JP2006290840A - Active oxygen species scavenging material containing platinum nanoparticle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an active oxygen species scavenging material which can scavenge active oxygen species included in a liquid such as blood and the like. <P>SOLUTION: The active oxygen species scavenging material is prepared by adhering platinum nanoparticles to a resin base. The active oxygen species scavenging material is also prepared by bringing a solution containing the platinum nanoparticles and a protection material into contact with the base material. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a reactive oxygen species removing material containing platinum nanoparticles and exhibiting excellent active oxygen removal ability. In the present specification, the term “platinum nanoparticle” refers to a nanometer-sized particle composed of a platinum group metal and / or a noble metal other than the platinum group metal and containing 25% by mass or more of platinum, and 90% or more of the particles. Is distributed in a particle size of 0.1 to 10 nm.

  2. Description of the Related Art Conventionally, treatment by blood purification such as hemodialysis has been performed for the treatment of a patient whose renal function is impaired, for example, a patient whose function of removing waste products in blood is impaired due to renal failure or the like. In blood purification treatment, low molecular weight substances such as urea, creatinine, uric acid, low molecular weight protein, and water accumulated in blood are mainly collected by contacting blood with dialysate through a semipermeable membrane in the dialyzer. Removed.

  In recent years, with the increase in long-term dialysis patients, long-term dialysis complications such as carpal tunnel syndrome have been attracting attention. Substances to be removed by hemodialysis are not only low-molecular substances such as urea and creatinine, but also low It extends from medium to high molecular weight substances such as molecular proteins. For this reason, dialysis membranes (high performance membranes) having a larger pore size than conventional ones have been used for treatment.

  By the way, it is known that not only when low molecular protein accumulates but also when reactive oxygen species accumulate in the body, it causes many diseases such as Alzheimer's in addition to aging and lifestyle-related diseases. More than 95% by mass of oxygen taken into the body by breathing becomes water through normal metabolic processes, while the remaining few percent become reactive oxygen species in the electron transport system of mitochondria and microsomes. The generated reactive oxygen species are often removed by antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. However, all of the reactive oxygen species cannot be removed from the body by these antioxidant enzymes, and some of the reactive oxygen species oxidize proteins, lipids, nucleic acids and the like. Among these oxidized substances, some are repaired by the biological defense mechanism, but some remain oxidatively damaged. Accumulation of this oxidatively damaged substance in the body is thought to lead to disease and aging.

  If reactive oxygen species in the blood can be removed by blood purification treatment, it is thought that it will lead to prevention of various diseases. However, reactive oxygen species are not present alone in the blood, but are adsorbed by large molecules and cannot pass through the pores of the dialysis membrane. Therefore, reactive oxygen species cannot be removed by blood purification treatment using osmotic pressure, and diseases induced by reactive oxygen species cannot be prevented.

  Accordingly, an object of the present invention is to provide a reactive oxygen species removing material that can remove reactive oxygen species contained in a liquid such as blood.

  As a result of intensive studies in view of the above object, the present inventors have found that the active oxygen species removing material in which platinum nanoparticles are attached to a resin substrate can remove active oxygen species by contact with a liquid containing active oxygen species. The present invention has been discovered.

  That is, the active oxygen species removing material of the present invention is characterized in that platinum nanoparticles are attached to a resin substrate.

  The active oxygen species removing material is preferably one in which a solution containing the platinum nanoparticles and the protective material is in contact with the resin substrate. The solution containing the platinum nanoparticles and the protective material comprises: (a) after refluxing a solution containing the platinum salt, the protective material, alcohol and water; and (b) from the solution to the alcohol and the water. It is preferable that (c) alcohol is added and (d) the alcohol and water are evaporated and diluted again. The protective material is preferably a polyacrylate.

  The active oxygen species removing material of the present invention is obtained by attaching platinum nanoparticles to a resin substrate. The platinum nanoparticles are excellent in scavenging ability of active oxygen species, and can act catalytically and remove active oxygen species simply by contacting the active oxygen species. Therefore, since the active oxygen species can be efficiently removed by allowing the liquid containing the active oxygen species to pass through the active oxygen species removing material, it is suitable for a dialysis membrane for blood purification treatment.

[1] Active oxygen species removing material The active oxygen species removing material comprises a resin substrate and platinum nanoparticles.
(1) Resin substrate The resin substrate is preferably porous. Since the porous resin substrate has a large surface area, many platinum nanoparticles can be attached. The shape of the resin substrate is not particularly limited, and may be granular, fibrous, or film-shaped.

  Examples of resin base materials include cellulose acetate, regenerated cellulose such as copper ammonia cellulose, cellulose derivatives, polymethyl methacrylate, polyether nylon, polyether amide, polysulfone, polyacrylonitrile, polyamide, cation exchange resin and anion exchange resin. Can be mentioned. Considering the melting temperature, it is preferably made of a thermoplastic resin such as polymethyl methacrylate, polyether nylon, polyacrylonitrile or polyamide.

  When the active oxygen species removing material is used for a dialysis membrane for blood purification treatment, the resin substrate needs to be a hollow fiber membrane. The thickness of the hollow fiber membrane for dialysis membrane is preferably 5 to 60 μm, more preferably 20 to 40 μm. The inner diameter of the hollow fiber is generally 180 to 250 μm, preferably 200 to 230 μm. Similar to a general artificial dialyzer, 5000 to 50,000 hollow fiber membranes are preferably accommodated in the housing.

(2) Platinum nanoparticles The average particle diameter of the platinum nanoparticles is 1 to 5 nm, preferably 1 to 3 nm, and particularly preferably 1.5 to 2.5 nm. Moreover, it is preferable that the particle size of platinum nanoparticles of 90% or more falls within the range of 1 to 3 nm. Thus, when the particle size distribution of platinum nanoparticles is narrow and the average particle size is in the range of 1 to 5 nm, it is considered that it is taken into the body and exhibits an antioxidant action. A solution containing platinum nanoparticles having such a narrow particle size distribution can be prepared by a manufacturing method described later.

Platinum nanoparticles exhibit a high ability to remove reactive oxygen species. The concentration ID ₅₀ of platinum nanoparticles required to halve the active oxygen species is preferably 200 μmol / L or less, and more preferably 180 μmol / L or less. Here, ID ₅₀ indicates a concentration that reduces the concentration of superoxide anion by 50%. ID ₅₀ can be determined, for example, by preparing platinum nanoparticle solutions of various concentrations, generating superoxide anions in each solution, and measuring the amount of decrease. Platinum nanoparticles can also remove reactive oxygen species (hydroxy radicals, hydrogen peroxide, etc.) other than superoxide anions.

As a method of generating a superoxide anion which is a kind of active oxygen species, a general method can be used. For example, hypoxanthine (HXN) as a reaction substrate, xanthine oxidase (XOD) as an oxidase, an enzymatic reaction method, or reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor and phenazine methosulfate (PMS) And a method using a chemical reaction in which is used as an electron transfer agent. And ID ₅₀ determined in HXN / XOD system, if the ID ₅₀ determined in NADPH / PMS system is different, it is preferred that at least one is not more than 200 [mu] mol / L, and more preferably both are less 200 [mu] mol / L .

Examples of reactive oxygen species that can be deactivated by platinum nanoparticles include superoxide anion (O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (.HO), singlet oxygen ( ¹ O ₂ ), Lipid peroxide radicals, alcohol peroxide radicals, and nitric oxide (NO). As shown in the following formula (1), platinum nanoparticles are considered to act catalytically to reduce active oxygen species and to generate water.

The platinum nanoparticles are preferably mainly attached to the inner surface of the hollow fiber membrane. The adhesion amount of platinum nanoparticles is preferably 10 μmol to 10000 mmol, more preferably 100 μmol to 1000 mmol, per 1 m ^{2 of the} hollow fiber membrane. Since platinum nanoparticles remove reactive oxygen species by a catalytic action, if platinum nanoparticles of 10 μmol to 10000 mmol adhere to the hollow fiber membrane, the active oxygen species in the blood are sufficiently removed. be able to.

[2] Production method of reactive oxygen species removing material Reactive oxygen species removal is possible as long as it is a method capable of adhering an effective amount of platinum nanoparticles to the hollow fiber membrane without including harmful substances. The method for producing the material is not particularly limited. For example, (a) platinum nanoparticles may be kneaded during the production of the resin substrate, or (b) a solution containing platinum nanoparticles may be brought into contact with the resin substrate. Among these methods, a method of bringing a solution containing platinum nanoparticles into contact with a resin substrate is preferable. In order to bring the solution containing the platinum nanoparticles into contact with the resin substrate, the solution may be impregnated or sprayed or passed through the resin substrate. Platinum nanoparticles can be attached to the surface of the resin substrate by contact with the solution. Hereinafter, a method for producing a reactive oxygen species removing material will be described by taking as an example a method of bringing a solution containing platinum nanoparticles into contact with a resin substrate.

(1) Platinum nanoparticle solution The platinum nanoparticle solution contains platinum nanoparticles and polyacrylate. Polyacrylate is coordinated to platinum and serves as a colloid protective agent that improves the solvophilicity of platinum. In the present specification, the platinum nanoparticle solution refers to a uniform dispersion of platinum nanoparticles. As the polyacrylate, sodium polyacrylate and potassium polyacrylate are preferable, and sodium polyacrylate is particularly preferable. Sodium polyacrylate is a food additive permitted by the Food Sanitation Law, and is known to have high safety. Therefore, when sodium polyacrylate is used as a protective colloid, a platinum nanoparticle solution that is safe even when used for a dialysis membrane or the like is obtained.

The R value in the platinum nanoparticle solution is preferably 180 or less, more preferably 10 to 150, and particularly preferably 20 to 130. The R value indicates the ratio between the number of moles of polyacrylate converted per monomer unit and the number of moles of platinum. When the R value is 80 to 180, the platinum nanoparticles can maintain a dispersed state. “Isotonic” refers to a state having the same osmotic pressure as body fluid. The osmotic pressure of the isotonic solution is specifically about 250 to 350 mOsm · kg ⁻¹ . When the active oxygen species removing material is used for blood purification treatment, since the active oxygen species removing material contacts the blood, the platinum nanoparticles preferably exhibit good dispersibility in an isotonic solution.

(2) Platinum nanoparticle solution manufacturing method
(a) Preparation of solution A solution containing a platinum salt, a polyacrylate, an alcohol, and water is prepared. The platinum salt is preferably soluble in a solvent comprising alcohol and water. Examples of the platinum salt include hexachloroplatinic acid, potassium hexachloroplatinate, and hydrates of these salts. Of these, hexachloroplatinic acid is preferred.

  Examples of polyacrylic acid salts include sodium salts and potassium salts. Of these, sodium polyacrylate is preferred.

  As the alcohol, methanol, ethanol, n-propyl alcohol, n-butyl alcohol, n-amyl alcohol and ethylene glycol are preferable, and ethanol is particularly preferable. By using ethanol, safe platinum nanoparticles can be obtained even when used for blood purification treatment.

  A method for producing a platinum nanoparticle solution using a platinum salt and a polyacrylate will be described. First, the polyacrylate is dissolved in water. The concentration of the polyacrylate in the solution is preferably 0.01 to 0.5 mol / L. An aqueous solution containing platinum salt is prepared in another container, and this platinum salt aqueous solution is added to the polyacrylate solution to obtain a platinum salt-polyacrylate solution. The concentration of the platinum salt in the platinum salt-polyacrylate solution is preferably 2 mmol / L or less, and more preferably 0.1 to 2 mmol / L.

The R value (R value) of the polyacrylate-platinum salt solution is preferably 80 to 180, more preferably 90 to 170, and particularly preferably 100 to 150. If the R value is less than 80, the dispersion capacity for the salting-out effect of the platinum nanoparticles when the osmotic pressure is set to 250 to 350 mOsm · kg ⁻¹ by adding a cation or the like to the colloidal solution is low and aggregates. It is easy to settle. If the R value exceeds 180, the polyacrylate is difficult to dissolve in a mixed solvent of water and alcohol, and a uniform solution is difficult to obtain.

  After adding the aqueous platinum salt solution, the platinum salt-polyacrylate solution is stirred. The stirring time is preferably about 30 to 40 minutes. After stirring, alcohol is added to the platinum salt-polyacrylate solution to obtain a reaction solution. The molar ratio of alcohol / platinum salt in the reaction solution is preferably 10-20.

(b) Reflux The reaction solution is refluxed. The reflux is preferably performed in an inert gas atmosphere. During reflux, alcohol such as ethanol reduces platinum ions to produce platinum nanoparticles, which are oxidized to aldehydes.

  Depending on the amount of platinum salt in the reaction solution, platinum ions are almost lost when platinum is refluxed for 2 to 3 hours to produce platinum nanoparticles. The disappearance of platinum ions and the formation of platinum nanoparticles can be confirmed by measuring the ultraviolet-visible absorption spectrum of the reaction solution.

(c) Preparation of alcohol solution By heating and / or reducing the pressure of the reaction solution after reflux, the alcohol and water are evaporated until the volume of the reaction solution reaches about 0.5 to 20% of the volume before heating and / or pressure reduction. To make a concentrated solution. For example, the reaction solution is put into an eggplant flask, and the eggplant flask is immersed in a hot water bath at room temperature to 30 ° C., and the pressure inside the flask is reduced by a water aspirator and held for 60 to 90 minutes. When the reaction solution before heating and / or decompression is 50 mL, it is preferable to reduce the pressure by a water aspirator for about 60 minutes so that the remaining amount is about 5 mL.

  Alcohol (for example, ethanol) is added to the concentrate obtained by evaporation of alcohol and water and stirred to obtain a platinum alcohol solution. The amount of alcohol added is preferably about 20 to 40 (alcohol / platinum salt molar ratio) with respect to the platinum salt of the raw material.

(d) Preparation of platinum nanoparticle solution When the solvent is evaporated by heating and / or depressurizing a platinum alcohol solution, paste-like platinum nanoparticles are obtained. By adding a solvent to the platinum nanoparticles and stirring, a uniform platinum nanoparticle solution can be prepared. Examples of the solvent include water, a mixture of water and ethanol, and the like. If all solvents are volatilized directly from the reaction solution without adding alcohol to the paste-like platinum nanoparticles, the resulting residue will be very viscous and sticky. . For this reason, a uniform platinum nanoparticle solution cannot be obtained. On the other hand, platinum nanoparticles having excellent dispersibility can be produced by preparing an alcohol solution and evaporating the alcohol again.

(3) Impregnation of platinum nanoparticle solution into resin substrate
(a) The resin substrate is immersed in the platinum nanoparticle solution, (b) the platinum nanoparticle solution is passed through the resin substrate, or (c) the platinum nanoparticle solution is sprayed on the resin substrate. Platinum nanoparticles adhere to the surface. When the solution is passed through, the concentration of the platinum nanoparticle solution is preferably 0.01 to 1000 mmol / L. When the platinum nanoparticles are attached, the hollow fiber membrane becomes black, so that the adhesion of the platinum nanoparticles can be visually confirmed.

  When used in an artificial dialyzer, the active oxygen species removing material may be accommodated in the housing, or the platinum nanoparticle solution is allowed to pass through an artificial dialyzer that does not contain platinum nanoparticles to attach the platinum nanoparticles to the hollow fiber membrane. May be. Even when water or blood is passed through the reactive oxygen species removing material, platinum nanoparticles are hardly detached. Accordingly, the blood passing through the dialyzer and the platinum nanoparticles can be brought into contact with each other during blood purification treatment, and the platinum nanoparticles can act as a catalyst to remove reactive oxygen species in the blood.

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

Example 1
(i) Preparation of platinum nanoparticles [protective agent: sodium polyacrylic acid (PAA), R value: 100]
A reaction system consisting of a 100 ml two-neck eggplant flask, an Allen condenser, and a three-way cock was assembled. In a two-necked eggplant flask, 0.31 g of sodium polyacrylate (manufactured by Aldrich) and milli-Q (registered trademark) water (purified water using Milli-Q manufactured by Millipore Japan Ltd.) are shown. ) 23 mL and dissolved, and stirred with a stirrer chip for 10 minutes. Hexachloroplatinic acid crystals (H ₂ PtCl ₆ · 6H ₂ O, manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in distilled water to a concentration of 1.66 × 10 ^-2 mol / L. 2 mL of this aqueous hexachloroplatinic acid solution was added to the two-necked eggplant flask, and the mixture was further stirred for 30 minutes.

  After substituting the reaction system with nitrogen, 25 mL of ethanol was added and refluxed at 100 ° C. for 2 hours while maintaining a nitrogen atmosphere. When the UV-visible absorption spectrum of the reaction solution was measured after the reflux, the peak of platinum ions disappeared and a peak due to scattering specific to metal solids appeared.

  Using a rotary evaporator, the flask was decompressed with a water flow aspirator and kept for about 60 minutes while the eggplant flask was immersed in a 30 ° C. hot water bath. When water and alcohol evaporated from the reaction solution and the reaction mixture in the flask reached about 5 mL, the vacuum was released and 50 mL of ethanol was added. When the solvent was evaporated again from the alcohol solution by reducing the pressure in the flask containing the alcohol solution with a water flow aspirator, paste-like platinum nanoparticles were obtained. 33 mL of milli-Q water was added to the paste-like platinum nanoparticles to obtain a 1 mmol / L platinum nanoparticle aqueous solution (PAA-Pt solution) using sodium polyacrylate as a protective agent.

(ii) Adhesion of platinum nanoparticles Add 200 μL of the 1 mmol / L PAA-Pt solution obtained in step (i) to a strong anion exchange resin column (Supelco Discovery DSC-SAX, 1 mL) connected to an aspirator. Then, the solution in the column was aspirated with an aspirator. When the total PAA-Pt solution was passed through a 2 mL column, it was confirmed that the solution in the suction bottle had turned light brown. The filler was black, and it was thought that platinum nanoparticles were attached by an ion exchange reaction. 2 mL of 125 mmol / L sodium polyacrylate aqueous solution was passed through another strong anion exchange resin column to obtain a control column.

(iii) Pass of O ₂ ^- radical solution In a test tube containing 400 μL of water, hypoxanthine (HXN) solution [5.5 mmol / L, 200 mmol / L phosphate buffer (pH 7.5), HXN: Wako Pure Chemical Industries, Ltd. company Ltd.] 200 [mu] L and, XOD solution [0.2 U / mL, 200 mmol / L phosphate buffer (pH 7.5), XOD: Roche] and 200 [mu] L was added, O ₂ ^- was generated radicals. 20 seconds after mixing of HXN solution and XOD solution, O ₂ ^- was added respectively to the PAA-Pt-containing column and the control column solution prepared in the step (ii) containing radicals were collected filtrate. The time for the HXN-XOD solution to pass through the column was 5-10 seconds.

(iv) Calculation of O ₂ ^- radical scavenging rate
HXN-XOD solution (HXN a solution containing the XOD) 60 seconds after the addition to PAA-Pt-containing column (O ₂ by mixing the HXN solution and XOD solution ^- 80 seconds after to generate radicals) In addition, 15 μL of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, manufactured by Dojin Chemical Laboratory Co., Ltd.) was added to the filtrate. The filtrate DMPO-OOH generated during (DMPO of O ₂ ^- radical capturer) to determine the amount of 140 seconds after the addition of DMPO. ESR was used for measurement. The measurement conditions for ESR were as follows.
Measuring equipment: JES-FA100
FREQ = 9423 MHz (Fluctuation range: +/− 1 MHz)
FIELD CENTER = 335.5 mT, Width = +/− 5.000 mT
POWER = 4.000000 mW, SWEEP TIME = 1.0 min
Mn (marker): 500

In the measurement, the same column was repeatedly used 4 times, and the filtrate was collected each time through the HXN-XOD solution, and the respective ESR spectrum, spin adduct ratio and elimination rate were obtained. DMPO-OOH is O ₂ produced by an oxidase XOD ^- considered to have produced by capturing radicals. The results are shown in Table 1. O ₂ ^- erasure rate of the radicals is around 50%, repeated reduction of scavenging ability be used was hardly observed.

Example 2
(i) Adhesion of platinum nanoparticles 200 µL of the 1 mmol / L PAA-Pt solution obtained in step (i) of Example 1 was passed through a commercially available dialyzer (1 mL). Turned pale brown. The hollow fiber membrane of the dialyzer was black, and it was confirmed that platinum nanoparticles were attached. Even when water was passed through the dialyzer, no color fading or coloration of the filtrate was observed. As a control, a 1 mmol / L sodium polyacrylate aqueous solution was passed through a dialyzer. The control hollow fiber membrane remained white.

the tubes containing the passage water 400μL of radical solution, mixed HXN solution and XOD solution obtained in Example 1 step (ii) each _{^{200μL, O 2 - - (ii}} ) O 2 caused the radical. 20 seconds after mixing of HXN solution and XOD solution, O ₂ ^- radical solution is passed through a PAA-Pt-containing dialyzer produced in step (ii) comprising, were collected filtrate.

80 seconds after to generate a radical ^- HXN-XOD solution O ₂ by mixing (HXN and containing a XOD solution) 60 seconds after the addition to PAA-Pt-containing dialyzer (HXN solution and XOD solution ), 15 μL of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, manufactured by Dojindo Laboratories) was added to the filtrate. The filtrate DMPO-OOH generated during (DMPO of O ₂ ^- radical capturer) to determine the amount of 140 seconds after the addition of DMPO. ESR was used for measurement, and the ESR measurement conditions were the same as in Example 1.

(iii) O ₂ ^- radical erasure rate calculation control (dialyzer containing no platinum nanoparticles), was passed through a mixed solution of HXN solution and XOD solution prepared in the same manner as step (ii), the filtrate It was added DMPO, O ₂ contained in the filtrate flowing out of the control by using the ESR ^- was measured radical scavenging body weight. As erasing ratio zero measure of control, O ₂ of the filtrate was passed through a dialyzer of Example 2 ^- was determined radical scavenging rate. The erasure rate was 47.9% or more.

Claims (4)

  A reactive oxygen species removing material, wherein platinum nanoparticles are attached to a resin substrate.   The active oxygen species removing material according to claim 1, wherein a solution containing the platinum nanoparticles and a protective material is in contact with the resin substrate.   The active oxygen species removing material according to claim 2, wherein the protective material is a polyacrylate.   The active oxygen species removing material according to claim 2 or 3, wherein the solution containing the platinum nanoparticles and the protective material comprises (a) a solution containing platinum salt, the protective material, alcohol and water. After refluxing, (b) the alcohol and the water are evaporated from the solution so that a part remains, (c) the alcohol is added, and (d) the alcohol and the water are evaporated again and diluted An active oxygen species removing material characterized by comprising: