, and carbonates such as sodium carbonate and sodium hydrogen carbonate. Further, as the catalyst, N-dimethyl-4-aminopyridine (DMAP) and the like can be mentioned. In addition, as the active ester, esters as reaction intermediates include 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide (NHS). When the base is added, it is preferable to add 0.5 to 1.5 equivalents to the carboxylic acid unit constituting the polycarboxylic acid. When the active ester is added, it is preferable to add 0.25 to 1.0 equivalent to the carboxylic acid unit constituting the polycarboxylic acid.

In the above condensation reaction, the modification ratio of the propargyl amino acid to the polycarboxylic acid can be changed by changing the amounts of the respective raw materials such as the propargyl amino acid and the dehydration condensation agent to be added. In the case of using a polycarboxylic acid, the modification ratio of the propargyl amino acid is preferably 1 to 55%, more preferably 5 to 50%, and still more preferably 10 to 50% per carboxylic acid unit. When the content exceeds 55%, the solubility in the solvent is poor. The size of the fine particles in the case of encapsulating the drug can be adjusted by adjusting the modification ratio of the propargyl amino acid to the polycarboxylic acid to the above range.

In the present invention, when the polycarboxylic acid derivative after the reaction is recovered, an organic solvent (poor solvent) in which the polycarboxylic acid derivative is insoluble is added to the reaction solution to precipitate the polycarboxylic acid derivative. The poor solvent is not particularly limited as long as it is a solvent in which the polycarboxylic acid derivative is insoluble and has good dispersibility, and examples thereof include acetone, methanol, ethanol, isopropanol, and acetate. The lean solvent is preferably in an amount of 3 to 10 times the reaction solvent. When the amount of the poor solvent added is too small, the polycarboxylic acid derivative may not be sufficiently precipitated. In addition, when the amount of the lean solvent added is too large, the cost for cleaning increases.

In the present invention, when removing impurities after recovering the polycarboxylic acid derivative, a method of purifying the polycarboxylic acid derivative by a so-called solution reprecipitation method in which the polycarboxylic acid derivative is dissolved in a good solvent such as water, and then the poor solvent is added while stirring at room temperature to reprecipitate the polycarboxylic acid derivative is exemplified. This operation is repeated as often as necessary 1 to 3 times to reduce the impurities to below the detection limit. The good solvent is not particularly limited as long as it can dissolve the polycarboxylic acid derivative, but is preferably water in view of the influence on safety and environment. The poor solvent is not particularly limited as long as it is a solvent in which the polycarboxylic acid derivative is hardly soluble, and preferable examples thereof include acetone, methanol, and ethanol.

The amount of the good solvent can be adjusted depending on the solubility of the polycarboxylic acid derivative, but a high concentration is desired from the viewpoint of industrial productivity. For example, the amount of the good solvent is preferably in the range of 10 to 60 wt%, and more preferably in the range of 10 to 30 wt%.

The amount of the lean solvent may be adjusted depending on the solubility of the polycarboxylic acid derivative, and may be the minimum amount of the polycarboxylic acid derivative to be precipitated. For example, the lean solvent is preferably 2 to 12 times the amount of the good solvent.

By repeating the above purification, impurities can be further reduced. The production cost can be reduced by 1 to 2 times.

The obtained polycarboxylic acid derivative is a compound obtained by condensing a carboxyl group of a polycarboxylic acid with a propargyl amino acid, and is an amphiphilic molecule composed of a hydrophilic site of the carboxyl group and a hydrophobic site of the propargyl amino group.

In the present invention, the polycarboxylic acid derivative and the chemical are allowed to coexist in a compatible solvent (in the reaction solution), whereby a complex in which the propargyl amino acid moiety of the polycarboxylic acid derivative is bonded to the chemical is formed, and the complex can be further self-organized into fine particles (fig. 1). The particle size of the fine particles can be adjusted by the modification ratio of the propargyl amino acid to the carboxyl group of the polycarboxylic acid derivative and the concentration of the drug. The average particle diameter of the fine particles is not particularly limited, but is preferably in the range of 0.05 μm to 0.7. mu.m. When the particle size is too large, the biofilm cannot penetrate into the inside thereof.

In the present invention, the amount of the chemical to be added to the reaction liquid for producing fine particles is preferably in the range of 0.1 to 0.75, more preferably 0.15 to 0.65, in terms of the molar ratio of the chemical to the propargylamino group of the polycarboxylic acid derivative, relative to the polycarboxylic acid derivative. When the molar ratio is too large, the fine particles tend to aggregate, and redispersibility in a solution may be poor. If the molar ratio is too small, fine particles effective for the biofilm may not be obtained.

In the present invention, the solvent used in the preparation of the fine particles is not particularly limited as long as both the polycarboxylic acid derivative and the drug are dissolved in the solvent, and water is preferred from the viewpoint of environment and safety. Additives such as a dispersant, a surfactant, and a solubilizer can be used as necessary.

In the present invention, the concentration of the drug in the reaction solution when the fine particles are prepared can be determined by referring to the values of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) when the antimicrobial (bacterial) agent is used. For example, when ε -PL is used, the content is preferably 0.01 to 1 wt%, more preferably 0.02 to 0.1 wt%. If the amount is more than 1 wt%, the composition may be disadvantageous in view of raw material components, and if the amount is less than 0.01 wt%, the biofilm resistance may be lowered.

In the present invention, the concentration of the polycarboxylic acid derivative in the reaction solution for producing the fine particles can be determined by the balance between the effective concentration of the above-mentioned chemical and the molar ratio. For example, the content is preferably in the range of 0.1 to 1 wt%, more preferably in the range of 0.5 to 1 wt%. When the amount exceeds 1 wt%, the amount is sometimes disadvantageous in terms of raw material cost. Less than 0.1 wt%, the concentration of the drug in the fine particles is reduced, and thus the biofilm resistance may be reduced.

The obtained microparticles are used as a microparticle dispersion for antimicrobial, microbe-killing, disinfection and removal of microbes and for removal of biofilm when water is used as a reaction solvent. Here, as shown in fig. 2, the fine particle dispersion means that fine particles are uniformly present in the dispersion without forming aggregates. The fine particle dispersion may be prepared by diluting or concentrating the biofilm formed, as necessary, and then spraying or scattering the biofilm. After the dispersion is sprayed or sprinkled on the biofilm, the biofilm can be left at room temperature for at least 1 minute. In addition, a surfactant, a proteolytic enzyme, a polysaccharide degrading enzyme, a DNA degrading enzyme, a bleaching agent, and the like may be added and used as necessary.

In the present invention, the fine particles may be used by spraying or misting the biofilm already formed, or may be applied in advance to the surface of a material on which the biofilm is not formed, thereby suppressing the adhesion of microorganisms to the surface of the material and the formation of the biofilm (biofilm-resistant coating). The coating film is not likely to flow out by water because of interaction between the carboxylic acid groups on the surface of the fine particles and the surface of the substrate or the fine particles are physically adsorbed on the surface of the substrate, and the particles are immobilized on the surface of the substrate, thereby maintaining the antimicrobial property and suppressing the formation of a biofilm for a long period of time.

In the present invention, when the fine particles are used as a biofilm-resistant coating layer, the amount of the agent adhered to the surface of the material by the fine particles is preferably 0.04 to 0.12. mu.g/cm²The range of (1). When the amount of the drug deposited is too small, the inhibition of the formation of the biofilm may not be sufficiently exhibited. In addition, when the amount of the drug deposited is too large, the present invention is disadvantageous in terms of cost effect.

When the surface of the above-mentioned material is subjected to a biofilm coating treatment, examples of substrates that can be coated include ceramics, metals, metal oxides, plastics, rubbers, minerals, and wood.

In the present invention, since the fine particles are made of a material having high safety against the ecosystem and can be dispersed in water, they are used not only in general households but also in many commercial applications such as environments in water areas for houses such as kitchens, toilets and bathtubs as a biofilm treatment in medical places such as hospitals and nursing places.

Examples

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

(preparation of Glycine-2-Propyn-1-yl-ester (GPE))

A mixture of 30mL of glycine (Nacalai Tesque, Ltd.) and 2.1g of 2-propyn-1-ol (Nacalai Tesque, Ltd.) was prepared, and 2.4mL of thionyl chloride (Nacalai Tesque, Ltd.) was added thereto at room temperature. The reaction solution was stirred at room temperature for 2 hours, and further stirred at 50 ℃ for 2 hours. The reaction solution was cooled to 5 ℃ and 90mL of ethyl acetate was added to obtain a precipitate. The precipitate was separated by filtration and washed 3 times with 30mL of ethyl acetate, and dried (50 ℃ C., 12 hours) to give 2-propyn-1-yl glycinate (2-propynyl aminoacetate, GPE)

(measurement of the modification ratio of propargyl amino acid)

Modification ratio of propargyl amino acid to carboxyl group constituting polycarboxylic acid derivative by measurement of D₂In O¹H-NMR spectroscopy (BRUKER, MR 400). For the calculation of the modification ratio, the integrated intensity ratio of the α hydrogens of the carboxyl group modified with a propargyl amino acid to the unmodified carboxyl group was measured and determined by the following formula.

Modification ratio (%) ([ α hydrogen of modified carboxyl group ]/[ (α hydrogen of unmodified carboxyl group) + (α hydrogen of modified carboxyl group) ] × 100

(measurement of particle diameter)

0.1mL of the fine particle dispersion was added to a fractionation tank to which pure water was added, and the diluted solution was stirred. The average particle size of the fine particles in the dispersion was measured by irradiating the dispersion with a laser beam from a nanometer particle size distribution measuring apparatus (SALD-7500 nano, Shimadzu corporation) to measure the refractive index.

(measurement of IR Spectrum)

The microparticle dispersion was centrifuged (4000rpm) and precipitated, then washed 3 times with 2mL of pure water, vacuum-dried at room temperature for 24 hours, and measured for IR spectrum by an infrared spectrometer (FT-IR device manufactured by Perkinelmer, model: SpectrumOne/MultiScope).

(measurement of biofilm amount)

After the incubation, the test piece was taken out, washed with physiological saline, and the amount of biofilm attached to the surface of the test piece was measured. The biofilm measurement method was performed by CV (crystal violet) staining. That is, after immersing the test piece in a 0.1 wt% CV aqueous solution for 30 minutes, the excess CV was washed with water, the stained portion was extracted with 1mL of 98% ethanol, and the absorbance at 500nm was measured.

(measurement of viable cell count)

The number of viable bacteria in the biofilm was determined by adding 1mL of physiological saline to the surface material to which the biofilm was adhered, disrupting the biofilm by an ultrasonic cleaner, collecting the eluate, and diluting the eluate to 10% with physiological saline²～10⁸Inoculating the strain into 3M Petrifilm^TMAnd (4) a culture medium. After 1 week of incubation at 30 ℃ the colonies were counted and the viable count determined.

Production example 1 production of GPE-processed Gamma-PGA (40)

0.5g of sodium polyglutamate (. gamma. -PGA) manufactured by Toyobo Co., Ltd, and 6mL of water were added thereto, and the mixture was stirred at room temperature. To this solution, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC) and 1-hydroxybenzotriazole (HOBt) were added in an amount of 0.75 equivalent to the carboxylic acid unit constituting γ -PGA, and the reaction was carried out at room temperature for 24 hours. After the reaction, 35mL of acetone was added to precipitate a polymer. The obtained polymer was washed with acetone and dried. After drying, the crude polymer was dissolved in 5.5mL of water, 60mL of acetone was added thereto, reprecipitation was performed, and the mixture was filtered and taken out. Vacuum drying at 60 deg.c for 12 hr to obtain GPE gamma-PGA as the target matter. By passing¹H-NMR(D₂O), it was confirmed that the modification ratio of GPE to the carboxylic acid unit of γ -PGA was 40%. The obtained polycarboxylic acid derivative was GPE-converted to γ -PGA (40).

Production example 2 production of GPE-modified Gamma-PGA (25)

The polycarboxylic acid derivative was produced by the method described in production example 1, except that GPE, WSC, and HOBt were used in an amount of 0.5 equivalent as a raw material preparation amount. By passing¹H-NMR(D₂O), it was confirmed that the modification ratio of GPE to the carboxylic acid unit of γ -PGA was 25%. The obtained polycarboxylic acid derivative was GPE-converted to γ -PGA (25).

Production example 3 production of GPE-modified Gamma-PGA (15)

As a raw materialPreparation of polycarboxylic acid derivative according to the method described in preparation example 1 was carried out except that 0.25 equivalents of GPE, WSC and HOBt were used. By passing¹H-NMR(D₂O) confirmed that the modification ratio of GPE to the carboxylic acid unit of γ -PGA was 15%. The obtained polycarboxylic acid derivative was GPE-converted to γ -PGA (15).

Production example 4 production of GPE-formed PAC (19)

Polycarboxylic acid derivatives were produced by the method described in production example 1 except that an aqueous sodium polyacrylate solution (PAC) produced by japan catalyst co. By passing¹H-NMR(D₂O) confirmed that the modification ratio of GPE to the carboxylic acid unit of PAC was 19%. The obtained polycarboxylic acid derivative was used as GPE-modified PAC (19).

Production example 5 production of GPE ALG 22

A polycarboxylic acid derivative was produced by the method described in production example 1, except that sodium Alginate (ALG) manufactured by Nacalai Tesque corporation was used as a raw material. By passing¹H-NMR(D₂O) confirmed that GPE had a modification ratio of 22% to the carboxylic acid unit of ALG. The obtained polycarboxylic acid derivative was converted into GPE-modified ALG (22).

(preparation of Fine particle Dispersion of GPE-modified. gamma. -PGA/. epsilon. -PL)

0.005g of GPE-made gamma-PGA obtained in production examples 1 to 3 was dissolved in 0.5mL of water at room temperature, 0.02 to 0.18 wt% of an aqueous solution of ε -PL (hydrochloride salt) manufactured by Toronto Research Chemicals, 0.5mL was added to the solution, and the mixture was stirred at room temperature for 1 minute to obtain a fine particle dispersion. The average particle size of the fine particles measured using the obtained dispersion is shown in table 1. It was confirmed that fine particles having an average particle diameter of 0.064 to 0.624 μm, wherein the molar ratio of ε -PL to GPE group of the polymer was 0.16 to 0.63, were obtained. When the molar ratio exceeds 0.75, the particles tend to aggregate and the dispersibility tends to deteriorate. When a mixture of gamma-PGA and epsilon-PL which had not been converted to GPE was prepared, aggregates were observed immediately, and no fine particles were obtained as described above. In order to analyze the components of the fine particles obtained in example 1, the fine particles were centrifuged by a centrifuge, washed with water, dried, and measured for an IR spectrum by an infrared spectrometer (shimadzu corporation). The IR spectrum of the centrifuged fine particles is shown in FIG. 3. The resulting microparticles observed absorption peaks derived from GPE-treated γ -PGA and ε -PL, and were confirmed to be compositions containing these two components.

[ Table 1]

Test example 1 preparation of P.fluoroscens bacterial suspension

Gram-negative bacilli p. fluoroscens, which have high biofilm productivity, were used for evaluation of biofilm resistance. Fluoroscens dry standards were purchased through NBRC No.14160 and recovered by the following method. 8.4g of Daigo 802 medium manufactured by Wako pure chemical industries, Ltd, and 300mL of pure water were weighed in a beaker, and suspended by stirring at room temperature for about 30 minutes. After sterilizing the resuscitation solution in a high pressure sterilizer, 0.2mL of the solution was taken with a micropipette, and added to an ampoule of a dry standard substance of p. The flask was charged with 25mL of SCD medium (Soybean-Casein Digest Broth) prepared separately and manufactured by Wako pure chemical industries, Ltd., and then 100. mu.L of the above suspended P.fluoroscens suspension was added thereto and cultured by a constant temperature shaking culture machine at 30 ℃ for 24 hours at a speed of 150 rpm. After the culture, the bacterial liquid was added to 80% glycerol and stored at-80 ℃.

(test example 2) test for coating of biofilm with Fine particle Dispersion

As a test solution for the anti-biofilm coating film, a fine particle dispersion with ε -PL was prepared by using the GPE-modified polycarboxylic acids of production examples 1 to 5. The fine particle dispersion was prepared by mixing the two aqueous solutions so that the concentration of the GPE-modified polycarboxylic acid in the dispersion was 0.5% by weight and the concentration of ε -PL in the dispersion was 0.045% by weight. For comparison, an aqueous solution of ε -PL monomer, an ethanol solution of IPMP (4-isopropyl-3-methylphenol) manufactured by Wako pure chemical industries, and an aqueous dispersion of silver nanoparticles (manufactured by Nippon Ion Co., Ltd.) were used to prepare a solution having a concentrationIs 0.045 wt%. Each antimicrobial component was applied to a test piece (1 cm made of vinyl chloride) as the amount of each test liquid applied²) And the amount of each antibacterial component attached is 0.04 to 0.12. mu.g/cm²And vacuum-dried at 30 ℃ for 12 hours. The test pieces coated with each chemical were subjected to washing conditions (25 ℃, 350rpm, 1 minute stirring) which were assumed to be the environment of the water area of the house. The washed test piece was immersed in a medium containing the bacterial suspension prepared in test example 1 (p. fluoroscens bacterial suspension was prepared to 10 using SCD medium)⁶cfu/mL) was cultured at 30 ℃ for 48 hours to form a biofilm. After the incubation, the test piece was taken out and washed with a physiological saline solution to measure the amount of biofilm adhering to the surface of the test piece. The biofilm inhibition rate was calculated as follows.

Biofilm inhibition ratio (%) (measured value of untreated test piece-measured value of test piece coated with test solution)/(measured value of untreated test piece) × 100

The results of the amount of each coating agent deposited and the biofilm inhibition ratio are shown in table 2 and fig. 4. The biofilm coating of the GPE polycarboxylic acid/epsilon-PL fine particles was confirmed to be inhibited by about 85% to the maximum extent relative to the untreated test piece. On the other hand, when the coating with the epsilon-PL monomer or silver nanoparticles is applied under water washing conditions, the biofilm inhibition rate, i.e., biofilm adhesion inhibition performance, is reduced. This is presumably because the agent contained in the coating layer is easily removed by washing with water. In addition, in the silver nanoparticle coating, the amount of attached biofilm tends to be higher as the amount of attached product is larger, and inactive components derived from the product are a starting point for promoting the adhesion of biofilm and bacterial cells. On the other hand, in the case of the GPE-modified polycarboxylic acid/epsilon-PL coating, the biofilm-inhibiting effect was maintained even under the water washing condition, and it was confirmed that the effect was not sustained by the epsilon-PL monomer and the silver nanoparticles.

[ Table 2]

(test example 3) biofilm removability test after drug immersion

A48-well plate (Sumitomo Bakelite Co., Ltd.) was charged with a bacterial solution (P. fluorescens, 10) containing test example 1⁶cfu/mL) at 30 ℃ for 1 week to form a biofilm on the plate, removing the supernatant, washing with sterilized physiological saline, and drying. The test solution was prepared by the same method as in test example 2. That is, a fine particle dispersion having a concentration of 0.5 wt% for the GPE-modified polycarboxylic acid and a concentration of 0.04 wt% for the ε -PL was prepared. The biofilm removability was examined by adding 1mL of each test agent to the microplate having the biofilm formed thereon, and immersing for 1 minute, 10 minutes, 30 minutes, and 60 minutes at intervals of each immersion (contact) time. After the immersion (contact) time, the test agent was removed, and the test agent was immediately washed 2 times with 1mL of physiological saline, and after drying, the biofilm amount was measured by the CV staining method described in test example 2. The removal rate of the biofilm was calculated by the following formula.

Biofilm removal rate (%) (untreated measurement value-measurement value after test solution immersion)/(untreated measurement value) × 100

The relationship between the contact (immersion) time of each test agent with the biofilm and the biofilm removal rate is shown in table 3 and fig. 5. The aqueous solution of ε -PL showed the highest biofilm removability, and GPE-modified polycarboxylic acid/ε -PL showed 70% biofilm removability. In addition, biofilm removability is on the same scale as IPMP known as a biofilm bactericide, and higher than silver nanoparticles. The result of silver nanoparticles is a decrease in biofilm removal with prolonged contact time. This is presumably because the silver nanoparticles are aggregated by impurities such as proteins derived from biofilms and salts of culture media.

Test example 4 biofilm inhibition test after immersion (contact) with drug

1mL of each test agent was added to the biofilm-formed microplate of test example 3, and the microplate was immersed for 1 minute, 10 minutes, 30 minutes, and 60 minutes, and after the contact time, the test agent was removed, and immediately washed 2 times with 1mL of physiological saline. Here, 1mL of SCD medium was added to the microplate, and the resulting mixture was incubated at 30 ℃ for 24 hours to form a biofilm again, and the re-inhibition of the biofilm was evaluated. After the culture, the amount of biofilm was measured. The amount of biofilm was measured by CV staining as described in test example 2. The biofilm inhibition rate was calculated by the following formula.

Biofilm inhibition ratio (%) (untreated measurement value-measurement value after test solution immersion)/(untreated measurement value) × 100

The relationship between each test drug and the inhibition ratio is shown in table 3 and fig. 6. The highest inhibitory effect on PAC-based biofilms was observed in the case of GPE-modified polycarboxylic acids/. epsilon. -PL. It is known that GPE-modified polycarboxylic acid/. epsilon. -PL adheres to the biofilm surface and maintains the biofilm inhibiting effect even in an environment where a biofilm is likely to form in a culture medium.

[ Table 3]

(test example 5) biofilm Fungicide Property test

After the test agent contacts the biofilm, the bactericidal properties of the bacteria in the biofilm were tested. The test agents were added to the biofilm-formed microplate in test example 3 in an amount of 1mL, immersed for 1 minute, 10 minutes, 30 minutes and 60 minutes, removed after the lapse of the contact time, and immediately washed 2 times with 1mL of physiological saline. 1mL of physiological saline was added to the microplate, and the microplate was treated with an ultrasonic cleaner for 1 minute to disrupt the biofilm and elute the bacteria into the physiological saline. Diluting the solution to 10 deg.C with sterilized normal saline²～10⁸Inoculating to Petrifilm made by 3M^TMThe medium was incubated at 30 ℃ for 1 week, colonies were counted, and viable cell count was measured. The relationship between the drug contact time and the viable cell count is shown in table 4 and fig. 7. In examples 37 to 39, the number of viable bacteria was reduced compared to 40000cfu/well, and it was confirmed that the number of bacteria in the biofilm was reduced by immersion in the fine particle dispersion.

[ Table 4]

Industrial applicability

The fine particles of the present invention are dispersed in water, and can effectively inhibit biofilm formation or remove a biofilm by coating or immersion, and therefore have a wide range of applications, and can be used as a biofilm measure because they use a material having high safety to living bodies and the environment and have substrate adhesion.