JP7470954B2 - Method for producing nanoparticles - Google Patents

Description

The present invention relates to a method for producing nanoparticles having a cumulant mean diameter of 10 to 100 nm or a volume-based median diameter of 10 to 100 nm.

Polymer nanospheres have attracted attention not only for their sustained release of encapsulated drugs, but also for their high tissue and membrane permeability, and for the possibility of targeting to diseased areas. Methods proposed for preparing polymer nanospheres include the use of high-pressure emulsifiers such as microfluidizers (Handbook of Pharmaceutical Controlled Release Technology edt by D.L.Wise, Marcel Dekker, Inc.,New York, 2000, pp 435-357) and the solvent diffusion method (Y.Kawashima, Handbook of powder technology vol.9 edt by D.Chulia,M.Deleuil,Y.Pourcelot, Elsevier,Amsterdam,1994,pp 493-512). However, the particle size is around 200 nm, and it is difficult to prepare polymer nanospheres with a particle size of 100 nm or less. In addition, it is difficult to encapsulate water-soluble drugs.

In general, it is difficult to sterilize polymeric microparticles. This is because in many cases, high-pressure steam sterilization or radiation sterilization causes decomposition of the polymer or aggregation of the microparticles. For this reason, in order to use polymeric microparticles as sterile preparations, they are generally manufactured using aseptic procedures that require advanced manufacturing control. On the other hand, in addition to the above-mentioned sterilization methods, sterilization can also be achieved by filtration, which removes bacteria. In order to apply filtration, the particle size of the microparticles must be 200 nm or less. In other words, by making the particle size of the microparticles 200 nm or less, sterilization by filtration can be applied, so when using microparticles as sterile preparations, it is of great significance in terms of manufacturability and reliability of ensuring sterility.

Handbook of Pharmaceutical Controlled Release Technology edt by D.L.Wise, Marcel Dekker, Inc., New York, 2000, pp 435-357 Y. Kawashima, Handbook of powder technology vol.9 edt by D. Chulia, M. Deleuil, Y. Pourcelot, Elsevier, Amsterdam, 1994, pp493-512

The microparticulation of 100 nm or less is not only important for sterility as mentioned above, but also for improving oral absorption. The inside of the intestinal tract is protected by a mucus layer, which has a mesh structure and its filtering function acts as a defense mechanism to prevent particles such as bacteria from being absorbed into the body. However, it has been reported that in this mechanism, the diffusion coefficient in the mucus layer increases when the particle diameter of the microparticles is 200 nm or less. By utilizing this, it is expected that the oral absorption rate of poorly absorbed drugs can be improved by nanoparticleization of 200 nm or less. The promotion of absorption of nanoparticles of 200 nm or less is not limited to oral absorption, but can also be expected to improve absorption rate through other mucous membranes such as the oral cavity, the nose, the lungs, and the skin.

As described above, nanoparticles in the 200 nm or smaller range have various advantages, but there are only limited methods for producing nanoparticles other than synthesis, and while these limited methods can produce particles of around 200 nm, it is difficult to produce particles of 100 nm or smaller.
An object of the present invention is to provide a method for producing nanoparticles having either a cumulant mean diameter or a volume-based median diameter of 10 to 100 nm.

The inventors therefore undertook the development of a method for producing polymeric fine particles, preferably 100 nm or less in size. We believed that it was necessary to lower the interfacial tension between oil and water during emulsification in the production of nanoparticles, and as a result of our investigation, we discovered that one of the emulsification methods used in the field of emulsion production is the D-phase emulsification method. The D-phase emulsification method is a method in which an oil phase is emulsified in a polyol solution such as butylene glycol containing a high concentration of surfactant to form an O/D emulsion, and then water is added to obtain an O/W type emulsion (Iwasawa et al., Interface Handbook, NTS, 90-92). This method is a method for producing emulsions with a micro-order dispersed phase, and its principle is to use a high concentration of surfactant and to use a polyol to lower the polarity of water, thereby lowering the interfacial tension.

On the other hand, nanoparticles, even if stable in the production solution, are difficult to handle because they aggregate when dried and the particle size before drying cannot be obtained. This, along with the difficulty in producing nanoparticles, is a factor that inhibits the expansion of applications.
Therefore, we have conducted extensive research with reference to the D-phase emulsification method, and have found that nanoparticles having a cumulant average diameter of 10 to 100 nm or a volume-based median diameter of 10 to 100 nm can be produced by emulsifying an organic solvent solution containing molecules with a molecular weight of 200 or more in polyvinyl alcohol and an aqueous solution of propylene glycol, butylene glycol, or polyethylene glycol, and then distilling off the organic solvent. This particle diameter allows for easy filtration sterilization, which is an industrially advantageous characteristic. Furthermore, we have found that when the emulsion is made into a thin film, the drying of the solvent in the oil phase is promoted, and at the same time, the water in the aqueous phase is also dried, forming a film as a solid, and that when the film is dissolved in water, nanoparticles having the same particle diameter as before drying are released, which is also advantageous in terms of handling.

That is, according to the present invention, the following inventions are provided.
<1> A method for producing nanoparticles, either of which has a cumulant mean diameter or a volume-based median diameter of 10 to 100 nm, comprising: step A of emulsifying an organic solvent solution containing molecules with a molecular weight of 200 or more in an aqueous solution of polyvinyl alcohol and a substance that forms a gel with polyvinyl alcohol at an aqueous solution concentration of 60% or more to obtain an emulsion; and step B of removing the organic solvent from the emulsion obtained in step A.
<2> The method according to <1>, wherein the substance that forms a gel with polyvinyl alcohol at an aqueous solution concentration of 60% or more is propylene glycol, butylene glycol or polyethylene glycol.
<3> The method according to <1> or <2>, wherein the molecule having a molecular weight of 200 or more is a biodegradable polymer.
<4> The method according to any one of <1> to <3>, wherein the molecule having 200 or more molecules is a lactic acid polymer, a lactic acid/glycolic acid copolymer, or a polymer containing acrylic acid.
<5> The method according to any one of <1> to <4>, wherein the organic solvent solution containing a molecule having a molecular weight of 200 or more is an organic solvent solution containing an oligolactic acid having a degree of polymerization of 3 to 19.
<6> The method according to any one of <1> to <5>, wherein the molecule having a molecular weight of 200 or more is a physiologically active substance.
<7> The method according to any one of <1> to <6>, wherein the organic solvent is methylene chloride.
<8> The method according to any one of <1> to <6>, wherein the 200 or more molecules include curcumin.
<9> The method according to any one of <1> to <8>, wherein the concentration of polyvinyl alcohol in the aqueous solution is more than 5% by mass and not more than 25% by mass.
<10> The method according to any one of <1> to <9>, wherein the concentration of propylene glycol or butylene glycol in the aqueous solution is more than 50% by mass and not more than 70% by mass.
<11> The method according to any one of <1> to <10>, wherein in step B, the emulsion obtained in step A is diluted with water in an amount equal to or greater than the saturation solubility of the organic solvent, thereby removing the organic solvent.
<12> The method according to any one of <1> to <11>, wherein in the step B, the emulsion is dried by air drying, heating, or reduced pressure to form a film.
<13> Nanoparticles produced by the method according to any one of <1> to <12>, having a cumulant average diameter or a volume-based median diameter of 10 to 100 nm.
<14> Janus fine particles, produced by the method according to any one of <1> to <12>, having a cumulant mean diameter and a volume-based median diameter of 10 to 100 nm.

According to the present invention, it is possible to produce nanoparticles having either a cumulant mean diameter or a volume-based median diameter of 10 to 100 nm.

Transmission electron microscope image of Example 14 Transmission electron microscope photograph after negative staining of Example 21

An embodiment of the present invention will be described.
The method for producing nanoparticles according to the present invention is a method for producing nanoparticles having a cumulant mean diameter or a volume-based median diameter of 10 to 100 nm, and includes the steps of: Step A of emulsifying an organic solvent solution containing molecules having a molecular weight of 200 or more in an aqueous solution of polyvinyl alcohol and a substance that forms a gel with polyvinyl alcohol at an aqueous solution concentration of 60% or more to obtain an emulsion; and Step B of removing the organic solvent from the emulsion obtained in Step A.

Regarding particle size, the average particle size according to cumulant analysis is 200 nm or less, preferably 180 nm or less, more preferably 150 nm or less, even more preferably 130 nm or less, and even more preferably 100 nm or less. There is no particular lower limit to the average particle size according to cumulant analysis, but it is generally 10 nm or more, or 20 nm or more.

The volume-based median diameter is 10 to 100 nm, preferably 10 to 80 nm, and more preferably 10 to 50 nm. Considering that the mesh size of the mucus mesh structure is 10 to 500 nm, and the size of the internal void of the N-terminal aggregate of MUC2, the main component of mucus, is reported to be 35 nm (D. Ambort et al., PNAS, 109, 5645-5650), the size required for mucus layer-penetrating microparticles is thought to be 50 nm or less.

The average particle size by cumulant analysis can be measured by a dynamic light scattering (DLS) method using a dynamic light scattering particle size measuring device.
The volume-based median diameter is a particle diameter corresponding to 50% of the cumulative particle size distribution value on a volume basis. The volume-based median diameter can be measured, for example, by dynamic light scattering (DLS), laser scattering, centrifugal sedimentation, field-flow fractionation, or electrical detector method, and is preferably measured by a dynamic light scattering particle size measuring device.

In the present invention, an organic solvent solution containing molecules with a molecular weight of 200 or more is emulsified in an aqueous solution of polyvinyl alcohol and a substance that forms a gel with polyvinyl alcohol at an aqueous solution concentration of 60% or more to obtain an emulsion (Step A).

Molecules with a molecular weight of 200 or more are the base for microparticles. The base for microparticles is preferably water-insoluble. From the viewpoint of water insolubility, molecules with a molecular weight of 200 or more are preferably polymers with a molecular weight of 500 or more, more preferably a molecular weight (weight average molecular weight) of 1000 or more.

The above-mentioned molecule serving as the base material of the microparticles is also preferably a biodegradable polymer. Specific examples of the above-mentioned molecule serving as the base material of the microparticles include lactic acid polymers, lactic acid-glycolic acid copolymers, and polymers containing acrylic acid. Another specific example of the above-mentioned molecule is oligolactic acid (with a degree of polymerization of, for example, 3 to 19). It has been revealed that linear and cyclic oligolactic acids in a specific molecular weight range exhibit various physiologically active effects such as anticancer effects, mitochondrial function enhancement effects, antiallergic effects, fibrosis suppression effects, and protection from radiation by inhibiting the anaerobic glycolysis, and are attracting attention as polymer materials with physiological activity.

The components constituting the microparticles may also be physiologically active. For example, the base molecule of the microparticles, which has a molecular weight of 200 or more, may be a physiologically active substance. In addition to the water-insoluble molecule with a molecular weight of 200 or more, the microparticles may also contain a physiologically active substance that is a water-soluble substance. Examples of physiologically active substances that can be used include vitamin B12 and curcumin.

The biologically active substance is not particularly limited, but the following ingredients can be used:
(Anti-inflammatory Agent)
Aspirin, acetaminophen, etodolac, mefenamic, meclofenamic, piroxicam, isopropylantipyrine, tranexamic acid, ibuprofen, etc. (hypnotics/sedatives)
Nitrazepam, triazolam, phenobarbital, amibarbital, allylisopropylacetylurea, bromwarenylurea, flunitrazepam, zolpidem tartrate, alprazolam, etizolam, paroxetine hydrochloride hydrate, lorazepam, ethyl loflazepate, escilopram oxalate, etc. (antiepileptic drugs)
Phenytoin, metharbital, primidone, clonazepam, carbamazepine, valproic acid, etc. (antidote)
Meclizine hydrochloride, dimenhydrinate, etc. (antidepressants)
Imipramine, noxiptiline, phenelzine, etc. (psychiatric drugs)
Haloperidol, meprobamate, chlordiazepoxide, diazepam, oxazepam, sulpiride, risperidone, aripiprazole, olanzapine, quetiapine fumarate, paliperidone, perospirone hydrochloride hydrate, duloxetine hydrochloride, paroxetine, sertraline hydrochloride, amoxapine, etc. (antispasmodics)
Papaverine, atropine, etomidorine, etc. (cardiac stimulants)
Digoxin, digitoxin, methyldigoxin, ubidecarenone, etc. (antiarrhythmic agents)
Pindolol, ajmaline, disopyramide, etc. (diuretics)
Hydrochlorothiazide, spironolactone, triamterene, furosemide, bumetanide, etc.

(Antihypertensive)
Reserpine, dihydroergotoxine mesylate, prazosin hydrochloride, metoprolol, propranolol, atenolol, candesartan lexetil, telmisartan, azilsartan, olmesartan, bisoprolol fumarate, carvedilol, valsartan, enalapril, imidapril, amlodipine besylate, diltiazem hydrochloride, doxacin, trichlormethiazide, etc. (coronary vasodilators)
Nitroglycerin, isosorbide dinitrate, diltiazem, nifedipine, dipyridamole, etc. (cough suppressants)
Noscapine, salbutamol, procaterol, tulopterol, tranilast, dextromethorphan hydrobromide, dihydrocodeine phosphate, codeine phosphate, etc. (expectorants)
Bromhexine hydrochloride, ambroxol hydrochloride, guaifenesin, etc. (agents for improving cerebral circulation)
Nicardipine, pinpocetine, etc. (sympathomimetics)
Methylephedrine hydrochloride, etc. (diabetes treatment drugs)
Glimepiride, voglibose, metformin, mitiglinide calcium hydrate, pioglitazone, vildagliptin, cedagliptin phosphate hydrate, trelagliptin succinate, etc. (antimicrobial agents)
Erythromycin, josamycin, chloramphenicol, tetracycline, rifampicin, griseofulvin, levofloxacin, cefditoren pivoxil, cefcapene pivoxil, tosufloxacin tosilate hydrate, cefdinir, azithromycin hydrate, amoxicillin, vancomycin, ofloxacin, metronidazole, acyclovir, valacyclovir, miconazole, itraconazole, etc. (antihistamines)
Diphenhydramine, promethazine, mequitazine, clemastine fumarate, bevotastine besilate, fexofenadine, olopatadine, cetirizine hydrochloride, loratadine, etc. (steroids)
Triamcinolone, dexamethasone, betamethasone, prednisolone, danazol, methyltestosterone, chlormadinone acetate, etc.

(Vitamin supplement)
Vitamin A, Vitamin B, Vitamin C (ascorbic acid, etc.), Vitamin D, Vitamin E, Vitamin K, folic acid (Vitamin M), etc. (treatment for digestive system disorders)
Tannic acid, albumin tannate, berberine, mesalazine, dimethicone, vonoprazan, famotidine, ranitidine, cimetidine, nizatidine, metoclopramide, famotidine, omeprazole, domperidone, sulpiride, trepibutone, sucralfate, active live bacteria (e.g., lactomin, bifidobacteria, etc.), antacids (e.g., aluminum hydroxide, synthetic hydrotalcite, magnesium oxide, magnesium aluminometasilicate, etc.), calcium polycarbophil, etc. (others)
Alendronate sodium hydrate, raloxifene, caffeine, dicoumarol, cinnarizine, clofibrate, gefarnate, probenecid, mercaptopurine, methotrexate, cyclosporine A, tacrolimus, ursodeoxycholic acid, dihydroergotamine mesylate, glucuronolactone, γ-aminobutyric acid, chondroitin, sodium chondroitin sulfate, lactoferrin, milk protein, cysteine, collagen, nucleic acid (DNA, si-RNA, RNA decoy, cDNA, antisense RNA, etc.), bioactive peptides (insulin, calcitonin, etc.), bioactive proteins (polyclonal antibodies, monoclonal antibodies, gamma globulin, growth hormone, interferon, etc.), etc.

Polyvinyl alcohol is used as a surfactant or emulsion stabilizer. The concentration of polyvinyl alcohol in the aqueous solution is preferably 5 to 25% by mass, and more preferably 5 to 15% by mass.

The substance that forms a gel with polyvinyl alcohol at an aqueous solution concentration of 60% or more is not particularly limited as long as it has such properties, but is preferably propylene glycol, polyethylene glycol, or butylene glycol, and more preferably propylene glycol. When using these substances in the continuous phase used for emulsification, from the viewpoint of operability, they are used in a state of an aqueous solution concentration that does not gel or solidify with polyvinyl alcohol, but preferably, they are used at a concentration that is close to the critical concentration that causes gel or solidification and does not cause gelation or solidification. The concentration of propylene glycol or butylene glycol in the aqueous solution is preferably 20 to 70% by mass, more preferably 60 to 70% by mass.

The organic solvent is not particularly limited, but examples include methylene chloride or a mixture of methylene chloride and methanol.

Emulsification can be performed using a commonly used emulsifier.

In step B, the organic solvent is removed from the emulsion obtained in step A.
The organic solvent can be removed by the following two methods.
The first method is to dilute the emulsion with water and then dry it in the liquid. The amount of water to be added may be set so as to give a viscosity that facilitates stirring. The amount of water to be added is preferably at least twice the amount of the emulsion. The amount of water to be added is preferably at least the saturation solubility of the organic solvent.

The second drying method is a method in which the emulsion is dried as is to remove the organic solvent. The emulsion may be made into a film by drying under heating or reduced pressure. That is, to increase the drying efficiency, the emulsion may be spread on an appropriate sheet to make it into a film, but in order to make the composition obtained after drying easier to use, it may be used as a substitute for a binder in the granulation of solid preparations and dried as a granule, or it may be applied to the surface of appropriate particles or tablets to form a film on the surface, or it may be impregnated into a porous material, preferably a film or granule, particularly preferably a porous microsphere, and then dried.

The present invention will be specifically explained by the following examples, but the scope of the present invention is not limited to the examples.

Example 1
0.5 g of a mixture of oligolactic acid (CPL, degree of polymerization 3-19, molecular weight 236-1360, Shumeido Co., Ltd.) was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of polyvinyl alcohol (PVA, J Poval JP-03, Nippon Vinyl Acetate Poval Co., Ltd.) in 60% butylene glycol (BG) aqueous solution was stirred at 20,000 rpm with an Ultra Turrax (T18 Digital, IKA), and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device (ELSZ, Otsuka Electronics).

<Comparative Example 1>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 10% Pluronic F127 in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The particle size of the obtained solution was measured using a laser diffraction particle size measuring device (SALD2200, Shimadzu Corporation).

<Comparative Example 2>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 10% polyethylene glycol 400 (PEG400, Wako Pure Chemical Industries) in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The particle size of the obtained solution was measured using a laser diffraction particle size measuring device.

<Comparative Example 3>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 10% polyoxyethylene hydrogenated castor oil 60 (HCO60, Nikko Chemical) in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The particle size of the obtained solution was measured using a laser diffraction particle size measuring device.

ａ）６０％ＢＧ水溶液中
ｂ）キュムラント径
ｃ）体積基準
平均±Ｓ．Ｄ．（３バッチ）

a) In 60% BG aqueous solution b) Cumulant diameter c) Volume-based average ± S.D. (3 batches)

The results in Table 1 show that by using PVA as an emulsifier, the volume-based median diameter becomes 100 nm or less.

Example 2
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 10% by mass of PVA in a 60% propylene glycol (PG) aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

Example 3
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 50% PVA and 10% PEG400 aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered with a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured with a dynamic light scattering particle size measuring device.

Example 4
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 20% PVA in a 50% PEG400 aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured with a dynamic light scattering particle size measuring device.

<Comparative Example 4>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 10% PVA in purified water was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The particle size of the obtained solution was measured using a laser diffraction particle size measuring device.

<Comparative Example 5>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 10% PVA in a 60% glycerin aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

<Comparative Example 6>
An oil phase was prepared by dissolving 0.5 g of CPL in 1.5 mL of methylene chloride. When 10 mL of a 10% solution of PVA in a 60% PEG400 aqueous solution was prepared, the solution solidified, and the oil phase could not be injected and emulsified.

a) Emulsifier 10% PVA
b) Cumulant diameter c) Volumetric basis
d) Emulsifier 20% PVA
Mean ± S.D. (3 batches), 1 batch for Example 4 only

From the results in Table 2, it was found that by using butylene glycol, propylene glycol, or polyethylene glycol as a solvent for dissolving PVA, nanoparticles with an average particle size of 100 nm or less by cumulant analysis and a volume-based median diameter of 100 nm or less could be obtained. On the other hand, nano-order particles could not be obtained with an emulsified aqueous phase that did not contain polyol. Also, when glycerin was used, nano-order particles could be obtained, but it was not possible to obtain particles with a cumulant average diameter or volume-based median diameter of 100 nm or less. When PEG400 was used as the polyol, as shown in Comparative Example 6, when the concentration of the PEG400 aqueous solution for dissolving PVA exceeded 60%, the PVA-polyol aqueous solution gelled and solidified at room temperature, making it impossible to emulsify.

<Comparative Example 7>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution in which PVA was 10% in a 50% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

<Comparative Example 8>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution in which PVA was 10% in a 40% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

Example 5
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution in which PVA was 10% in a 70% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

<Comparative Example 9>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution in which PVA was 10% in a 20% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

<Comparative Example 10>
An oil phase was prepared by dissolving 0.5 g of CPL in 1.5 mL of methylene chloride. A 10% solution of PVA in an 80% butylene glycol aqueous solution was prepared (10 mL). However, this solution solidified, and therefore injection into the oil phase and emulsification were not possible.

(a) 10% PVA
Mean ± S.D. (n=3 batches)

As with PEG 400 in Comparative Example 6, the butylene glycol solution in which PVA is dissolved also gelled and solidified at 80% or more, and emulsification was not possible (Comparative Example 10). At BG concentrations below that, it was found that nanoparticles of the desired particle size could be obtained at concentrations exceeding 20%.

Example 6
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution of 10% PVA in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

Example 7
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 15 mL of a solution in which PVA was 10% in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

Example 8
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 3 mL of a solution of 10% PVA in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

(a) 10% PVA-60% BG aqueous solution Mean ± S.D. (n=3 batches)

Table 4 shows the effect of the volume of the PVA-polyol aqueous solution during emulsification. Considering that the volume of the oil phase solvent is 1.5 mL, it can be seen that there is no variation in particle size when the oil phase/aqueous phase ratio is 1/2 or more, and it can be said that this manufacturing method is highly robust to the factor of the oil phase/aqueous phase ratio.

<Comparative Example 11>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution in which PVA was 5% in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

<Example 9>
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 20% PVA in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

Example 10
0.5 g of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 10 mL of a solution of 25% PVA in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

(a) Mean ± S.D. in 60% BG aqueous solution (n = 3 batches)

As shown in Table 5, the desired particle size was obtained at a PVA concentration of over 5%, and the particle size became smaller as the concentration increased. At a PVA concentration of 25% or more, the viscosity of the PVA-polyol aqueous solution was high, making it difficult to stir the oil phase during emulsification, and this was considered to be unsuitable.

Example 11
0.195g of lactic acid-glycolic acid copolymer (PLGA5020 molecular weight 20K, Wako Pure Chemical Industries) and 5mg of vitamin _B12 (molecular weight 1355) were dissolved in a mixture of 2mL methylene chloride and 1.1mL methanol to prepare an oil phase. 10mL of a solution of 20% PVA in 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100mL purified water, and methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45μm membrane filter to remove dust, and then particle size measurement was performed with a dynamic light scattering particle size measuring device. The obtained solution was also filtered through a 5μm membrane filter to remove dust, and dehydrated and washed by ultrafiltration to obtain a fine particle suspension. 50 mg of mannitol was dissolved per 1 mL and freeze-dried. The vitamin B12 content in the microparticles was quantified by dissolving the microparticles in 1 mL of methylene chloride, extracting with 5 mL of purified water, and measuring the absorbance of the supernatant at 360 nm.

Example 12
0.195g of PLGA5020 and 5mg of vitamin _B12 were dissolved in a mixture of 2mL of methylene chloride and 1.1mL of methanol to prepare an oil phase. 10mL of a solution of 20% PVA in 60% PG aqueous solution was stirred at 20,000rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was spread on a Teflon (registered trademark) sheet and dried overnight. The dried film was dissolved in 100mL of purified water. The dispersed liquid was filtered through a 0.45μm membrane filter to remove dust, and then the particle size was measured with a dynamic light scattering particle size measuring device. The obtained liquid was also filtered through a 5μm membrane filter to remove dust, and dehydrated and washed by ultrafiltration to obtain a microparticle suspension. 50mg of mannitol was dissolved per mL and freeze-dried. The vitamin B12 content in the microparticles was quantified by dissolving the freeze-dried microparticles in 1 mL of methylene chloride, extracting with 5 mL of purified water, and measuring the absorbance of the supernatant at 360 nm.

<Comparative Example 12>
Solvent diffusion method: 0.195 g of PLGA5020 and ₅ mg of vitamin B12 were dissolved in a mixture of 10 mL of acetone and 2.2 mL of methanol to prepare an oil phase. The prepared oil phase was poured into 100 mL of 0.5% PVA, stirred with a stirrer, and dried under reduced pressure for 3 hours under airflow to remove the solvent. The obtained liquid was filtered through a 0.45 μm membrane filter to remove dust, and then the particle size was measured using a dynamic light scattering particle size measuring device. The obtained liquid was also filtered through a 5 μm membrane filter to remove dust, and dehydrated and washed by ultrafiltration to obtain a microparticle suspension. 50 mg of mannitol was dissolved per mL and freeze-dried. The vitamin B12 content in the microparticles was quantified by dissolving the freeze-dried microparticles in 1 mL of methylene chloride, extracting them with 5 mL of purified water, and measuring the absorbance of the supernatant at 360 nm.

Example 13
0.195 g of Eudragit RLPO (Rohm) and 5 mg of vitamin B12 (molecular weight 1355) were dissolved in a mixture of 2 mL of methylene chloride and 1.1 mL of methanol to prepare an oil phase. 10 mL of a solution in which PVA was 20% by mass in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was spread on a Teflon (registered trademark) sheet and dried overnight. The film obtained by drying was dissolved in 100 mL of purified water. The particle size of the dispersed liquid was measured using a dynamic light scattering particle size measuring device. The obtained liquid was also filtered through a 5 μm membrane filter to remove dust, and dehydrated and washed by ultrafiltration to obtain a fine particle suspension. 50 mg of mannitol was dissolved per mL and freeze-dried. The vitamin B12 content in the microparticles was quantified by dissolving the freeze-dried microparticles in 1 mL of methylene chloride, extracting with 5 mL of purified water, and measuring the absorbance of the supernatant at 360 nm.

In the preparation of drug-encapsulated nanospheres using lactic acid/glycolic acid copolymer, nanoparticles with a median diameter based on weight of 30 nm or less were obtained (Examples 11 and 12). In addition, the median diameter based on weight of Eudragit RLPO was 20 nm or less (Example 13). On the other hand, in the solvent diffusion method, even though an excess amount of solvent was used to prepare finer nanoparticles, the median diameter based on volume could not be made 100 nm or less (Comparative Example 12). The inclusion rate was low, but since water-soluble vitamin B ₁₂ does not dissolve in a 20% PVA-60% propylene glycol aqueous solution, it is believed that vitamin B ₁₂ did not leak into the continuous phase during emulsification and film formation in Examples 12 and 13, but it took one day to wash with water, and it is believed that vitamin B ₁₂ leaked during that time. In Example 12, the emulsion was dried and the solidified mixture was dissolved and dispersed in purified water. Even after drying, the particle size obtained was equivalent to that of Example 11 in the dispersion state, proving that Example 12 is a solid preparation capable of releasing nanoparticles.

<Example 14>
60 mg of Eudragit RSPO (Rohm) and 120 mg of LABRAFIL M2130CS (Gatefose) were dissolved in 1.5 mL of methylene chloride and 0.2 mL of ethanol to prepare an oil phase. A mixture of 10 mL of a 20% solution of PVA in a 60% BG aqueous solution and 0.1 mL of a 0.1 M hydrochloric acid solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the mixture, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the solvent was distilled off for 3 hours under stirring with a stirrer. The obtained liquid was subjected to ultrafiltration, and the solvent was replaced with 0.0001 M hydrochloric acid and concentrated. The liquid was subjected to particle size measurement using a dynamic light scattering particle size measuring device. The obtained liquid was also filtered through a 5 μm membrane filter to remove dust, and was dehydrated and washed by ultrafiltration to obtain a fine particle suspension. The cumulant average particle size was 89.3 nm, the scattering intensity D50 was 94.6 nm, the volume distribution D50 was 55.3 nm, and the number distribution D50 was 43.5 nm. Observation with a transmission electron microscope confirmed that Janus particles were formed (FIG. 1).

Example 15
10 mg of curcumin (molecular weight 368.38, Nacalai Tesque) and 90 mg of PLGA5020 were dissolved in a mixture of 0.5 mL of ethanol and 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution in which PVA was 20% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove dust, and then the particle size was measured with a dynamic light scattering particle size measuring device (immediately after preparation). In addition, the solution was filtered through a 0.8 μm membrane filter to remove dust, and then ultrafiltered using a 500 KDa filtration membrane, and concentrated to 7 mL after solvent replacement with purified water. 350 mg of mannitol was added, and the mixture was freeze-dried. The freeze-dried product was also resuspended in purified water and filtered through a 0.45 μm membrane filter to remove foreign matter, and then the particle size was measured using a dynamic light scattering particle size measurement device.

<Example 16>
10 mg of curcumin and 90 mg of PLGA5020 were dissolved in a mixture of 0.5 mL of ethanol and 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution of 20% PVA in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was spread on a Teflon (registered trademark) sheet and dried for 3 days. The film obtained by drying was dissolved in 100 mL of purified water. The obtained solution was filtered through a 0.45 μm membrane filter to remove dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

Drug-loaded nanospheres were prepared using lactic acid/glycolic acid copolymer with curcumin, a water-insoluble compound. As a result, curcumin-containing nanoparticles with a volume-based median diameter of approximately 40 nm were obtained. In Example 15, it was found that redispersible nanoparticles could be obtained by freeze-drying with mannitol, which is commonly used in the past. Example 16 is a solid preparation that was dried immediately after emulsification, but even after being dried, the same particle diameter was obtained as in Example 15 when redispersed, proving that Example 16 is a solid preparation that can release nanoparticles.

<Example 17>
10 mg of curcumin and 10 mg of Eudragit E100 (Rohm) were dissolved in a mixture of 0.5 mL of ethanol and 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution of 10% PVA in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The liquid was measured using a dynamic light scattering particle size analyzer.

<Example 18>
10 mg of curcumin and 90 mg of hypromellose phthalate (HP55, Shin-Etsu Chemical) were dissolved in a mixture of 0.5 mL of ethanol and 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution in which PVA was 10% in a 60% BG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was spread on a Teflon (registered trademark) sheet and dried for 3 days. The film obtained by drying was dissolved in 10 mL of purified water. The obtained solution was filtered through a 0.45 μm membrane filter to remove dust (product immediately after preparation). The particle size of the solution was measured using a dynamic light scattering particle size measuring device. On the other hand, the solution was adjusted to pH 6.8 with a phosphate buffer solution (treated product), filtered through a 0.45 μm membrane filter to remove dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

<Example 19>
10 mg of curcumin and 90 mg of Eudragit L100 (Rohm) were dissolved in a mixture of 0.5 mL of ethanol and 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution in which PVA was 20% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was spread on a Teflon (registered trademark) sheet and dried for 3 days. The film obtained by drying was dissolved in 10 mL of purified water. The obtained solution was filtered through a 0.45 μm membrane filter to remove dust (product immediately after preparation). The particle size of the solution was measured using a dynamic light scattering particle size measuring device. On the other hand, the solution was adjusted to pH 6.8 with a phosphate buffer solution (treated product), filtered through a 0.45 μm membrane filter to remove dust, and then the particle size was measured using a dynamic light scattering particle size measuring device.

When this formulation was produced using the preparation method of the present invention together with an enteric polymer (HP55, Eudragit L100), the desired nanoparticles could not be obtained when redispersed in water. However, when a phosphate buffer solution of pH 6.8 was added to the liquid, the volumetric D50 became 11.2 nm in Example 18 and 25 nm in Example 19. This suggests that when this formulation is taken orally, the enteric polymer dissolves in the small intestine, releasing the nanosized curcumin contained therein.

<Example 20>
150 mg of CPL and 50 mg of Eudragit E100 were dissolved in 1.5 mL of methylene chloride to prepare an oil phase. The prepared oil phase was poured into 5 mL of a 10% solution of 60% PVA and PG aqueous solution while stirring the oil phase at 20,000 rpm with an Ultra Turrax, and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and methylene chloride was distilled off under stirring with a stirrer for 3 hours. The particle size of the obtained liquid was measured using a dynamic light scattering particle size measuring device.

<Example 21>
500 mg of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution of 60% PVA and 15% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered with a 0.45 μm membrane filter to remove aggregates and dust. The particle size and ζ potential of the solution were measured using a dynamic light scattering particle size measuring device.

<Example 22>
Eudragit RSPO (Rohm) 100 mg and CPL 400 mg were dissolved in methylene chloride 1.5 mL to prepare an oil phase. 5 mL of a solution in which PVA was 15% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution and emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust. The particle size and ζ potential of the solution were measured using a dynamic light scattering particle size measuring device.

<Example 23>
250 mg of Eudragit RSPO and 250 mg of CPL were dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution in which PVA was 15% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove dust. The particle size and ζ potential of the solution were measured using a dynamic light scattering particle size measuring device.

<Example 24>
Eudragit RSPO 400mg and CPL 100mg were dissolved in methylene chloride 1.5mL to prepare an oil phase. 5mL of a solution in which PVA was 15% in a 60% PG aqueous solution was stirred at 20,000rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100mL purified water, and methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45μm membrane filter to remove dust. The particle size and ζ potential of the solution were measured using a dynamic light scattering particle size measuring device.

The cationic polymer Eudragit RSPO was mixed with oligolactic acid CPL and nanoparticles were formed in the present invention. There was no effect on the particle size up to Eudragit RSPO/CPL = 0/500 to 100/400, but a decrease in particle size was observed when the amount of Eudragit RSPO was increased to 250/250 and 400/100. Figure 2 shows a transmission electron microscope photograph of Example 21 after negative staining. It was confirmed that the particles were spherical, and the number-based D ₅₀ calculated from the electron microscope photograph was 25.6 nm. When the ζ potential was measured, the potential was increased to 14.68 mV and 20.10 mV at Eudragit RSPO/CPL = 250/250 and 400/100, respectively, and it is presumed that the particle size was reduced due to increased stability and suppression of aggregation during production.

<Example 25>
500 mg of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution in which PVA was 15% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of a 0.14 M aqueous sodium hydroxide solution, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered with a 0.45 μm membrane filter to remove aggregates and dust. The particle size of the solution was measured with a dynamic light scattering particle size measuring device.

<Example 26>
500 mg of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution in which PVA was 20% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of a 0.14 M aqueous sodium hydroxide solution, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust. The particle size of the solution was measured using a dynamic light scattering particle size measuring device.

<Example 27>
500 mg of CPL was dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution in which PVA was 10% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of a 0.14 M aqueous sodium hydroxide solution, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust. The particle size of the solution was measured using a dynamic light scattering particle size measuring device.

When the emulsion was poured into purified water, the pH became acidic at 3.3 due to the acidic substance contained in the CPL. In other words, it is considered that the CPL particles having a negative charge have a low electric potential under acidic conditions, and tend to aggregate with each other. Therefore, the pH of the final dispersion was adjusted to 6.8 to 7.8 by adding sodium hydroxide in advance for the purpose of neutralization. As a result, it was found that the amount of aggregates decreased, and at the same time, the volume-based D ₅₀ and the number-based D ₅₀ decreased. It is considered that the neutral pH of the dispersion increased the negative electric potential of the particles and strengthened the repulsive force between the particles, which resulted in suppressing the fusion and aggregation of the particles during production.

<Example 28>
150 mg of CPL and 50 mg of Eudragit L100 were dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 3 mL of a solution in which PVA was 10% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust. The particle size of the solution was measured using a dynamic light scattering particle size measuring device.

<Example 29>
150 mg of CPL and 50 mg of PLGA5020 were dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 3 mL of a solution in which PVA was 10% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust. The particle size of the solution was measured using a dynamic light scattering particle size measuring device.

<Example 30>
150 mg of CPL and 50 mg of PEG6000 were dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 3 mL of a solution in which PVA was 10% in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was then emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust. The particle size of the solution was measured using a dynamic light scattering particle size measuring device.

平均±Ｓ．Ｄ．（ｎ＝３ バッチ）

Mean ± S.D. (n=3 batches)

<Example 31>
50 mg of cyclosporine and 50 mg of Eudragit E100 were dissolved in 1.5 mL of methylene chloride to prepare an oil phase. 5 mL of a solution of 15% PVA in a 60% PG aqueous solution was stirred at 20,000 rpm with an Ultra Turrax, and the prepared oil phase was poured into the solution, which was emulsified for 5 minutes to obtain an emulsion. The obtained emulsion was immediately poured into 100 mL of purified water, and the methylene chloride was distilled off under stirring with a stirrer for 3 hours. The obtained solution was filtered through a 0.45 μm membrane filter to remove aggregates and dust. The particle size of the solution was measured using a dynamic light scattering particle size measuring device.

Claims (6)

A method for producing nanoparticles, either of which has a cumulant average diameter or a volume-based median diameter of 10 to 100 nm, comprising: step A of emulsifying a methylene chloride solution containing a lactic acid polymer or a lactic acid/glycolic acid copolymer in an aqueous solution of polyvinyl alcohol and propylene glycol, butylene glycol or polyethylene glycol to obtain an emulsion; and step B of removing the organic solvent from the emulsion obtained in step A,
The concentration of polyvinyl alcohol in the aqueous solution is more than 5% by mass and not more than 25% by mass,
The concentration of propylene glycol or butylene glycol in the aqueous solution is 60% by mass or more and 70% by mass or less, or the concentration of polyethylene glycol in the aqueous solution is 50% by mass.
Method. The method according to claim 1, wherein the lactic acid polymer is an oligolactic acid having a degree of polymerization of 3 to 19. The method of claim 1 or 2, wherein the methylene chloride solution further contains a biologically active substance. The method of any one of claims 1 to 3, wherein the methylene chloride solution further contains curcumin. The method according to any one of claims 1 to 4, wherein in step B, the organic solvent is removed by diluting the emulsion obtained in step A with water in an amount equal to or greater than the saturation solubility of the organic solvent. The method according to any one of claims 1 to 5, wherein in step B, the emulsion is formed into a film by drying by air drying, heating, or reduced pressure.