Method for producing polymer microparticles

Abstract

To provide a method for manufacturing a polymer fine particle with narrower particle size distribution, having an average particle diameter of 200 nm or less.SOLUTION: A manufacturing method of a polymer fine particle includes: a step of mixing styrene, glycidyl methacrylate and a cross-linking agent to obtain a mixed liquid A; a step of mixing a water solution of a surfactant to the mixed liquid A to obtain a mixed liquid B; and a step of mixing a polymerization initiator to the mixed liquid B. A concentration of the surfactant in the mixed liquid B is smaller than a critical micelle concentration of the surfactant. The surfactant is a nonion-based surfactant having critical micelle concentration of 5 to 30 mM.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing submicron-sized polymer microparticles.

Background Art

[0002] In recent years, it has been widely practiced to purify or quantify target molecules using polymer microparticles having a ligand adsorbed on the surface thereof and having an affinity for the target molecule.

[0003] In this regard, Patent Document 1 discloses a method for purifying a specific protein using polymer microparticles having a DNA strand having a base sequence specifically binding to the specific protein adsorbed on the surface.

[0004] Here, the polymer microparticles used in Patent Document 1 are those obtained by performing a polymerization reaction without soap (in the absence of a surfactant) using both monomers of styrene and glycidyl methacrylate, and coating the surface of the copolymer with polyglycidyl methacrylate, and having a particle size of about 200 nm. The particles composed of styrene and glycidyl methacrylate (hereinafter referred to as "SG particles") can reduce non-specific adsorption of proteins by chemical modification via glycidyl groups present on the surface.

[0005] However, from the viewpoint of new utilization of polymer microparticles, such as improving the purification efficiency of target factors and improving the sensing accuracy by using them as probes in biosensors, further miniaturization of polymer microparticles has been desired.

[0006] Regarding this point, in Patent Document 2, in order to further miniaturize polymer microparticles, attention was paid to the type of surfactant, and a method of copolymerizing styrene and glycidyl methacrylate in the presence of a polyoxyethylene alkyl ether surfactant of a non-alkylphenol ethoxylate (APE) type was disclosed. As a means for miniaturizing SG particles, a method of polymerizing by reducing the polymer concentration in emulsion polymerization is generally known. However, according to the method of Patent Document 2, it is possible to miniaturize without reducing the polymer concentration, which is effective from the viewpoint of improving productivity.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] According to the methods of Patent Documents 1 and 2, polymer microparticles with an average particle diameter of 200 nm or less can be obtained, but although slightly, the particle size distribution becomes wide. For example, when used as a probe in a biosensor, the sensing accuracy may decrease.

[0009] The present invention has been made in view of the above problems, and an object thereof is to provide a novel method for producing submicron-sized polymer microparticles with reduced non-specific adsorption and a narrow particle size distribution.

Means for Solving the Problems

[0010] In order to improve the particle size distribution of polymer microparticles, the inventors focused on the type of surfactant and reexamined the method for producing polymer microparticles. As a result, it was found that by performing emulsion polymerization of styrene and glycidyl methacrylate using a surfactant having a critical micelle concentration within a specific range and suppressing the concentration of the surfactant below the critical micelle concentration, polymer microparticles with a good particle size distribution can be obtained, leading to the present invention.

[0011] In emulsion polymerization, monomers are sequentially supplied from the oil phase to the water phase, and polymerization is initiated by the reaction of the monomers dissolved in water with a polymerization initiator. During emulsion polymerization, when a surfactant is added at a concentration that forms micelles, micelles containing oil droplets may be formed. If polymerization proceeds in a state where micelles containing oil droplets are formed, the particle size distribution of the polymer microparticles produced by the polymerization may become broad.

[0012] As a result of intensive studies on a method for forming polymer microparticles without generating micelles containing oil droplets, the inventors have reached the present invention.

[0013] That is, the present invention is a method for producing polymer microparticles, comprising a step of mixing styrene and glycidyl methacrylate to obtain a mixed solution A, a step of mixing an aqueous solution of a surfactant with the mixed solution A to obtain a mixed solution B, and a step of mixing a polymerization initiator with the mixed solution B, wherein the concentration of the surfactant in the mixed solution B is below the critical micelle concentration of the surfactant, and the surfactant is a nonionic surfactant having a critical micelle concentration of 5 mM or more and 30 mM or less.

Effect of the Invention

[0014] According to the present invention, a method for producing polymer microparticles with a narrow particle size distribution and an average particle size of 200 nm or less is provided.

Mode for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these embodiments. The method for producing polymer microparticles according to this embodiment includes a step of mixing styrene and glycidyl methacrylate to obtain a mixed solution A, a step of mixing an aqueous solution of a surfactant with the mixed solution A to obtain a mixed solution B, and a step of mixing a polymerization initiator with the mixed solution B. Further, the concentration of the surfactant in the mixed solution B is equal to or lower than the critical micelle concentration of the surfactant, and the surfactant is a nonionic surfactant having a critical micelle concentration of 5 mM or more and 30 mM or less. As described above, in the method for producing polymer microparticles according to this embodiment, by using a nonionic surfactant at a predetermined concentration, polymer microparticles having a narrow particle size distribution and an average particle diameter of 200 nm or less can be obtained. Also, non-specific adsorption of the protein after chemical modification is reduced without reducing productivity. Details will be described below.

[0016] 1. Surfactant The surfactant used in the production method of the present invention is a nonionic surfactant. If an anionic surfactant or a cationic surfactant generally used in emulsion polymerization is present as a residue, there is a concern that the antibody will be denatured when the polymer microparticles are formulated into pharmaceuticals. At the antibody site with a deformed three-dimensional structure, a specific antibody-antigen reaction does not occur, and there is also a concern that it will not function as a desired biosensor or particle for bioseparation. Therefore, a nonionic surfactant is used as the surfactant of the present invention.

[0017] In addition, the nonionic surfactant used in the production method of the present invention is characterized in that the critical micelle concentration is 5 mM or more and 30 mM or less. More preferably, the critical micelle concentration of the surfactant is 15 mM or more and 30 mM or less, still more preferably 15 mM or more and 25 mM or less, and particularly preferably 7 mM or more and 25 mM or less. Further, the molecular weight of the surfactant is preferably 100 or more and 600 or less, more preferably 292 or more and 335 or less. When a surfactant is added to an emulsion polymerization system, the surfactant forms micelles in an aqueous solution, and thus oil droplets are included in the micelles. Therefore, in the production of polymer microparticles, the surfactant contributes to the deterioration of the particle size distribution. In the present invention, by using a surfactant having a high critical micelle concentration range, that is, a surfactant that hardly forms micelles in an aqueous solution, inclusion of oil droplets in the micelles is suppressed. As a result, in the production method of the present invention, it is possible to narrow the particle size distribution of the polymer microparticles. Examples of the structure of the surfactant used in the present invention include sugar-based nonionic surfactants (alkyl glucosides). By using a sugar-based nonionic surfactant, since a sugar structure having high hydrophilicity is slightly present on the surface layer of the SG particles, the non-specific adsorption property of the protein of the particles obtained by chemically modifying the SG particles is improved (non-specific adsorption is reduced). Specifically, the compound represented by the following general formula (1) is preferable. R1(OR2)sGt (1) In formula (1), R1 is a linear or branched alkyl group, alkenyl group or phenylalkyl group having 8 to 18 carbon atoms, preferably an alkyl group. In formula (1), R2 is an alkylene group having 2 to 4 carbon atoms. s is an integer of 0 or more and 5 or less. In formula (1), G represents a residue derived from a reducing sugar having 5 or 6 carbon atoms. t represents an integer of 1 or more and 5 or less.

[0018] G, which is a residue derived from a reducing sugar, may be derived from either an aldose or a ketose. Examples of G include residues derived from pentoses having 5 carbon atoms and hexoses having 6 carbon atoms. Specific examples of pentoses include apiose, arabinose, lyxose, xylose and the like. Specific examples of hexoses include galactose, glucose, mannose, gulose, idose, talose, fructose, glucosamine and the like.

[0019] The bond between R1 or R2 and G can be a glycosidic bond, a thioglycosidic bond, an N-glycosidic bond, an ester bond, a thioester bond, an amide bond, etc.

[0020] Specific examples of the sugar-based nonionic surfactant include n-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglycoside, n-decanoyl-N-D-glucosamine, etc.

[0021] 2. Monomer The monomers used in the method for producing the polymer microparticles of the present invention are styrene and glycidyl methacrylate. By containing styrene as a repeating unit of the polymer, physical properties with a high glass transition temperature and excellent mechanical strength can be imparted to the polymer. Therefore, when purifying polymer microparticles containing styrene as a repeating unit by methods such as centrifugation or ultrafiltration, the effect of suppressing damage such as cracks and chips in the polymer microparticles is exhibited. Also, by containing glycidyl methacrylate as a repeating unit of the polymer, it is possible to reduce non-specific adsorption of proteins and the like by chemical modification of various reactive functional groups via the glycidyl group. When introducing a reactive functional group, the glycidyl group is ring-opened to become a glycol group, thereby expressing hydrophilicity due to the hydroxyl group and reducing non-specific adsorption. In the present invention, styrene and glycidyl methacrylate are copolymerized by radical polymerization to form a polymer. In order to carry out chemical modification of various reactive functional groups via the glycidyl group, it is preferable that the glycidyl group is localized on the surface layer of the particles. As a method for localizing the glycidyl group on the surface layer of the particles, there is a method of post-adding glycidyl methacrylate.

[0022] 3. Crosslinking agent In the method for producing polymer microparticles of the present invention, a radically polymerizable monomer having crosslinkability is used. Examples of the radically polymerizable monomers having crosslinkability include diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, 1,6 - hexanediol diacrylate, neopentyl glycol diacrylate, tripropylene glycol diacrylate, polypropylene glycol diacrylate, 2,2'-bis(4-(acryloxydiethoxy)phenyl)propane, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3 - butylene glycol dimethacrylate, 1,6 - hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polypropylene glycol dimethacrylate, 2,2'-bis(4-(methacryloxydiethoxy)phenyl)propane, 2,2'-bis(4-(methacryloxypolyethoxy)phenyl)propane, trimethylolpropane trimethacrylate, tetramethylolmethane tetramethacrylate, divinylbenzene, divinylnaphthalene, and divinyl ether. However, the present invention is not limited thereto as long as the object of the present invention can be achieved. Also, two or more radically polymerizable monomers having crosslinkability may be used in combination. The addition amount of the radically polymerizable monomer having crosslinkability is preferably in the range of 0.1 part by mass to 5 parts by mass with respect to 100 parts by mass of the total amount of styrene and glycidyl methacrylate.

[0023] 4. Polymerization initiator As the polymerization initiator used in the method for producing polymer microparticles of the present invention, it is preferable to use a water - soluble polymerization initiator. For example, peroxides such as ammonium persulfate, potassium persulfate, hydrogen peroxide, and water - soluble azo polymerization initiators such as 2,2'-azobis(2 - methylpropionamidine) dihydrochloride, and other conventionally known radical polymerization initiators can be preferably used.

[0024] The amount of the polymerization initiator used can be appropriately adjusted according to the reaction conditions. The amount of the polymerization initiator used is preferably 0.1% by mass or more and 20% by mass or less, more preferably 5% by mass or more and 15% by mass or less, based on the total mass of styrene, glycidyl methacrylate and the crosslinking agent.

[0025] 5. Method for measuring the particle size of polymer microparticles The particle size of the polymer microparticles produced by the production method of the present invention can be measured based on the principle of dynamic light scattering (DLS). The particle size in this example is measured using a Zetasizer NS manufactured by Malvern.

[0026] As the measurement sample, a sample solution in which polymer microparticles are dispersed in an ultrapure water solvent at 0.005% by weight is used, and the measurement is carried out using a disposable plastic cell. As the analysis parameters, the refractive index of the polymer microparticles is 1.59 equivalent to latex, the absorbance of the polymer microparticles is 0.01, the refractive index of the dispersion medium is 1.33 equivalent to water, the viscosity of the dispersion medium is 0.8872 cP equivalent to water, and the measurement temperature is 25°C. The number of measurements is 3 times, and the average value is taken as the average particle size of the polymer microparticles. In this specification, the average particle size is represented by the Z-average particle size.

[0027] 6. Production method Styrene and glycidyl methacrylate are added to water and mixed and stirred for a sufficient time to be emulsified to obtain a mixed solution A. Separately, a surfactant is added to water and mixed and stirred for a sufficient time to obtain an aqueous surfactant solution. As the water, any water can be used as long as it does not interfere with the polymerization reaction shown below, but deionized water, ion-exchanged water, distilled water, ultrapure water, etc. are preferably used.

[0028] Subsequently, a mixed solution A and an aqueous surfactant solution are mixed and stirred for a sufficient time to obtain a mixed solution B. In one embodiment of the present invention, the concentration of the surfactant in the mixed solution B is set to be equal to or lower than the critical micelle concentration of the surfactant. A polymerization initiator is added to the mixed solution B and mixed and stirred for a sufficient time, so that styrene and glycidyl methacrylate are copolymerized to form granular copolymer. Thereafter, glycidyl methacrylate is further added to the reaction system and mixed and stirred for a sufficient time, so that polymer fine particles having a surface of the granular copolymer coated with polyglycidyl methacrylate are formed. Finally, the polymer fine particles are separated and washed.

[0029] As described above, according to the production method according to one embodiment of the present invention, polymer fine particles having a narrow and controlled particle size distribution can be obtained with a small number of washing times.

[0030] By applying the produced polymer fine particles to a carrier for purification of a physiologically active substance or a biosensing probe, an improvement in the purification efficiency or sensing accuracy of the physiologically active substance can be expected.

[0031] Hereinafter, the method for producing the polymer fine particles of the present invention will be described more specifically using examples, but the present invention is not limited to the examples described below.

Examples

[0032] (Example 1) As a surfactant, 0.3 g of n-octyl-β-D-glycopyranoside (manufactured by Tokyo Chemical Industry Co., Ltd., critical micelle concentration 25 mM) was added to 70 ml of ultrapure water, and the mixture was stirred at 70 °C and 200 rpm for 30 minutes to obtain an aqueous surfactant solution. Separately, 1.2 g of styrene, 1.8 g of glycidyl methacrylate, and 0.04 g of divinylbenzene were added to 40 ml of ultrapure water and emulsified to obtain a mixed solution A. This mixed solution A was added to the aqueous surfactant solution, and the mixture was stirred at 70 °C and 200 rpm for 30 minutes to obtain a mixed solution B. To the mixed solution B, an aqueous solution prepared by dissolving 0.06 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Fujifilm Wako Pure Chemical Corporation) as a polymerization initiator in 10 ml of ultrapure water was added, and the mixture was stirred at 70 °C and 200 rpm for 1 hour. Then, 0.33 g (305 μl) of glycidyl methacrylate was further added to this polymerization reaction solution, and the mixture was stirred at 70 °C and 200 rpm for 24 hours. Finally, the polymerization reaction solution was centrifuged to remove the supernatant, and then ultrapure water was added, and the operation of redispersing was repeated 5 times (hereinafter, this operation is referred to as a washing operation) to obtain a suspension of polymer microparticles. After centrifuging the obtained suspension of polymer microparticles to remove the supernatant, the particles were dried under reduced pressure at 40 °C for 48 hours to obtain polymer microparticles (hereinafter, this operation is referred to as an "isolation operation"). It was confirmed that the same results were obtained even when only the GMA / styrene ratio was changed in the range of 1.4 to 1.7 without changing the polymer concentration. It was also confirmed that the same results were obtained when the temperature was changed in the range of 65 to 75 °C.

[0033] (Example 2) To 70 ml of ultrapure water, 1.2 g of styrene, 1.8 g of glycidyl methacrylate, and 0.04 g of divinylbenzene were added, and the mixture was emulsified by stirring at 70 °C and 200 rpm for 30 minutes to obtain mixture A. To mixture A, an aqueous surfactant solution prepared by dissolving 0.3 g of n-octyl-β-D-thioglucoside (manufactured by Dojindo Laboratories, critical micelle concentration 9 mM) in 40 ml of ultrapure water was added, and the mixture was stirred at 70 °C and 200 rpm for 30 minutes to obtain mixture B. To mixture B, an aqueous polymerization initiator solution prepared by dissolving 0.06 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Fujifilm Wako Pure Chemical Corporation) in 10 ml of ultrapure water was added, and the mixture was stirred at 70 °C and 200 rpm for 1 hour. Then, 0.33 g (305 μl) of glycidyl methacrylate was further added to this polymerization reaction solution, and the mixture was stirred at 70 °C and 200 rpm for 24 hours. Furthermore, the washing operation was repeated 5 times to obtain a suspension of polymer microparticles. Finally, an isolation operation was performed on the obtained suspension of polymer microparticles to obtain polymer microparticles.

[0034] (Example 3) To 70 ml of ultrapure water, 1.2 g of styrene, 1.8 g of glycidyl methacrylate, and 0.04 were added, and the mixture was emulsified by stirring at 70 °C and 200 rpm for 30 minutes to obtain mixture A. To mixture A, an aqueous surfactant solution prepared by dissolving 0.3 g of n-decanoyl-N-D-glucosamine (manufactured by Dojindo Laboratories, critical micelle concentration 7 mM) in 40 ml of ultrapure water was added, and the mixture was stirred at 70 °C and 200 rpm for 30 minutes to obtain mixture B. To mixture B, an aqueous polymerization initiator solution prepared by dissolving 0.06 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Fujifilm Wako Pure Chemical Corporation) in 10 ml of ultrapure water was added, and the mixture was stirred at 70 °C and 200 rpm for 1 hour to obtain mixture B. Then, 0.33 g (305 μl) of glycidyl methacrylate was added to this polymerization reaction solution, and the mixture was stirred at 70 °C and 200 rpm for 24 hours. Furthermore, the washing operation was repeated 5 times to obtain a suspension of polymer microparticles. Finally, an isolation operation was performed on the obtained suspension of polymer microparticles to obtain polymer microparticles.

[0035] (Comparative Example 1) To 110 ml of ultrapure water, 1.2 g of styrene, 1.8 g of glycidyl methacrylate, and 0.04 of divinylbenzene were added, and the mixture was emulsified by stirring at 70 °C and 200 rpm for 30 minutes to obtain a mixed solution A. To the mixed solution A, an aqueous solution of a polymerization initiator prepared by dissolving 0.06 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Fuji Film Wako Pure Chemical Corporation) in 10 ml of ultrapure water was added, and the mixture was stirred at 70 °C and 200 rpm for 1 hour. Thereafter, 0.33 g (305 μl) of glycidyl methacrylate was added to this polymerization reaction solution, and the mixture was stirred at 70 °C and 200 rpm for 24 hours. Further, the washing operation was repeated 5 times to obtain a suspension of polymer microparticles. Finally, an isolation operation was performed on the obtained suspension of polymer microparticles to obtain polymer microparticles.

[0036] (Comparative Example 2) An aqueous surfactant solution was prepared by adding 0.5 g of a non-alkylphenol ethoxylate-based polysorbate surfactant Tween 20 (manufactured by Nacalai Tesque) to 70 ml of ultrapure water and stirring at 70 °C and 200 rpm for 30 minutes. Polymer microparticles were obtained in the same production method as in Example 1, except that the washing operation was repeated 6 times.

[0037] (Comparative Example 3) An aqueous surfactant solution was prepared by adding 0.5 g of an alkylphenol ethoxylate-based surfactant NP-40 (polyoxyethylene octylphenyl ether: manufactured by Nacalai Tesque) to 70 ml of ultrapure water and stirring at 70 °C and 200 rpm for 30 minutes. Polymer microparticles were obtained in the same production method as in Example 1, except that the washing operation was repeated 6 times.

[0038] (Comparative Example 4) An aqueous surfactant solution was prepared by adding 0.5 g of a polyoxyethylene alkyl ether surfactant (trade name: Emulgen 1150S-60, manufactured by Kao Corporation), which is not an alkylphenol ethoxylate type, to 70 ml of ultrapure water and stirring at 70 °C and 200 rpm for 30 minutes. Polymer microparticles were obtained by the same production method as in Example 1, except that the washing operation was repeated 7 times.

[0039] (Experimental results) The average particle diameters of the polymer microparticles produced in Examples 1 to 3 and Comparative Examples 1 to 4 were measured. Table 1 summarizes the average particle diameters and particle size distributions of the polymer microparticles.

[0040]

Table 1

[0041] As shown in Table 1, in the production methods of Examples 1 to 3 using nonionic surfactants with a critical micelle concentration of 5 mM or more and 30 mM or less, polymer microparticles with an average particle diameter of 200 nm or less were obtained with a narrow particle size distribution, and the detergency was also good. Also, as shown in Table 1, in Comparative Example 1 without adding a surfactant, polymer microparticles with an average particle diameter of 202 nm were obtained. Furthermore, in the production method of Comparative Example 2 using the non-alkylphenol ethoxylate type polysorbate surfactant Tween 20, which is not a nonionic surfactant with a critical micelle concentration of 5 mM or more and 30 mM or less, the average particle diameter of the obtained polymer microparticles varied significantly, and the particle size distribution could not be measured (indicated as "-" in Table 1). Additionally, in the production method of Comparative Example 3 using the alkylphenol ethoxylate type surfactant NP-40, the average particle diameter of the obtained polymer microparticles was as large as 410 nm, and the particle size distribution was also wide. Moreover, in the production method of Comparative Example 4 using a non-alkylphenol ethoxylate type polyoxyethylene alkyl ether surfactant, polymer microparticles with an average particle diameter of 114 nm, which were sufficiently small, were obtained, but the particle size distribution was wide, and a large number of washing times were required.

Claims

1. A step of mixing styrene, glycidyl methacrylate, and a crosslinking agent to obtain a mixed solution A; A step of mixing an aqueous solution of a surfactant with the mixed solution A to obtain a mixed solution B; A step of mixing a polymerization initiator with the mixed solution B, and having, The concentration of the surfactant in the mixed solution B is equal to or lower than the critical micelle concentration of the surfactant, The surfactant is a nonionic surfactant having a critical micelle concentration of 5 mM or more and 30 mM or less, A method for producing polymer microparticles, wherein the structure of the surfactant is a compound represented by the general formula (1). R1(OR2)sGt (1) (In the formula (1), R1 is a linear or branched alkyl group, alkenyl group, or phenylalkyl group having 8 or more and 18 or less carbon atoms, R2 is an alkylene group having 2 or more and 4 or less carbon atoms, s is an integer of 0 or more and 5 or less, G represents a residue derived from a reducing sugar having 5 or 6 carbon atoms, t represents an integer of 1 or more and 5 or less.)

2. The method for producing polymer microparticles according to claim 1, wherein the critical micelle concentration of the surfactant is 7 mM or more and 25 mM or less.

3. The method for producing polymer microparticles according to claim 1 or 2, wherein the molecular weight of the surfactant is 292 or more and 335 or less.

4. The method for producing polymer microparticles according to any one of claims 1 to 3, wherein the polymerization initiator is any one of ammonium persulfate, potassium persulfate, and 2,2'-azobis(2-methylpropionamidine) dihydrochloride.

5. The method for producing polymer microparticles according to any one of claims 1 to 4, wherein the crosslinking agent is a radically polymerizable monomer having crosslinking properties.

6. The method for producing polymer microparticles according to any one of claims 1 to 5, wherein the crosslinking agent is divinylbenzene.

7. The method for producing polymer microparticles according to any one of claims 1 to 6, wherein the surfactant is any one of n-octyl-β-D-glycopyranoside, n-octyl-β-D-thioglycoside, and n-decanoyl-N-D-glucosamine.

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