JP7693330B2 - Method for producing polymer microparticles - Google Patents

Description

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

In recent years, polymer microparticles with ligands that have affinity for target molecules adsorbed on their surfaces have been widely used to purify and quantify target molecules.

In this regard, Patent Document 1 discloses a method for purifying a specific protein using polymer microparticles on whose surface DNA strands having a base sequence that specifically binds to the specific protein are adsorbed.

Here, the polymer microparticles used in Patent Document 1 are produced by performing a soap-free (surfactant-free) polymerization reaction using both monomers, styrene and glycidyl methacrylate, and coating the surface of the copolymer with polyglycidyl methacrylate, and have a particle size of approximately 200 nm. These particles composed of styrene and glycidyl methacrylate (hereinafter referred to as "SG particles") can reduce nonspecific adsorption of proteins by chemical modification via the glycidyl groups present on the surface.

However, from the perspective of new applications of polymer microparticles, such as improving the efficiency of target factor purification and improving sensing accuracy by using them as probes in biosensors, further miniaturization of polymer microparticles was desired.

In this regard, Patent Document 2 focuses on the type of surfactant in order to further reduce the size of polymer microparticles, and discloses a method of copolymerizing styrene and glycidyl methacrylate in the presence of a non-alkylphenol ethoxylate (APE) polyoxyethylene alkyl ether surfactant. A commonly known method of reducing the size of SG particles is to polymerize by reducing the polymer concentration in emulsion polymerization, but the method of Patent Document 2 makes it possible to reduce the size of the particles without reducing the polymer concentration, which is also effective in terms of improving productivity.

Japanese Patent Application Publication No. 4-46193 JP 2014-193972 A

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

The present invention has been made in consideration of the above problems, and aims to provide a new method for producing submicron-sized polymer microparticles with a narrow particle size distribution while reducing nonspecific adsorption.

In order to improve the particle size distribution of polymer microparticles, the inventors focused on the type of surfactant and reconsidered the manufacturing method of polymer microparticles. As a result, they discovered that polymer microparticles with a good particle size distribution can be obtained by performing emulsion polymerization of styrene and glycidyl methacrylate using a surfactant with a critical micelle concentration in a specific range and suppressing the surfactant concentration below the critical micelle concentration, which led to the present invention.

In emulsion polymerization, monomers are sequentially fed from the oil phase to the water phase, and polymerization is initiated when the monomers dissolved in water react with the polymerization initiator. If a surfactant is added in a concentration that forms micelles during emulsion polymerization, the surfactant may form micelles that encapsulate oil droplets. If polymerization proceeds in a state where the surfactant has formed micelles that encapsulate oil droplets, the particle size distribution of the polymer microparticles produced by polymerization may become broader.

The inventors of the present invention arrived at this invention after extensive research into methods for forming polymer microparticles without generating micelles containing oil droplets.

That is, the present invention is a method for producing polymer microparticles, which comprises the steps of mixing styrene and glycidyl methacrylate to obtain mixed liquid A, mixing the mixed liquid A with an aqueous solution of a surfactant to obtain mixed liquid B, and mixing a polymerization initiator into the mixed liquid B, wherein the concentration of the surfactant in the mixed liquid 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.

The present invention provides a method for producing polymer microparticles with a narrow particle size distribution and an average particle size of 200 nm or less.

Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this embodiment.
The method for producing polymer microparticles according to the present 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. The concentration of the surfactant in the mixed solution B is equal to or less than the critical micelle concentration of the surfactant, and the surfactant is a nonionic surfactant having a critical micelle concentration of 5 mM to 30 mM. As described above, the method for producing polymer microparticles according to the present embodiment can obtain polymer microparticles having a narrow particle size distribution and an average particle size of 200 nm or less by using a nonionic surfactant at a predetermined concentration. In addition, nonspecific adsorption of proteins after chemical modification is reduced without reducing productivity. Details will be described below.

1. Surfactant The surfactant used in the manufacturing method of the present invention is a nonionic surfactant. If an anionic surfactant or a cationic surfactant, which is generally used in emulsion polymerization, is present as a residue, there is a concern that the antibody may be denatured when the polymer microparticles are made into a pharmaceutical product. There is also a concern that a specific antibody-antigen reaction does not occur at the antibody site where the three-dimensional structure is deformed, and the desired function as a biosensor or bioseparation particle is not expressed. Therefore, a nonionic surfactant is used as the surfactant of the present invention.

The nonionic surfactant used in the production method of the present invention is characterized in that it has a critical micelle concentration of 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, even more preferably 15 mM or more and 25 mM or less, and particularly preferably 7 mM or more and 25 mM or less.
The molecular weight of the surfactant is preferably 100 to 600, more preferably 292 to 335. When a surfactant is added to an emulsion polymerization system, the surfactant forms micelles in an aqueous solution, so that oil droplets are contained 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, a surfactant having a high critical micelle concentration range, that is, a surfactant that does not easily form micelles in an aqueous solution, is used to suppress the inclusion of oil droplets in the micelles. This makes it possible to narrow the particle size distribution of the polymer microparticles in the production method of the present invention. The structure of the surfactant used in the present invention can include a sugar-based nonionic surfactant (alkyl glucoside). By using a sugar-based nonionic surfactant, a small amount of a highly hydrophilic sugar structure is present on the surface layer of the SG particle, so that the nonspecific adsorption of proteins of the particles obtained by chemically modifying the SG particle is improved (nonspecific adsorption is reduced). Specifically, a 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, an alkenyl group, or a phenylalkyl group having 8 to 18 carbon atoms, and is preferably an alkyl group.
In formula (1), R2 is an alkylene group having 2 to 4 carbon atoms. s is an integer of 0 to 5.
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.

The residue G derived from a reducing sugar may be derived from either an aldose or a ketose. Examples of G include residues derived from a pentose having five carbon atoms or a hexose having six carbon atoms. Specific examples of pentose include apiose, arabinose, lyxose, and xylose. Specific examples of hexose include galactose, glucose, mannose, gulose, idose, talose, fructose, and glucosamine.

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.

Specific examples of sugar-based nonionic surfactants include n-octyl-β-D-glycopyranoside, n-octyl-β-D-thioglycoside, and n-decanoyl-N-D-glucosamine.

2. Monomer The monomers used in the method for producing polymer microparticles of the present invention are styrene and glycidyl methacrylate. By containing styrene as a repeating unit of the polymer, the polymer can be given physical properties such as a high glass transition temperature and excellent mechanical strength. Therefore, when polymer microparticles containing styrene as a repeating unit are purified by a method such as centrifugation or ultrafiltration, the effect of suppressing damage such as cracking and chipping of the polymer microparticles is exhibited. In addition, 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 glycidyl groups. When introducing reactive functional groups, the glycidyl group is ring-opened to become a glycol group, thereby expressing hydrophilicity due to the hydroxyl group, and non-specific adsorption is reduced. 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 glycidyl groups, it is preferable that the glycidyl group is localized on the surface layer of the particle. As a method for localizing the glycidyl group on the surface layer of the particles, there is a method of post-adding glycidyl methacrylate.

3. Crosslinking Agent In the method for producing polymer microparticles of the present invention, a radically polymerizable monomer having crosslinking properties is used. Examples of radically polymerizable monomers having crosslinking properties 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, glycidyl methacrylate, 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, but are not limited to these as long as the object of the present invention can be achieved. In addition, two or more types of radical polymerizable monomers having crosslinking properties may be used in combination. The amount of the radical polymerizable monomer having crosslinking properties added is preferably in the range of 0.1 to 5 parts by mass relative to 100 parts by mass of the total amount of styrene and glycidyl methacrylate.

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, conventionally known radical polymerization initiators such as peroxides such as ammonium persulfate, potassium persulfate, and hydrogen peroxide, and water-soluble azotization polymerization initiators such as 2,2'-azobis(2-methylpropionamidine)dihydrochloride can be suitably used.

The amount of the polymerization initiator used can be adjusted appropriately depending on 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.

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 dynamic light scattering (DLS) principle. The particle size in this example is measured using a Zetasizer NS manufactured by Malvern.

The measurement sample is a sample solution in which polymer microparticles are dispersed at 0.005% by weight in ultrapure water solvent, and is measured using a disposable plastic cell. The analysis parameters are: refractive index of polymer microparticles 1.59 equivalent to latex, absorbance of polymer microparticles 0.01, refractive index of dispersion medium 1.33 equivalent to water, viscosity of dispersion medium 0.8872 cP equivalent to water, and measurement temperature 25°C. Measurements are performed three times, and the average value is taken as the average particle size of the polymer microparticles. In this specification, the average particle size is expressed as the Z-average particle size.

6. Production method Styrene and glycidyl methacrylate are added to water, and the mixture is mixed and stirred for a sufficient time to emulsify, thereby obtaining a mixed solution A. Separately, a surfactant is added to water, and the mixture is 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 it is preferable to use demineralized water, ion-exchanged water, distilled water, ultrapure water, etc.

Next, the mixed solution A is mixed with the aqueous surfactant solution and stirred for a sufficient time to obtain the 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 the mixture is mixed and stirred for a sufficient time to copolymerize styrene and glycidyl methacrylate to produce a granular copolymer. Thereafter, glycidyl methacrylate is further added to the reaction system, and the mixture is mixed and stirred for a sufficient time to form polymer microparticles in which the surface of the granular copolymer is coated with polyglycidyl methacrylate.
Finally, the polymer particles are separated and washed.

As described above, according to the manufacturing method of one embodiment of the present invention, polymer microparticles with a narrowly controlled particle size distribution can be obtained with fewer washing steps.

By applying the produced polymer microparticles as carriers for purifying physiologically active substances or as biosensing probes, it is expected that the purification efficiency or sensing accuracy of physiologically active substances can be improved.

The method for producing polymer microparticles of the present invention will be explained in more detail below using examples, but the present invention is not limited to the examples described below.

Example 1
0.3 g of n-octyl-β-D-glycopyranoside (Tokyo Chemical Industry Co., Ltd., critical micelle concentration 25 mM) as a surfactant was added to 70 ml of ultrapure water, and the mixture was stirred at 70 ° C. and 200 rpm for 30 minutes to obtain a surfactant aqueous 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 surfactant aqueous 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 of 0.06 g of 2,2'-azobis (2-methylpropionamidine) dihydrochloride (trade name: V-50, Fujifilm Wako Pure Chemical Industries, Ltd.) as a polymerization initiator dissolved 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 further added to this polymerization reaction liquid, and the mixture was stirred at 200 rpm at 70° C. for one day and night. Finally, the polymerization reaction liquid was centrifuged to remove the supernatant, and then ultrapure water was added and the redispersion operation was repeated five times (hereinafter, this operation is referred to as washing operation) to obtain a suspension of polymer fine particles.
The obtained suspension of polymer particles was centrifuged to remove the supernatant, and the particles were then dried under reduced pressure at 40° C. for 48 hours to obtain polymer particles (hereinafter, this operation is referred to as “isolation operation”).
It was also confirmed that similar results were obtained when the polymer concentration was not changed and only the GMA/styrene ratio was changed within the range of 1.4 to 1.7. It was also confirmed that similar results were obtained when the temperature was changed within the range of 65 to 75°C.

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 a mixed solution A. To the mixed solution A, a surfactant aqueous solution in which 0.3 g of n-octyl-β-D-thioglycoside (manufactured by Dojindo Laboratories, critical micelle concentration 9 mM) was dissolved in 40 ml of ultrapure water was added, 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, a polymerization initiator aqueous solution in which 0.06 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved 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 further added to the polymerization reaction solution, and the mixture was stirred at 70° C. and 200 rpm for one day and night. The washing operation was repeated five times to obtain a suspension of polymer particles. Finally, the obtained suspension of polymer particles was subjected to an isolation operation to obtain polymer particles.

Example 3
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 a mixed solution A. To the mixed solution A, a surfactant aqueous solution in which 0.3 g of n-decanoyl-N-D-glucosamine (manufactured by Dojindo Laboratories, critical micelle concentration 7 mM) was dissolved in 40 ml of ultrapure water was added, 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, a polymerization initiator aqueous solution in which 0.06 g of 2,2'-azobis (2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 10 ml of ultrapure water was added as a polymerization initiator, and the mixture was stirred at 70 ° C. and 200 rpm for 1 hour to obtain a mixed solution B. Then, 0.33 g (305 μl) of glycidyl methacrylate was added to the polymerization reaction solution, and the mixture was stirred at 70° C. and 200 rpm for 24 hours. The washing operation was repeated five times to obtain a suspension of polymer particles. Finally, the obtained suspension of polymer particles was subjected to an isolation operation to obtain polymer particles.

(Comparative Example 1)
To 110 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 a mixed solution A. To the mixed solution A, a polymerization initiator aqueous solution in which 0.06 g of 2,2'-azobis (2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 10 ml of ultrapure water was added as a polymerization initiator, 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 the polymerization reaction liquid, and the mixture was stirred at 70 ° C. and 200 rpm for 24 hours. Furthermore, the washing operation was repeated five times to obtain a suspension of polymer fine particles. Finally, an isolation operation was performed on the obtained suspension of polymer fine particles to obtain polymer fine particles.

(Comparative Example 2)
A surfactant aqueous solution was prepared by adding 0.5 g of a non-alkylphenol ethoxylate 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 by the same manufacturing method as in Example 1, except that the washing operation was repeated six times.

(Comparative Example 3)
A surfactant aqueous solution was prepared by adding 0.5 g of an alkylphenol ethoxylate 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 by the same manufacturing method as in Example 1, except that the washing operation was repeated six times.

(Comparative Example 4)
A surfactant aqueous solution was obtained by adding 0.5 g of a non-alkylphenol ethoxylate-based polyoxyethylene alkyl ether surfactant (product name: Emulgen 1150S-60, manufactured by Kao Corporation) to 70 ml of ultrapure water and stirring at 70°C and 200 rpm for 30 minutes. Polymer microparticles were obtained by the same manufacturing method as in Example 1, except that the washing operation was repeated seven times.

(Experimental Results)
The average particle size of the polymer fine particles produced in Examples 1 to 3 and Comparative Examples 1 to 4 was measured. Table 1 shows the average particle size and particle size distribution of the polymer fine particles.

As shown in Table 1, in the manufacturing methods of Examples 1 to 3, which used a nonionic surfactant with a critical micelle concentration of 5 mM or more and 30 mM or less, polymer microparticles having an average particle size of 200 nm or less were obtained with a narrow particle size distribution in all cases, and the cleaning properties were also good.
Furthermore, as shown in Table 1, in Comparative Example 1 in which no surfactant was added, polymer fine particles having an average particle size of 202 nm were obtained.
Furthermore, in the production method of Comparative Example 2 using the non-alkylphenol ethoxylate 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 size of the obtained polymer microparticles significantly varied, and the particle size distribution could not be measured (represented as "-" in Table 1). Furthermore, in the production method of Comparative Example 3 using the alkylphenol ethoxylate surfactant NP-40, the average particle size of the obtained polymer microparticles was large at 410 nm, and the particle size distribution was also wide.
Furthermore, in the manufacturing method of Comparative Example 4 using a non-alkylphenol ethoxylate-based polyoxyethylene alkyl ether surfactant, the average particle size of the resulting polymer microparticles was 114 nm, which was sufficiently small, but the particle size distribution was broad and many washing steps were required.

Claims (7)

A step of mixing styrene, glycidyl methacrylate, and a crosslinking agent to obtain a mixed solution A;
mixing the mixed solution A with an aqueous solution of a surfactant to obtain a mixed solution B;
and mixing a polymerization initiator into the mixed solution B,
a concentration of the surfactant in the mixed solution B is equal to or lower than a 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,
The method for producing polymer fine particles , wherein the structure of the surfactant is a compound represented by general formula (1) .
R1(OR2)sGt (1)
In formula (1), R1 is a linear or branched alkyl group, alkenyl group, or phenylalkyl group having from 8 to 18 carbon atoms, and R2 is an alkylene group having from 2 to 4 carbon atoms.
s is an integer of 0 to 5, G is a residue derived from a reducing sugar having 5 or 6 carbon atoms,
t is an integer between 1 and 5. The method for producing polymer microparticles according to claim 1, characterized in that the critical micelle concentration of the surfactant is 7 mM or more and 25 mM or less. The method for producing polymer microparticles according to claim 1 or 2, characterized in that the molecular weight of the surfactant is 292 or more and 335 or less. 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. The method for producing polymer microparticles according to claim 1 , wherein the crosslinking agent is a radical polymerizable monomer having crosslinkability. The method for producing polymer microparticles according to claim 1 , wherein the crosslinking agent is divinylbenzene. 7. The method for producing polymer microparticles according to claim 1, wherein the surfactant is any one of n-octyl-β-D-glycopyranoside, n-octyl-β-D-thioglycoside, and n-decanoyl-N-D-glucosamine.