JPS5829962B2 - Method for producing hydrophilic water-insoluble fine particles - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing novel hydrophilic insoluble fine particles.

In recent years, synthetic polymer compound latex has often been used for the purpose of medical diagnosis.

The most commonly used is polystyrene latex.

For example, the phagocytic ability of leukocytes is measured by phagocytizing leukocytes with polystyrene fine particles.

In addition, immunological tests are often performed by adsorbing immunoactive substances on the surface of polystyrene fine particles and using the agglutination reaction or non-adherence reaction to cells.

However, a problem when using polystyrene fine particles is that polystyrene is hydrophobic and therefore adheres non-selectively to cells and induces physiological reactions.

Furthermore, since immunologically active proteins such as antibodies to be immobilized on the polystyrene surface are not strongly adsorbed, satisfactory results can only be obtained in limited cases even with agglutination reactions.

In addition, hydrophilic gels such as agarose, cross-linked dextran, and polyacrylamide are often used as carriers for immunoadsorption chromatography because of their low nonspecific adsorption to proteins.

In this case, immobilization of immunologically active substances such as antigens or antibodies to the carrier is carried out by covalent bonding.

However, these conventionally known chromatographic pieces have a diameter of 10 μm or more and are not suitable for use in measuring phagocytosis, markers for cell adhesion, or agglutination reactions.

For the measurement of oligophagocytosis, fine particles with an average diameter in the range of 0.1 to 5 μm and which do not non-specifically adhere to cells are desirable.

It would also be advantageous if it could be labeled with staining, fluorescence or radioactivity.

The preferred carrier for micropieces for cell attachment or agglutination reactions is a diameter in the range of 0.1 to 10μ, which does not adhere to cells or non-specifically adsorb serum proteins, and which does not chemically remove proteins or fluorescent substances. It has the necessary functional groups to bond to.

macromolecule, 9, 328 (19
76), by polymerizing 2-hydroxyethyl methacrylate (hereinafter abbreviated as HEMA) in water using radiation, PHEMA fine particles with a particle size ranging from 0.3 to 3μ were obtained, and the particles were applied as a cell labeling marker. It is proposed to do so.

These PHEMA fine particles have a relatively narrow particle size distribution and can be used for immunological tests other than cell labeling, but a major limitation is that they cannot be manufactured without radiation polymerization.

On the other hand, Japanese Patent Publication No. 53-16037 proposes that PHEMA fine particles with a diameter of 10 μm or less can be obtained by polymerizing HEMA and a cross-binder in an inert diluent such as an aromatic hydrocarbon. .

However, the problem with these PHEMA particles is that they
It is prone to spontaneous agglutination in urine or serum.

One of the main objects of the present invention is to bring microparticles on which an immunoactive substance (e.g., an antigen) is immobilized into contact with another immunoactive substance (e.g., an antibody) that specifically reacts with the immobilized immunoactive substance (e.g., an antibody). It is an object of the present invention to provide fine carrier particles that can be suitably used for performing immunological tests by utilizing the phenomenon of aggregation.

It is clear that when used for such purposes, the intended test cannot be performed if spontaneous aggregation occurs regardless of the immunological reaction.

The present inventors have arrived at the present invention as a result of intensive research to develop microparticles useful for immunological or cytological tests such as agglutination reactions, cell labeling, and phagocytosis measurements.

That is, the present invention provides an addition polymerizable monomer containing 50 mol% or more of glycidyl methacrylate and a crosslinkable monomer containing at least two polymerizable carbon-carbon double bonds in the molecule in the range of 0.1 to 30 mol%. The monomer mixture is polymerized in an organic solvent that dissolves the monomer mixture but precipitates the polymer produced by polymerization, and hydrolyzes the precipitated particulate polymer. The repeating units are at least 50 mole% of the total repeating units
It is a crosslinked polymer that accounts for a
The present invention provides a method for producing hydrophilic water-insoluble fine particles having an average diameter in a water-containing state of o,i to 10μ.

It can be said that the microparticles according to the present invention do not non-specifically adhere to human or animal cells, and do not substantially non-specifically adsorb proteins from serum.

In addition, it does not agglutinate in physiological saline or urine, and substantially does not agglutinate in serum if it is diluted 4 times or more.

Furthermore, since it contains hydroxyl groups and ester bonds, its reactivity can be used to immobilize physiologically active substances and immunologically active substances, including antigens and antibodies, through covalent bonds.

Also, fluorescent labeling and staining are easy. The polymer constituting the fine particles of the present invention is a crosslinked polymer in which 2,3-dihydroxypropyl methacrylate accounts for at least 50 mol% of the repeating units, and in this case, the copolymer component is allowed to be less than 50 mol%. This includes non-crosslinking copolymerization components and crosslinking copolymerization components, and examples of non-crosslinking copolymerization components include methyl methacrylate, methacrylamide, acrylamide, 2-oxyethyl meccrylate, and methacrylic acid. , diethylaminoethyl methacrylate, diethylaminoethyl methacrylate, and styrene.

Although the addition of these non-crosslinking copolymer components is not necessarily necessary, the hydrophilicity, water content, dispersion stability, etc. of the particles can be adjusted by introducing the non-crosslinking copolymer component.

The moisture content of the particles can also be adjusted by the degree of crosslinking.

Crosslinking of the polymer is an essential condition.

This is because the polymer of 2°3-dihydroxypropyl methacrylate, which is the main component, dissolves in water in a non-crosslinked state and cannot maintain its particle form.

If the proportion of the non-crosslinkable copolymer component exceeds 50 mol%, the fine particles will not be able to achieve the desired performance of the present invention, that is, to maintain a stable dispersion state without aggregating even when mixed with serum or urine, and to maintain a stable dispersion state even when mixed with serum or urine. It cannot have features such as not adhering non-specifically and not adsorbing serum proteins non-specifically.

As is obvious from the expression, the fine particles of the present invention have a sufficient amount of polymers to maintain a solid state, but since they have a crosslinked structure, it is impossible to measure their specific value by normal means. It is.

The fine particles of the present invention need to have a moisture content of 10 to 90%.

Moisture content is defined by the following formula and is measured at 25°C.

If the water content is less than 10%, there are problems such as particles tending to non-specifically adhere to cells and tend to aggregate spontaneously, and are not suitable for the purpose of the present invention.

Furthermore, when the water content exceeds 90%, the difference in refractive index between the hydrous gel particles and the aqueous medium becomes extremely small, making it difficult to observe phenomena such as aggregation, cell adhesion, and phagocytosis both macroscopically and microscopically. It becomes difficult.

Dyeing particles to make them easier to observe is a technique that is often used, but it is difficult to dye them in deep colors at high moisture contents of 90% or more.

It is necessary that the average diameter of the fine particles of the present invention is within the range of 0.1 to 10μ.

When the particle size is 0.1 μm or less, it becomes difficult to observe aggregation with the naked eye and to observe adhesion to cells with an optical microscope.

If the particle size is 10 μm or more, the dispersion stability of the particles will be poor and the particles will also be relatively large compared to the cells, making them unsuitable for observing cell adhesion.

As can be understood from the above explanation, it is desirable that the particle size distribution be as narrow as possible.

Specifically, for example, the standard deviation of particle size is calculated as the average diameter (number average).
If the value divided by is 0.2 or less, it can be said that it is suitable for the purpose of the present invention.

The method for producing the polymer fine particles of the present invention having the above-mentioned characteristics is to polymerize glycidyl methacrylate alone or together with other ethylenically uncelled monomers in a suitable diluent in the coexistence of a crosslinking agent. It consists of precipitating fine particles of a methacrylate homopolymer or copolymer and treating the fine particles with an acidic or alkaline aqueous solution to hydrolyze the epoxy ring in the glycidyl methacrylate unit.

The appropriate diluent here refers to a medium in which the glycidyl methacrylate monomer is easily dissolved, but the polyglycidyl methacrylate produced by polymerization is not dissolved but precipitated, such as ethyl acetate, n-propyl acetate, etc. ,
Isopropyl acetate, butyl acetate isomers, esters such as the above-mentioned corresponding esters of propionic acid, methyl n-
These include ketones such as propyl ketone, methyl isopropyl ketone, and methyl isobutyl ketone.

The above polymerization media may be used in combination.

Examples of crosslinking agents mixed with halogenated hydrocarbons such as carbon tetrachloride, chloroform, and bromoform include ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, and H(-OCH2-CH2
), -0H (n is an integer of 3 or more), dimethacrylate of polyethylene glycol, divinylbenzene, or methylenebisacrylamide, etc. are preferably used.

The amount of crosslinking monomer added is usually 0.1 to 30% of the total monomers.
It is mole%.

If the amount is less than 0.1 mol %, it is impossible to prevent the polymer from dissolving in water when the epoxy groups in the polymer are subsequently hydrolyzed.

Moreover, if the amount is 30 mol % or more, the dispersion state of particles during polymerization becomes poor and the tendency to agglomerate becomes significant.

The significance of adding a non-crosslinking copolymer component and the type of suitable non-crosslinking copolymer component have already been discussed, but another major effect of using a non-crosslinking copolymer component is the particle size of the polymer particles. It is an adjustment of

The average diameter of the polymer particles can vary greatly depending on the type of copolymerizable component.

Another major factor influencing average particle size is the type of polymerization medium.

That is, the diameter of the produced polymer particles is mainly controlled by the type and amount of the polymerization medium and copolymer components.

However, the copolymerization component, including crosslinkable and non-crosslinkable components, cannot exceed 50 mol% of the total monomers.

If the copolymerization component is in a range of 50 mol □ or more, the resulting particles will lose their dispersion stability in physiological saline, urine, serum, etc., and their non-adsorption of proteins, which will not meet the purpose of the present invention.

As the polymerization initiator, common radical polymerization initiators such as 2,2'-azobisisobutyronitrile, 2,2'-
Azobis(2,4-dimethylvaleronitrile), 2,2
'-azobis(2,4-dimethyl4-methoxyvaleronitrile), 2,2'-azobis(4-methoxy-4,2
-dimethylvaleronitrile), benzoyl peroxide, dilauroyl peroxide, ter
Peroxides such as t-butyl peroxide can be used.

The polymerization temperature is also within the normal radical polymerization temperature range of 20℃.
C. to 80.degree. C. is particularly preferred.

The concentration of the polymerization initiator in the polymerization mixture is usually about 0.001 to 0.03 mol/l.

The concentration of monomer in the polymerization mixture is usually 5 to 50% by weight.
A range of is preferred.

When the monomer concentration exceeds 50% by weight, the resulting polymer particles tend to aggregate.

Although the present invention can be carried out even if the monomer concentration is less than 5% by weight, productivity will be lower because fewer polymer particles will be obtained.

Note that the polymerization is preferably carried out by replacing the atmosphere with an inert gas such as nitrogen or argon.

Next, the polymer fine particles containing glycidyl methacrylate as a main component produced as described above are subjected to a hydrolysis treatment.

This hydrolysis is usually carried out by treating the polymer particles with an acidic or alkaline aqueous solution, and the epoxy groups in the polymer are hydrolyzed and converted into diols.

At this time, if the reaction conditions are harsh, even the ester bonds will be hydrolyzed, so it is desirable to choose conditions as mild as possible to avoid this.

Since alkali has a strong tendency to cleave esters, it is generally preferable to use acidic conditions.

The acid is preferably a dilute aqueous solution of an inorganic acid such as sulfuric acid, hydrochloric acid, nitric acid, or phosphoric acid, or an organic acid such as benzenesulfonic acid or toluenesulfonic acid.

The acid concentration is preferably in the range of 0.01 to 1N.

The hydrolysis temperature is preferably in the range of 0°C to 50°C.

At higher temperatures, side reactions cannot be ignored.

As mentioned above, since the reaction conditions are mild, the reaction rate is low, and it usually takes a considerable amount of time to complete the reaction even with sufficient stirring.

Therefore, the reaction can be accelerated by adding to the reaction system an organic solvent that is soluble in water and has the ability to swell the polymer, such as acetone, tetrahydrofuran, or dioxane.

It is understood that these solvents help the polymer to swell and the acid to diffuse into the interior of the polymer particles, thereby shortening the hydrolysis reaction time.

After hydrolysis is complete, the particles are usually washed with a sufficient amount of water.

Further, washing may be performed with an aqueous sodium hydrogen carbonate solution or the like.

In this way, the epoxy group can be converted into 1,2-diol with almost no side reactions, and polymer particles containing 2,3-dioxypropyl methacrylate as a main component can be produced.

For example, Example 1 shows that the desired polymer is produced.
This can be confirmed by infrared absorption spectra as described in detail in .

The polymer fine particles produced in this manner and containing 2,3-dioxypropyl methacrylate as a main component have an average diameter in a water-containing state in the range of 0.1 to 10 μm, and have a relatively narrow particle size distribution.

Moreover, the equilibrium moisture content at 25° C. is in the range of 10 to 90%, and it is usually stored in a dispersed state in water, but it may be stored in a dry state.

Dry fine particles can be easily redispersed by adding water and shaking at the time of use.

Example 1 Glycidyl methacrylate and triethylene glycol dimethacrylate were mixed at a molar ratio of 95:5, and 23 parts of this monomer mixture (based on weight, the same hereinafter) was dissolved in 77 parts of ethyl propionate.2. 2'-Azobis (2,
0.1 part of 4-dimethyl-4-methoxyvaleronitrile) was added and polymerized.

The initial concentration of polymerization initiator is 3.6 mmol/12. Polymerization was carried out under an argon atmosphere at 40° C. for 1.5 hours.

After a certain period of time, the cloudy polymerization mixture was poured into acetone.
The mixture was centrifuged at 1500X for 9 and 10 minutes, and the precipitated particles were redispersed and washed with ethanol, followed by centrifugation again.

It was dried under reduced pressure to obtain 20 parts of a particulate polymer.

One part of this particulate polymer was suspended in 100% N/20 sulfuric acid and stirred at 30° C. for 14 days to effect hydrolysis.

It was observed that as time elapsed, the opacity of the suspension decreased, and the dispersed state of the fine particles became stable, making it difficult for them to settle even after the stirring was stopped.

After completion of hydrolysis, the fine particles were washed with water by repeating centrifugation and dispersion.

A portion of the dispersion was dried and its infrared absorption spectrum was measured.

Separately, 2,3-dioxypropyl methacrylate monomer and triethylene glycol dimethacrylate were mixed in a ratio of 95:5.
The infrared absorption spectrum of the standard polymer prepared by mixing in a molar ratio of 1 and 2 and polymerizing in water using ammonium persulfate as a polymerization initiator by a conventional method matched the infrared absorption spectrum of the above-mentioned particulate polymer.

The particulate polymer obtained here is thus poly(2,3-dioxypropyl methacrylate) crosslinked with triethylene glycol dimethacrylate.

When the polymer particles were observed with an optical microscope at a magnification of 1000 times, the diameter of the particles was in the range of 1.6μ to 3.2μ, with an average diameter of 2.7μ.

Next, add 0.5 g of dry particles to 5 my/m of blue dextran.
l! The blue dextran concentration was measured again after adding it to 2 ml of the solution and dispersing it and leaving it overnight at 25°C.The water content of the particles was calculated from the change in the blue dextran concentration due to the addition of dry particles. It was 48%.

Pharmacia Fi as blue dextran
BlueDextran manufactured by ne Chemicals
2000 (average molecular weight 2,000,000), and the concentration in the solution was 620m! It was measured by absorption of i.

Actual example 2゜3 of glycidyl methacrylate, 2-oxyethyl methacrylate and ethylene glycol dimethacrylate
24 parts of the monomer mixture and 76 parts of ethyl propionate were mixed in a molar ratio of 85.7:9.5:4.8.
and 2,2'-azobis(2,4-dimethyl-4-
methoxyvaleronitrile) 0.13 parts (4.7 mmol
/11) was polymerized in the same manner as in Example 1.

However, only the polymerization time was changed to 3 hours*.

Similarly, it was washed with acetone and ethanol and dried.

The yield of particulate polymer was 47 parts.

One part of this polymer fine particle was dispersed in a mixed solution of 50 parts of water, 50 parts of acetone, and 0.2 part of concentrated sulfuric acid, and the mixture was stirred at 30° C. for 7 days to perform hydrolysis.

When this polymer particle was observed with an optical microscope at a magnification of 1000 times, the particle diameter distribution was as shown in Table 1.

The mean diameter is 3.52μ and the standard deviation is 0.447μ, so standard deviation/mean=0.126.

When the water content was measured by the blue dextran method in the same manner as in Example 1, the water content at 25°C was 46%.

Alternatively, the water content was determined from the ratio of the diameters of the particles swollen with water and the particles dried and dehydrated.

In other words, assuming that the volume of the hydrogel is additive with its constituent polymer and water, the following formula (
1) is derived.

However, Sw = swollen particle diameter Sd - dry particle diameter d,
= Specific gravity of polymer = 1.15 In the case of this example, the average diameter of the dry particles was 2.89μ, so the water content was 45%, which was in good agreement with the blue dextran method.

Example 3 A mixture of 9.47 parts of glycidyl methacrylate, 0.96 parts of 2-oxyethyl methacrylate and 0.06 parts of triethylene glycol dimethacrylate (molar ratio is 8
9.9 : 9.9 : 0.27) mixture at 30
2,2'-Azobis(2,4-dimethyl-4-methoxyvaleronitrile) was dissolved in ethyl propionate.
．． 03 parts were added and polymerized at 40° C. for 3 hours under a nitrogen atmosphere.

The concentration of 2,2'-azobis(2゜4-dimethyl-4-methoxyvaleronitrile) in the polymerization solution is 2.2 liters.
/l.

The cloudy polymerization mixture was poured into acetone, centrifuged, washed once with acetone, once with methanol, and twice with petroleum ether, and then filtered and dried.

The yield of dried polymer particles was 2.91 parts.

It was further hydrolyzed with dilute sulfuric acid in the same manner as in Example 2.

The microparticles after hydrolysis were observed under a microscope, and the average diameter of the particles was 3.0μ, with a standard deviation of 0.43μ.

According to the blue dextran method, the water content of these fine particles is 6.
1%, and 59% according to formula (1).

Examples 4 to 18゜ By the operation according to Example 2, conditions such as non-crosslinking copolymerization component, crosslinking copolymerization component, polymerization medium, polymerization initiator, polymerization temperature, and polymerization time were changed. Hydrogel microparticles were prepared.

The polymerization conditions, yield, average diameter and water content of the hydrogel fine particles after hydrolysis are summarized in Table 2.

HEMA: 2-hydroxyethyl meccrylate, M
MA: Methyl methacrylate, TEGDM nitriethylene glycol dimethacrylate, EGDM: Ethylene glycol dimethacrylate, V-70: 2,2'-azobis(4-methoxy-4,2-dimethacrylate) Example 19゜Execution The dispersion stability in saline was compared for the hydrogel microparticles according to the present invention prepared according to Examples 1, 2, 3, and 18 and the PHEMA microparticles made by the method of Richard Chimerosek et al.

PHEMA fine particles obtained by polymerization in toluene by the method of Chimerosek et al. have an average diameter of about 1.5 μm and are stably dispersed in distilled water.

Its moisture content was 38%. The five types of fine particles mentioned above were all dispersed in distilled water with the polymer concentration adjusted to 5% by weight.

Next, this fine particle dispersion liquid and the same volume of physiological saline (0.9%
When PHEMA fine particles were mixed with sodium chloride aqueous solution on a glass plate, they agglomerated strongly, but Example 1
, 2, 3 and 18 did not aggregate.

Example 20 A similar comparative experiment was conducted using serum and diluted serum in place of the physiological saline in Example 19.

Healthy human serum was used as the serum, and the dilution was 0.01M.
pH 7.4 with phosphate and 0.9% sodium chloride
buffered saline solution was used.

The results are shown in Table 3.

Example 21 A comparative experiment regarding serum protein adsorption was conducted between the hydrogel microparticles prepared in Examples 1, 2.3, and 18, and sheep red blood cells fixed with commercially available polystyrene latex and formalin.

As polystyrene latex, Po 1ysc i
Monodisperse Mi cr manufactured by ences
osphere +cat-41=7765 (diameter 0.
620μ, standard deviation 0.0021μ), and as the fixed sheep red blood cells, unsensitized blood cells (treated with formalin and tannic acid, dried) in the syphilis HA antigen kit manufactured by Fuji Organ Pharmaceutical Co., Ltd. were used.

Polystyrene latex was used as it was commercially available as a 2.5% dispersion, and hydrogel microparticles and sheep red blood cells were suspended in distilled water at 2.5%.

Mix 0.2 ml of each of the above microparticles or blood cell suspensions with 0.2 ml of healthy human serum in a test tube, and mix at 37°C for 1 hour.
Incubated for 5 minutes.

The microparticles or blood cells were then centrifuged and washed twice with saline adjusted to pH 7.2 with phosphate.

The washed microparticles or blood cells were placed in physiological saline containing 1% bovine serum albumin and adjusted to pH 7.2 with phosphate.
．． 2rul! was redispersed.

On a glass plate, add 1 drop of this redispersed suspension and 1 drop of a 10-fold dilution of anti-human I, yG antiserum (rabbit, titer 2.4 m97 ml) in phosphate buffered saline (pH 7.2). Mixed and observed.

In the case of polystyrene latex and sheep red blood cells, agglutination was observed to occur within 3 minutes, but in actual cases 1, 2.3
In the case of the hydrogel fine particles prepared in Example 1 and No. 18, no aggregation was observed even after 10 minutes.

As is clear from the above experiment, polystyrene latex and fixed red blood cells adsorb at least gG from serum;
The hydrogel microparticles according to the present invention do not adsorb.

Claims (1)

[Claims] 1 An addition-polymerizable monomer mixture containing 50 mol% or more of glycidyl methacrylate and a crosslinkable monomer containing at least two polymerizable carbon-carbon double bonds in the molecule in the range of 0.1 to 30 mol%. , at least 50 repeating units characterized in that the monomer mixture is dissolved but the polymer produced by polymerization is polymerized in an organic solvent such that the polymer formed by the polymerization is precipitated, and the precipitated fine particulate polymer is hydrolyzed. It consists of a cross-linked polymer with a mole % of 2゜3-dioxyprobyl meccrylate units, an equilibrium moisture content at 25°C of 10 to 90%, and an average diameter of 0.1 to 0.1 in the water-containing state.
A method for producing hydrophilic water-insoluble fine particles having a size within the range of 10μ.