JP5718095B2 - Gold nanocomposite - Google Patents

Description

  The present invention relates to a gold nanocomposite used for a drug delivery system, a fluorescent sensor or the like. More specifically, the present invention relates to a gold nanocomposite and a drug delivery system using the gold nanocomposite that release an included organic molecule by thermal stimulation only under a predetermined condition.

  Drug delivery systems are actively studied as ideal drug administration systems and for various types of carriers. Nanoparticle materials having a particle size of 100 nm or less have characteristics that the particle size is suitable for the size of the drug carrier and that the surface area per unit mass available for interaction is large. For this reason, utilization in a wide range of fields such as bioseparation, various assays, diagnosis, and drug delivery systems is expected from the perspective of practicality, such as inclusion of functional substances and improvement in analytical sensitivity.

  Among them, many stimuli-responsive polymers whose functions and physical properties change discontinuously and reversibly in response to external stimuli such as temperature and pH are highly functional materials that have advanced functions such as sensing, judgment and movement. It has been studied.

  A stimulus-responsive polymer is a polymer that senses and responds to various physical and chemical information (temperature, heat, solvent, composition, pH, electric field, light, etc.) in the external environment, and is particularly sensitive to heat. Is called a thermoresponsive polymer. Currently, four basic interactions (hydrogen bonding, hydrophobicity, van der Waals force, electrostatic interaction) and polymer phase transitions are shown.

  This phase transition is induced by intermolecular interactions in the gel (between the polymer chain and the solvent molecule, between the polymer chain and the polymer chain). Can be caught. Conventionally well-known thermoresponsive polymers include poly (acrylaminide) analogs such as poly-N-isopropylacrylamide.

  As described above, the cross-linked thermoresponsive polymer can change the conformation of the polymer chain in response to an external thermal stimulus. Therefore, by using a cross-linked thermoresponsive polymer that undergoes a phase transition at a temperature close to body temperature, the drug is reliably administered to the necessary part of the body, and the on-off of the drug administration is controlled at the optimum temperature. It has been studied as a carrier for drug delivery.

  However, in such a drug delivery system using a cross-linked thermoresponsive polymer, the encapsulated drug is released due to the heat phase transition of the polymer (that is, the drug release is activated by thermal stimulation). Therefore, there remain problems to be overcome such as stability problems due to release of the drug from the polymer due to environmental temperature changes and leakage due to drug self-diffusion.

  On the other hand, gold nanoparticles are nanoparticles that have been studied for a long time with new physical properties and structures that have not been seen in bulk, and have been used as lead-free red paints and inorganic paint pigments in stained glass and ceramics. For example, a bright wine red color can be obtained by preparing gold nanoparticles in the liquid phase. Since the color development of the gold nanoparticles is caused by surface plasmon resonance (SPR), various colors are given depending on the shape and particle diameter. A solution having a particle size of about several tens of nanometers in a dispersed state exhibits a maximum absorption wavelength near 520 nm and exhibits a red color. When these particles are aggregated, a blue shift occurs, a new absorption band is generated on the long wavelength side, and the solution turns blue. Gold nanoparticles do not easily return to a dispersed state once they are in an aggregated state.

  In recent years, using such a dramatic change in color tone, it has been attracting attention as a next-generation sensing material and photonics material such as a biosensor and an optical switch, and many studies have been conducted.

  For example, in Patent Document 1 by the present inventor, as a color difference measurement method capable of simply, rapidly and accurately measuring a thiol group-containing compound such as glutathione or cysteinylglycine, a specific heat is applied to gold nanoparticles. A method using a gold nanoparticle composite formed by combining a responsive polymer has been proposed. According to this method, a gold nanoparticle composite obtained by combining gold nanoparticles and a specific thermoresponsive polymer is aggregated in a solution and exhibits a blue color. It does not settle in solution by chemisorbing on the particles. Then, by mixing a specific thiol group-containing compound such as glutathione or cysteinyl glycine into this complex solution, glutathione or cysteinyl glycine is adsorbed on the gold nanoparticles instead of the polymer being released, and It becomes a dispersed state and turns red by the action of a carboxyl group possessed by glutathione, cysteinyl glycine or the like.

  Similarly, Patent Document 2 by the present inventor includes a gold nanocluster having a particle size of 1 to 5 μm and a thermoresponsive polymer (excluding a thermoresponsive polymer containing an anion group such as a carboxyl group). Disclosed is a method for producing gold nanoparticles, characterized by having a step of preparing a mixed solution and a step of heating and cooling the mixed solution. According to this method, the gold (III) ion is not reduced to produce gold nanoparticles or rod-shaped gold nanoparticles having a particle size of more than 50 nm, but a gold nanocluster having a particle size of 1 to 5 μm and thermal response. Has proposed a new method of obtaining gold nanoparticles having a particle size in the range of 50 to 100 nm by a simple operation of heating and cooling a mixed solution with a conductive polymer. Further, in Patent Document 2, the thiol compound is adsorbed on the gold nanoclusters constituting the aggregate, thereby preventing contact / fusion between the gold nanoclusters, and as a result, the production of the gold nanoparticles is inhibited, It has been proposed that the solution for producing gold nanoparticles can be applied as a selective detection solution for a thiol compound because the absorption peak at 550 nm in absorbance becomes small and redness becomes weak.

  Non-Patent Document 1 discloses that an aqueous solution of gold nanoparticles on which Nile Red is adsorbed noncovalently is used for detection of thiols. This is because the addition of thiols such as cysteamine and homocysteine increases the fluorescence of the aqueous solution of gold nanoparticles on which the Nile Red is adsorbed non-covalently. It has been reported that it does not occur due to the addition of amines, acids, alcohols, bovine serum albumin, hemoglobin and the like.

  Non-Patent Document 2 discloses that gold nanoparticles coated with a thermoresponsive block copolymer having a narrow polydispersity exhibit reversible thermal response.

  In Non-Patent Document 3, 4 nm gold nanoparticles having a 1,10-phenanthroline ligand selectively bind to lithium ions to form a 2: 1 ligand-metal complex. Have been shown to cause the gold nanoparticles to agglomerate and cause a shift in the excitation spectrum with a color change, which is a useful optical solution for lithium ions in aqueous solution. It is disclosed that it becomes a detection method.

  Gold nanoparticles are a kind of nanoparticle materials as described above, and they are also being studied as carriers for drug delivery. However, drugs are transported in an adsorbed state on gold nanoparticles and generally have a binding force. Therefore, it is difficult to control the release of the drug from the outside.

  In addition, nanocomposites in which fluorescent molecules are physically adsorbed on gold nanoparticles have been studied as nanoparticle fluorescent sensors. When a substance to be analyzed is added to the nanoparticle fluorescent sensor solution, the fluorescent molecules adsorbed on the nanocomposite are released, so that the fluorescence resonance energy transfer (hereinafter referred to as FRET) by the nanocomposite is eliminated and the fluorescence intensity is restored. However, in this nanoparticle fluorescent sensor, aggregation may occur due to the addition of a substance to be analyzed, which causes a decrease in reproducibility.

  Thus, both gold nanoparticles and cross-linked thermoresponsive polymers have been studied as drug delivery carriers. However, no gold nanoparticles coated with a thermoresponsive polymer have been put into practical use as a drug delivery carrier.

JP 2009-229147 A Japanese Patent Application No. 2010-44792

Analytical Chemistry, 76, 3727-3734 (2004). Colloids and Surfaces, A, 317, 764-767 (2008). Langmuir, 26, 6818-6825 (2010).

  In order to construct an effective drug delivery system, it is necessary to develop a carrier that can accurately release a drug at a target site. As described above, when a cross-linked thermoresponsive polymer is used, it is possible to control the release of the drug contained by external heat stimulation. However, leakage due to drug self-diffusion has been a problem to be overcome in the past. On the other hand, when gold nanoparticles are used as a carrier, the drug is carried while adsorbed on the gold nanoparticles, but in this case, there is a problem that the release of the drug cannot be controlled from the outside.

  An object of the present invention is to provide a novel gold nanocomposite for use in drug delivery systems, fluorescent sensors and the like. In particular, the present invention is to provide a gold nanocomposite capable of accurately releasing a drug at a target site, and a drug delivery system using the same.

  As a result of intensive studies and studies to solve the above problems, the present inventor has paid attention to the fact that thiols exhibit strong adsorptivity to gold nanoparticles, and adsorbs drugs (organic substances) on the surface of gold nanoparticles. Thus, a gold nanocomposite coated with a thermoresponsive polymer was prepared. When this gold nanocomposite is brought into contact with a thiol compound such as glutathione, the thiol compound causes substitution of adsorbed molecules on the surface of the gold nanoparticle, but the drug is a gold nanocomposite covered with a thermoresponsive polymer. Stay inside. When a heat stimulus is applied in this state, the drug is released from the complex by contraction of the thermoresponsive polymer. As a result, the gold nanocomposite establishes a drug delivery system that can release a drug by thermal stimulation only in the presence of a thiol compound such as glutathione, and can accurately release the drug at a target site. As a result, the present inventors have found that a fluorescent sensor with good reproducibility can be obtained when a fluorescent substance is used.

  That is, the gold nanoparticle composite according to the present invention for solving the above-described problem has gold nanoparticles having a drug adsorbed on the surface and a thermoresponsive polymer formed by coating the gold nanoparticles. Features.

  According to this invention, it has gold nanoparticles having a drug adsorbed on the surface and a thermoresponsive polymer coated with the gold nanoparticles, so that the drug can be produced by thermal stimulation in the presence of a thiol compound such as glutathione. Can be efficiently released. Such a gold nanocomposite has no fear of self-diffusion because the surface of the gold nanoparticle that is the core adsorbs the drug. The mechanism of drug release is considered to be performed in the following steps. That is, the thiol compound causes substitution of the adsorbed molecule on the surface of the gold nanoparticle, but the drug remains in the gold nanocomposite covered with the thermoresponsive polymer. When a thermal stimulus is applied in this state, the drug is released from the complex due to the shrinkage of the thermoresponsive polymer. Thus, by coating a gold nanoparticle with a thermoresponsive polymer, it is possible to construct a drug delivery carrier or a nanoparticle fluorescent sensor that overcomes problems that could not be overcome when these were used alone. .

In the gold nanocomposite according to the present invention, the thermoresponsive polymer is preferably a polymer represented by the following chemical formula 2. In the formula, R ¹ and R ² each independently represents an alkyl group. R ³ and R ⁴ each independently represents hydrogen or a methyl group. Moreover, x / y is 95/5-70/30, and n is 1-4.

  Of these, poly (N-isopropylacrylamide-co-acryloyldiethylenetriamine) [poly (NIPAAm-co- (AC-DETA))] and poly (N-isopropylacrylamide-co-acryloyltriethylenetetramine) [poly (NIPAAm- co- (AC-TETA)) is preferred.

  In the gold nanocomposite according to the present invention, a fluorescent substance, a physiologically active substance, or a pharmaceutical compound can be used as the drug.

  The method for producing a gold nanocomposite according to the present invention for solving the above-described problem is to add a drug aqueous solution and a thermoresponsive polymer solution in this order to the colloidal solution of gold nanoparticles, It is characterized in that the drug is adsorbed on and then coated with a thermoresponsive polymer.

  The drug delivery system drug according to the present invention for solving the above-mentioned problems is characterized by using the above-described gold nanocomposite according to the present invention by heating in the presence of thiols or cystine.

  According to the gold nanocomposite according to the present invention, the gold nanoparticle having a drug adsorbed on the surface and a thermoresponsive polymer formed by coating the gold nanoparticle, the coexistence of a thiol compound such as glutathione. In this case, the drug can be efficiently released by thermal stimulation. Such a gold nanocomposite has no fear of self-diffusion because the drug is adsorbed on the surface of the gold nanoparticle as the core.

It is a schematic diagram which shows the mechanism of the chemical | medical agent discharge | release from a gold nanocomposite. It is drawing which shows the excitation and fluorescence spectrum of fluorescent substance Nile Red. It is drawing which shows the fluorescence spectrum intensity | strength which shows the influence of the chemical | medical agent release characteristic by the difference in the thermoresponsive polymer used in the gold nanocomposite. It is a figure which shows the influence of the fluorescence spectrum intensity by the density | concentration of the thermoresponsive polymer (NIP-DETA) which coat | covers a gold nanoparticle. It is a figure which shows the influence of the fluorescence spectrum intensity by the density | concentration of the thermoresponsive polymer (NIP-TETA) which coat | covers a gold nanoparticle. It is drawing which shows the fluorescence spectrum intensity | strength which shows the influence of the chemical | medical agent release characteristic of a gold nanocomposite by pH environment change. It is drawing which shows the fluorescence spectrum intensity | strength which shows the influence of the chemical | medical agent release characteristic of the gold nanocomposite by the change of heating temperature. It is drawing which shows the structural formula of the molecular switch compound used in the Example. It is drawing which shows the fluorescence-spectrum intensity | strength which shows the influence of the chemical | medical agent release characteristic of a gold nanocomposite by the difference in the added molecular switch compound. It is drawing which shows the fluorescence spectrum intensity | strength which shows the influence of the chemical | medical agent release characteristic of a gold | metal nanocomposite by the addition density | concentration change of a molecular switch compound. It is drawing which shows the fluorescence spectrum intensity | strength which shows the influence of the chemical | medical agent release characteristic of a gold | metal nanocomposite by the addition density | concentration change of a molecular switch compound. It is drawing which shows the fluorescence-spectrum intensity | strength which shows the influence of the chemical | medical agent release characteristic of a gold nanocomposite by presence of the coexisting substance at the time of addition of a molecular switch compound.

  Hereinafter, the gold nanoparticle composite according to the present invention will be described with reference to the drawings.

[Gold nanoparticle composite]
The gold nanoparticle composite according to the present invention includes gold nanoparticles having a drug adsorbed on a surface and a thermoresponsive polymer formed by coating the gold nanoparticles. First, prior to describing the gold nanocomposite according to the present invention, important components of the present invention will be described individually.

(Gold nanoparticles)
As the gold nanoparticles, those having various shapes can be used, but it is desirable to have a particle shape or a spherical shape. The particle size of the gold nanoparticles depends on the preparation method, and various particle sizes can be obtained. The range of the particle size of the gold nanoparticles that can be used in the present invention is not particularly limited. However, if the particle size is too small, it is difficult to produce, and on the other hand, if the particle size is too large, the particles may settle. Select with consideration. In general, those having a particle size of about 10 to 100 nm, particularly about 10 to 50 nm are often used. The particle size of the gold nanoparticles can be obtained from the observation result with a transmission electron microscope (TEM).

  Gold nanoparticles can be prepared as a gold nanoparticle solution. This gold nanoparticle solution can be prepared by sodium citrate reduction. Specifically, an example in which a trisodium citrate aqueous solution is added to a predetermined amount of tetrachloroauric acid aqueous solution is given. it can. Alternatively, a commercially available gold nanoparticle solution having a predetermined size may be used.

  As described in Patent Document 1, a gold nanoparticle composite containing gold nanoparticles is produced by heating and cooling a mixed solution of gold nanoclusters having a particle size of 1 to 5 μm and a thermoresponsive polymer. It is also possible to prepare by application. This preparation method will be described in detail below.

Here, the gold nanocluster is a gold particle having a particle diameter of 1 to 5 μm as measured by a transmission electron microscope image (TEM image). The shape of the gold nanocluster is desirably a grain shape or a spherical shape. Gold nanoclusters can be produced by various methods, for example, by a method using a sodium tetrahydroborate solution. Specifically, after adding a polyoxyethylene solution to a chloroauric acid solution, water is added, and after adjusting the pH, a NaBH ₄ (sodium tetrahydroborate) solution is added and then reacted to synthesize. Examples of the polyoxyethylene solution include tetraethylene glycol. By such a synthesis method, a gold nanocluster having a particle size of 1 to 5 μm can be obtained.

(Thermo-responsive polymer)
Examples of the thermoresponsive polymer include a polymer containing at least N-isopropylacrylamide as a structural unit, poly (methyl vinyl ether), hydroxypropyl cellulose, and the like.

  Among the thermoresponsive polymers, examples of the polymer containing N-isopropylacrylamide as a structural unit include those represented by the following general formulas 1-6.

The polymer of Chemical Formula 1 is a poly (N-isopropylacrylamide). In Chemical Formula 1, R ¹ and R ² each independently represents an alkyl group, preferably a linear alkyl group having about 1 to 4 carbon atoms, more preferably a methyl group. R ³ each independently represents hydrogen or a methyl group, preferably hydrogen. n is an integer of 1000 to 100,000. Poly (N-isopropylacrylamide) in which R ¹ and R ² are methyl groups and R ³ is hydrogen is represented by “P-NIP”.

The polymer of Chemical Formula 2 is a poly (N-isopropylacrylamide-co-acryloyl-amine). In Chemical Formula 2, R ¹ and R ² each independently represents an alkyl group, preferably a linear alkyl group having about 1 to 4 carbon atoms, more preferably a methyl group. R ³ and R ⁴ each independently represent hydrogen or a methyl group, preferably hydrogen. Moreover, x / y is 95 / 5-70 / 30, Preferably it is the range of 90 / 10-85 / 15. n is 1-4. Poly (N-isopropylacrylamide-co-acryloyl-amine) in which R ¹ and R ² are methyl groups and R ³ and R ⁴ are hydrogen is represented by “pNIP-AC”.

  The right side chain of the polymer of Formula 2 is acryloylamine (hereinafter referred to as AC) containing a repeating unit of an ethyleneimine chain. More specifically, when n = 0 which is a repeating unit of the ethyleneimine chain on the right side chain, poly (N-isopropylacrylamide-co-acryloylethylenediamine) [hereinafter, poly (NIPAAm-co- (AC -EDA)). When n = 1, it is referred to as poly (N-isopropylacrylamide-co-acryloyldiethylenetriamine) [hereinafter referred to as poly (NIPAAm-co- (AC-DETA)). In the case of n = 2, it is referred to as poly (N-isopropylacrylamide-co-acryloyltriethylenetetramine) [hereinafter referred to as poly (NIPAAm-co- (AC-TETA)). When n = 3, it is referred to as poly (N-isopropylacrylamide-co-acryloyltetraethylenepentamine) [hereinafter referred to as poly (NIPAAm-co- (AC-TEPA)). In the case of n = 4, it is referred to as poly (N-isopropylacrylamide-co-acryloylpentaethylenehexamine) [hereinafter referred to as poly (NIPAAm-co- (AC-PEHA)). ].

The polymer of Chemical Formula 3 is a poly (N-isopropylacrylamide-co-acryloyl-cystamine) class. In Chemical Formula 3, R ¹ and R ² and R ⁷ and R ⁸ each independently represent an alkyl group, preferably a linear alkyl group having about 1 to 4 carbon atoms, more preferably a methyl group. R ³ and R ⁴ and R ⁵ and R ⁶ each independently represent hydrogen or a methyl group, preferably hydrogen. Further, x / y / z is 95 / 2.5 / 2.5 to 80/10/10. Poly (N-isopropylacrylamide-co-acryloyl-amine) in which R ¹ and R ² and R ⁷ and R ⁸ are methyl groups and R ³ and R ⁴ and R ⁵ and R ⁶ are hydrogen is “pNIP-Cys”. Represented by

The polymer of Chemical Formula 4 is poly (N-isopropylacrylamide-co-acryloyl-alanine). In Chemical Formula 4, R ¹ , R ² and R ⁵ each independently represent an alkyl group, preferably a linear alkyl group having about 1 to 4 carbon atoms, more preferably a methyl group. R ³ and R ⁴ each independently represent hydrogen or a methyl group, preferably hydrogen. Moreover, x / y is 90 / 10-80 / 20. Poly (N-isopropylacrylamide-co-acryloyl-amine) in which R ¹ , R ² and R ⁵ are methyl groups and R ³ and R ⁴ are hydrogen is represented by “pNIP-AC”.

The polymer of Chemical Formula 5 is poly (N-isopropylacrylamide-co-glycidylmethacrylic acid-lactic acid). In Chemical Formula 5, R ¹ , R ² and R ⁵ each independently represents an alkyl group, preferably a linear alkyl group having about 1 to 4 carbon atoms, more preferably a methyl group. R ³ and R ⁴ each independently represent hydrogen or a methyl group, preferably hydrogen. Moreover, x / y is 95/5 to 80/20. Poly (N-isopropylacrylamide-co-acryloyl-amine) in which R ¹ , R ² and R ⁵ are methyl groups and R ³ and R ⁴ are hydrogen is represented by “pNIP-GMA-lactic acid”.

The polymer of Chemical Formula 6 is poly (N-isopropylacrylamide-co- (meth) acrylic acid). In Chemical Formula 6, R ¹ and R ² each independently represent an alkyl group, preferably a linear alkyl group having about 1 to 4 carbon atoms, more preferably a methyl group. R ⁴ represents hydrogen or a methyl group. Moreover, x / y is 95 / 5-70 / 30, Preferably it is the range of 90 / 10-85 / 15. Poly (N-isopropylacrylamide-co-methacrylic acid) in which R ¹ and R ² are methyl groups and R ⁴ is a methyl group is represented by “pNIP-MA”.

  In addition, like the polymer represented by the chemical formula 6, the thermoresponsive polymer containing an anion group such as a carboxyl group is a mixed solution of the gold nanocluster and the thermoresponsive polymer described in Patent Document 1 described above. It cannot be used for the production method of preparing gold nanoparticles from

  Also, polyethylene glycol (PEG) and polyacrylamide which are not thermoresponsive polymers cannot produce gold nanoparticles.

  As the molecular weight of the polymers represented by Chemical Formulas 1 to 6, those having a weight average molecular weight of 8000 to 100,000 can be used, and those having a molecular weight of 10,000 to 20,000 are preferred. Each polymer is usually used alone, but two or more may be used in combination. In addition, other heat-responsive polymers that exhibit the same effects as the polymers of Chemical Formulas 1 to 6, such as poly (methyl vinyl ether), hydroxypropyl cellulose, etc. may be used within the scope of the present invention. Good.

  The polymers of the above chemical formulas 1 to 6 can be synthesized by the method shown in the experimental examples described later. For example, the polymer of Chemical Formula 1 may be obtained by dissolving N-isopropylacrylamide as a monomer component in water, adding a polymerization initiator and a polymerization accelerator, polymerizing, purifying the product, and then freeze-drying. it can. Also, the polymers of Chemical Formulas 2-6 can be polymerized by dissolving the monomer components to be polymerized in a solvent, adding a polymerization initiator and a polymerization accelerator, purifying the product, and then freeze-drying to obtain each polymer. it can.

  Although not particularly limited, among the polymers of Chemical Formulas 1 to 6, pNIP-AC of Chemical Formula 2, preferably poly (NIPAAm-co- (AC-DETA), poly (NIPAAm-co- (AC-TETA) )), Particularly preferably poly (NIPAAm-co- (AC-DETA). Poly (NIPAAm-co- (AC-DETA)) is adsorbed on gold nanoparticles by thermal stimulation in the presence of thiols. It is particularly preferable because it exhibits excellent drug release characteristics and exhibits stable drug retention in the state of thermal stimulation in the absence of thiols or in the absence of thermal stimulation in the presence of thiols. .

The thermoresponsive polymer is a polymer represented by the above chemical formula 2 having a large number of repeating units of the ethyleneimine chain on the right side chain, specifically, poly (NIPAAm- co- If (AC-TETA) ethyleneimine units than those is large, many NH group and NH ₂ group, when thermally responsive polymer is thermally contracted by heat stimulation, such groups are gold nanoparticles In the presence of thiols, a certain amount of the drug is released by thermal stimulation even in the absence of thiols. Compared with the case of thermal stimulation, the degree of release is significantly different, and the degree of release is the highest among the thermoresponsive polymers used in the experiment, as shown in the examples described later. Therefore, it can be exemplified as a preferable thermoresponsive polymer.

(Drug)
The drug adsorbed by gold nanoparticles is released better than gold nanocomposites by thermal stimulation in the presence of thiols, while in the absence of thermal stimulation in the absence of thiols or in the presence of thiols As long as it can be stably held in the gold nanocomposite, various materials can be used without particular limitation.

  As a drug, it is considered as having a specific function such as physiological activity, drug activity, catalytic activity, optical resolution, antioxidant activity, surface activity, fragrance, or optical activity in a state where it is not supported or attached. Is done. Among them, a physiologically active substance or a pharmaceutical compound is exemplified as a representative one.

  Examples of the physiologically active substance or pharmaceutical compound include various drugs shown in the latest edition of Goodman and Gilman's “The Pharmacological of Basis of Therapeutics” or the latest edition of The Merck Index. For example, drugs that act on the central nervous system; drugs that act on peripheral synapses; drugs that affect the inflammatory response; drugs that affect the composition of body fluids; drugs that affect kidney function and electrolyte metabolism; cardiovascular agents, gastrointestinal Drugs that affect vascular function; drugs that affect uterine motility; chemotherapeutic agents for parasitic infections, chemotherapeutic agents for microbial diseases; antitumor or anticancer agents; immunosuppressants, blood and blood Drugs affecting the forming organs; hormones and hormone antagonists; skin agents; heavy metal antagonists, vitamins and nutrients, vaccines, oligonucleotides and gene therapy agents. In addition, amine-based drugs such as amitriptyline hydrochloride, imipramine hydrochloride, clomipramine hydrochloride, trimipramine maleate, nortriptyline hydrochloride, amoxapine, dosrepin hydrochloride, lofepramine hydrochloride and the like can also be mentioned. Further, it may be a complex drug such as cisplatin.

  A typical example of a fluorescent substance representative of a drug having a function of optical properties is Nile Red.

  In the gold nanocomposite according to the present invention, the drug adsorbed by the gold nanoparticle is substituted in the presence of a thiol as described later in order for the thiol to exhibit strong adsorptivity to the gold nanoparticle. It is thought that it is liberated from the particles. If the drug has a stronger adsorptivity to gold nanoparticles than thiol compounds, it will be less likely to be released from the gold nanocomposite even by thermal stimulation in the presence of thiols. For this reason, although it depends on the type of thiols to be used, in general, a compound that exhibits a certain degree of adsorptivity to gold nanoparticles while exhibiting an adsorptivity lower than that of thiols is used. . Examples of such compounds include functional groups or skeletal structures that exhibit weaker interactions with the gold nanoparticle surface than thiol groups possessed by thiol compounds, such as alkyl groups such as ethyl groups and methyl groups, phenyl groups, and hydroxyl groups. , A compound having an appropriate carboxyl group, amino group and the like.

  In addition, a polymer compound that forms a so-called protective colloid with respect to gold nanoparticles, that is, a polymer compound in which one molecule is adsorbed on the gold nanoparticle surface at multiple points (multi-point adsorption), etc. It is considered that desorption is hardly caused by addition. For this reason, there are many drugs that are not generally suitable for use in the nanocomposite according to the present invention.

[Gold nanocomposite production method 1]
The gold nanocomposite according to the present invention is basically added to a colloidal solution of gold nanoparticles in the order of an aqueous drug solution and a thermoresponsive polymer solution in order to adsorb the drug to the gold nanoparticles first. Then, it can be manufactured by coating a thermoresponsive polymer.

  Unlike the above, after adding the thermoresponsive polymer solution to the colloidal solution of gold nanoparticles, and then preparing the gold nanocomposite in the order of adding the aqueous drug solution, The thermoresponsive polymer is coated. From this, in the resulting gold nanocomposite, the drug molecule is not directly adsorbed to the gold nanoparticle, but is incorporated into the thermoresponsive polymer that coats the gold nanoparticle. Is guessed. According to the present inventors, in this case, drug molecules are released by thermal stimulation regardless of whether or not a molecular switch such as thiols described below is added or whether or not the organic compound molecule to be added contains a thiol group. Is seen. From this, as described above, it is considered important for the method for producing a gold nanocomposite according to the present invention to contact a colloidal solution of gold nanoparticles with a pharmaceutical aqueous solution first.

  The heat-responsive polymer to be used is usually one kind of heat-responsive polymer, but may be two or more kinds of heat-responsive polymers as long as a gold nanocomposite can be produced. . Moreover, although the pH of a mixed solution at the time of manufacturing a gold nanocomposite is dependent on the kind of the chemical | medical agent and thermoresponsive polymer to contain, it is preferable that it is the range of 2.9-10.0 normally.

The mixed solution preferably contains gold nanoparticles in the range of 0.0018 to 0.0090% by mass, and the thermoresponsive polymer depends on the type of the thermoresponsive polymer to be used. It is preferable that it is contained in the range of 25-2.0 mass%. The concentration of the drug, is also dependent on the type of agent used, for example, with respect to gold 1g, can be 0.2 to 20 × 10 ^-5 M concentration of about. With this level of drug concentration, the added drug can be efficiently adsorbed to the gold nanoparticles, and the drug concentration dependence on the mechanism of drug release by thermal stimulation in the presence of thiols as described below. There is no influence by.

[Gold nanocomposite production method 2]
As a manufacturing method of the gold nanocomposite according to the present invention, a method applying the manufacturing method described in Patent Document 1 can be exemplified.

  That is, a gold nanocomposite is prepared by sequentially adding an aqueous solution of a drug and a thermoresponsive polymer (excluding a thermoresponsive polymer containing an anion group such as a carboxyl group) to the gold nanocluster solution. A gold nanocomposite is manufactured by heating and cooling the mixed solution.

The mixed solution preferably contains gold nanoclusters in a range of 0.0018 to 0.0090% by mass, and preferably contains a thermoresponsive polymer in a range of 0.25 to 2.0% by mass. . Further, when the gold nanocluster and the thermoresponsive polymer are expressed by mass ratio, the mass ratio of gold nanocluster / thermoresponsive polymer is preferably in the range of 9/10000 to 36/1000. Further, the concentration of the drug depends on the type of the drug to be used, but can be set to a concentration of about 0.2 to 20 × 10 ⁻⁵ M with respect to 1 g of gold, for example. With this level of drug concentration, the added drug can be efficiently adsorbed to the gold nanoparticles, and the drug concentration dependence on the mechanism of drug release by thermal stimulation in the presence of thiols as described below. There is no influence by.

  The thermoresponsive polymer to be contained in the mixed solution is usually one type of thermoresponsive polymer. However, if gold nanoparticles can be produced, two or more types of thermoresponsive polymers can be used. Also good. Further, although the pH of the mixed solution depends on the kind of the thermoresponsive polymer to be contained, it is usually preferably in the range of 2.9 to 10.0.

  The gold nanocomposite is produced by heating and cooling the prepared gold nanocomposite production solution (mixed solution). Heating is an operation that causes a phase transition in the thermoresponsive polymer and fuses the gold nanoclusters. The heating temperature is 40 to 95 ° C, preferably 70 to 95 ° C. The higher the temperature, the larger the gold nanoparticles can be obtained. The heating time is preferably 30 minutes to 2 hours. As the heating time increases, the number of large particles increases and the average particle size increases apparently. However, the particles do not grow into large particles. Further, the change in absorbance does not change much after 30 minutes.

  Cooling is an operation of re-transparenting a turbid solution that has been heated to deposit a thermoresponsive polymer. The cooling temperature is 0 to 30 ° C, preferably 4 to 10 ° C. The lower the cooling temperature, the faster the solution becomes clear. A cooling time of 30 minutes or more is sufficient, and the upper limit is not particularly limited (about 1 hour or 2 hours).

  By such heating and cooling means, a gold nanocomposite can be produced from the mixed solution. The manufactured gold nanoparticles have a particle size in a range of 50 to 100 nm that can be measured from a TEM image, and show a peak at a particle size of 100 to 500 nm as measured by a dynamic light scattering method. In addition, the measurement from a TEM image takes a TEM photograph and measures from the photograph. On the other hand, the measurement of the particle size by the dynamic light scattering method is measured by a dynamic light scattering measurement device.

[Drug release from gold nanocomposites]
Release of the adsorbed drug molecule from the gold nanocomposite according to the present invention is performed by adding a thiol such as glutathione or cystine as described later and performing thermal stimulation (heating → cooling).

  FIG. 1 is a schematic diagram showing the mechanism of drug release from a gold nanocomposite. That is, as shown in FIG. 1, an organic compound serving as a molecular switch such as a thiol is added to a gold nanocomposite formed by adsorbing drug molecules on the gold nanoparticle surface and coating a thermoresponsive polymer. Then, although the organic compound molecule causes substitution of the adsorbed molecule on the surface of the gold nanoparticle, the drug remains in the gold nanocomposite covered with the thermoresponsive polymer. When heat stimulation (heating → cooling) is applied in this state, the thermoresponsive polymer undergoes phase transfer and once contracts, thereby being released from the gold nanocomposite and released.

(Thiols)
Next, thiols used for drug release from the gold nanocomposite according to the present invention as described above will be described.

  Due to the adsorption of thiols to the gold nanocomposite, drug molecules adsorbed on the gold nanoparticle are desorbed from the gold nanoparticle. That is, when a thiol group having a thiol group is added to the aggregated gold nanoparticle composite, it exchanges with the functional group of the drug molecule on the surface of the gold nanoparticle and has a strong binding property to the gold nanoparticle surface. The thiol group is coordinated and thiols are adsorbed.

  Thiols that effectively cause the elimination of drug molecules as described above by addition of thiols include (1) presence of thiol groups, (2) molecular size and structure, and (3) carboxyl groups and amino groups. It is thought that factors such as the presence of hydrogen bonds between them are necessary.

  It is considered that the thiol group (1) is adsorbed on the surface of the gold nanoparticle and causes an exchange reaction with a functional group of the drug molecule. Unlike a compound having a thiol group, a compound having a disulfide bond such as oxidized glutathione and a compound having a sulfide bond such as methionine do not appear to undergo an exchange reaction with a functional group of a drug molecule. However, according to experiments conducted by the present inventors, even in the case of a compound having a disulfide bond, in the case of cystine (a dimer of cysteine), when a thermal stimulus is given after the addition of the compound, a high drug molecule release effect Exceptionally, cystine is a compound that effectively acts on the gold nanocomposite according to the present invention.

  Regarding the molecular size and structure of (2), the substitution reaction with drug molecules by adsorption of thiols on the gold nanoparticle surface is considered to occur generally faster as the thiols have a lower molecular weight. For example, although the protective effect of gold nanoparticles is large when the molecular size is large like glutathione, substitution with drug molecules or thermoresponsive polymers adsorbed on the surface of the gold nanocomposite is slow. A thiol group-containing compound having a low molecular weight carboxyl group such as L-cysteine (L-Cys) or homocysteine (Hcys) has little protective effect on gold nanoparticles, and the reaction is fast. Cysteinyl glycine, glutamyl cysteine, etc., which are intermediate in size between glutathione and L-cysteine, have a protective effect on gold nanoparticles, and the reaction rate of substitution with drug molecules and thermoresponsive polymers is relatively fast. Conceivable.

  Therefore, when a low molecular weight thiol group-containing compound such as L-cysteine or Hcys is used, a considerable amount of drug is released from the gold nanocomposite only by adding the compound, and the drug is released by thermal stimulation. There is a high possibility that the starting action of the gold nanocomposite according to the present invention does not work effectively. Since such a compound has a small molecular size, when it is added, the release of drug molecules is not sufficient to inhibit the redispersion of gold nanoparticles due to thermal stimulation (heating → cooling). The molecular weight seems to decrease.

  Even in the case of cysteinyl glycine, glutamyl cysteine, etc., although this tendency is seen to some extent, the redispersion of gold nanoparticles is promoted after receiving heat stimulation, and the drug release is more than before applying heat stimulation. Can be said to be a preferred compound. In the case of glutathione, it can be said that the addition of the compound alone is a particularly preferable compound because the degree of drug release is the lowest and the rate of drug release after thermal stimulation is extremely high.

(3) Regarding the electrostatic repulsion of the carboxyl group, it is considered that the electrostatic repulsion of the acid-dissociated carboxyl group of the thiol group-containing compound is largely involved. The pKa of the two carboxyl groups of glutathione is pKa ₁ = 2.16 and pKa ₂ = 3.55, and the pH of the complex solution at a thermoresponsive polymer concentration of 0.2% by mass under consideration is about Since it is 8, the carboxyl group is COO-. On the other hand, the NH group and NH ₂ group are positively charged with some hydrogen ions added. First, gold nanoparticles are in an anionic state by citric acid and are dispersed by electrostatic repulsion. By adding a polymer having an NH group or NH ₂ group to this state, the charge on the surface of the gold nanoparticle is partially canceled, and the NH group or NH ₂ group is adsorbed on the gold, thereby aggregating the gold nanoparticle. To do. At this time, the gold nanoparticles are coated with the polymer, and the polymer is in a state of entering between the aggregated particles. When glutathione, which is a thiol group-containing compound, is added to the gold nanoparticles in this state, the thiol group, the NH group, and the NH ₂ group are exchanged, and the thiol group-containing compound is bonded to the surface of the gold nanoparticle. At this time, the functional group is very crowded on the surface of the gold nanoparticle, but the polar group (functional group) of the thiol group-containing compound or the thermoresponsive polymer is present in a random state. It becomes disadvantageous in terms of energy. Arrangement of polar groups (functional groups) by hydrogen bonding eliminates the disadvantageous state of energy. As a result, it is considered that the distance between the gold nanoparticles increases.

The presence of the above thiol groups, molecular size and structure, presence of carboxyl groups of thiol group-containing compounds, non-covalent protective effects by polymers having NH groups and NH ₂ groups affect the redispersion of gold nanoparticles. In addition, it is considered that the charge of other functional groups of the thiol group-containing compound, the structure of the compound present in the solution, the charge of each functional group, etc. influence the redispersion of the gold nanoparticles as a whole.

  From the above points, the thiols used as the molecular switch for drug release by the gold nanocomposite according to the present invention are not particularly limited, but include glutathione (GSH) and cysteinylglycine (Cys-Gly). , Glutamic acid cysteine (Glu-Cys) and the like can be exemplified as preferable ones, and among these, glutathione is particularly preferable. As described above, cystine can also be exemplified as a preferable compound as a molecular switch for drug release.

In addition, as the addition concentration of thiols used as such a molecular switch for drug release, generally, the higher the addition concentration of thiols, the greater the amount of drug released after thermal stimulation is applied. However, as the additive concentration increases, the amount of drug released in a state where no thermal stimulation is applied tends to increase. In addition, if the added concentration is not more than a certain level, there is no significant difference in the amount of released drug before and after thermal stimulation. For this reason, since it depends on the type of thiols to be used, it cannot be defined unconditionally. For example, when glutathione is used, the concentration is preferably about 4 to 10 × 10 ⁻⁶ M.

  In addition, when preferable thiols such as glutathione are used as a molecular switch for drug release, even if a coexisting substance such as L-Cys, Hcys, glycine, and glutamic acid is present, after the addition of the glutathione (molecular switch), heat stimulation The activation action of the gold nanocomposite according to the present invention that releases the drug by the function effectively functions. This is because the presence of amino acids such as L-Cys, Hcys, glycine, and glutamic acid, peptides, and the like is naturally assumed in vivo, and thus the drug delivery system using the gold nanocomposite according to the present invention is provided. It seems to function effectively in vivo.

(Thermal stimulation)
With respect to the gold nanocomposite according to the present invention, the drug is released from the gold nanocomposite by heating and cooling after the addition of thiols as molecular switches. Heating is an operation for causing a phase transition in the thermoresponsive polymer. The heating temperature is 40 to 95 ° C, preferably 70 to 95 ° C. The heating time is preferably 30 minutes to 2 hours. The change in the amount of drug released estimated from the fluorescence intensity does not change much after 30 minutes.

  Cooling is an operation in which the thermally responsive polymer that has been heated to cause a phase transition and contracted is dispersed again to release the drug from the gold nanocomposite. The cooling temperature is 0 to 30 ° C, preferably 4 to 10 ° C. A cooling time of 30 minutes or more is sufficient, and the upper limit is not particularly limited (about 1 hour or 2 hours).

  The phenomenon in which gold nanocomposites release adsorbed drug molecules upon addition of a molecular switch and thermal stimulation occurs almost uniformly within a range of pH 5 to 9, which is a general pH environment, and does not depend much on the pH value. The adsorbed drug molecules are not substantially released before the heat stimulation or remain in a very small amount, and a large release occurs after the heat stimulation. More desirably, use in a weakly acidic or neutral region having a pH of about 5 to 7.5 is preferable for suppressing the amount of adsorbed drug molecules released before heat stimulation and causing large release after heat stimulation.

(Drug delivery system)
Since the drug that has been adsorbed can be released by applying thermal stimulation to the gold nanocomposite according to the present invention in the presence of thiols or cystine as described above, the drug can be released at the target site. It is suitably used in a drug delivery system that can be accurately released.

  There are no particular limitations on the route of administration of the gold nanocomposite in vivo, depending on the type of drug to be carried and the body site or tissue to which the drug is to be administered, oral, nasal, vaginal, intravenous It can be introduced into a living body through an appropriate administration route such as subcutaneous or intraperitoneal.

  In vivo, thiols, which are molecular switches of gold nanocomposites, such as glutathione, cysteinylglycine, and cysteine glutamate, which are suitable as molecular switches, are naturally present, and the concentration of such glutathione is high. In the case of drug administration targeting a living body site or tissue, a drug delivery system drug using the gold nanocomposite according to the present invention can be obtained by applying thermal stimulation without separately administering thiols from outside the system. It can be accurately activated at the target site. Of course, as necessary, thiols as such molecular switches can be introduced from the outside into a target site or tissue in the living body.

  The means for performing thermal stimulation (heating) on the living body is not particularly limited, and various methods can be used. For example, far infrared from two directions (typically, orthogonal directions) If laser heating is performed, the target site can be locally heated.

(Fluorescence sensor)
When a fluorescent substance such as Nile Red as described above is used as the drug to be carried on the gold nanocomposite according to the present invention, it can be used as a nanofluorescent sensor with good reproducibility.

  When a substance to be analyzed such as glutathione, which is a thiol compound, is added to the nanoparticle fluorescent sensor solution and thermal stimulation is applied, the fluorescent substance is released from the gold nanocomposite with the phase transition of the thermoresponsive polymer. As a result, the fluorescence resonance energy transfer (hereinafter referred to as FRET) due to the gold nanocomplex is eliminated, and the fluorescence intensity is recovered. Since the recovered fluorescence intensity is proportional to the concentration of the analyte to be added, the analyte can be quantified. If no thermal stimulation is applied, the fluorescent substance is not substantially released from the gold nanocomposite, so that the fluorescent substance released by the addition of the analyte is not aggregated, and quantitative analysis is possible with high reproducibility.

  The present invention will be described in more detail by the following experiments, but the present invention is not limited to the following examples. In particular, in the following examples, in order to facilitate the confirmation of the degree of release of the adsorbing drug, an example using the fluorescent substance Nile Red as the adsorbing drug will be mainly described. The present invention is not limited to such fluorescent substances, and various kinds of drugs as described above can be used, and such various kinds of drugs are also effective as in the case of the fluorescent substances shown in the following examples. It seems that those skilled in the art can easily understand that the drug release phenomenon occurs.

[Devices and reagents]
First, the apparatus and reagents used in this example were as follows. For the measurement of the absorption spectrum, a JASCO V-560 type UV-visible spectrophotometer equipped with a 1 cm optical path length plastic cell was used. For the fluorescence spectrum measurement, an FP-6300 spectrofluorometer manufactured by JASCO Corporation equipped with a quartz cell having an optical path length of 1 cm was used.

  For ultrafiltration of the synthesized thermo-responsive polymer, Advantech's ultrafilter for plastic ultrafilter equipped with Millipore's ultrafiltration membrane (exclusion molecular weight: 5000) MODEL-UHP-K used. For the freeze-drying of the synthesized thermo-responsive polymer, ULVAC KIKOU direct-coupled oil rotary vacuum pump equipped with ULVAC KOKI OMT-050A type oil mist trap, Tokyo Science Instruments UT-1000 UNITRAP, And the FD-5N type freeze dryer made by Tokyo Science Instruments equipped with the CMW-1 type moisture trap made by Tokyo Science Instruments was used. For measurement of pH, HORIBA pH-METER F-13 was used.

  α, α'-azobisisobutyronitrile (AIBN, Fw: 164.21), potassium hydroxide (Fw: 56.11), sodium hydroxide (Fw: 40.00), diethyl ether (Fw: 74.12), 1,4-dioxane (Fw: 88.1), potassium peroxodisulfate (Fw: 270.32), and diethylenetriamine (DETA, Fw: 103.17) manufactured by Kanto Chemical Co., Ltd. were used. Pyrogallol (Fw: 126.11) used the first grade made by Kanto Chemical. Methacrylic acid (MA, Fw: 86.09), grade 1 manufactured by Kanto Chemical, was used after removing the polymerization inhibitor by the freezing method. Triethylenetetramine (TETA, Fw: 146.24) was manufactured by Kanto Chemical. N-isopropylacrylamide (NIPAAm, Fw: 113.16) was used after recrystallizing Wako Pure Chemical's special grade using Kanto Chemical's special grade hexane (Fw: 86.18). N, N, N ', N'-tetramethylethylenediamine (TEMED, Fw: 116.21) was a Wako Pure Chemicals special grade. Wako Pure Chemicals Grade 1 was used for acryloyl chloride (AC, Fw: 90.51). Tetrachloroauric acid tetrahydrate (Fw: 411.85), trisodium citrate dihydrate (Fw: 294.1), and acetone (Fw: 58.08) were special grades manufactured by Kanto Chemical. Nile Red (NR, Fw: 318.57) used was the first grade made by Tokyo Chemical Industry. Glycine (Gly, Fw: 75.07), glutamic acid (Fw: 147.03), L-cysteine (L-Cys, Fw: 121.16), 1-butanethiol (Fw: 90.19), 1-octanethiol (Fw: 146.29), 2 -Mercaptoethanol (Fw: 78.13) and 2,3-dimercapto-1-propanol (Fw: 124.23) are special grades manufactured by Kanto Chemical, reduced glutathione (GSH, Fw: 307.32), 3-mercapto-1,2 -Propanediol (Fw: 108.16) used the first grade made by Kanto Chemical. 3-mercaptopropionic acid (3-MPA, Fw: 106.14) and 1,2-ethanedithiol (Fw: 94.2) are special grades made by Tokyo Chemical Industry, oxidized glutathione (GSSG, Fw: 612.63), n-dodecyl mercaptan (Fw: 202.4), ethyl 3-mercaptopropionate (Fw: 134.2) used the first grade made by Tokyo Kasei, cystamine sulfate (Fw: 250.36), and Hcys (Fw: 135.19) used the one made by Tokyo Kasei. . Glutamylcysteine (Glu-Cys, Fw: 250.27), cysteinyl glycine (Cys-Gly, Fw: 178.2), DL-penicillamine (Fw: 149.2) manufactured by Sigma were used.

  Water used in the experiment was distilled and ion-exchanged, and then manufactured using a Milli-Q ultrapure water production system manufactured by Millpore. Methanol (Fw: 32.04) was used after distillation of a special grade manufactured by Kanto Chemical.

[Synthesis Example 1 (Preparation of gold nanoparticles)]
The gold nanoparticle solution was prepared by a sodium citrate reduction method. The instruments used were all soaked in aqua regia for 30 minutes, then washed with ultrapure water and dried. The preparation procedure is shown below. A 500 mL round bottom flask was charged with 250 mL of a 1 mM tetrachloroaurate solution and heated to just before boiling with vigorous stirring. While boiling, 25 mL of 38.8 mM trisodium citrate aqueous solution was added, and it was confirmed that the color of the solution changed from pale yellow to deep red. After further heating for 10 minutes with stirring, stirring was continued for 15 minutes at room temperature. The solution was subjected to suction filtration using a membrane filter having a pore size of 1 μm, and the gold nanoparticle solution was transferred to a glass reagent bottle and stored in a cool dark place (4 ° C.) (0.18 g-Au / L gold colloid solution).

[Synthesis Example 2 (Synthesis of Chemical Formula 1 / P-NIP)]
In a 500 mL three-necked separable flask, 30 g of N-isopropylacrylamide (NIPAAm) was taken and dissolved in 100 mL of water. While maintaining the flask at 0 ° C. using an ice bath and stirring, 1.0 g of potassium peroxodisulfate as a polymerization initiator and N, N, N ′, N′-tetramethylethylenediamine (TEMED) as a polymerization accelerator 5 mL was added and polymerized for 4 hours. The structural formula of the synthesized P-NIP is one in which R ¹ and R ² in the above chemical formula 1 are methyl groups and R ³ is a hydrogen atom. After completion of the polymerization, 100 mL of methanol was added and the precipitated polymer was taken out and dissolved in 100 mL of methanol. 100 mL of water was added to this solution, and the precipitated polymer was taken out and dissolved in 100 mL of water. This operation was repeated twice, and purified P-NIP was dissolved in water and lyophilized. Furthermore, the obtained polymer was dissolved in water, freeze-dried, and stored in a cool dark place. The lower critical solution temperature (LCST) of this thermoresponsive polymer was 34.1 ° C.

Synthesis Example 3 (Formula 2 / Synthesis of Poly (N-isopropylacrylamide-co-acryloyltriethylenetetramine))]
When triethylenetetramine (TETA) is used as a monomer, it is necessary to introduce a vinyl group in advance. Therefore, first, acryloyltriethylenetetramine [hereinafter referred to as AC-TETA] was synthesized from TETA and acryloyl chloride (AC). Reaction of a mixed solution of AC 1.625 mL (0.02 mol) and 1,4-dioxane 50 mL dropwise to a mixed solution of TETA 29.73 mL (0.2 mol) and 1,4-dioxane 200 mL while cooling and stirring. I let you. After completion of the dropwise addition, the white precipitate was filtered off and dissolved in a small amount of methanol. 100 mL containing a potassium hydroxide-methanol solution (potassium hydroxide 1.18 g (0.02 mol)) was added thereto, stirred, and the filtrate obtained by filtration was used for copolymerization with NIPAAm.

  In a 500 mL three-necked separable flask, 20.37 g (0) of N-isopropylacrylamide (NIPAAm) so that the monomer supply ratio (NIPAAm substance amount: TETA substance amount) was 90:10 (mol%) in molar ratio. .18 mol) and AC-TETA in methanol solution was added. While heating and stirring the flask to 60 ° C. using an oil bath, 3-mercaptopropionic acid (3-MPA) (0.1 mL with respect to 20 mL of methanol) as a polymerization accelerator and α, α ′ as a polymerization initiator. -0.8211 g (0.005 mol) of azobisisobutyronitrile (AIBN) was added and vented to a deoxygenation line using 100 mL of 5 w / v% pyrogallol-10 w / v% alkaline solution (alkali: NaOH). Polymerization was performed for 4 hours under a nitrogen atmosphere.

The obtained polymer was purified by the following procedure. First, after removing methanol contained in the polymer solution with an evaporator, the obtained polymerization reaction product was poured into 1.0 L of cold diethyl ether. The supernatant was removed by decantation and redissolved with methanol. After repeating this process twice, water was added to the precipitate obtained after decantation to 300 mL. This solution was dialyzed 6 times using an ultrafiltration membrane having a molecular weight of 5000 to remove unreacted monomers. Thereafter, the obtained polymer solution was freeze-dried and stored in a cool dark place. The structural formula of the synthesized poly (N-isopropylacrylamide-co-acryloyltriethylenetetramine) [hereinafter referred to as NIP-TETA] is that R ¹ and R ² in the above chemical formula ² are methyl groups, R ³ and R ⁴ are hydrogen atoms, n is 2. The lower critical solution temperature (LCST) of the obtained NIP-TETA was 38.3 ° C.

Synthesis Example 4 (Formula 2 / Synthesis of Poly (N-isopropylacrylamide-co-acryloylditriethylenetetramine))
When diethylenetriamine (DETA) is used as a monomer, it is necessary to introduce a vinyl group into DETA. Therefore, first, acryloyldiethylenetriamine [hereinafter referred to as AC-DETA] was synthesized from DETA and acryloyl chloride (AC) by the following procedure. A mixed solution of AC and 1,4-dioxane (1.625 mL / 50 mL) is cooled and stirred in a mixed solution of DETA-containing 1,4-dioxane (20.63 g / 200 mL) and allowed to react dropwise. (DETA substance amount: AC substance amount = 10: 1). After completion of the dropwise addition, the white precipitate was filtered off and dissolved in a small amount of methanol. A KOH-methanol solution was added to this, stirred and filtered, and the obtained filtrate was used for copolymerization with NIPAAm.

In a 500 mL three-necked separable flask, N-isopropylacrylamide (NIPAAm) and AC-DETA were added so that the monomer supply ratio (substance amount of NIPAAm: substance amount of DETA) was 90:10 (mol%). The containing methanol solution was added. 3-mercaptopropionic acid (3-MPA) (0.1 mL with respect to 20 mL of methanol) was used as a polymerization accelerator, and α, α′-azobisisobutyronitrile (AIBN) 0.3284 g (0. 005 mol) was added. While agitating the solution in the flask in an oil bath, nitrogen that was passed through a deoxygenation line (5 w / v% pyrogallol-10 w / v% NaOH solution 100 mL) was vented into the flask and deoxygenated for 30 minutes under a nitrogen atmosphere. went. Then, it heated at 60 degreeC, ventilating nitrogen, and superposed | polymerized in nitrogen atmosphere for 4 hours. The obtained polymer was purified by the following procedure. First, after removing methanol contained in the polymer solution as much as possible with an evaporator, the obtained polymerization reaction product was poured into 1.0 L of cold diethyl ether. The supernatant was removed by decantation and redissolved with methanol. After repeating this process twice, water was added to the precipitate obtained after decantation to 300 mL. This solution was dialyzed 6 times using an ultrafiltration membrane having a molecular weight of 5000 to remove unreacted polymer. Thereafter, the obtained polymer solution was freeze-dried and stored in a cool dark place. The structural formula of the synthesized poly (N-isopropylacrylamide-co-acryloylditriethylenetetramine) [hereinafter referred to as NIP-DETA] is that R ¹ and R ² in the above chemical formula ² are methyl groups, R ³ and R ⁴ are hydrogen atoms, n is 1. The lower critical solution temperature (LCST) of the obtained NIP-DETA was 38.4 ° C.

[Synthesis Example 5 (Synthesis of Chemical Formula 6 / pNIP-MA)]
In a 500 mL three-necked separable flask, NIPAAm 20.37 g (0.18 mol) was supplied so that the monomer supply ratio (NIPAAm substance amount: MA substance amount) was (90:10) (mol%) in molar ratio. Methacrylic acid (MA) 1.72 g (0.02 mol) was added. 65.2 mL of methanol was added to dissolve the monomer so that the content of NIPAAm and MA was 30% by mass as the sum of both. While heating the flask to 60 ° C. using an oil bath and stirring, 3-MPA (0.1 mL with respect to 20 mL of methanol) was used as a polymerization accelerator, and AIBN 0.8211 g (0.005 mol) was used as a polymerization initiator. The nitrogen which was added and passed through the deoxygenation line (5 w / v% pyrogallol-10 w / v% sodium hydroxide solution 100 mL) was passed through the flask, and polymerization was performed for 4 hours under a nitrogen atmosphere. After completion of the polymerization, the methanol solution containing the product is cooled to room temperature. Using 2 L of cold diethyl ether per 100 mL of methanol, a methanol solution containing the product is slowly dropped dropwise into cold diethyl ether and stirred. Thus, the precipitated gummy product is recovered and dissolved again in about 50 mL of methanol. After performing this purification twice, the polymer obtained was dissolved in water and the solution was frozen. After the solution was completely frozen, it was lyophilized and stored in a cool dark place. The structural formula of the synthesized pNIP-MA is ^{one in} which R ¹ and R ² in the above chemical formula 6 are methyl groups and R ³ and R ⁴ are hydrogen atoms. The lower critical solution temperature (LCST) of the obtained pNIP-MA was 35.7 ° C.

[Reference Example 1 (Characteristics of fluorescent material)]
The fluorescent substance Nile Red was used as an adsorbing drug for evaluating the drug release characteristics of the gold nanocomposite according to the present invention. Therefore, first, the fluorescence spectrum of Nile Red was measured. 0.0159 g (5.0 × 10 ⁻⁵ mol) of Nile Red was taken, dissolved in a small amount of acetone, and then made up to 20 mL with acetone (2.5 × 10 ⁻³ M). 0.04 mL of this solution was taken into a 10 mL volumetric flask and made up to volume with water (1.0 × 10 ⁻⁵ M). This Nile Red aqueous solution was taken up in a 10 mL volumetric flask at 0 to 0.100 mL, and fixed to 10 mL with water (0 to 1.0 × 10 ⁻⁷ M). The fluorescence spectra of these solutions were measured with the excitation wavelength fixed at 580 nm and the fluorescence wavelength fixed at 652 nm. As a result, an increase in fluorescence intensity was confirmed depending on the concentration of Nile Red.

Since Nile Red was mixed with the thermoresponsive polymer and gold nanoparticles, the final concentration of Nile Red was 1.0 × 10 ⁻⁷ M in the following experiment. As detailed later, this concentration of Nile Red has no effect on redispersion. FIG. 2 shows the excitation and fluorescence spectrum of Nile Red at a concentration of 1.0 × 10 ⁻⁷ M.

[Examples 1 to 4 and Comparative Example 1]
(Preparation of gold nanocomposites and evaluation of drug release by differences in thermoresponsive polymers)
Each 0.1 mL of 0.18 g-Au / L gold nanoparticle solution was added to a 10 mL volumetric flask, and 0.100 mL of 1.0 × 10 ⁻⁵ M Nile Red solution was added. 3.75 mL of a 2.0 mass% solution of thermoresponsive polymer was added, and the volume was adjusted to 10 mL with water.

  In order to evaluate drug release from the prepared gold nanocomposites (Examples 1 to 4), the fluorescence wavelength was measured with the excitation wavelength fixed at 580 nm and the fluorescence wavelength fixed at 652 nm. Then, it heated at 90 degreeC for 30 minutes, cooled at 4 degreeC for 1 hour, and measured the fluorescence spectrum on the same conditions.

Moreover, after adding 250 microliters of 1.0 * 10 < ^-3 > M glutathione (GSH) solutions as thiols to each prepared gold nanocomposite solution, the fluorescence spectrum measurement was performed on the conditions similar to the above. Then, it heated at 90 degreeC for 30 minutes, cooled at 4 degreeC for 1 hour, and measured the fluorescence spectrum on the same conditions.

In addition, for comparison, 0.18 g-Au / L gold nanoparticle solution 0-5.0 mL was added to a 10 mL volumetric flask, and 0.100 mL of 1.0 × 10 ⁻⁵ M Nile Red solution was added to add water. To 10 mL.

  For the obtained comparative gold nanocomposite (Comparative Example 1), as in Examples 1 to 4, fluorescence spectrum measurement before and after heating in the GSH-free state, fluorescence spectrum before and after heating in the GSH-added state Measurements were made.

  The obtained results are shown in FIG. In FIG. 3, Au + NR represents the comparative gold nanocomposite of Comparative Example 1 in which Nile Red was adsorbed on gold nanoparticles, and “P-NIP” represents the P in Synthesis Example 2 in which Nile Red was adsorbed on gold nanoparticles. -The gold nanocomposite of Example 1 coated with NIP, "TETA" represents the gold nanocomposite of Example 2 in which Nile Red was adsorbed on gold nanoparticles and NIP-TETA of Synthesis Example 3 was coated. “DETA” is the gold nanocomposite of Example 3 in which Nile Red is adsorbed on gold nanoparticles and coated with NIP-DETA of Synthesis Example 4, and “MA” is the synthesis example 5 in which Nile Red is adsorbed on gold nanoparticles. The gold nanocomposites of Example 4 coated with pNIP-MA are respectively shown.

  As shown in FIG. 3, the gold nanocomposites of Examples 1 to 4 showed higher fluorescence intensity after heating in the system to which GSH was added than in the system to which GSH was not added, and GSH was added. It was revealed that Nile Red is effectively released from the gold nanocomposite by applying thermal stimulation. In addition, the fluorescence intensity after heating in the system to which GSH was added was higher than that in Comparative Example 1, and the drug release by thermal stimulation in the presence of thiols by the gold nanocomposite according to the present invention. It was shown that the effect was excellent.

  In addition, as shown in FIG. 3, when NIP-TETA was coat | covered (Example 2), it turned out that Nile Red is partially liberated also in the system which does not add GSH. This seems to occur because Nile Red was exchanged when the TETA group was adsorbed on the gold nanoparticle surface. On the other hand, no release of Nile Red was observed when other thermoresponsive polymers were added.

  On the other hand, in the system to which GSH is added, it can be observed that GSH has a thiol group and thus reacts specifically with gold nanoparticles and has an effect of promoting redispersion without heating and cooling. From this, the fluorescence intensity of NIP-TETA (Example 2) and NIP-DETA (Example 3), which cause re-dispersion by thermal stimulation, recovered significantly due to the effective release of Nile Red by the addition of GSH. it is conceivable that. Also in NIP-MA (Example 4) and P-NIP (Example 1) that do not cause re-dispersion, it seems that the release of Nile Red was caused to some extent by the added GSH, and relatively high fluorescence after heat stimulation. It is thought that it showed strength.

[Example 5]
(Preparation of gold nanocomposites and evaluation of drug release by the concentration of thermoresponsive polymer)
A 0.18 g-Au / L gold nanoparticle solution is added to a 10 mL volumetric flask, 0.100 mL of 1.0 × 10 ⁻⁵ M Nile Red solution is added, and the final concentration of the thermoresponsive polymer is 0-1. When 0% by mass, 0-5 mL of 2.0% by mass thermoresponsive polymer solution is added, and when the final polymer concentration is 1.25 and 1.50% by mass, 5.0% by mass thermoresponsive Gold nanocomposites having different thermoresponsive polymer concentrations were prepared by adding 2.5 mL and 3 mL of the molecular solution to a final volume of 10 mL with water. Note that NIP-DETA of Synthesis Example 4 was used as the thermoresponsive polymer used.

  About each of these solutions, it measured on the conditions similar to Examples 1-4 in the state without GSH addition. The obtained results are shown in FIG. As shown in FIG. 4, in the system in which GSH was not added, release of Nile Red hardly occurred even when heat stimulation was applied, regardless of the amount of coated NIP-DETA, while GSH was added. In some systems, large Nile Red release was shown to occur after thermal stimulation. When the addition concentration of NIP-DETA is 0.25% by mass or more, there is a significant difference in the amount of Nile Red released after thermal stimulation compared to the case where NIP-DETA is not added. A great effect was obtained in the range of about 0.5 to about 1.0% by mass.

[Example 6]
(Preparation of gold nanocomposites and evaluation of drug release by the concentration of thermoresponsive polymer)
As in Example 5, except that NIP-TETA of Synthesis Example 3 was used instead of NIP-DETA of Synthesis Example 4 as the thermoresponsive polymer to be used, gold having a different thermoresponsive polymer concentration was used. Nanocomposites were prepared, and for each of these solutions, fluorescence spectra before and after heating in the GSH-added state were measured in the same manner as in Example 5.

  The obtained results are shown in FIG. As shown in FIG. 5, in the system to which GSH was added, it was shown that a large release of Nile Red occurs after heat stimulation. Even when the addition concentration of NIP-TETA was relatively low, a significant difference was observed in the amount of Nile Red released after thermal stimulation compared to the case where NIP-TETA was not added. When the concentration exceeded about 1.0% by mass, there was a tendency that the amount of Nile Red released before heat stimulation increased compared to the case where NIP-TETA was not added. From the viewpoint of the difference in fluorescence intensity before and after thermal stimulation, a great effect was obtained when the addition concentration was in the range of about 0.25 to about 1.25% by mass, particularly about 0.25% by mass.

[Example 7]
(Preparation of gold nanocomposites and evaluation of drug release by changes in environmental pH conditions)
Add 0.18 g-Au / L gold nanoparticle solution to a 10 mL volumetric flask, add 0.100 mL of 1.0 × 10 ⁻⁵ M Nile Red solution, and add 3.73 mL of 2 mass% NIP-DETA solution. To a final volume of 10 mL with water. The pH was adjusted between 5.0 and 9.0 by adding small amounts of 1.0 M NaOH aqueous solution and 1 M HCl aqueous solution to this solution. After adding 250 μm μL of a 1.0 × 10 ⁻³ M glutathione (GSH) solution to each solution, fluorescence spectra were measured under the same conditions as in Examples 1 to 4. Then, it heated at 90 degreeC for 30 minutes, cooled at 4 degreeC for 1 hour, and measured the fluorescence spectrum on the same conditions.

  The obtained result is shown in FIG. As shown in FIG. 6, the phenomenon of releasing the adsorbed drug molecule by thermal stimulation in the GSH addition system occurs almost uniformly within the measured pH range of 5 to 9 without depending much on the pH value. Previously it was shown that adsorbed drug molecules remained in a very small amount of release and a large release occurred after thermal stimulation. In particular, good results were obtained in the range of pH 5.5 to pH 7.5.

[Example 8]
(Evaluation of drug release by fluctuation of thermal stimulation temperature)
Add 0.18 g-Au / L gold nanoparticle solution to a 10 mL volumetric flask, add 0.100 mL of 1.0 × 10 ⁻⁵ M Nile Red solution, and add 3.73 mL of 2 mass% NIP-DETA solution. To a final volume of 10 mL with water.

After adding 250 μL of 1.0 × 10 ⁻³ M glutathione (GSH) solution to the obtained gold nanocomposite solution, fluorescence spectrum measurement was performed under the same conditions as in Examples 1-4. . Then, set the heating temperature to 30, 40, 50, 60, 70, 80, 90 ° C, heat for 30 minutes, cool at 4 ° C for 1 hour, and measure the fluorescence spectrum under the same conditions It was.

  The obtained results are shown in FIG. As shown in FIG. 7, when the heating temperature was about 40 ° C. or higher, particularly about 70 ° C., good release of Nile Red due to thermal stimulation occurred.

[Examples 9 to 12 and Reference Examples 2 to 7]
(Evaluation of molecular switch)
Add 0.18 g-Au / L gold nanoparticle solution to a 10 mL volumetric flask, add 0.100 mL of 1.0 × 10 ⁻⁵ M Nile Red solution, and add 3.73 mL of 2 mass% NIP-DETA solution. To a final volume of 10 mL with water.

After adding 250 μL of each 1.0 × 10 ⁻³ M compound solution to the obtained gold nanocomposite solution, fluorescence spectrum measurement was performed under the same conditions as in Examples 1 to 4. . Thereafter, heating was performed at 90 ° C. for 30 minutes, followed by cooling at 4 ° C. for 1 hour, and fluorescence spectrum measurement was performed under the same conditions.

  The added compounds were glutathione (GSH) (Example 9), cysteinyl glycine (Cys-Gly) (Example 10), cystine (Example 10), cysteine glutamate (Glu-Cys) (Example 11). ), Glycine (Gly) (Reference Example 2), glutamic acid (Reference Example 3), oxidized glutathione (GSSG) (Reference Example 4), L-cysteine (L-Cys) (Reference Example 5), Hcys (Reference Example 6) ) And methionine (Reference Example 7). The structural formula of the compound used is shown in FIG.

  The obtained results are shown in FIG. As shown in FIG. 9, it was confirmed that Nile Red was liberated in all cases where thiol compounds of GSH, Cly-Cys, Cys-Gly, L-Cys, and Hcys were used. Among them, GSH, Glu-Cys, and Cys-Gly were able to confirm the recovery of very large fluorescence intensity. GSH, Glu-Cys, and Cys-Gly have a large molecular size. Therefore, GSH, Glu-Cys, and Cys-Gly can promote re-dispersion and can be sufficiently re-dispersed by applying thermal stimulation. . Since the molecular size of L-Cys and Hcys is small, when L-Cys and Hcys are added, Nile Red is not sufficiently released to inhibit redispersion. As a result, the fluorescence intensity is sufficiently high even when heated and cooled. It is thought that it did not recover.

  Since Gly and glutamic acid are compounds that do not contain a thiol group, they did not react with gold nanoparticles, and Nile Red was not liberated even after heating, indicating that it was not suitable as a molecular switch. Nile Red was not released even after heating for GSSG with disulfide bond and methionine with sulfide bond, but for cystine with disulfide bond, the fluorescence intensity was greatly recovered after heating and cooling. It was shown to be applicable as a molecular switch.

[Examples 13 to 16 and Reference Examples 8 to 9]
(Evaluation of molecular switch)
From the results in Examples 9 to 12 and Reference Examples 2 to 7, the concentrations of GSH, Glu-Cys, Cys-Gly, and cystine that are considered to be suitable as molecular switches were examined. For comparison, the same study was performed for L-Cys and Hcys.

In the experiment, with respect to the gold nanocomposite solution obtained in the same manner as in Examples 9 to 12 and Reference Examples 2 to 7, 1.0 × 10 ⁻³ M of each compound solution was in the range of 0 to 250 μL. After changing the addition amount, fluorescence spectrum measurement was performed under the same conditions as in Examples 1 to 4. Thereafter, heating was performed at 90 ° C. for 30 minutes, followed by cooling at 4 ° C. for 1 hour, and fluorescence spectrum measurement was performed under the same conditions.

The obtained result is shown in FIG. As shown in FIG. 10, it was confirmed that GSH, Glu-Cys, Cys-Gly, and cystine function effectively as molecular switches in the measured concentration range of 0.5 to 2.5 × 10 ⁻⁵ M. did it.

In addition, in GSH, since a significant effect was seen even at a relatively low addition concentration, a finer concentration change was given within a concentration range of 0 to 1 × 10 ⁻⁶ M, and the difference in effect due to concentration fluctuations I investigated. The obtained results are shown in FIG. As shown in FIG. 11, it was found that when the concentration was about 4 × 10 ⁻⁶ M or more, the fluorescence intensity was greatly recovered after heating and cooling.

[Example 17]
(Examination of the influence of coexisting substances)
From the results of Examples 13 to 16 and Reference Examples 8 to 9, it was found that GSH is one of the suitable molecular switches, so whether the function of this GSH molecular switch is affected by coexisting substances. Study was carried out.

Add 0.18 g-Au / L gold nanoparticle solution to a 10 mL volumetric flask, add 0.100 mL of 1.0 × 10 ⁻⁵ M Nile Red solution, and add 3.73 mL of 2 mass% NIP-DETA solution. The final volume was made up to 10 mL with water.

50 μL of 1.0 × 10 ⁻³ M GSH solution and 50 μL of each 1.0 × 10 ⁻³ M solution of L-Cys, Hcys, Gly, and glutamic acid as coexisting substances to the obtained gold nanocomposite solution After the addition, fluorescence spectrum measurement was performed under the same conditions as in Examples 1 to 4. Thereafter, heating was performed at 90 ° C. for 30 minutes, followed by cooling at 4 ° C. for 1 hour, and fluorescence spectrum measurement was performed under the same conditions. As a comparative control, a system in which only 50 μL of a 1.0 × 10 ⁻³ M GSH solution was added without adding any coexisting substances was separately prepared and subjected to the same measurement.

  The obtained result is shown in FIG. As shown in FIG. 12, it was confirmed that GSH functions effectively as a molecular switch even in the presence of coexisting substances.

Claims (4)

  It is characterized by adding a drug aqueous solution and a thermoresponsive polymer solution in this order to a colloidal solution of gold nanoparticles, adsorbing the drug on the gold nanoparticles first, and then coating the thermoresponsive polymer. A method for producing a gold nanocomposite. A gold nanocomposite comprising a gold nanoparticle having a drug adsorbed on its surface and a thermoresponsive polymer formed by coating the gold nanoparticle is used by heating in the presence of thiols or cystine. Drug delivery system drug. The drug delivery system drug according to claim 2 , wherein the thermoresponsive polymer is a polymer represented by the following chemical formula 2 (wherein R ¹ and R ² each independently represents an alkyl group) R ³ and R ⁴ each independently represents hydrogen or a methyl group, and x / y is 95/5 to 70/30, and n is 1 to 4.

The drug delivery system drug according to claim 2 or 3 , wherein the drug is a fluorescent substance, a physiologically active substance, or a pharmaceutical compound.

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