JP7346998B2 - Surface-modified magnetic particles and method for producing the same - Google Patents

Description

The present invention relates to surface-modified magnetic particles and a method for producing the same.

In recent years, magnetic particles have been increasingly applied to diagnostic reagents and pharmaceutical research applications because they can provide an excellent reaction field for immune reactions between antigens and antibodies as well as hybridization between DNAs or DNA and RNA. Magnetic particles used in this field are required to have high ability to bind biologically related substances, ability to suppress nonspecific adsorption, and stability against pH fluctuations.

In order to satisfy the above required characteristics, it has been proposed to introduce saccharides onto the particle surface to suppress non-specific adsorption of physiologically active substances such as antibodies (see, for example, Patent Documents 1 and 2). However, although these methods suppress non-specific adsorption, they also suppress the binding of physiologically active substances such as antibodies to particles, resulting in problems such as decreased sensitivity and the elution of magnetic substances when the pH decreases. The problem was that it could not be suppressed.

On the other hand, it has been proposed to form a polymer brush on the particle surface and introduce a reactive functional group to the end thereof (see, for example, Patent Document 3). According to this method, by selecting a hydrophilic polymer as the polymer brush and introducing a carboxyl group at the end, a high amount of antibody introduction and suppression of nonspecific adsorption have been achieved, but the elution of the magnetic substance when the pH decreases. Suppression was insufficient.

Japanese Patent Application Publication No. 2006-321932 Japanese Patent Application Publication No. 2007-145985 Japanese Patent Application Publication No. 2016-57106

An object of the present invention is to provide surface-modified magnetic particles that have high ability to bind biologically related substances and suppress non-specific adsorption, which were difficult to achieve with conventional techniques, and also have high stability against pH fluctuations.

As a result of intensive studies to solve the above problems, the present inventors have found that by forming three layers on the surface of magnetic particles, consisting of an acidic polysaccharide coating layer, a polyamine crosslinked layer, and a polycarboxylic acid coating layer, the The present inventors have discovered that surface-modified magnetic particles can be obtained that have a high ability to bind related substances, a high ability to suppress non-specific adsorption, and are highly stable against pH fluctuations, leading to the completion of the present invention.

That is, the present invention
1. A surface-modified magnetic particle characterized in that the surface of the magnetic particle has three layers in this order: an acidic polysaccharide coat layer, a polyamine crosslinked layer, and a polycarboxylic acid coat layer.
2. A method for producing surface-modified magnetic particles, the method comprising at least the following four steps.
(First step)
A process of introducing amino groups onto the surface of magnetic particles.
(Second process)
A step of adsorbing or reacting an acidic polysaccharide with the magnetic particles obtained in the first step to form a coat layer made of the acidic polysaccharide on the surface of the magnetic particles.
(Third step)
A step of adsorbing or reacting a polyamine to the magnetic particles having the acidic polysaccharide coat layer obtained in the second step and then crosslinking them to form a polyamine crosslinked layer on the surface of the magnetic particles.
(Fourth step)
A step of adsorbing or reacting polycarboxylic acid to the magnetic particles having the polyamine crosslinked layer obtained in the third step to form a polycarboxylic acid coat layer on the surface of the magnetic particles.
This will be explained in detail below.

Various particles can be used as the magnetic particles serving as the base material coated in the present invention. For example, as described in JP-A No. 2015-192995, an ionic functional group on the surface of a fine particle and an ionic functional group on the surface of a magnetic material having an opposite charge to the ionic functional group on the surface of the fine particle. Magnetic particles obtained by reacting with a group, or fine particles coated with a metal oxide such as iron oxide, as described in JP 2016-184703 A, and having an ionic functional group on the surface. Preferably used are magnetic particles obtained by reacting a magnetic material with a wet method. There are no particular restrictions on the diameter of the magnetic particles, but it is preferably about 1 to 10 μm, particularly preferably 1 to 5 μm. Furthermore, an amino group is introduced onto the surface of the magnetic particles, and the amount of the amino group introduced is preferably in the range of 50 to 500 μmol/g.

The surface-modified magnetic particles of the present invention have three layers on the surface of the magnetic particles: an acidic polysaccharide coating layer, a polyamine crosslinked layer, and a polycarboxylic acid coating layer, in this order.

In the present invention, the acidic polysaccharide coat layer is a coat layer formed of a polysaccharide having an acidic functional group, and has the function of suppressing nonspecific adsorption of proteins and the like. Since polysaccharides are extremely hydrophilic and do not have hydrophobic parts, they can prevent non-specific adsorption of proteins and the like caused by hydrophobic adsorption. The acidic functional group referred to here refers to a functional group exhibiting acidity such as a carboxyl group, a sulfonic acid group, and a phenolic hydroxyl group, and is introduced into the polysaccharide via a covalent bond. These acidic functional groups bond with the amino groups introduced on the surface of the magnetic particles as a base material through covalent bonds or ionic bonds, and play the role of immobilizing the acidic polysaccharide coat layer on the surface of the magnetic particles. , plays the role of immobilizing the polyamine cross-linked layer on the surface of the acidic polysaccharide coat layer by bonding with the amino groups in the polyamine cross-linked layer formed on the acidic polysaccharide coat layer through covalent bonds or ionic bonds. ing. Some examples of acidic polysaccharides used in the present invention include carboxymethyl cellulose, carboxymethyl dextran, carrageenan, pectin, gum arabic, xanthan gum, gellan gum, agar, gum tragacanth, alginic acid, hyaluronic acid, chondroitin sulfate, and the like. The thickness of the acidic polysaccharide coat layer is not particularly limited, but can be appropriately selected within the range of 5 nm to 50 nm. When the coating layer thickness is 5 nm or more, non-specific adsorption of proteins and the like caused by hydrophobic adsorption can be sufficiently suppressed. On the other hand, if the thickness is 50 nm or less, it does not adversely affect the magnetic collecting property.

In the present invention, the polyamine crosslinked layer is a polyamine crosslinked with a crosslinking agent such as isocyanate, and suppresses permeation of acid components such as citric acid to the magnetic particles and prevents iron elution from the magnetic material. Polyamine is a linear or branched aliphatic polymer in which three or more amino groups are bonded, and some examples thereof include polyethyleneimine, polyallylamine, polydimethyldiallylammonium chloride, and copolymers thereof. It will be done. These polyamines are adsorbed to the acidic polysaccharide coat layer or fixed via ionic bonds or covalent bonds, and then crosslinked with a crosslinking agent to form a polyamine crosslinked layer. There is no particular restriction on the structure of the crosslinked portion in the polyamine crosslinked layer. For example, when isocyanate is used as a crosslinking agent, the polyamine is crosslinked via urethane bonds. The thickness of the polyamine crosslinked layer is also not particularly limited, and can be appropriately selected within the range of 5 nm to 20 nm. It is preferable that the crosslinked layer thickness is 5 nm or more because permeation of the acid component can be sufficiently suppressed. On the other hand, if the thickness is 20 nm or less, there is less concern about hydrophobic adsorption due to increased hydrophobicity, which is preferable.

In the present invention, the polycarboxylic acid coat layer is a coat layer formed of a polymer containing polycarboxylic acid, and has the function of providing a bonding site with an antibody. Since the antibody has an amino group, it reacts with the carboxyl group in the polycarboxylic acid and is immobilized on the surface of the magnetic particle via an amide bond. On the other hand, the carboxyl groups in the polycarboxylic acid coat layer are also bonded to the amino groups in the polyamine crosslinked layer via ionic or covalent bonds, thereby stably immobilizing the polycarboxylic acid coat layer on the magnetic particle surface. Some examples of polycarboxylic acids used in the present invention include polyacrylic acid, polymethacrylic acid, polyitaconic acid, polyvinyl benzoate, polyfumaric acid, polymaleic acid, styrene-maleic anhydride copolymer, isobutene-maleic anhydride. Examples include acid copolymers and salts thereof. The thickness of the polycarboxylic acid coating layer is not particularly limited, but can be appropriately selected within the range of 5 nm to 20 nm. A coating layer thickness of 5 nm or more is preferable because the amount of carboxyl groups introduced is sufficient and the amount of antibody introduced does not decrease. On the other hand, it is preferable that the thickness is 20 nm or less because there is no concern about hydrophobic adsorption due to increased hydrophobicity. Furthermore, since the polycarboxylic acid coating layer is introduced into the outermost layer of the magnetic particles, about 5 to 200 μmol/g of carboxyl groups are introduced to the surface of the magnetic particles.

Next, a method for producing surface-modified magnetic particles of the present invention will be explained. The feature of the method for producing surface-modified magnetic particles of the present invention is that it includes at least the following four steps. Each step will be explained below.
(First step)
This is a step of introducing amino groups onto the surface of magnetic particles. The method of introducing amino groups is not particularly limited, but a method of reacting aminosilane to magnetic particles and introducing them is simple and preferred. The aminosilanes used include 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, N-2-(aminoethyl)-3- Aminopropylmethyldimethoxysilane or mixtures thereof can be used. The reaction solvent is preferably a hydrous alcohol such as methanol or ethanol in which water is added, and ammonia or the like may be added to promote the reaction. The reaction conditions can be set over a wide range, and the reaction temperature can be set arbitrarily within the range of room temperature to 80°C, and the reaction time can be set arbitrarily within the range of 1 hour to 24 hours. The amount of amino groups introduced into the magnetic particles varies depending on the surface area of the magnetic particles as a base material, but can be about 50 to 500 μmol/g.
(Second process)
This is a step in which an acidic polysaccharide is adsorbed or reacted with the magnetic particles obtained in the first step to form a coat layer made of the acidic polysaccharide on the surface of the magnetic particles. It is preferable to use a condensing agent in the reaction in which the amino groups on the surface of the magnetic particles and the carboxyl groups in the acidic polysaccharide are reacted to immobilize the acidic polysaccharide on the surface of the magnetic particle via an amide bond. The condensing agents used include N,N-dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (hereinafter abbreviated as EDC), and EDC hydrochloride (hereinafter EDC).・Carbodiimide condensing agents such as HCL; imidazole condensing agents such as N,N'-carbonyldiimidazole and 1,1'-carbonyldi(1,2,4-triazole); 4 (4,6- Triazine-based condensing agents such as dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (hereinafter abbreviated as DMT-MM); 1H-benzotriazol-1-yloxytris (dimethylamino ) Phosphonium-based condensing agents such as phosphonium hexafluorophosphate and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; O-(benzotriazol-1-yl)-N,N,N' , N'-tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, etc. Examples include condensing agents. When using a condensing agent, active intermediates such as acylisourea produced by the reaction between the condensing agent and the carboxyl group are unstable and side reactions are likely to proceed due to hydrolysis, etc., so it is not recommended to use N-hydroxysuccinimide in combination. preferable. N-hydroxysuccinimide (hereinafter abbreviated as NHS) reacts with acylisourea and can convert the active intermediate into a more stable NHS ester, contributing to improved yield. The solvent used is preferably water in which the acidic polysaccharide is dissolved, and particularly preferably a buffer solution that can suppress pH changes during the reaction. Although there are no restrictions on the reaction conditions, it is preferable to carry out the reaction at 10 to 60°C for 5 to 24 hours.
(Third step)
This is a step in which the magnetic particles having the acidic polysaccharide coat layer obtained in the second step are adsorbed or reacted with a polyamine and then crosslinked to form a polyamine crosslinked layer on the surface of the magnetic particles. The polyamine cross-linked layer is created by adsorbing polyamine on the surface of acidic polysaccharide-coated magnetic particles in a water layer, dissolving the cross-linking agent in the oil layer side, and performing a cross-linking reaction at the (water/oil) interface to form a polyamine cross-linked thin film on the magnetic particles. Form on the surface. By using an oil-soluble emulsifier as an emulsifier and producing a w/o emulsion, a polyamine crosslinked thin film with a high crosslinking density can be formed. As the crosslinking agent used in this step, polyisocyanate is preferably used. The reason is that polyisocyanates have both high reactivity and stability against water. Examples of the polyisocyanate used in the present invention include toluene diisocyanate, diphenylmethane diisocyanate, hexamethylene diisocyanate, pentamethylene diisocyanate, isophorone diisocyanate, and the like. The emulsifier used in this step is an oil-soluble emulsifier and has an HLB of 3 to 6. Some specific examples include sorbitan monooleate, sorbitan trioleate, sorbitan monostearate, sorbitan tristearate, sorbitan monopalmitate, and the like. The reaction proceeds at a low temperature and in a short time, and a reaction temperature of 0° C. to room temperature and a reaction time of about 5 to 30 minutes are sufficient.
(Fourth step)
This is a step in which polycarboxylic acid is adsorbed or reacted with the magnetic particles having the polyamine crosslinked layer obtained in the third step to form a polycarboxylic acid coat layer on the surface of the magnetic particles. In order to stably hold the polycarboxylic acid coating layer on the surface of the magnetic particles, it is necessary to react the amino groups in the polyamine crosslinked layer and the carboxyl groups in the polycarboxylic acid to bond and immobilize them through an amide bond. preferable. This reaction is similar to the reaction in the second step, and it is preferable to use a condensing agent. The condensing agent used is the same as that used in the second step, and the reaction conditions are also the same.

According to the present invention, it is possible to provide surface-modified magnetic particles that have a high ability to bind biologically related substances, a high ability to suppress nonspecific adsorption, and a high stability against pH fluctuations.

The surface-modified magnetic particles of the present invention exhibit high ability to bind biologically related substances, ability to suppress nonspecific adsorption, and high stability against pH fluctuations, so they can be applied to the fields of diagnostic drug carriers and pharmaceutical research applications, and are of practical use. It is excellent.

EXAMPLES The present invention will be explained in more detail below based on Examples, but the present invention is not limited to these Examples.

Example 1
(First step) Introduction of amino groups to the surface of magnetic particles 0.5 g of magnetic particles (average particle size 2.5 μm, iron content 22%) manufactured by the method described in JP 2015-192995A The mixture was dispersed in 10 g of ethanol, 0.3 g of 3-aminopropyltrimethoxysilane was added, and the mixture was stirred at room temperature for 5 minutes. Next, 85 μL of 28% ammonia water and 31 μL of pure water were added, and the mixture was reacted at 60° C. for 3 hours under sealed conditions. After the reaction was completed, the magnetic particles were collected by magnetic collection, washed with ethanol, and isolated by drying under reduced pressure.

The introduced amino groups were quantified according to the method described in the previous literature (Kiyohisa Imada, Journal of the Chemical Society of Japan 407 (1990)). The amount of amino groups in the magnetic particles before introduction of amino groups was 30 μmol/g, whereas the amount of amino groups after introduction was 185 μmol/g.

(Second Step) Formation of Acidic Polysaccharide Coat Layer Using alginic acid as the acidic polysaccharide, a coat layer was formed by the following method. 1.0 g of sodium alginate (I-3G, manufactured by Kimika Co., Ltd.) was dissolved in 100 ml of MES buffer, and then 0.96 g of EDC/HCL and 0.58 g of N-hydroxysuccinimide were added and dissolved. Furthermore, a slurry prepared by dispersing 0.2 g of the amino group-introduced magnetic particles in 20 ml of pure water was gradually added, and the reaction was carried out overnight at room temperature. After the reaction was completed, the magnetic particles were collected by magnetic collection and washed with pure water. When the zeta potential of this particle was measured, it showed a negative value of -40.7 mV, confirming that an alginic acid coat layer was formed on the surface of the magnetic particle. On the other hand, the amount of amino groups after the formation of the acidic polysaccharide coat layer decreased to 60 μmol/g, confirming that the amino groups on the surface of the magnetic particles and sodium alginate reacted to form the acidic polysaccharide coat layer. Ta.

(Third step) Formation of polyamine crosslinked layer The above alginate-coated magnetic particles were dispersed in 120 ml of a 4% aqueous solution of polyethyleneimine (molecular weight 70,000, manufactured by Wako Pure Chemical Industries, Ltd.), stirred at room temperature for 30 minutes, and collected by collecting the magnetic particles. did. Dissolve 5ml of Span85 in 100ml of n-hexane, add and disperse the magnetic particles collected and collected above, cool to 5°C, and dropwise add a solution of 0.25g of toluene diisocyanate dissolved in 50ml of n-hexane. The mixture was further stirred and reacted for 30 minutes. After the reaction was completed, it was washed successively with ethanol and pure water and isolated. The amount of introduced amino groups measured by the same method as above (Kiyohisa Imada, Journal of the Chemical Society of Japan 407 (1990)) was 270 μmol/g, and it was confirmed that a polyamine crosslinked layer was formed.

(Fourth step) Formation of polycarboxylic acid coating layer Dissolve 0.36 g of polyacrylic acid (average molecular weight approximately 5,000, manufactured by Wako Pure Chemical Industries, Ltd.) in 100 ml of MES buffer, add 2.77 g of DMT-MM, and leave at room temperature. Stir to obtain a homogeneous solution. Thereto, 0.1 g of the polyamine crosslinked layer-introduced magnetic particles obtained in the above step was added as a slurry dispersed in 20 ml of pure water, and the mixture was reacted overnight at room temperature. After the reaction was completed, it was washed with pure water and isolated. The introduced carboxyl group was quantified according to the method described in the previous literature (Bing Yan et al., Anal. Chem. 71, 4564 (1999)). The amount of carboxyl groups introduced was 17 μmol/g, and it was confirmed that a polycarboxylic acid coating layer was formed on the outermost layer of the magnetic particles.

Evaluation of surface-modified magnetic particles; magnetic responsiveness The magnetic responsiveness of the surface-modified magnetic particles was evaluated by the following method.

After dispersing 5 mg of the surface-modified magnetic particles obtained by the above method in 10 ml of pure water, 2 ml was dispensed into a cell. The cell was set in an absorption photometer equipped with a magnet, and the absorbance was measured every 2 seconds at a wavelength of 550 nm, and the time required for the absorbance to become 1/10 of the initial absorbance (magnetic collection time) was measured. The magnetism collection time was 79 seconds, indicating good magnetic response.

Evaluation of surface-modified magnetic particles; citric acid elution test In order to evaluate durability against pH fluctuations, a citric acid elution test was conducted using the following method.

10 mg of the surface-modified magnetic particles obtained by the above method was placed in an Eppendorf microtube, 1 ml of 0.1M citric acid solution was added thereto, and the mixture was stirred at 37° C. for 17 hours using a mix rotor. After the stirring was completed, the magnetic particles were collected and removed, the solution portion was transferred to a cell, and the absorbance was measured at a wavelength of 460 nm. The absorbance was 0.023, and almost no elution of the magnetic substance by citric acid was observed.

Example 2
In the fourth step, the same operation as in Example 1 was performed, except that polyacrylic acid with an average molecular weight of 25,000 (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of polyacrylic acid with an average molecular weight of 5,000. Surface-modified magnetic particles were obtained. The amount of carboxyl groups introduced onto the surface of the magnetic particles was 33 μmol/g. The magnetism collection time was 54 seconds, indicating good magnetic response. Further, the absorbance in the citric acid elution test was 0.019, and no elution of the magnetic material by citric acid was observed.

Example 3
In the third step, hexamethylene diisocyanate was used instead of toluene diisocyanate as a crosslinking agent, and polyacrylic acid with an average molecular weight of 25,000 (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of polyacrylic acid with an average molecular weight of 5,000. Surface-modified magnetic particles were obtained by carrying out the same operation as in Example 1, except for using the following methods. The amount of amino groups after forming the polyamine crosslinked layer was 450 μmol/g, and the amount of surface carboxyl groups after forming the polycarboxylic acid coat layer was 86 μmol/g. The magnetism collection time was 68 seconds, indicating good magnetic response. Further, the absorbance in the citric acid elution test was 0.012, and no elution of the magnetic material by citric acid was observed.

Comparative example 1
Magnetic collection property evaluation and citric acid elution test were conducted on uncoated magnetic particles, which were the starting materials used in the examples. The magnetic collection time was 97 seconds, indicating good magnetic responsiveness, but the absorbance in the citric acid elution test was 0.050, indicating elution of the magnetic material by citric acid. It can be seen that if surface modification is not performed, citric acid will come into contact with the magnetic material and the magnetic material will be eluted.

Comparative example 2
The reaction of introducing amino groups into the magnetic particles and the formation of the acidic polysaccharide coat layer were carried out in the same manner as in Example 1, but the polyamine crosslinked layer and the polycarboxylic acid coat layer were not formed, and magnetic collection property evaluation and citric acid A dissolution test was conducted. The magnetic collection time was 97 seconds, indicating good magnetic responsiveness, but the absorbance in the citric acid elution test was 0.052, indicating elution of the magnetic material by citric acid. It can be seen that the acidic polysaccharide coating layer alone cannot prevent citric acid from permeating and diffusing into the magnetic particles, and the magnetic material and citric acid come into contact and the magnetic material is eluted.

The surface-modified magnetic particles of the present invention exhibit high ability to bind biologically related substances, ability to suppress nonspecific adsorption, and high stability against pH fluctuations, so they can be used as biochemical carriers such as carriers for diagnostic reagents, especially in automatic diagnostic systems. It can be applied to carriers for diagnostic drugs.

Claims (2)

A surface-modified magnetic particle characterized in that the surface of the magnetic particle has three layers in this order: an acidic polysaccharide coat layer, a polyamine crosslinked layer, and a polycarboxylic acid coat layer. A method for producing surface-modified magnetic particles, the method comprising at least the following four steps.
(First step)
A process of introducing amino groups onto the surface of magnetic particles.
(Second process)
A step of adsorbing or reacting an acidic polysaccharide to the magnetic particles obtained in the first step to form an acidic polysaccharide coat layer on the surface of the magnetic particles.
(Third step)
A step of adsorbing or reacting a polyamine to the magnetic particles having the acidic polysaccharide coat layer obtained in the second step and then crosslinking them to form a polyamine crosslinked layer on the surface of the magnetic particles.
(Fourth step)
A step of adsorbing or reacting polycarboxylic acid to the magnetic particles having the polyamine crosslinked layer obtained in the third step to form a polycarboxylic acid coat layer on the surface of the magnetic particles.