JP2006151943A - Radical scavenger and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a metal colloid dispersion, especially a platinum colloid dispersion, which is applied to fields of medicines, foods, and cosmetics, has a radical compound-catching ability, and has high dispersion stability, and to provide a method for producing the liquid phase. <P>SOLUTION: This metal colloid dispersion obtained by irradiating a solution containing a metal ion and polyethylene glycol derivatives: R-PEG-R'-SX, R-PEG-S-S-PEG-R', R-PEG/PAMA, R-PEG-NY, and/or R-PEG-R'-Z (R, R', R<SB>1</SB>and R<SB>2</SB>are each a functional group selected from the group consisting of acetal, aldehyde, OH, amino, carboxyl, methacryloyl, acryloyl and the like) with electromagnetic waves to reduce the metal ion, or heating a solution containing the polymer, the metal ion and an alcohol to reduce the metal ion, and having a radical compound-catching ability and high dispersion stability, and the method for producing the same. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention provides a polyethylene glycol derivative having a functional group capable of stably binding to or adsorbing to a metal and a reactive functional group for biological substances, such as an acetal group that generates a reactive aldehyde in a protein at the ω-terminus, and an α-terminus. Manufactured by reducing metal ions by liquid phase reduction, more specifically by photoreduction or alcohol reduction in the presence of a heterochelic polyethylene glycol derivative having a mercapto group, pyridylthio group, oligoamino group or polyamino group The present invention relates to a dispersion-stable metal colloidal radical scavenger having a radical scavenging ability and a method for producing the same. According to the present invention, one end of the polyethylene glycol is physically and / or chemically adsorbed to the surface of the metal particle, and the other is bound to the biological compound directly or via a linker, and radical scavenging specifically and / or at a local site. An effect can be expressed.

In recent years, active research has been conducted on the effects of free radicals on living bodies. Examples of radicals include O ₂ −, H ₂ O ₂ , OH ·, ¹ O ₂ , and particularly substances that affect living tissue include “hydrogen peroxide H ₂ O ₂ ” and “singlet oxygen ¹ O ₂ ”. In addition, there are two types of free radicals that are particularly important among the active oxygen species, namely “superoxide O ₂ ⁻ ” and “hydroxyl radical OH.”. It is said that the “hydroxyl radical” accounts for about 95% of the influence on the living body, and it is important for the living body to control the “hydroxyl radical”.
These active oxygens and free radicals are excessively generated by external stimuli such as smoking, eating and drinking, and stress, causing damage to cells.
Now that genetic technology has advanced, the relationship between active oxygen and free radicals and biological injury has been extensively studied. Although it is known that SOD (Super Oxide Dimutase) in cells decreases with aging, there are still many unclear points regarding the relationship between SOD and life span and SOD and tissue damage. For this reason, development of a functional compound that expresses a radical scavenging action at a specific and / or local site is required.
Here, “SOD activity” refers to the ability to remove the active oxygen compound.
It has been reported by Esumi et al. That colloidal gold has the ability to trap hydroxy radicals (Non-patent Document 1). However, the colloidal gold prepared by this method uses chitosan as a polymer protective material, and its dispersion stability is insufficient under a high ionic strength environment at the living body level. Nonspecific adsorption and homochelic chitosan are There is a problem that the reaction specificity is poor in expressing the radical scavenging action.
The production of metal colloids is roughly classified into a gas phase production method (hereinafter referred to as a gas phase method) and a liquid phase production method (hereinafter referred to as a liquid phase method). Specifically, the gas phase method is disclosed in Patent Document 1. However, the gas phase method has a drawback that it is difficult to control the particle size distribution and the particle size distribution becomes wide. In addition, the vapor phase synthesis method requires a large-scale apparatus and has a drawback that the cost is increased.

  The liquid phase method is a method of reducing metal ions in a solution, and it is possible to obtain a colloidal solution having a narrower particle size distribution than particles obtained by a gas phase method. However, in the liquid phase method, the growth process and / or the completed particles may be aggregated by electrostatic interaction and / or van der Waals force interaction. The liquid phase method is further roughly divided into a physical method and a chemical method. As a physical method, ultraviolet rays (Non-Patent Document 2), ultrasonic waves (Non-Patent Document 3), γ rays (Non-Patent Document 4), and the like are disclosed. The chemical method is generally a reduction using a reducing agent such as sodium borohydride, sodium ascorbate, or sodium citrate in a solution. Specifically, for example, Non-Patent Document 5 discloses a liquid phase method using sodium citrate. In the chemical reduction method, generally 3 to 5 times the molar amount of reducing agent is added to gold ions, and the mixture is heated to reflux as necessary. Here, sodium citrate functions as a reducing agent and a dispersing agent. Citrate ions are adsorbed on the surface of the gold particles generated by reducing the gold ions, and aggregation of the particles is suppressed by electrostatic repulsion.

Many compounds having a mercapto group have been reported as effective dispersants because they have a strong adsorptive power to the particle surface (Non-patent Document 6). However, since the mercapto group has a strong coordinating power to metal ions, a strong reducing agent such as sodium borohydride disclosed in Patent Document 2 is required (Non-Patent Document 7). Patent Document 2 discloses a method for preparing a gold colloid when sodium borohydride is prepared as a reducing agent and acetal-PEG-SH is used as a polymer protective material. Sodium borohydride used in the method is disclosed It must be added very rapidly with cooling because it is extremely unstable in aqueous solution, generates bubbles and decomposes violently and has a very strong reducing power. For this reason, it is difficult for this method to prepare a colloid with good reproducibility.
Further, it has been reported that platinum colloid can be synthesized by irradiating ultraviolet / visible light with a high-pressure mercury lamp in the presence of an ionic surfactant (sodium dodecylsulfate, dodecyltrimethylammonium chloride) (Non-patent Document 8). However, as is well known, colloids using an ionic surfactant as a protective agent cannot be dispersed under ionic strength at a biological level and easily aggregate.

  JP 58-186967 A

  JP 2002-080903 A

  Esumi, Langmuir, 2004, 20, 2536-2538

  Sau et al. Nanopart. Res. 2001, 3, 257-261

  Okitsu et al. Ultrasonic Chemistry 1996, 3, 249-251

  Arnim et al. Phys. Chem. B, 1999, 103, 9533-9539

  Turkevich et al. Discuss. Faraday Soc. 1951, 11, 55-75

  Royce Murray et al. Am. Chem. Soc. 1998, 120, 12696-12697

  Quarterly Chemistry Review Inorganic organic nanocomposites, No. 42, 148-149

  Toshima et al., Chem. Lett. 1985, 1245-1248

  In view of the above, an object of the present invention is to provide a metal colloid radical scavenger having a radical scavenging ability and target directivity (reaction specificity) and a high dispersion stability by a liquid phase method, and a method for producing the same. To do.

As a result of intensive studies in view of the above-described situation, the present inventor has obtained a metal colloid dispersion having a radical scavenging ability and a target directivity (reaction specificity) in a liquid phase method, and a high dispersion stability. It is intended to provide a manufacturing method. The present inventor has developed a colloidal metal dispersion that is stable even under ionic strength above the living body level by irradiating a solution containing metal ions with ultraviolet rays and / or visible rays in the presence of a polyethylene glycol derivative having a mercapto group at the terminal. It was discovered that can be prepared and that the metal colloid dispersion has radical scavenging ability.
That is, the present invention relates to polyethylene glycol derivative R-PEG-R′-SX and / or polyethylene glycol derivative R-PEG-SS—PEG-R ′ and / or polyethylene glycol derivative R-PEG / PAMA and / or polyethylene glycol derivative R— PEG-NY and or R-PEG-R′-Z (R and R ′ are acetal, aldehyde, hydroxyl group, amino group, carboxyl group, active ester azide group, biotin group, monosaccharide, oligosaccharide, amino acid, nucleic acid, A functional group selected from the group consisting of an allyl group, a vinylbenzyl group, a methacryloyl group and an acryloyl group, PEG is — (CH ₂ CH ₂ O) _n —, X is hydrogen or a pyridylthio group, and PEG / PAMA is represented by structural formula 1 with polyethylene glycol. A block polymer with a methacrylic acid polymer, NY represents an amine derivative segment represented by structural formula 2, and Z represents a cyclic amine derivative, a cyclic sulfide derivative or a dendrimer) A metal colloid dispersion characterized in that the metal colloid is dispersed, and the metal ions are selected from the group consisting of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum The metal colloid dispersion is at least one kind of metal ion. Here, that the polyethylene glycol derivative is bonded to the metal surface means that a part of the polyethylene glycol derivative is physically adsorbed and / or chemically adsorbed on the metal surface.

  Further, the metal colloid dispersion liquid, wherein the solvent of the liquid is one or more solvents selected from the group consisting of water and an organic solvent, and the amount of the polyethylene glycol derivative is a molar ratio with respect to the amount of metal. "Molar amount of metal": "Mole amount of polyethylene glycol derivative" = 1: 0.001 to 1: 100.

The metal colloidal dispersion of the present invention comprises polyethylene glycol derivatives R-PEG-R'-SX and / or R-PEG-SS-PEG-R 'and or R-PEG / PAMA, R-PEG-NY and or R- PEG-R′-Z (R and R ′ are acetal, aldehyde, hydroxyl group, amino group, carboxyl group, active ester azide group, biotin group, monosaccharide, oligosaccharide, amino acid, nucleic acid, allyl group, vinylbenzyl group, A functional group selected from the group consisting of a methacryloyl group and an acryloyl group, PEG is — (CH ₂ CH ₂ O) _n — (n is an arbitrary integer of 2 to 10, 0000), X is hydrogen or a pyridylthio group, NY is An amine derivative segment represented by Structural Formula 2 is shown, and Z represents a cyclic amine derivative, a cyclic sulfide derivative, or a dendrimer. A colloidal metal dispersion formed without agglomeration during the reduction reaction, in which a polyethylene glycol derivative is adsorbed or bonded to the surface of the metal particles. It is a metal colloid dispersion.
R-PEG-R'-SX and / or R-PEG-SS-PEG-R 'and / or R-PEG / PAMA and / or R-PEG-NY and or R-PEG-R'-Z are used It may also be used as a mixture. In aqueous solution, it is considered that R-PEG-R'-SX is oxidized by dissolved oxygen and dissolved in a state of dissolution equilibrium with R-PEG-SS-PEG-R '.

The metal in the present invention is not particularly limited, but preferable metals include gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum. In particular, gold, silver and platinum are mercapto groups or disulfides. It is the most suitable metal because it binds to a group or amino group very stably.
Further, the metal compound which is a metal salt is not particularly limited, but chloroauric acid, silver nitrate, silver acetate, silver perchlorate, chloroplatinic acid, potassium chloroplatinate, palladium chloride dihydrate. Barium nitrate, rhodium nitrate, rhodium acetate, ruthenium acetate, hexanitroiridate, osmium oxide and the like can be preferably applied.

  Examples of the light irradiated to reduce metal ions include infrared rays, visible rays, ultraviolet rays, x-rays, γ-rays, γ-rays, radio waves, etc. Ultraviolet light and / or visible light is a simple device. Since it can irradiate, it can be used suitably.

  The molecular weight of R-PEG-R'-SX or R-PEG-SS-PEG-R 'or R-PEG / PAMA or R-PEG-NY or R-PEG-R'-Z is 100-100,000. The range is used. It is preferably in the range of 300 to 20,000. If it is less than 200, the dispersion stability due to the steric repulsive force is insufficient and the particles may aggregate. On the other hand, when it exceeds 20, 0000, the viscosity becomes too high, for example, sufficient stirring is difficult, and the particle size distribution becomes wide. Further, n in Chemical Formula 2 is an arbitrary integer of 1 to 30, and more preferably in the range of 1 to 10.

As the solvent used in the photoreduction method and / or alcohol reduction method, water can be preferably used from the viewpoint of the environment, but may be a mixed solvent in which water and an organic solvent soluble in water are mixed. It does not specifically limit as a solvent soluble in water, C1-C4 alcohols, such as methanol and ethanol, Ketones, such as acetone, Esters, such as ethyl acetate, etc. can be mentioned. Alcohols preferably have about 1 to 4 carbon atoms because the boiling point of the alcohol increases as the number of carbon atoms increases, making it difficult to distill off.
The solvent used in the alcohol reduction method is preferably in a volume ratio of water: alcohol = 5: 95 to 95: 5, more preferably in the range of 20:80 to 80:20.

  The amount of R-PEG-R'-SX or R-PEG-SS-PEG-R 'or R-PEG / PAMA or R-PEG-NY is relative to the amount of metal (ion). The molar ratio of “molar amount of metal”: “molar amount of polyethylene glycol derivative” = 1: 0.001 to 1: 100 is preferable, and more preferably 1: 0.01 to 1:50. Range. When the molar ratio is 0.01 or less, it is difficult to prepare a colloidal dispersion having a stable dispersion and / or narrow particle size distribution. On the other hand, when the molar ratio is 50 or more, the viscosity becomes too high.

The metal colloid dispersion obtained in the present invention is stable without agglomeration even under an ionic strength of a living body level or more, and further functions as a radical scavenger capable of imparting target directivity.
Further, the dispersion has a narrow particle size distribution and a particle size of 50 nm or less, and has a functional compound reactive functional group at one end of PEG as described above, so that it can be used for medical diagnosis, optical materials, catalyst materials, etc. It can be preferably used.

  Examples and the like specifically showing the configuration and effects of the present invention will be described below.

Example 1 Synthesis of Platinum Colloid by Photoreduction Method In a Schlenk tube, 24.5 ml of an α-3,3-diethylpropyl-ω-mercaptopolypropylene aqueous solution (10.2 mg / ml) and 500 μl of an aqueous solution of potassium chloroplatinate (0.01 mmol / ml) And degassing with a vacuum pump and argon gas purging were repeated three times. Next, while stirring, the solution was irradiated with ultraviolet light / visible light for 2 hours at a power of 500 W by UI501C manufactured by USHIO. It was measured with the transmission electron microscope CAREO ZEISS LEO922 that the nanoparticle was produced | generated by irradiation of the ultraviolet visible light (FIG. 1).

Comparative Example 1
α-3,3-diethylpropyryl-ω-mercaptopolyoxyethylene was prepared in the same manner as in Example 1 except that purified water was used instead of the aqueous solution (10.2 mg / ml). Was purified.

Example 2 Radical scavenging capacity of colloidal platinum synthesized by the photoreduction method of Example 1, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) 15 μl and hypoxanthine (HPX) (5 mM, prepared with purified water) 50 μl And dimethylenepentaacetic acid (DTPA) (5.5 mM, prepared with purified water), 35 μl and 50 μl of the aqueous colloidal platinum solution of Example 1 were mixed in this order, and finally xanthine oxidase (XOD) (0.40 U / ml, pH 7. (Prepared with 100 mM phosphate buffer of No. 4) 50 μl was added, and after 1 minute, radical scavenging ability was measured by JE-RE1X by JEOL Data Corporation by electron spin resonance. The marker used was solid manganese, and the measurement conditions were a central magnetic field of 336 mT, a sweep width of 5 mT, a modulation width of 0.079 mT, a gain of 1000 times, a time constant of 0.1, a sweep time of 1 minute, an output of 5 mW, and a frequency of 9.438 GHz ( Figure 2).

Comparative Example 2
The same operation as in Example 2 was performed except that purified water was added instead of the platinum colloid aqueous solution (FIG. 2).

Comparative Example 3
The same operation as in Example 2 was carried out except that an α-3,3-diethylpropyl-ω-mercaptopolyoxyethylene aqueous solution (10.2 mg / ml) was added instead of the platinum colloid aqueous solution (FIG. 2).

Example 3 Preparation of platinum colloid by alcohol reduction method Acetal-PEG-SH, Acetal-PEG-PAMA, PAA, or MeO-PEG-b-PEPA with the weights listed in Table 1 were dissolved in 12 ml of purified water, and platinum chloride was added. 500 μl of an aqueous potassium acid solution (0.01 mmol / ml) and 12.5 ml of ethanol were mixed and stirred for 30 minutes. And it heated and refluxed for 2 hours. After heating to reflux, the “↓” sample produced a precipitate, and the “◯” sample became a brown solution indicating the formation of yellow platinum nanoparticles.

Example 4 Radical scavenging ability of platinum colloid synthesized by the ethanol reduction method of Example 3 DMPO 15 μl, HPX 50 μl, DTPA 35 μl, and “μ” platinum colloid aqueous solution 50 μl in Table 1 were mixed in this order, and finally XOD 100 μl was added. One minute later, the radical scavenging ability was measured by JE-RE1X using an electron spin resonance method. The marker used was solid manganese, and the measurement conditions were a central magnetic field of 336 mT, a sweep width of 5 mT, a modulation width of 0.079 mT, a gain of 16000 times, a time constant of 0.1, a sweep time of 1 minute, an output of 20 mW, and a frequency of 9.438 GHz. IC50 values (generated _{O 2} ^- The amount of the radical scavenger was required to remove 50% of the radical) and K. Mitsuta, Y.M. Mizuta, M.M. Kohno, M .; Hiramatsu and A.A. Mori, Bull. Chem. Soc, Jpn. , 63, 187 (1990), and are shown in Table 2. As shown in Table 2, the platinum colloid synthesized by the method of Example 3 has a radical scavenging ability. On the other hand, the aqueous solution containing only the polymer containing no platinum nanoparticles did not have radical scavenging ability. These experimental facts suggest that a radical scavenging reaction occurs on the metal surface of the platinum nanoparticles.

Comparative Example 4 Preparation of Platinum Colloid by Alcohol Reduction Method An α-3,3-diethylpropyryl-ω-mercaptopolyoxyethylene aqueous solution was synthesized in the same procedure as in Example 3 except that the concentration was (0.0104 mg / ml). In Comparative Example 4, a black precipitate was formed during the reflux, and a dispersion-stable platinum colloid solution was not obtained. The molar ratio of platinum ion and α-3,3-diethylpropylyl-ω-mercaptopolyoxyethylene in the comparative example is platinum ion: α-3,3-diethylpropylyl-ω-mercaptopolyethylene = 1: 0.0005.

  FIG. 1 shows a photograph of a transmission electron microscope in Example 1.

  FIG. 2 shows the electron spin resonance spectra described in Example 2, Comparative Example 2, and Comparative Example 3.

Claims (10)

A radical scavenger having a radical compound scavenging ability formed by binding a polyethylene glycol derivative to the surface of metal particles. 2. The radical scavenger according to claim 1, wherein the metal particles have a group in which the metal particle is bonded to the metal by a covalent bond and / or an ionic bond and / or a coordinate bond and / or a hydrophobic bond and / or a hydrogen bond. Dispersion-stable metal colloidal radical scavengers stabilized by The radical scavenger according to claim 1, wherein the polyethylene glycol derivative is at least a polyethylene glycol derivative R-PEG-R'-SX and / or a polyethylene glycol derivative R-PEG-SS-PEG-R 'and / or Polyethylene glycol derivative R-PEG / PAMA and / or polyethylene glycol derivative R-PEG-NY and or R-PEG-R′-Z (R, R ′, R ₁ and R ₂ are acetal, aldehyde, hydroxyl group, amino group , Carboxyl group, active ester azide group, biotin group, monosaccharide, oligosaccharide, amino acid, nucleic acid, allyl group, vinylbenzyl group, methacryloyl group, and acryloyl group, and PEG is-( CH ₂ CH _{2 O) n} - and is, X is hydrogen also Is a pyridylthio group, PEG / PAMA is a block polymer of polyethylene glycol and a methacrylic acid polymer represented by the following structural formula 1, NY represents an amine derivative segment represented by the following structural formula 2, and Z Is a metal colloid radical scavenger formed by binding at least one selected from the group consisting of cyclic amine derivatives, cyclic sulfide derivatives, and dendrimers to the surface of metal particles.
(In formula 1, m represents an arbitrary integer of 1 to 10, and n represents an arbitrary integer of 1 to 100)
(N in Chemical Formula 2 represents an arbitrary integer of 1 to 30) The metal colloid radical scavenger, wherein the metal ion is at least one kind of metal ion selected from the group consisting of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum. The metal colloid radical scavenger, wherein the solvent is one or more solvents selected from the group consisting of water and organic solvents. Metal colloid in which the amount of polyethylene glycol derivative is in the range of “molar amount of metal ion”: “molar amount of polyethylene glycol derivative” = 1: 0.001 to 1: 100 in terms of molar ratio with respect to the amount of metal ion. Radical scavenger. Polyethylene glycol derivatives R-PEG-R'-SX and / or R-PEG-SS-PEG-R and / or polyethylene glycol derivatives R-PEG / PAMA and / or polyethylene glycol derivatives R-PEG-NY and / or polyethylene glycol derivatives R -The method for producing a metal colloidal radical scavenger described above, wherein metal ions are reduced by irradiating a solution containing PEG-R'-Z and a metal salt with ultraviolet rays and / or visible rays. Polyethylene glycol derivatives R-PEG-R'-SX and / or R-PEG-SS-PEG-R and / or polyethylene glycol derivatives R-PEG / PAMA and / or polyethylene glycol derivatives R-PEG-NY and / or polyethylene glycol derivatives R -Preparation method of said metal colloid radical scavenger characterized by preparing in the environment purged with PEG-R'-Z and a gas which is oxidizing and / or reducing inert with respect to a metal ion. Polyethylene glycol derivatives R-PEG-R'-SX and / or R-PEG-SS-PEG-R and / or polyethylene glycol derivatives R-PEG / PAMA and / or polyethylene glycol derivatives R-PEG-NY and / or polyethylene glycol derivatives R -A method for producing the metal colloid radical scavenger, wherein a solution containing PEG-R'-Z, a metal salt and an alcohol is heated to reduce metal ions. A pharmaceutical and / or food and / or cosmetic comprising a metal colloid formed by adsorbing the polyethylene glycol derivative on a metal surface.