JP2014162785A - Nano-dispersion method of hardly-soluble organic substance, and nanoparticle colloid of hardly-soluble organic substance obtained by the dispersion method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology for nanoparticle colloid of hardly-soluble organic substance with high purity which has excellent dispersion stability and storage stability, and no impurity, a technology for nanoparticle colloid which maintains a structure of original hardly-soluble organic substance and has improved functions, such as activity and reactivity, compared with, for example, a solution of the organic substance, and a technology for nanoparticle colloid in which nanoparticles have high surface modification performance.SOLUTION: In a nano-dispersion method of hardly-soluble organic substance, the hardly-soluble organic substance is irradiated with an ultrashort pulse laser beam in a liquid or solid medium and is subjected to laser ablation, the hardly-soluble organic substance is refined to nanoparticles, and the nanoparticles are dispersed in the liquid or solid medium. Nanoparticle colloid is obtained by the nano-dispersion method of hardly-soluble organic substance.

Description

  The present invention relates to a nano-dispersion method of a hardly soluble organic substance and a nanoparticle colloid of the hardly soluble organic substance obtained by the dispersing method.

  A nanoparticle refers to an ultrafine particle having an average particle diameter of less than 1 micrometer (μm), and generally having a particle diameter of about 1 to 100 nanometers (nm). Compared to a solid having a normal size or a solution in which the solid is dissolved, the nanoparticles have increased dispersibility and diffusivity due to their high surface area, and can further improve functions such as activity and reactivity. Applications to a wide range of technical fields such as medicine, agricultural chemicals, cosmetics, the environment, electronic devices, and chemical reaction catalysts are expected. For this reason, research and development are being carried out to utilize the characteristics of nanoparticles. In addition, nanoparticle colloids in which nanoparticles are dispersed in water, an organic solvent, or the like are expected to be applied to substances whose use has been restricted due to their poor solubility, assuming that they do not cause precipitation or phase separation.

  A method for producing a nanoparticle colloid in which nanoparticles are dispersed in another substance has been reported. For example, a method for producing a nanoparticle colloid of an inorganic metal using a reverse micelle method has been reported ( For example, see Patent Document 1). Specifically, Patent Document 1 includes water, a hydrophobic organic solvent, a surfactant, and a hydrophilic organic solvent having a solubility parameter difference of 0 to 5 based on the hydrophobic organic solvent. Reverse micelle solution containing 2 to 300 millimoles (mmol) per liter (L) of total volume of organic solvent, and water / hydrophobic organic solvent, surfactant, and inorganic value based on the surfactant / A reverse micelle solution containing a hydrophilic organic solvent having a difference in organic value ratio within ± 1.5 and containing 2 to 300 millimoles (mmol) per 1 liter (L) of the total volume is disclosed. . And the method of producing the inorganic nanoparticle of a metal, an alloy, or its oxide using the said reverse micelle liquid is disclosed. In addition, a technique for encapsulating a low molecular weight antioxidant in a polymer micelle (see, for example, Patent Document 2) has been reported. Specifically, Patent Document 2 discloses a composition in which a low molecular weight antioxidant is encapsulated in a polymerized cyclic nitroxide radical compound in a polymer micelle form. Examples of low molecular weight antioxidants encapsulated in polymer micelles include poorly water-soluble drugs such as vitamin E, β-carotene, ubiquinone, and bilirubin, which can be solubilized in an aqueous medium, It is disclosed that it is possible to expand the use of antioxidants whose fields of use are limited.

  Here, the application of a poorly water-soluble drug to a living body has a problem that the expected medicinal effect cannot be obtained because it hardly penetrates into biological membranes and cells. For example, when administered orally, the absorbability from the gastrointestinal mucosa is poor and the bioavailability is significantly reduced. Also for parenteral administration, for example, drugs administered by intramuscular or subcutaneous injection must pass through one or more biological membranes to reach the systemic circulation, thus limiting bioavailability improvements. Is. Furthermore, it was not transported to the inside of the target cell and was discharged outside the body without exhibiting a desired drug effect. Therefore, improvement of the solubility, absorbability, and intracellular permeability of a poorly water-soluble drug is an important issue in formulation design. In particular, due to changes in drug discovery strategies in recent years, a large amount of drug candidate compounds have been synthesized and evaluated in large quantities by using combinatorial chemistry, high-throughput screening, and the like. Therefore, poorly water-soluble compounds are increasing in the field of drug discovery, and the formulation design has become a big issue.

JP 2009-82828 A JP 2011-184429 A

  However, although the technique of Patent Document 1 is a technique for producing a nanoparticle colloid, there is no disclosure of functions such as activity and reactivity when such a nanoparticle colloid is compared with the original solid. Moreover, the technique of patent document 1 ionizes an inorganic metal once and prepares it to a nanoparticle by reducing this, In the preparation, various kinds of hydrophobic and hydrophilic organic solvents, surfactants, and the like are used. A simple reagent is used. For this reason, there is a problem that it is difficult to apply the technology to the fields of medicine, cosmetics, food, etc. intended for living body application.

  The technique of Patent Document 2 uses micelles similarly to the technique of Patent Document 1, and is a technique of encapsulating a drug in polymer micelles. And the result of having examined the application to Alzheimer's disease as a biological application of the composition indicated by patent documents 2 is indicated. However, since various reagents including a polymerized cyclic nitroxide radical compound are used in the preparation of the composition, the application range is expected to be limited. Therefore, it still does not solve the problem of Patent Document 1.

  Therefore, in view of the problems of the prior art, the present invention is a technology that can provide a nanoparticle colloid of a highly pure hardly soluble organic material that has excellent dispersion stability and storage stability and does not contain impurities. With the goal. And it aims at construction of the technique which can provide the nanoparticle colloid which retained the structure of the original poorly soluble organic substance, and improved functions, such as activity and reactivity, compared with the solution etc. of the said organic substance. Moreover, it aims at construction | assembly of the technique in which the containing nanoparticle can provide the nanoparticle colloid which has high surface modification ability.

  As a result of intensive research to solve the above problems, the inventors of the present application have achieved excellent dispersion stability by irradiating a poorly soluble organic substance with ultrashort pulsed laser light having a pulse width of femtosecond level, etc. It was also found that a high-purity nanoparticle colloid having storage stability and free of impurities can be provided. In addition, laser ablation is a physical pulverization technique that is performed without heat treatment, so that the nanoparticles have a high surface modification ability and retain the structure of the original organic substance, The present inventors have found that a colloidal nanoparticle with improved functions such as activity and reactivity can be provided compared with a solution and the like, and have completed the present invention based on these findings.

That is, the invention shown in the following [1] to [7] is provided.
[1] A nano-dispersion method of a hardly soluble organic substance, wherein the hardly soluble organic substance is nano-particled by irradiating the hardly soluble organic substance with an ultrashort pulse laser beam in a liquid or solid medium to perform laser ablation. A nano-dispersion method of a sparingly soluble organic material, wherein the nano-particles are dispersed into the liquid or solid medium.
[2] The ultrashort pulse laser beam is a laser beam having a pulse time width of 1 femtosecond or more and less than 1 picosecond.
[3] The medium is a liquid.
[4] The poorly soluble organic substance is selected from an antioxidant, an anti-inflammatory agent, and an antineoplastic agent.
[5] The hardly soluble organic substance is a fat-soluble vitamin.
[6] The fat-soluble vitamin is vitamin E.
[7] A nanoparticle colloid of a hardly soluble organic material obtained by the nano-dispersion method of the hardly soluble organic material.

  According to the above configurations [1] to [6], a method for nano-dispersing a sparingly soluble organic material is provided by irradiating a sparingly soluble organic material with an ultrashort pulsed laser beam in a liquid or solid medium to perform laser ablation. can do. In this method, a hardly soluble organic substance is physically pulverized to a nano size to be uniformly finely dispersed in a medium. Therefore, since it is non-thermal processing, it can be nano-dispersed without destroying the chemical structure of the hardly soluble organic substance. In addition, it can be carried out more easily than conventionally known chemical treatment methods such as adding a hydrophilic group, and the surface thereof is not subjected to chemical modification such as coating. There is an advantage that particles can be obtained. Furthermore, there is an advantage that nano-dispersion of a hardly soluble organic substance is possible without mixing impurities such as a reaction reagent such as a dispersing agent in the production process. And since the nanoparticle colloid obtained by the said method is a particulate form, the inside of particle | grains is protected from light. As a result, unlike a solution, there is an advantage that an organic substance can be prevented from being oxidized and a nanoparticle colloid excellent in storage stability can be provided. And when the nanoparticle colloid obtained by the said method is administered in the living body, the said organic substance is compared with the state of the solution or suspension which dissolved or suspended the original poorly soluble organic substance in the solvent. It can be transported into cells at a high concentration. Furthermore, it is possible to provide a technique that can contribute to greatly expanding the use of a hardly soluble organic substance that has been difficult to administer in vivo due to its poor solubility or has been restricted in dosage form. There are also advantages.

  In particular, according to the configuration [2] above, a femtosecond laser that irradiates an ultrashort pulse laser beam of 1 femtosecond or more and less than 1 picosecond during laser ablation is used. This makes it possible to irradiate the sparingly soluble organic substance with extremely strong laser light within an extremely short time, and as a result of a small heat-affected zone, the laser ablation efficiency can be increased.

  Moreover, according to the configuration of [3] above, since the hardly soluble organic substance is finely dispersed in the liquid medium, a nanoparticle colloidal solution of the hardly soluble organic substance can be provided. Such a nanoparticle colloidal solution has improved functions such as activity and reactivity compared to the case of a solution or suspension, and can be used in various technical fields such as pharmaceuticals and chemical catalysts.

  Furthermore, according to the configuration of [4] to [6] above, it is possible to provide a nano-dispersing method for a poorly soluble drug, and to improve the bioavailability of the hardly soluble drug. In other words, when the nanoparticle colloid obtained by the method is administered into a living body, the organic substance is expressed in cells compared with a solution or suspension in which the original poorly soluble organic substance is dissolved or suspended in a solvent. It can be transported at a high concentration in the inside, and its medicinal effects can be sufficiently exerted. Furthermore, conventionally, since it is hardly soluble, the use of the hardly soluble organic substance, which is difficult to administer to a living body or whose administration form is limited, can be greatly expanded. The use value is very high nowadays when the hardly soluble organic substances are increasing.

  According to the configuration of [7] above, a nanoparticle colloid of a hardly soluble organic substance can be provided. The nanoparticle colloid is a colloid obtained by finely dispersing a sparingly soluble organic substance into a nanosize by physical pulverization. And since it is prepared by non-thermal processing, a nano-dispersed nano-particle colloid can be provided without destroying the chemical structure of the hardly soluble organic substance. In addition, the nanoparticle colloids are different from those prepared by chemical treatment, because the surface of the nanoparticles is not subjected to chemical modification such as coating, and therefore contains nanoparticles with high surface modification ability. And have the advantage. Furthermore, it is a nanoparticle colloid of a highly pure hardly soluble organic substance that does not contain impurities such as reaction reagents. Further, since the nanoparticle colloid is in the form of particles and the inside of the particles is protected from light, unlike the solution, there is an advantage that the oxidation of the organic substance is suppressed and the storage stability is excellent. When the nanoparticle colloid is administered into a living body, the organic colloid is increased in the cell compared to a solution or suspension obtained by dissolving or suspending the original poorly soluble organic substance in a solvent. Can be transported to a concentration. Furthermore, conventionally, since it is hardly soluble, the use of the hardly soluble organic substance, which is difficult to administer to a living body or whose administration form is limited, can be greatly expanded.

It is a figure which shows roughly the laser type | system | group system suitable for utilization of the nano dispersion | distribution method of this invention. It is a graph which shows the result of Example 1 which performed the verification of the antioxidant and anti-inflammatory ability using the nanoparticle colloid of vitamin E, and shows the relationship between the VE amount (microgram) added to the macrophage cell, and fluorescence intensity. The fluorescence intensity correlates with the amount of reactive oxygen species produced, and the same concentration of VE nanoparticle colloid is compared with the solution. It is a graph which shows the result of Example 2 which verified the antioxidant and anti-inflammatory ability using the nanoparticle colloid of vitamin E, and represents the amount of TNF-α mRNA as a relative amount ratio with the amount of β-actin mRNA, and the same concentration Compare VE nanoparticle colloids and solutions.

  Hereinafter, the present invention will be described in detail.

  The dispersion method of the sparingly soluble organic material of the present invention is to refine the sparingly soluble organic material into nanoparticles by irradiating the sparingly soluble organic material with an ultrashort pulse laser beam in a liquid or solid medium and performing laser ablation. In addition, the nanoparticles are dispersed in the liquid or solid medium.

  Here, the dispersion means a phenomenon in which other substances are dispersed in the form of fine particles in a continuous homogeneous phase substance. A substance in which fine particles are dispersed can be referred to as a dispersion medium, and the dispersed fine particles can be referred to as a dispersoid. The nano-dispersion means a state in which the dispersoid is dispersed in the form of nano-sized fine particles, that is, as nanoparticles. At this time, it is preferable that the dispersoid is stably dispersed in the dispersion medium without settling. And in this way, what has the dispersoid nano-dispersed in a dispersion medium is called a nanoparticle colloid.

  Nanoparticles refer to ultrafine particles having an average particle size of less than 1 μm, and generally having a particle diameter of about 1 to 100 nm. Here, the particle size is preferably 20 to 1000 nm, More preferably, it is ˜300 nm. If the particle size is larger than 1000 nm, the nanoparticles will settle out in a few hours, causing problems in dispersibility. To ensure stable dispersibility, the particle size should be 500 nm or less, and It is preferable that the size of the prepared particles does not vary.

  A substance that is a target of the nano-dispersing method of the present invention, that is, a substance that can be a dispersoid is a hardly soluble organic substance. Here, poorly soluble means poorly water-soluble and corresponds to drugs classified as BCS class II or class IV. It means a property that is difficult to dissolve in a medium such as water. Preferably, it is poorly water soluble. In the present specification, the poor solubility includes so-called insolubility that hardly or not dissolves in the solvent. In addition, in the case where it is hardly soluble in a solvent to be dispersed, the substance can be a target of the dispersion method of the present invention even if it is easily soluble in other solvents.

  An organic substance means a compound containing at least a carbon atom as a constituent element in its structure. In addition to carbon atoms, hydrogen, nitrogen, oxygen, sulfur, phosphorus, halogen atoms and the like can be contained. In addition, an organic substance having an unshared electron pair is a metal complex in which a metal or a metal ion is coordinated, an organometallic compound including a direct bond between a carbon atom and a metal atom in the structure, an oxygen, nitrogen, or phosphorus atom in the structure. Includes those bonded to a metal, metal salts of organic acids, and the like.

  The hardly soluble organic substance is preferably a solid, particularly preferably a solid at room temperature. Moreover, the shape may be any of a block shape, a granular shape, a powder shape, and the like.

  Examples of poorly soluble organic substances include medical and cosmetic drugs, agricultural chemicals, dyes, reagents and the like. Preferably, it is a medical drug, antioxidant, anti-inflammatory agent, antineoplastic agent, analgesic, antitussive, diuretic, antibacterial agent, antiviral agent, muscle relaxant, gastrointestinal therapeutic agent, neuropsychiatric therapeutic agent, Examples thereof include, but are not limited to, diabetes therapeutic agents, cardiovascular therapeutic agents, nutritional supplements, and the like. Antioxidants, anti-inflammatory agents, and anti-malignant tumor agents are preferred. Specifically, plant-derived components are exemplified as antioxidant and anti-inflammatory agents. For example, fat-soluble vitamins such as vitamin A, vitamin D, vitamin E, vitamin K and the like can be mentioned. In addition, α-carotene, β-carotene, γ-carotene and δ-carotene, which are provitamins A, and carotenoids including carotenes such as lycopene, xanthophylls such as lutein and astaxanthin, curcuminoids such as curcumin, isoflavones, quercetin and rutin And polyphenols including flavonoids such as Furthermore, anti-inflammatory agents such as fluorobrofen, indomethacin, ibuprofen, acetaminophen, steroidal anti-inflammatory agents such as glucocorticoid, fluorometholone, betamethazone, antineoplastic agents such as paclitaxel, docetaxel, etoposide, tamoxifen, camptothecin, Examples include immunosuppressive agents such as cyclosporine, antibacterial agents such as clarithromycin and miconazole, gastrointestinal therapeutic agents such as rebamipide, cardiovascular therapeutic agents such as nifedipine, and neuropsychiatric therapeutic agents such as phenytoin. However, the present invention is not limited to these, and a wide range of poorly soluble drugs can be targeted.

  Preferably, the poorly soluble organic substance is a fat-soluble vitamin. Fat-soluble vitamins include vitamin A, vitamin D, vitamin E, vitamin K and the like. As vitamin A, retinol, 3-dehydroretinol and derivatives thereof, vitamin D as cholecalciferol, ergocalciferol and derivatives thereof, vitamin E as α, β, γ-tocopherol, α, β, γ -Tocotrienol and derivatives thereof, and vitamin K include phylloquinone, methoquinone, menadione and derivatives thereof, but are not limited thereto. Examples of the derivatives include esters such as succinic acid, acetic acid, and fatty acids. In addition, poorly soluble provitamins can also be targeted for dispersion in the present invention.

  Among these, vitamin E is preferable, and succinic acid ester of vitamin E that is solid at room temperature is particularly preferable. Vitamin E has antioxidant, anti-inflammatory and anti-aging effects and is reported to be useful for the prevention and treatment of bacterial infections, arteriosclerosis, cardiovascular diseases, pressure ulcers, frostbite and aging It has a medicinal effect.

  In other words, the poorly soluble organic substance that is a suitable target for the nano-dispersing method of the present invention has an excellent medicinal effect and is poorly soluble while having an excellent function. It is a substance that could not be fully demonstrated. By finely dispersing these substances by the method of the present invention, their functions can be sufficiently exhibited and enhanced.

  The dispersion medium may be a liquid or a solid medium, but is preferably a liquid having a fluidity. And when a dispersion medium is a solid medium, it is preferable that it is a powder so that a dispersoid can disperse | distribute uniformly. Further, it may be in a gel or sol state in which a solid and a liquid are mixed. Examples of the dispersion medium include salt solutions such as water, distilled water, physiological saline, sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium bicarbonate, ammonium nitrate, phosphate buffer, borate buffer, and tris buffer. Solution, aqueous media such as buffer solutions such as citric acid and fumarate buffer, ethanol, methanol, isopropyl alcohol, aryl alcohol, ethylene glycol, glycerol and other alcohols, diethyl ether and other ethers, chloroform, benzene, acetone, Organic media such as hexane, acetonitrile, castor oil, olive oil, soybean oil, linseed oil, palm oil, sesame oil, carnauba wax and other animal oils, beeswax, squalane, lanolin and other animal oils, paraffin, petroleum jelly and other mineral oils, Inorganic acid salts such as calcium carbonate, lactose, sucrose Examples include, but are not limited to, sugars such as glucose, starch, cellulose, gelatin, carboxymethylcellulose, and dextran, and synthetic polymer polymers such as polyvinyl chloride, polystyrene, polypropylene, and silicon. A liquid or solid medium that can be used as a medium can be used. And a dispersion medium is suitably selected according to the use and objective of the nanoparticle colloid which is a product of the nano dispersion method of this invention. However, since it is not desirable that the poorly soluble organic substance that becomes the dispersoid dissolves, it is important to consider the solubility of the hardly soluble organic substance that becomes the dispersoid when selecting the dispersion medium. A medium exhibiting properties is selected as a dispersion medium. For example, when the object of dispersion is a poorly water-soluble organic substance, it is preferable to use water or an aqueous medium as the dispersion medium.

  The amount of the dispersion medium is not particularly limited as long as the hardly soluble organic substance serving as a dispersoid can be dispersed as nanoparticles.

  Next, laser ablation is a phenomenon in which particles are explosively peeled and released from the surface of the material by irradiating the surface of the material with extremely strong laser light in a short time. In the nano-dispersion method of the present invention, it is preferable to use femtosecond laser ablation in which the pulse width oscillates at a femtosecond level. For example, refer to Japanese Unexamined Patent Application Publication No. 2012-13668.

  FIG. 1 is a diagram schematically showing a part of a laser system suitable for use in the nano-dispersing method of the present invention. Here, a case where a lump-like hardly soluble organic substance (target) is finely dispersed in a liquid dispersion medium (liquid) is exemplified. The laser beam 1 is received from an ultrashort pulse source (not shown), focused by a lens 2, and guided to a target 4 by a mechanism for rapid beam movement, for example, a vibrating mirror 3. The target 4 is submerged several millimeters below the surface of the liquid 5 contained in the container 6, preferably less than 1 cm. The container 6 is disposed on a motion stage 7, for example, a moving stage. A liquid flow is introduced into the container so that the nanoparticles 8 are swept away and collected elsewhere. The liquid flow also cools the laser focal volume. The liquid is preferably deionized water having a specific resistance of more than 1 M ohm · cm, but can be appropriately selected according to the purpose of use and application as described above. A controller (not shown) is operably coupled to the pulse source, motion system and / or circulation system. The controller regulates beam emission, liquid flow and motion. The controller is also configured to be coupled to a system computer, user interface, telecommunications equipment and / or other standard device and programmed from a remote location.

  Lasers for the production of nanoparticles have a wavelength of about 1.03 μm (a layer of a few millimeters of liquid has negligible absorbance at this wavelength), a pulse in the range of about 1-20 microjoules, preferably less than about 10 microjoules. May have energy. Pulse widths of about 500 fs and up to about 10 ps can be used without significantly affecting nanoparticle formation. The pulse repetition rate may range from about 100 kHz to 5 MHz. Preferred laser systems are further described below. Liquid flow, beam movement, or both can be used to avoid heat storage at high repetition rates.

  For example, the oscillating mirror 3 is configured for rapid rastering or other movement of the laser beam onto the target surface. The mirror frequency is preferably greater than 10 Hz and the angular amplitude is preferably greater than 1 mrad. A raster speed on the target surface of greater than 0.01 m / s can be provided. Such a mirror may be a piezo-driven mirror, a galvanometer mirror, or other suitable device for beam movement.

  The liquid flow can be introduced into the container by a circulation system, and the flow rate is preferably greater than 10 mL / s. If a circulation system is not available, the flow of liquid across the ablation spot can be caused locally by introducing transverse vibrational movement. For example, the motion stage 7 can be moved in a direction perpendicular to the laser beam as shown in FIG. The vibration stage preferably has a frequency of several Hz and an amplitude of several millimeters.

  Stable and chemically pure nanoparticles are produced by controlling both laser parameters and liquid flow rate. Laser parameters include pulse width, pulse energy, pulse repetition rate, and beam movement.

  In the present invention, it is preferable to employ an ultrashort pulse width. In many laser processing applications, for example, ultrashort pulse widths in the femtosecond range increase ablation efficiency as a result of very high maximum power and small heat affected zone. Therefore, the pulse width is preferably 1 femtosecond or more and less than 1 picosecond.

  Research for application in nanoparticle generation (Reference 1: B. Liu, ZD Hu, Y. Che, YB Chen, XQ Pan, "Nanoparticle generation in ultrafast pulsed laser ablation of nickel", Applied Physics Letters, Vol. 90, 044103 (2007), Reference 2: B. Liu, ZD Hu, Y. Che, "Ultrafast sources: ultrafast lasers produce nanoparticles", Laser Focus World, Vol. 43, 74 (2007)) It has been found that energy (more precisely, low fluence) is preferred for nanoparticle production. Ablated materials exist mainly in the form of nanoparticles with a narrow particle size distribution. US Patent Publication No. 2008/0006524 also teaches a method for producing colloidal particles in vacuum and in atmospheric gases based on these studies.

  Preferably, a high pulse repetition rate of, for example, at least about 10 kHz, more preferably at least about 100 KHz is used for at least three reasons. The first is the multipulse effect in high repetition rate pulsed laser ablation. With a pulse interval of less than 10 microseconds (ie, a high repetition rate above 100 kHz), the ablated material undergoes multiple laser firings and becomes highly charged before leaving the laser focal volume. Stable colloidal particles can be made at such high repetition rates without the addition of additional chemical stabilizers. The second reason is that large particles may be fragmented during multipulse ablation, resulting in a particle size distribution of the colloidal particles. The third reason is a high production rate that benefits from a high repetition rate.

  Also, a rapid raster of the laser beam during ablation is beneficial with high repetition rate operation. For example, without such a rapid raster of the laser beam, the colloidal particle stream produced by the preceding laser pulse will eventually block subsequent laser pulses by scattering and absorption. More importantly, the accumulated heating to water with a high repetition rate can also induce agglomeration of colloidal particles.

  As described above, according to the nano-dispersing method of the present invention, there is provided a nano-dispersing method of a hardly soluble organic material by irradiating the hardly soluble organic material with an ultrashort pulse laser beam in a liquid or solid medium and performing laser ablation. can do. In this method, a hardly soluble organic substance is physically pulverized to a nano size to be uniformly finely dispersed in a medium. And since it is non-thermal processing using ultra-short pulse laser light such as femtosecond laser light, nano-dispersion is possible without destroying the chemical structure of the sparingly soluble organic substance to be dispersed.

  Further, according to this method, it can be carried out more easily than conventionally known chemical treatment methods such as adding a hydrophilic group, and since the surface is not chemically modified such as coating, it is high. There is an advantage that nanoparticles having surface modification ability can be obtained. Furthermore, according to the method, there is an advantage that nano-dispersion of only a hardly soluble organic substance can be performed without mixing impurities such as a reaction reagent such as a dispersant in the production process. And since the nanoparticle colloid obtained by the said method is a particulate form, the inside of particle | grains is protected from light. As a result, unlike a solution, it is possible to provide a nanoparticle colloid in which oxidation of an organic substance is suppressed and storage stability is excellent. The nanoparticle colloid obtained by this method can be used to inject a drug into a cell as compared with a solution or suspension obtained by dissolving or suspending an original poorly soluble organic substance in a solvent. Can be transported to high concentration. Furthermore, since it is hardly soluble in the prior art, it can contribute to greatly expanding the use of hardly soluble organic substances that are difficult to administer to living bodies or whose administration forms are limited.

  A nanoparticle colloid which is a dispersion obtained by the nanodispersion method of the present invention is also included in the present invention and constitutes a part of the present invention. The nanoparticle colloid of the present invention is one in which a hardly soluble organic substance is uniformly and stably dispersed as nanoparticles.

  Furthermore, the nanoparticle colloid of the present invention can be put into practical use as it is, but can be formulated according to its purpose and use. The form of the preparation may be liquid or solid, but is preferably liquid. In formulation, an appropriate carrier can be included, regardless of whether it is solid or liquid, as long as the properties such as the activity of the finely dispersed hardly soluble organic substance can be stably maintained, There is no particular limitation.

  When formulated as a liquid form, for example, it can be prepared by diluting or substituting the nanoparticle colloid of the present invention into a liquid such as water, an appropriate salt solution or buffer, an organic solvent, or an oil. As such a liquid, those exemplified as the dispersion medium can be used, but the liquid is not limited thereto. Further, the hydrogen ion concentration (pH) may be set as appropriate according to the application. Furthermore, auxiliary agents such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers, and solubilizing agents may be included.

  In the case of formulation as a solid form, in addition to the case where the nanoparticle colloid dispersion medium is a solid, the nanoparticle colloid dispersion medium is a liquid. It can also be prepared by subjecting it to drying or the like to form solid nanoparticles. And it can contain a suitable carrier and is mixed with excipients, binders, lubricants, disintegrants, etc., and tablets, powders, fine granules, granules, capsules, pills, etc. by conventional methods It can also be prepared in the form of Here, as the solid carrier, those exemplified as the above-mentioned dispersion medium can be used, and can be appropriately selected according to the purpose of use and application. For example, as an excipient, starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc., as a binder, starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, etc., as a lubricant, stearic acid, Examples of disintegrants such as magnesium stearate and calcium stearate include carboxymethyl cellulose and talc. However, it is not limited to these, and a known material can be appropriately selected and used.

  As described above, the nanoparticle colloid of the present invention is a colloid obtained by finely dispersing a hardly soluble organic substance into a nanosize by physical grinding. Since it is prepared by non-thermal processing, a nano-dispersed nano-particle colloid can be provided without destroying the chemical structure of the hardly soluble organic substance. In addition, the nanoparticle colloids are different from those prepared by chemical treatment methods, because the surface of the nanoparticles is not subjected to chemical modification such as coating, and therefore contains nanoparticles with high surface modification ability. There are advantages. Therefore, it can be modified with various substances depending on the purpose of use and application, and for example, a desired labeling substance or coating can be applied. Furthermore, it is possible to provide a nanoparticle colloid of a highly pure hardly soluble organic substance that does not contain impurities such as reaction reagents. Therefore, even when applied in vivo, it is safe without inducing undesirable side reactions. Further, since the nanoparticle colloid is in the form of particles and the inside of the particles is protected from light, unlike the solution, there is an advantage that the oxidation of the organic substance is suppressed and the storage stability is excellent.

  And when the nanoparticle colloid is administered in vivo, the concentration of the drug in the cell is higher than that of the solution or suspension obtained by dissolving or suspending the original poorly soluble organic substance in a solvent. Can be transported. Furthermore, conventionally, since it is hardly soluble, the use of the hardly soluble organic substance, which is difficult to administer to a living body or whose administration form is limited, can be greatly expanded.

  That is, the nanoparticle colloid of the present invention can be used for the production of various products, and can contribute to improving the performance of the products. For example, it can be used for pharmaceuticals, agricultural chemicals, cosmetics, dyes, reagents such as labels and reaction catalysts, electronic materials such as displays and recording media, and the like.

  In particular, its usefulness can be exhibited in the pharmaceutical field, and the insoluble drug can be uniformly dispersed as nano-sized particles, so that the permeability and absorbability of the drug can be improved. That is, since the drug can be transported into the cell at a high concentration and can penetrate deep into organs, tissues, and cells, the drug effect can be sufficiently exerted. Even when compared with the state of a solution or suspension obtained by dissolving or suspending the original poorly soluble organic substance in a solvent, very high medicinal effects can be exhibited. Further, the absorbability of the drug from the mucous membrane such as the oral mucosa and the digestive tract mucous membrane and the skin can be improved. Thus, conventionally, it is possible to greatly expand the use of hardly soluble organic substances that have been difficult to administer to living bodies due to poorly soluble properties and whose administration forms are limited. Therefore, the present invention can be applied to any dosage form of oral administration and parenteral administration. Therefore, any known dosage form such as intramuscular administration, subcutaneous administration, transdermal administration, intranasal administration, rectal administration, etc. may be used, and since high dispersion stability can be realized, there is a possibility for intravenous administration. It is.

  Nanoparticles are known to have a property of accumulating in phagocytic cells such as macrophages that are present abundantly in the reticuloendothelial system such as liver, spleen and bone marrow. In addition, since the vascular permeability of tumor cells is significantly enhanced compared to normal tissues, the nanoparticles are more likely to flow out of the blood vessels. On the other hand, because the lymphatic system has not developed, the nanoparticles that have reached the tumor tissue Accumulate in tumor cells. In general, when a fat-soluble drug is administered orally, the drug absorbed in the intestine is metabolized in the liver, chemically changed to hydrophilic substances by the action of enzymes, etc., excreted in the urine, and not absorbed in the intestine The drug is discharged as stool as it is. Therefore, when a fat-soluble drug is administered as the nanoparticle colloid of the present invention, it is stored in macrophages and tumor cells, so that discharge from the kidney and the like is reduced. Therefore, the nanoparticle colloid of the present invention is particularly suitable for use as a therapeutic agent for inflammatory diseases and cancers associated with macrophages.

  Moreover, since the nanoparticle which comprises the nanoparticle colloid of this invention has high surface modification activity, it can modify with various substances according to a use purpose and an application. For example, a drug delivery system that modifies the target site with a substance with high specific affinity and effectively delivers the drug to the target site, or modifies it with an appropriate labeling substance that can be detected from the outside, and observes the pharmacokinetics of the drug However, it is not limited to these. Accordingly, the nanoparticle colloid of the present invention is expected to open up new applications due to its excellent physical properties.

  Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples.

Example 1. Verification of antioxidant and anti-inflammatory activity using nanoparticle colloid of vitamin E-1
In this example, the antioxidant ability of vitamin E was compared with that of vitamin E in a solution state. The antioxidant ability was verified by detecting reactive oxygen species produced by inflammation-induced macrophage cells.

(reagent)
Since vitamin E (hereinafter sometimes referred to as “VE”) is oily at room temperature, the following examination was performed using a VE derivative, which is a powder at room temperature, and a VE succinate.
VE nanoparticle colloid was prepared by finely dispersing VE succinate using IMRA laser (femtosecond fiber laser) (IMRA AMERICA D-1000, irradiation energy: 1W, dispersion medium: ion exchange) water). The nanoparticle colloid prepared here contained VE nanoparticles having an average particle size of 189 nm.
The nanoparticle colloid of VE was diluted with a cell culture medium solution so as to be 0, 1.25, 2.5, 5, 10, 20 μg / well, and prepared to contact macrophage cells.

(Method)
After inoculating a macrophage cell line (Raw 264.7) in a petri dish, 10 μg / ml lipopolysaccharide (hereinafter referred to as “LPS”) was added to induce inflammation. After induction, each amount of VE nanoparticle colloid was added to the petri dish. Subsequently, 2'7'-dichlorofluorescein diacetate was added, and then fluorescence was measured to measure antioxidant capacity. Here, 2'7'-dichlorofluorescein diacetate is used as an index of intracellular reactive oxygen species because it becomes dichlorofluorescein that fluoresces when oxidized by reactive oxygen species. The experiment was performed with n = 3.

  Further, as a comparative example, the same amount of solution VE (solvent: cell medium solution) was added to another petri dish instead of VE nanoparticle colloid, and fluorescence was measured to measure antioxidant ability (Comparative Example 1). . As controls, the fluorescence derived from dichlorofluorescein was measured in the same manner for untreated macrophage cells (control, untreated) and macrophage cells only subjected to inflammation induction by LPS (control, LPS inflammation induction only).

(result)
The results are shown in FIG. FIG. 2 is a graph showing the relationship between the amount of VE added to macrophage cells (μg) and the fluorescence intensity, and the fluorescence intensity correlates with the production amount of reactive oxygen species. It is a graph comparing the production amount of reactive oxygen species in the solution with the same amount of VE nanoparticle colloid. As a result, the colloid of VE nanoparticles suppresses the production of reactive oxygen species produced by macrophages of reactive oxygen species (Example 1). And it was confirmed that the production | generation inhibitory effect of a reactive oxygen species is higher than a VE solution (comparison with the comparative example 1). Thus, it can be understood that the nanoparticle colloid of VE can effectively suppress the production of reactive oxygen species that can be a cause of exacerbation of inflammation.

Example 2 Verification of antioxidant and anti-inflammatory ability using nanoparticle colloid of vitamin E-2
In this example, the anti-inflammatory ability of vitamin E was compared and verified with solution vitamin E. The anti-inflammatory ability was verified by measuring the amount of TNF-α mRNA, which is an index of inflammation, by real-time RT-PCR.

(reagent)
VE nanoparticle colloid was prepared in the same manner as in Example 1. The nanoparticle colloid of VE and the nanoparticle colloid of VE were diluted with a cell medium solution so as to be 5 μg / well in the same manner as in Example 1 and prepared so as to contact macrophage cells.

(Method)
In the same manner as in Example 1, VE nanoparticle colloids were added to macrophage cells in which inflammation was induced by LPS and cultured. After culture, total RNA was extracted from the cells using RNeasy mini kit (Quiagen), and then cDNA was synthesized using Reverse transcriptase (Invitorogen). Then, the gene expression level of TNF-α was measured by real-time PCR (Applied & Biosystem) using PCR primer and probes (Universal Probe Library: designed with a primer design tool manufactured by Roche) as a primer. The experiment was performed with n = 3.

  As a comparative example, TNF-α gene expression level was measured by RT-PCR using VE in the same volume (cell medium solution) instead of VE nanoparticle colloid and added to another petri dish. (Comparative Example 2). As a control, the TNF-α gene expression level was also measured in the same manner for untreated macrophage cells (control, untreated) and macrophage cells that had only undergone inflammation induction by LPS (control, LPS inflammation induction only). .

(result)
The results are shown in FIG. FIG. 3 is a graph showing the amount of TNF-α mRNA as a relative amount ratio to the amount of β-actin mRNA, and is a graph comparing the amount of mRNA in the same concentration of VE nanoparticle colloid with that of a solution. As a result, it was confirmed that the nanoparticle colloid of VE suppressed the production of TNF-α induced by LPS to about half (Example 2). And it was confirmed that the production | generation of TNF- (alpha) is suppressed effectively compared with solution form VE (comparison with the comparative example 2). That is, it can be understood that the nanoparticle colloid of VE effectively suppresses the production of inflammatory cytokines and exhibits a high anti-inflammatory effect.

  From the results of Example 1 and Example 2, it was confirmed that the nanoparticle colloid of VE exhibits higher antioxidant and anti-inflammatory effects than the solution. The nanoparticle colloid is recognized by macrophages and can contribute to improvement of medicinal effect. In particular, it is expected to be used as a therapeutic agent for macrophage-related diseases.

  The present invention can be used in all technical fields that require the use of sparingly soluble organic substances. For example, it can be used for pharmaceuticals, agricultural chemicals, cosmetics, dyes, reagents such as labels and reaction catalysts, electronic materials such as displays and recording media, and the like.

DESCRIPTION OF SYMBOLS 1 Laser beam 2 Lens 3 Vibrating mirror 4 Target 5 Liquid 6 Motion stage 7 Nanoparticle

Claims (7)

A method of nano-dispersing a sparingly soluble organic substance,
The hardly soluble organic substance is made into nanoparticles by irradiating the hardly soluble organic substance in a liquid or solid medium with laser ablation by irradiating the ultrashort pulse laser beam, and the nanoparticles are contained in the liquid or solid medium. A nano-dispersion method for sparingly soluble organic substances.   The method for nano-dispersing a sparingly soluble organic material according to claim 1, wherein the ultrashort pulse laser beam is a laser beam having a pulse time width of 1 femtosecond or more and less than 1 picosecond.   The method for nano-dispersing a sparingly soluble organic substance according to claim 1 or 2, wherein the medium is a liquid.   The method for nano-dispersing a sparingly soluble organic substance according to any one of claims 1 to 3, wherein the sparingly soluble organic substance is selected from an antioxidant, an anti-inflammatory agent, and an antineoplastic agent.   The method for nano-dispersing a sparingly soluble organic substance according to claim 4, wherein the anti-inflammatory agent is a fat-soluble vitamin.   The method for nano-dispersing a sparingly soluble organic substance according to claim 5, wherein the fat-soluble vitamin is vitamin E.   A nanoparticle colloid of a sparingly soluble organic material obtained by the nanodispersing method of a sparingly soluble organic material according to any one of claims 1 to 6.

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