JP2005225772A5 - - Google Patents

Description

The present invention relates to a composite of fine particles in which a closed shell carbon compound having 30 to 120 carbon atoms, excluding graphite and diamond, is enclosed in a structure (or polymer micelle) derived from a block copolymer, and the fine particles further complexes of fine small particles ultrafine particles are enclosed in metal, and to the use of these complexes.

Furthermore, when fullerene is added to an aqueous solution of an acryloylmorpholine oligomer or N, N-dimethylacrylamide oligomer having a fluoroalkyl chain added at the end and sufficiently stirred, the amount of solubilized fullerene becomes about 100 μg / ml. and this self-organization product (self assembly) is obtained has been reported (see non-Patent Document 1). The present inventors also have specific block copolymers in which C ₆₀ fullerene is used as a hydrophilic polymer chain segment and has a poly (ethylene glycol) chain, and a tertiary amino group is supported on a side chain as a functional polymer chain. It was reported that C ₆₀ fullerene can be solubilized in water by adding it to an aqueous solution and thoroughly stirring (see Non-Patent Document 2).
JP-A-7-048302 JP-A-8-143478 Journal of Colloid and Interface Science, 263, (2003), 1-3 52nd (2003) Polymer Symposium Proceedings of the Society of Polymer Science, Japan, 112 pages

On the other hand, a block copolymer capable of forming a structure (or polymer micelle) encapsulating such a carbon compound includes a segment derived from poly (ethylene glycol) as a hydrophilic polymer chain segment, and a tertiary amino group And / or a polymer chain segment containing a repeating unit carrying a secondary amino group on a side chain, and the object of the present invention, for example, C ₃₀ -C ₁₂₀ fullerene can be efficiently solubilized in water Any molecular species can be used. However, preferably the general formula (A):

[Wherein R ¹ represents a hydrogen atom or a C _1-6 alkyl group, R ² and R ³ independently represent a C _1-6 alkyl group, or R ² and R ³ are bonded to each other. Together with the nitrogen atom may form a 5- or 6-membered heterocycle which may contain an additional 1 or 2 nitrogen atoms, 1 oxygen or sulfur atom,
X ′ represents —O— or —NH—, and p ′ is an integer of 2 to 6;
L represents C _1-6 alkylene or a valence bond,
Y represents a hydrogen atom, a hydroxyl group, a carboxyl group, an amino group, an acetalized formyl group or a formyl (or aldehyde) group,
m is an integer from 1 to 10,000;
n is an integer from 10 to 20,000, and p 'is an integer from 2 to 6]
Can be conveniently used in the present invention.

The structure (or polymer micelle) encapsulating the carbon compound is configured such that the surface of the carbon compound is sufficiently covered with the block copolymer as described above, and the carbon compound is solubilized in water. Means an object. Although not limited, such a structure contains a repeating unit carrying a tertiary amino group and / or a secondary amino group of a block copolymer in a side chain so that a carbon compound can be encapsulated as a core. It is preferably a molecular assembly in which block copolymer molecules are associated with each other in an aqueous medium so that the polymer chain segment forms an inner part or a central part, and the poly (ethylene glycol) chain segment covers the outer part. Such molecular assemblies may also be referred to herein as polymer micelles). Used in the above and herein, the aqueous medium, water, an organic solvent miscible with water (e.g., ethanol, acetone, N, N-di-methyl formamide (DMF), di-methyl sulfoxide (DMSO), acetonitrile, etc. And a mixed solution of water and water, or a solution that may further contain a buffer and / or an osmotic pressure adjusting agent in the mixed solution.

As the dipolar aprotic solvent that can be used in such a production method, any solvent can be used as long as it can provide high solubility as described above. Preferably, DMF and DMSO can be mentioned. . First, a carbon compound including fullerene and a block copolymer are dissolved in such a solvent. The dissolving order may be to dissolve the carbon compound and then the block copolymer, and conversely, the block copolymer and then the carbon compound, respectively, or may be dissolved simultaneously. Upon dissolution, the solution may be heated to the boiling point of the solvent as necessary, but may be treated at room temperature. Also, the solution may be sonicated during dissolution and mixing. The solution thus obtained is allowed to stand at room temperature for a certain time (several hours to overnight) after removing insolubles by filtration, if necessary. Thereafter, it is dialyzed against water, preferably distilled water, through a dialysis membrane having a desired molecular weight cut off. Dialysis is usually between 2:00 to around 10 times more water relative to the untreated solution, and more than three times, and finally dialyzed overnight. Moreover, it is preferable to use a dialysis membrane having a molecular weight cut off of 12000 to 14000, and the dialysis membrane should be sufficiently swollen with distilled water before use. All of the above operations can be performed at room temperature, but can be performed under cooling (for example, 0 to 5 ° C.) or under heating (40 to 80 ° C.) as necessary. Thus, a composite of fine particles in which a carbon compound is encapsulated in a polymer micelle and having a solubility in distilled water at room temperature, for example, 25 ° C., of 500 μg / ml or more can be efficiently obtained. The concentration of the carbon compound and the block copolymer in the mixed solution is not limited as long as it can be dissolved in the solvent used, but the carbon compound is usually not in the range of 0.001 wt% to 10 wt% per volume of the solution. Is conveniently used in the range of 0.001 wt% to 2 wt%.

Therefore, the above-mentioned composite of microparticles characterized in that a metal element or an ultrafine particle of a metal in its ionic form is encapsulated in the closed shell structure of the carbon compound described above, and the present invention in another aspect. Offered as. Such a metal may be any metal as long as it is used in a technical field whose usefulness is enhanced by being able to be solubilized in an aqueous medium, for example, in the medical, diagnostic, and food technical fields. . Therefore, the metal that is used as a contrast medium for use in screening or diagnosis of injury any organ disease or organism, e.g., Gad linear um (Gd), a europium (Eu), terbium (Tb) and erbium (Er) normal magnetic metal derived from elements selected from the group consisting, other, zinc, copper, magnesium, iron, platinum, metals derived from the elements capable of providing an oxide etc., etc. is used as a metal in the present invention The These metals can also be in the form of ions. These metals can be encapsulated in the carbon compound in the form of ultrafine particles. The ultrafine particles typically have a size that can be encapsulated in the fullerene. Such fullerenes encapsulating metal are partly commercially available and can be used as they are. However, as described in a method known per se, Patent Document 2, for example, the purpose is A metal oxide can be obtained by laser evaporation together with graphite under high temperature and high pressure.

The molecular weight of the purified polymer was PEG / PAMA = 4,500 / 5,500.
Dispersion stabilizer C ₆₀ fullerenes of the fullerene in water by dialysis method <Production Example 1 of the complex>: mixing ratio of acetal -PEG- PA MA block copolymer (PEG / PAMA = 4,500 / 5,500 ) (F: Add 1 mg of fullerene and 13.8 mg of block copolymer to 25 mL of solvent dimethylformamide (DMF) so that P) becomes 1: 0, 1: 0.5, 1: 1 (when F: P = 1: 1) After sonication for 6 hours, the mixture was allowed to stand overnight. Then, the solution was put into a dialysis membrane having a molecular weight cut off of 12000-14000 which was swollen overnight with distilled water, and dialyzed (3 times of water exchange). The solvent of the 30 mL fullerene-containing microparticle solution thus obtained was removed by lyophilization, and then 5 mL of distilled water was added and redispersed. Thereafter, DLS measurement was performed again.

Soluble liquid after the concentration thus exhibited a blackish brown fullerene-specific. In the absence of the block copolymer, turbidity is produced by concentration, but the complex according to the present invention was very easily dispersed and dissolved in water even after lyophilization. When DLS measurement was performed again for a mixture ratio of F: P = 1: 1, 1: 0.5, the photon count increased compared to before the concentration redispersion, but compared with the value measured previously. it was not possible to obtain a sufficient photon count. Thus, the fullerene particles prepared under these conditions are dispersed in a form close to a molecular dispersion having a particle size (approximately 3 nm) or less that can be detected by light scattering.
<Production Example 2 of Composite> Dispersion Stabilization of C ₆₀ Fullerene in Water by Bubbling Evaporation Method 1 mg of fullerene was mixed with 25 mL of methylene chloride solvent and dissolved in methylene chloride by applying ultrasonic waves. Thereafter, 139 mg of acetal-PEG- PA MA block copolymer (PEG / PAMA = 4,500 / 5,500) was put in a solvent, subjected to ultrasonic waves for 2 hours, and then allowed to stand overnight.

The methylene chloride solution thus prepared was added dropwise to 40 mL of distilled water with argon bubbling. The solution thus prepared provided a microparticle complex that gave a pale yellow transparent solution, similar to that described in Example 1. As a result of DLS measurement of the solution of the obtained dispersion-supported fullerene complex in water, it was confirmed that nanoparticles having a particle size of about 170 nm were formed.
<Production Example of Composite> Production Example of High Concentration Fullerene Aqueous Solution 100 mg of C ₆₀ fullerene was mixed with 150 mL of DMF solution at room temperature, and subjected to ultrasonic treatment for 3 hours. Thereafter, the solution was removed with a 0.45 μm (Millipore) hydrophobic filter to remove substances insoluble in the solvent, and then the acetal-PEG- PA MA block copolymer (with a polymer concentration of 5 mg / ml with respect to the solvent). PEG / PAMA = 5,000 / 5,900) was added and allowed to stand overnight. Thereafter, the solution was put into a bag made of a dialysis membrane having a molecular weight cut off of 12000-14000 which was swollen overnight in distilled water, and dialyzed against 2 liters of DMF: distilled water (150: 2000) (water exchange: 5 Times, 2, 4, 6, 8, 10 hours, exchange after 24 hours).

When subsequently, a solution of dialysis processed lyophilized, after the powder, the sample is redissolved with distilled water 400 [mu] L, performs UV-Vis spectrum measurement, was calculated solubility absorbance of fullerene, Fullerene could be solubilized in water at a high concentration of 2.9 mg / mL .
<Confirmation of active oxygen scavenging action of fullerene encapsulated microparticle composite>
A spin trapping agent 5,5-dimethyl-1-pyrroline N- oxide 30μ what was diluted 2-fold (DMPO) in MiIIiQ L, 5 mM hypoxanthine prepared in 100mM phosphate buffered solution prepared in MiIIiQ (PBS) (HPX) to 50μ L, 9.625mM diethylenetriamine over N, N, N ', N ", N" - pentaacetic acid (DTPA) and 20 [mu] L samples (. 1.PBS, 2 fullerene solution) each 50μ L, 0. 50 mL of 4 U / mL L xanthine oxysidase (XOD) was put into a container in order, and spectrum measurement was performed using ESR 90 seconds after XOD addition.

In the relaxation time measurement ( T ₁ measurement), first, a T ₁ null point was obtained using the Null method, T ₁ was estimated based on the value, and measurement conditions were set for each measurement.

Index relaxivity representing the relaxation time-shortening effect is (or relaxivity, relaxiv ili ty (R)) , the contrast agent concentration C (m mol / L), the longitudinal relaxation time of the pre-contrast (i.e., longitudinal relaxation of the solvent R) is defined by the following equation, where T ₁₀ is the time) and T ₁ を is the relaxation time after administration of the contrast agent (the relaxation time in the presence of the sample).

Here, 1 / T _1, which is the reciprocal of the relaxation time, is called a relaxation rate and is an index indicating the speed of relaxation as the name suggests. The degree of relaxation (R) indicates how much the relaxation rate is increased when the contrast agent is distributed in the tissue to a unit concentration. That is, the larger R is, the greater the relaxation rate promoting effect (relaxation time shortening effect) of the contrast agent is .

A relaxation degree R was calculated based on the concentration and T ₁ of each sample and the relaxation time of the solvent used, and a comparative study was performed. The results are shown in Table 1. From Table 1, the Gd @ C ₈₂ / polymer complex has a significantly high R value and is promising as a contrast agent for MRI.

Claims (14)

A composite of fine particles in which a carbon compound having a closed shell structure consisting essentially of carbon atoms having 30 to 120 carbon atoms is coated with a polymer chain,
The microparticles are derived from a block copolymer comprising a polymer chain segment and a poly (ethylene glycol) chain segment containing repeating units bearing tertiary amino groups and / or secondary amino groups on the side chains; A composite of microparticles, wherein the carbon compound is enclosed in a structure having the former segment as a core and the latter segment as a shell. Solubility in distilled water at 25 ° C. is 0 . It is 5 mg / ml or higher, according to claim 1 conjugate of microparticles according. A polymer chain segment containing a repeating unit carrying a tertiary amino group and / or a secondary amino group on the side chain is represented by the general formula (A):

[Wherein, R ^1a represents a hydrogen atom or a C _1-6 alkyl group, R ^2a and R ^3a independently represent a C _1-6 alkyl group, or R ^2a and R ^3a are bonded to each other. Together with the nitrogen atom may form a 5- or 6-membered heterocycle which may contain an additional 1 or 2 nitrogen atoms, 1 oxygen or sulfur atom,
X represents -O- or -NH-, and p is an integer of 2-6. ] And the solubility in distilled water at 25 ° C. is 0 . It is 5 mg / ml or more, a complex of fine particles according to claim 1. The block copolymer has the general formula (A-1):

[Wherein R ¹ represents a hydrogen atom or a C _1-6 alkyl group, R ² and R ³ independently represent a C _1-6 alkyl group, or R ² and R ³ are bonded to each other. Together with the nitrogen atom may form a 5- or 6-membered heterocycle which may contain an additional 1 or 2 nitrogen atoms, 1 oxygen or sulfur atom,
X ′ represents —O— or —NH—, and p ′ is an integer of 2 to 6;
L represents C _1-6 alkylene or a valence bond,
Y represents a hydrogen atom, a hydroxyl group, a carboxyl group, an amino group, an acetalized formyl group or a formyl (or aldehyde) group,
m is an integer from 1 to 10,000;
The complex of microparticles according to any one of claims 1 to 3, wherein n is an integer of 10 to 20,000, and p 'is an integer of 2 to 6. The composite of microparticles according to any one of claims 1 to 4, wherein the carbon compound is C _30-120 fullerene composed of only carbon atoms.   A closed chain carbon compound consisting essentially of carbon atoms having 30 to 120 carbon atoms and a polymer chain segment containing a repeating unit having a tertiary amino group and / or a secondary amino group supported on a side chain and poly ( Formed by dissolving and mixing a block copolymer comprising an ethylene glycol) chain segment in a dipolar aprotic solvent and then dialyzing against an aqueous solvent through a dialysis membrane having a molecular weight cut off of 12000 to 14000. 2. The method for producing a composite of microparticles according to claim 1, wherein the carbon particles obtained are encapsulated in a structure derived from a block copolymer.   The active oxygen scavenger which uses the composite of the microparticles of any one of Claims 1-5 as an active ingredient.   The active oxygen scavenger according to claim 7, which is used in the fields of food, medicine, dermatology or cosmetics. A composite of fine particles in which a carbon compound having a closed shell structure consisting essentially of carbon atoms having 30 to 120 carbon atoms is coated with a polymer chain,
The microparticles are derived from a block copolymer comprising a polymer chain segment and a poly (ethylene glycol) chain segment containing repeating units bearing tertiary amino groups and / or secondary amino groups on the side chains; Further, the carbon compound is enclosed in a structure having the former segment as a core and the latter segment as a shell, and a metal element or an ionic form of a metal in the closed shell structure of the carbon compound. A complex of microparticles, characterized in that it contains small particles.   The composite of fine particles according to claim 9, wherein the metal element or the metal in its ionic form is a paramagnetic metal. Normal magnetic metal is, Gad linear um, europium, composite microparticle according to claim 10 is derived from elements selected from the group consisting of terbium and erbium. A polymer chain segment containing a repeating unit carrying a tertiary amino group and / or a secondary amino group on the side chain is represented by the general formula (A):

[Wherein, R ^1a represents a hydrogen atom or a C _1-6 alkyl group, R ^2a and R ^3a independently represent a C _1-6 alkyl group, or R ^2a and R ^3a are bonded to each other. Together with the nitrogen atom may form a 5- or 6-membered heterocycle which may contain an additional 1 or 2 nitrogen atoms, 1 oxygen or sulfur atom,
X represents -O- or -NH-, and p is an integer of 2-6. The composite of microparticles according to any one of claims 9 to 11, which is derived from a monomer represented by the formula: The block copolymer has the general formula (A-1):

[Wherein R ¹ represents a hydrogen atom or a C _1-6 alkyl group, R ² and R ³ independently represent a C _1-6 alkyl group, or R ² and R ³ are bonded to each other. Together with the nitrogen atom may form a 5- or 6-membered heterocycle which may contain an additional 1 or 2 nitrogen atoms, 1 oxygen or sulfur atom,
X ′ represents —O— or —NH—, and p ′ is an integer of 2 to 6;
L represents C _1-6 alkylene or a valence bond,
Y represents a hydrogen atom, a hydroxyl group, a carboxyl group, an amino group, an acetalized formyl group or a formyl (or aldehyde) group,
m is an integer from 1 to 10,000;
The composite of microparticles according to claim 12, wherein n is an integer of 10 to 20,000, and p ′ is an integer of 2 to 6.   A contrast agent comprising the composite of fine particles according to claim 11 or 12 as an active ingredient.