JP2006316237A - Nano-cluster that disperses stably in water and has fluorescence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nano-cluster that can be used for excluding a problem of a nano-particle already on the market as a fluorescent marker, i.e., for excluding a danger of exposing production workers to a poisonous material such as cadmium, caused by using the poisonous material, and for reducing a burden to the environment arising from its production process and a disposal process after use of the same. <P>SOLUTION: The nano-cluster is produced by using one or more of titanium, silicon, germanium, and zirconium as a raw material, and by modifying its surface in such a manner that the modified surface is stable in an aqueous solution for a long term, and can easily bind with an antibody or a chemical. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nanocluster that can be used as a fluorescent marker for testing or research in the medical field, biochemical field, and the like, and a method for producing the same.

  Nanoparticles that can be used as fluorescent markers in water have been conventionally synthesized using cadmium (see, for example, Patent Document 1).

  In order to obtain water dispersibility, the nanoparticles reported in Patent Document 1 have a surface treatment, an alkyl chain having a thiol group on one side and a water-soluble substituent such as a carboxyl group on the other end. It is designed to cover the whole by a coordinate bond between the surface sulfur atom and the thiol group of the alkyl chain.

  However, the surface modification group of the nanoparticles produced by this method is considered to be bound by coordination bond, and the binding force is weak, and the ligand may be detached even by oxidation or pH change of the solution. is there.

  In addition, when manufacturing the nanoparticles, toxic heavy metals such as cadmium are used, which is not only a concern for health problems of manufacturing workers, but also in manufacturing and disposal processes. There are concerns about adverse environmental impacts.

On the other hand, as reported in Non-Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4, synthesis examples of organic solvent-soluble particles containing silicon and germanium as constituent components have already been reported.

  However, the nanoparticles obtained by this method are soluble in organic solvents, and it is difficult to convert this surface modifying group and maintain a stable state in water for a long time. Is impossible.

  As described in Patent Document 5, taxol has been confirmed to have an excellent effect on solid cancers such as breast cancer and advanced ovarian cancer, that is, anticancer activity, as its physiological activity.

  Various synthetic examples of antibodies that are composed of a polypeptide chain and specifically bind to an antigen have been reported. As an example, a peptide consisting of the amino acid sequence Cys-Gly-Phe-Glu-Cys-Val-Arg-Gln-Cys-Pro-Glu-Arg-Cys described in Non-Patent Document 2 can be mentioned.

US6,251,303B1 JP-A-11-14841 JP-A-11-130867 JP 2002-105207 A JP 2000-44523 Chem. Mater. 1999, 11, 3366-3670 Proceedings of the National Academy of Sciences of the United States of America, 2002, 99, 20, 12617-12621

  The object of the present invention is to eliminate the risk of exposure of toxic substances such as cadmium to the manufacturing worker, which is caused by using toxic substances such as cadmium, which is a problem of nanoparticles already marketed as fluorescent markers. And to reduce the environmental load generated in the manufacturing process and the disposal process after use.

  Furthermore, by performing a surface treatment by stable bonding in an aqueous solution, it is easy to chemically bond biological substances, physiologically active substances, or two or more organic substances thereof to nanoclusters.

  The present invention relates to a nanocluster having fluorescence emission characteristics, comprising silicon, titanium, germanium, zirconium, or a mixture thereof, which is considered to be a material having low environmental impact and low environmental impact, and a simple production method thereof It is.

  First, one or more of silicon tetrahalide, titanium tetrahalide, germanium tetrahalide and zirconium tetrahalide are reduced with an alkali metal or an alkaline earth metal in an organic solvent to produce a nanocluster.

  At this time, a portion of silicon tetrahalide, titanium tetrahalide, germanium tetrahalide, tetrahalogenated zirconium, trihalogenated silicon, trihalogenated titanium, trihalogenated germanium, trihalogenated zirconium, silicon dihalide, Titanium dihalide, germanium dihalide, or zirconium dihalide may be used.

  As the organic solvent used for producing the nanocluster, an aprotic solvent represented by tetrahydrofuran, diethyl ether, 1,4-dioxane, normal hexane and the like is used, but a non-high-boiling point such as triethylene glycol dimethyl ether is used. It is preferred to use a protic solvent.

  Next, in order to stabilize the surface of the nanocluster, an organic solvent-soluble stable nanocluster is obtained by adding only an alkylzinc halide having an ester group or a cyano group to perform surface treatment.

  The alkyl zinc halide having an ester group or a cyano group referred to in the present invention has an ester group, a cyano group and a halogen group in an alkyl group having 2 to 22 carbon atoms regardless of the presence or absence of a double bond or a branched side chain. An organic metal reagent in which halogen is substituted using zinc.

  At this time, instead of an alkyl zinc halide having an ester group or a cyano group, a trialkyl halogenated silicon, a trialkyl titanium halide, a trialkyl halogenated germanium, a dialkyl halogenated silicon, a dialkyl titanium halide, a dialkyl halogenated germanium, Any ratio of monoalkyl halogenated silicon, monoalkyl titanium halide, monoalkyl halogenated germanium, or a combination of any two or more of these in any proportion and an alkyl zinc halide having an ester group or a cyano group Even if the surface treatment is performed by adding a mixture of the above, it is possible to obtain a stable nanocluster soluble in an organic solvent.

  A water-dispersible nanocluster can be obtained by hydrolyzing an ester group-containing nanocluster in the presence of a base such as an alkali metal hydroxide or an alkaline earth metal hydroxide. This has a fluorescence emission characteristic stably in physiological saline without aggregation and precipitation for one month or more.

  The nanocluster having a cyano group can be obtained as a water-dispersible nanocluster by reducing the cyano group to an amino group by a known technique to form a salt of a mineral acid or carboxylic acid. This nanocluster also has a fluorescence emission property stably in physiological saline without aggregation and precipitation for one month or more.

  The nanocluster obtained by the above-described method is surface-modified with an alkyl carboxylic acid or an alkyl amine, and this carboxylic acid or alkyl amine is a biological substance, a physiologically active substance, or two of them directly or via a linker. The above can be combined and chemically bonded.

  The linker referred to in this invention is an organic substance having at least two functional groups from the group consisting of thiol group, amino group, hydroxyl group, carboxyl group, sulfenyl group, sulfinyl group, sulfonyl group, and disulfide group.

  The biological substance referred to in the present invention is a protein, nucleic acid, polypeptide, polysaccharide, thiol group, sulfonyl group, or a protein, nucleic acid, polypeptide, polysaccharide modified with a combination of two or more, or An organic substance combining two or more.

  The present invention uses silicon tetrahalide, titanium tetrahalide, germanium tetrahalide, zirconium tetrahalide, or silicon dihalide, titanium dihalide, germanium dihalide and zirconium dihalide as starting materials. As shown in the examples, even in the manufacturing process, it is possible to suppress the generation of waste with a large environmental load as much as possible, and the load applied to the environment is suppressed as much as possible even when the manufactured nanocluster is discarded after use. It is possible to do.

  In addition, the nanocluster produced according to the present invention has a surface covered with an organic substance having an alkyl group and an ester group or a cyano group with a stable chemical bond.

  This ester group can be chemically reacted by a known method such as hydrolysis, amidation, esterification or reduction.

  The cyano group can be reduced by a known method and converted to an amino group.

  When the nanocluster having an ester group is hydrolyzed with a base such as an alkali metal hydroxide or an alkaline earth metal hydroxide, a nanocluster having an alkyl carboxylate is obtained.

  Further, when a mineral acid or carboxylic acid represented by hydrochloric acid or the like is added to the nanocluster having an amino group, a nanocluster having an amine salt can be obtained.

  This nanocluster alkylcarboxylate or alkylamine salt does not coagulate and precipitate in physiological saline without adding any dispersant or surfactant and without performing antioxidant treatment such as nitrogen substitution for one month. As described above, it has the characteristic of stably emitting fluorescence.

  These nanocluster alkylcarboxylates or alkylamine salts can be chemically bonded in combination with biological substances, bioactive substances or two or more of them directly or via linkers as necessary. It is.

  Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

17 g of methyl 3-bromopropionate was dissolved in 500 ml of triethylene glycol dimethyl ether, 7 g of zinc powder was added and heated at 100 ° C. for 2 hours to obtain methyl 3-zinc bromopropionate.

10 g of tetrachlorotitanium was dissolved in 300 ml of triethylene glycol dimethyl ether, 6 g of zinc powder was added under a nitrogen atmosphere, and the mixture was heated and stirred at 170 ° C. for 100 hours to obtain nanoclusters.

Heated at 00 ° C. for 1 hour. After cooling to 20 ° C., 15 g of trimethylchlorosilane was added, and the mixture was heated at 20 ° C. for 30 minutes, further heated to 60 ° C. and stirred for 1 hour. The obtained reaction mixture was dispersed in water and extracted with normal hexane. Normal hexane was removed under reduced pressure to obtain 0.3 g of a slightly yellow oily substance. When this nanocluster was measured with a fluorescence spectrophotometer RF-5300PC manufactured by Shimadzu Corporation at an excitation wavelength of 300 nanometers, a fluorescence emission maximum was observed in the vicinity of 440 nanometers.

  Hereinafter, the fluorescence emission analysis was performed by the above method.

0.3 g of the oily substance obtained in Example 3 was added to a solution obtained by dissolving 30 mg of sodium hydroxide in a mixed solvent of 100 ml of ion exchange water and 100 ml of ethanol and stirred at 50 ° C. for 1 hour, and then water and ethanol were removed under reduced pressure. 0.4 g of a slightly yellow solid was obtained.

In the solution obtained by dissolving the slightly yellow solid obtained in Example 4 in physiological saline at a weight ratio of 0.1%, a fluorescence emission maximum was observed at around 440 nanometers.

The solution obtained in Example 5 was subjected to a stability test for 1 month at 35 ° C. in an air atmosphere. However, no change was observed in the fluorescence intensity and the fluorescence wavelength, and no aggregation precipitation occurred.

Dissolve 5 mg of the slightly yellow solid obtained in Example 4 in 100 ml of ion-exchanged water, add hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and add it at 10 ° C. or lower for 2 hours. Stir to active esterify. L-cystine was added to this aqueous solution and stirred at 10 ° C. or lower for 12 hours. After further reduction by adding dithiothreitol at the same temperature, the low molecular reagent was removed using an ultrafiltration membrane and lyophilized to obtain 4 mg of a water-dispersible nanocluster having N-propionylcysteine bound to the particle surface. It was.

5 mg of the slightly yellow solid obtained in Example 4 was dissolved in 100 ml of ion-exchanged water, hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added, and the mixture was stirred at 10 ° C. or lower for 2 hours. And then active esterified. L-Serine was added to this aqueous solution and stirred at 10 ° C. or lower for 12 hours. When the low molecular reagent was removed from the reaction solution using an ultrafiltration membrane and freeze-dried, 4 mg of a water-dispersible nanocluster having N-propionylserine bound to the particle surface was obtained.

Even when tetrachlorotitanium was replaced with tetrachlorosilane by the same method as in Example 2 and Example 3, nanoclusters having fluorescence emission characteristics were obtained.

Even when tetrachlorotitanium was replaced with tetrachlorogermanium by the same method as in Example 2 and Example 3, nanoclusters having fluorescence emission characteristics were obtained.

Even when tetrachlorotitanium was replaced with tetrachlorozirconium by the same method as in Example 2 and Example 3, nanoclusters having fluorescence emission characteristics were obtained.

Even when tetrachlorotitanium was replaced with tetrachlorogermanium by the same method as in Example 2 and Example 3, nanoclusters having fluorescence emission characteristics were obtained.

10 mg of the slightly yellow solid obtained in Example 4 was dissolved in 100 ml of DMF, 0.1 mg of taxol was added, (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added, and the mixture was stirred at 10 ° C. or lower for 12 hours to give an ester. Turned into. DMF was concentrated under reduced pressure, dissolved in 50 ml of 0.01 mol aqueous sodium hydroxide solution, low molecules were removed by ultrafiltration, and freeze-dried to obtain 8 mg of product. From the obtained product, a spectrum derived from a taxol skeleton was observed by 1H-NMR analysis.

The product obtained in Example 13 was dissolved in 100 ml of 50% water-containing DMF and subjected to active esterification in the same manner as in Example 8. Then, the amino acid sequence Cys-Gly-Phe-Glu-Cys-Val-Arg-Gln-Cys- A peptide consisting of Pro-Glu-Arg-Cys was added to bind the peptide chain. DMF and water were concentrated under reduced pressure, dissolved in 50 ml of 0.01 mol aqueous sodium hydroxide solution, low molecules were removed by ultrafiltration, and then lyophilized to obtain 7 mg of product. In the obtained product, the amino acid component was confirmed by amino acid analysis.

Methyl 3-bromopropionate was prepared in the same manner as in Example 1.

In the same manner as in Example 1, methyl 3-bromopropionate was replaced with 3-bromopropionitrile to obtain 3-zinc bromopropionitol.

By reacting 3-zinc bromopropionitrile obtained in Example 15 in the same manner as in Example 2 and Example 3, an organic solvent-soluble nanocluster having a cyanoethyl group bonded to the surface was obtained.

The nanocluster obtained in Example 16 was reduced with lithium aluminum hydride in tetrahydrofuran to obtain a nanocluster converted to an aminopropyl group.

  By using silicon, titanium, germanium, zirconium or two or more raw materials, it can be expected that the safety against direct introduction into the body for medical use will be dramatically improved.

  In addition, since a stable surface state can be established and a physiologically active substance or biological substance can be chemically bonded to the nanocluster, it can be used not only for medical purposes but also in the biochemical field.

Claims (14)

  A nanocluster having a fluorescence emission characteristic, which is composed of a mixed composition of titanium, germanium, zirconium and silicon, or a single composition.   The method for producing a nanocluster according to claim 1.   The nanocluster according to claim 1, wherein the organic substance is directly chemically bonded to the nanocluster according to claim 1.   The organic substance according to claim 3 is an alkyl group having 2 to 22 carbon atoms, whether or not a straight chain, branched or double bond, or a combination thereof, and a carboxyl group, a cyano group or the like. The nanocluster according to claim 3, wherein the nanocluster has 2 or more.   The manufacturing method of the nanocluster of Claim 4.   5. The nanocluster according to claim 4, wherein the carboxyl group is converted to a hydrophilic functional group by converting the carboxyl group to an alkali metal or alkaline earth metal salt, and stably dispersed without aggregation and precipitation in water. Nanoclusters characterized by   The method for producing a nanocluster according to claim 6.   The cyano group of the nanocluster according to claim 4 is reduced to an amino group, and the amino group is converted into a mineral acid or a salt of a carboxylic acid, thereby stably dispersing without aggregation and precipitation in water. Nanoclusters characterized by emitting fluorescence.   The method for producing a nanocluster according to claim 8 or 9.   The organic substance is a carboxyl group of the nanocluster according to claim 6, or an alkali metal salt or an alkaline earth metal salt, or an amino group of the nanocluster according to claim 8, or a mineral acid of the amino group, or a salt of a carboxylic acid. The nanocluster according to claim 6 or 8, wherein the nanocluster is chemically bonded directly or via a linker.   The organic substance according to claim 10 is a biomolecule, and has at least one of protein, nucleic acid, polypeptide and polysaccharide, and protein, nucleic acid, polypeptide and polysaccharide modified with thiol group or sulfonyl group. 11. Nanocluster according to claim 10, characterized.   The nanocluster according to claim 10, wherein the organic substance according to claim 10 is a physiologically active substance.   The linker according to claim 10 is an organic substance having one or more functional groups among thiol group, amino group, hydroxyl group, carboxyl group, sulfenyl group, sulfinyl group, sulfonyl group, and disulfide group. The nanocluster according to claim 10.   The organic substance according to claim 10 is a biomolecule according to claim 11 or a physiologically active substance according to claim 12, and one or both of the biomolecules according to claim 8, The nanocluster according to claim 10, wherein the nanocluster is bonded or chemically bonded via the linker according to claim 11.