JP6771122B2 - Composite particles - Google Patents

Description

The present invention relates to composite particles, specifically composite particles having a specific binding ability to a specific molecule.

In order to give particles useful for detection and separation of biomolecules biochemical functions and biocompatibility, technological development of modifying the particle surface with a polymer has been actively carried out for a long time (Non-Patent Document 1). As a development system of these technologies, many research examples have also been reported for the development of technologies that impart an affinity ligand such as an antibody to a polymer portion to have a function of specifically binding to a specific molecule.

As one of the particle surface modification techniques using such a polymer, there is a technique of grafting a polymer having a uniform length onto the particle surface at an extremely high density by utilizing surface-initiated living radical polymerization (Patent Document 1, Non-Patent). Document 2). The polymer-imparted particles synthesized by this technique exhibit uniform particle size and high dispersion stability. In addition, when the polymer is hydrophilic, it shows the characteristic that non-specific adsorption of proteins is suppressed, and there are examples of research reports that make use of this in bio-medical applications (Patent Documents 2, 3 and Non-Patent Documents). 3, 4).

However, immobilizing a new substance such as an affinity ligand on the particle surface leads to modification of the structure and properties of the particle surface, so that the uniformity of particle size and surface structure and the maintenance of dispersion stability (aggregation) (Avoidance) has a negative effect (Non-Patent Document 5). Affinity ligands are immobilized on polymer moieties under appropriate control, demonstrating that the ligand-immobilized polymer-imparted particles specifically bind to specific molecules, and uniform granules under physiological conditions. There is no example that has cleared the technical hurdle of maintaining stable dispersion under radial and physiological conditions.

International Publication No. 2005-108451 JP-A-2010-280594 International Publication No. 2012-073588

Kango S. et al., "Progress in Polymer Science", 2013, Vol. 38, pp. 1232-1261 Ohno K. et al., "Macromolecules", 2005, Vol. 38, pp. 2137-2142 Ohno K. et al., "Macromolecules", 2012, Vol. 13, pp. 927-936 Ohno K. et al., "Macromolecules", 2013, Vol. 14, pp. 3453-3462 Laurent S. et al., "Chem. Rev.", 2008, Vol. 108, pp. 2064-2110

Therefore, an object of the present invention is to provide particles useful for detection and separation of biomolecules, which have the characteristics of having a uniform particle size, high dispersion stability, and specifically binding to a specific molecule. And.

The present invention is a composite particle containing a polymer graft chain, fine particles and an affinity ligand.
One end of the polymer graft chain is bonded to the surface of the fine particles at a graft density of 0.1 double chain / nm ² or more.
A composite particle in which the affinity ligand is bound to another end of the polymer graft chain, and the affinity ligand is bound to less than 20% of the total length of the polymer graft chain.

The composite particle of the present invention has an advantage that it has a specific binding ability to a specific molecule. Further, the composite particle of the present invention has an advantage that it has a controlled immobilization affinity ligand, and also has a uniform particle size under physiological conditions and a stable dispersion retention under physiological conditions. ..

FIG. 1 is a calibration curve for quantifying the amount of antibody immobilized by the ELISA method. FIG. 2 is an overlay of bound sensorgrams of composite particles. FIG. 3 is an overlay of bound sensorgrams of another composite particle. FIG. 4 shows the fluorescence intensity distribution of antigen-expressing cells. FIG. 5 shows the fluorescence intensity distribution of another antigen-expressing cell. FIG. 6 shows the fluorescence intensity distribution of another antigen-expressing cell. FIG. 7 shows the fluorescence intensity distribution of each composite particle.

The present invention is a composite particle containing a polymer graft chain, fine particles and an affinity ligand.
One end of the polymer graft chain is bonded to the surface of the fine particles at a graft density of 0.1 double chain / nm ² or more.
A composite particle in which the affinity ligand is bound to another end of the polymer graft chain, and the affinity ligand is bound to less than 20% of the total length of the polymer graft chain.

In the present specification, the grafted synthetic polymer chain is referred to as "polymer graft chain" or simply "graft chain". The polymer graft chain means a polymer chain formed by extending two or more monomers from the surface of fine particles by a polymerization reaction. In addition, in this specification, "polymer" is basically synonymous with polymer.

As used herein, the term "affinity ligand" refers to the selective collection (binding) of a target molecule from a set of molecules based on the specific intermolecular affinity represented by the binding between an antigen and an antibody. ) Is a term that refers to a substance (functional group), and even when simply expressed as "ligand", it is synonymous with "affinity ligand". Typical ligands include antibodies (immunoglobulins), but the substance is not particularly limited as long as it has the ability to specifically bind to a specific molecule, and is either a protein, a sugar, a nucleic acid, or a synthetic compound. It may be.

As used herein, "immobilization" refers to the attachment of an affinity ligand to a polymeric graft chain. The bond is preferably a covalent bond, but is not limited to this, and may be, for example, an ionic bond or a hydrophobic bond.

In the present invention, the size, structure, and substance of the fine particles are not particularly limited. In this specification, when it is difficult to distinguish from composite particles, it is referred to as "core particles". The size of the core particles is preferably a size of so-called "fine particles", more preferably a size of so-called "nanoparticles", but is not limited to this. The size of the core particles (diameter equivalent to a projected area circle (average particle size)) is preferably 1 nm or more, more preferably 2 nm or more, still more preferably 5 nm or more, still more preferably 10 nm or more. The size of the core particles is preferably 150 μm or less, more preferably 100 μm or less, even more preferably 50 μm or less, and even more preferably 10 μm or less. As the structure of the core particles, spherical particles are preferable, but the structure is not limited to this, and for example, hollow particles or porous particles may be used. The material of the core particles is preferably inorganic particles, but is not limited to this, and may be, for example, polymer particles. Examples of the substance constituting the inorganic particles include, but are not limited to, metals, metal oxides, silicon oxides, and the like.

In the present invention, the "specific binding ability to a specific molecule" can be confirmed by a general biochemical method. For example, "specific binding ability to a specific molecule" can be determined by analysis using a surface plasmon (SPR) device. In the case of large particles that are difficult to add to the channel, it is possible to analyze the "specific binding ability to a specific molecule" using, for example, an isothermal titration calorimetry (ITC) device. Regarding specific binding to a specific molecule, cells that highly express the specific molecule may be used for analysis using a flow cytometry (FACS) device, or evaluated in vivo using various imaging devices. It doesn't matter.

A feature of the composite particle in the present invention is that the polymer graft chain is bonded to the particle surface at a density of 0.1 double chain / nm ² or more. In the present specification, "density" is the number of graft chains bonded to the surface per unit area (nm ² ) of the particle surface, and "graft density" is also synonymous with this. The graft density is preferably 0.1 strand / nm ² or more, and more preferably 0.2 strand / nm ² or more. The graft density is preferably 1.5 strands / nm ² or less, more preferably 1.0 strands / nm ² or less, and even more preferably 0.5 strands / nm ² or less. For the graft density (s), determine the amount of polymer graft chains (graft amount, w) formed by extending from the surface of fine particles by elemental analysis, etc., and determine the value, the surface area of core particles (S, nm ² ), and the graft. It is calculated from the following formula using the number average molecular weight (Mn) and Avogadro's number (Av) of the polymer.

Calculation method: s = (w / Mn) Av / S

Another feature of the composite particle in the present invention is that the affinity ligand is bound to another end of the polymer graft chain, and the affinity ligand is bound to less than 20% of the total length of the polymer graft chain. That is what has been done. "Another one end of the polymer graft chain" means the end opposite to the end bonded on the surface of the core particle of the graft chain, and means the outer side or the surface layer of the composite particle. The ratio of the portion on which the affinity ligand is immobilized to the total length of the polymer graft chain is less than 20%, preferably less than 15%, and more preferably less than 10%. When the ratio is less than 20%, the highly elongated structure of the polymer graft chain can be retained, and the affinity ligand of the composite particle of the present invention can efficiently bind to the molecule capable of binding to the affinity ligand. This is because they can be combined. This is because, in the composite particle obtained by the present invention, the affinity ligand is present only on the outside or the surface layer of the composite particle.

The polymer graft chain can be obtained, for example, by living radical polymerization. "Living radical polymerization (LRP)" is a type of living polymerization in which the molecular weight distribution can be controlled, and it is possible to polymerize by reversibly generating radical species from stable covalent bond species (Dormant species). Become. It is synonymous with polymerization called "precision radical polymerization (CRP)". A technique for applying this polymerization method to the surface of a material is "surface-initiated living radical polymerization", in which a polymerization-initiating group can be introduced on the surface of core particles to allow the graft polymerization to proceed while maintaining a constant graft density. That is, the amount of graft can be increased in proportion to the number average molecular weight of the graft chains, the polymerization can proceed in a living room, and all the graft chains can be grown almost evenly. As a result, a polymer chain having a regulated chain length and chain length distribution can be grown on the particle surface with an unprecedented high surface density.

The type of monomer used for this polymerization is preferably one or more selected from the group consisting of acrylic acid derivatives, methacrylic acid derivatives, acrylamide derivatives, methacrylamide derivatives and styrene derivatives, but is not limited thereto and is recognized by those skilled in the art. It may be appropriately selected within the range to be used. For example, in the present invention, the methacrylic acid derivative is one of the preferred monomers. In the present invention, in order to improve the selectivity of binding, it is better that non-specific adsorption of protein can be suppressed, and this can be achieved by using a hydrophilic monomer. For this purpose, for example, polyethylene glycol monomethacrylate (PEGMA), which is a methacrylic acid derivative whose side chain is oligopolyethylene glycol (PEG), is one of the preferred monomers.

In the composite particle of the present invention, since the affinity ligand is immobilized at a position close to the tip of the polymer graft chain, it is preferable that the monomer composition of the portion near the tip and the other portion are different. As one of the preferred forms, preferably the affinity ligand-binding monomer is only near the tip, eg, less than 20%, preferably less than 15%, more preferably less than 10% from the end. The chain may further comprise a monomer that is binding to the affinity ligand. Therefore, the polymer graft chain in the present invention is preferably a copolymer (copolymerized type) composed of two or more blocks having different monomer compositions. The block containing the affinity ligand and the monomer binding to the affinity ligand does not have to be limited to one block near the tip. For example, when the polymer graft chain consists of three or more blocks, not only the block near the tip but also the block close to the core particles may contain a monomer binding to the affinity ligand. In this case, for example, the monomer of the block adjacent to the core particle may be used for cross-linking between the polymer graft chains via a covalent bond, or may be used for immobilization of dyes for detection.

The structure of the monomer binding to the affinity ligand used for immobilizing the affinity ligand on the polymer graft chain is not particularly limited. When the affinity ligand is covalently immobilized, it is preferable that the side chain of the monomer has a reactive functional group capable of forming a covalent bond with the ligand. To form a covalent bond with a proteinaceous ligand such as an antibody, it is preferred to have a functional group that reacts with the amino, carboxyl and / or thiol groups of the protein. For example, for the amino group of the proteinaceous ligand, a monomer having benzyloxycarbonyl or N-hydroxysuccinimide in the side chain can be used, and the proteinaceous ligand is immobilized by a coupling reaction between the active ester and the amino group. it can. Immobilization of the affinity ligand is not limited to immobilization via a covalent bond. For example, a monomer having biotin in the side chain is used to obtain an avidin-biotin-fused proteinaceous affinity ligand. It may be immobilized by binding. Other such combinations include the binding of glutathione to glutathione-S-transferase (GST).

Specific examples of the monomer binding to the affinity ligand include nitrophenyloxycarbonyl polyethylene glycol methacrylate (MEONP) and N-hydroxysuccinimide methacrylate (NHSMA).

The composition of the tip portion that immobilizes the affinity ligand on the polymer graft chain does not have to be a monomer that is all binding to the ligand, but rather is preferably contained in a constant proportion. This ratio depends on the size of the affinity ligand and the like, and is not particularly limited. For example, when the ligand is an antibody, the antibody can be immobilized in a polymerization reaction using two or more types of monomers. The monomer content (molar ratio) is preferably less than 20%, more preferably less than 10%. The ligand-binding monomer does not need to be contained over the entire length of the polymer graft chain, but by utilizing this technology, one or more ligand immobilization points per graft chain are limited to only the tip portion. It is possible to form. The tip portion means, for example, a portion of less than 20%, preferably less than 15%, more preferably less than 10% from the end of the polymer graft chain. This technique is more efficient and effective (for binding) than the technique of forming immobilization points only at the ends of polymer chains and the technique of randomly forming immobilization points on polymer chains. It has the advantage of being able to immobilize the ligand (in an advantageous state).

That is, in the composite particle of the present invention, the polymer graft chain is selected from the group consisting of an acrylic acid derivative, a methacrylic acid derivative, an acrylamide derivative, a methacrylamide derivative and a styrene derivative based on the polymerization initiator group on the particle surface. Obtained by living radical polymerization of one or more monomers and
It is preferable that a monomer binding to the affinity ligand is further contained at a site of less than 20%, preferably less than 15%, more preferably less than 10% of the total length of the polymer graft chain.

The method for analyzing the amount of ligand immobilized on the polymer graft chain is not particularly limited. For example, the amount of immobilization can be calculated back from the concentration of the ligand in the reaction solution before and after the immobilization reaction, and it can also be calculated by performing ELISA in a system in which the molecule binding to the ligand is immobilized on a solid phase. Is.

In the present invention, the number average molecular weight (Mn) of the polymer graft chain of the composite particle is not particularly limited. As the lower limit of the number average molecular weight (Mn) of the polymer graft chains of the composite particles, 10,000 or more is preferable, 30,000 or more is more preferable, 50,000 or more is even more preferable, and 70,000 or more is further. More preferably, 100,000 or more is even more preferable. The upper limit of the number average molecular weight (Mn) of the polymer graft chains of the composite particles is preferably 320,000 or less, more preferably 240,000 or less. In order to excel in both dispersion stability and specific binding ability to a specific molecule, the number average molecular weight (Mn) of the polymer graft chain of the composite particle is preferably about 30,000 to 150,000. In this range, the larger the molecular weight, the better their performance. In the present invention, for example, when the number average molecular weight (Mn) of the polymer graft chain is 100,000, the number average molecular weight (Mn) of the tip portion for immobilizing the ligand immobilization is 20 of 100,000. It falls within the percentage of 20,000 or less.

That is, in the composite particles of the present invention, the number average molecular weight (Mn) of the polymer graft chain is preferably 10,000 or more, 320,000 or less, and more preferably 30,000 or more. And 320,000 or less, more preferably 50,000 or more, and 240,000 or less, even more preferably 70,000 or more, still more preferably 100,000 or more. And it is 240,000 or less.

Since the number average molecular weight (Mn) of the polymer graft chain depends on the molecular weight of the monomer, it may be better to express it by the number average degree of polymerization of the polymer graft chain. The preferred range of the number average degree of polymerization is about 10 to 100,000. For example, when the molecular weight of the monomer is 500, the number average degree of polymerization of the polymer graft chain is preferably 20 to 640, and 60 to 640. 480 is more preferable. The number average molecular weight Mn of the polymer graft chain can be measured by, for example, gel filtration column analysis, and the number average degree of polymerization of the polymer graft chain can be calculated by the number average molecular weight / monomer molecular weight.

When the polymer graft chain is a copolymer (copolymerized type) composed of two or more blocks having different monomer compositions, the molecular weight distribution (Mw / Mn) of the copolymer in each block is 1.0 to 1.5. It is preferable to have it. In this case, the composite particles have a homogeneous graft surface. The molecular weight Mw of the polymer graft chain can be determined by analysis such as gel filtration.

As described above, the composite particle of the present invention has an advantage that it has a specific binding ability to a specific molecule. Further, the composite particle of the present invention has an advantage that it has a controlled immobilization affinity ligand, and also has a uniform particle size under physiological conditions and a stable dispersion retention under physiological conditions. ..

In the composite particles of the present invention, not only the immobilization site of the antibody is controlled at the tip portion, but also the binding force to the target molecule depends on the chain length of the polymer graft chain, and the longer the graft chain length, the better. The target molecular binding ability of composite particles is greatly improved. This is because it is important in binding to the target molecule that the ligand immobilized on the particle is not only immobilized at a position easily accessible to the target molecule but also the degree of freedom is secured. It is shown that the composite particles of are compatible with each other. On the other hand, the binding force of the composite particle of the present invention to the target molecule (expressing cell) does not depend on the amount of antibody immobilization. This is a great advantage when immobilizing a ligand that is costly to manufacture, such as an antibody.

The composite particles of the present invention can be produced, for example, as follows. First, the compound as the polymerization initiator-containing coupling agent is fixed to the particle surface by a chemisorption method. Then, one or more kinds of monomers are supplied to perform living radical graft polymerization. By fixing the polymerization initiator group-containing coupling agent to the surface of the fine particles in advance, the graft polymerization can proceed while maintaining the graft density constant on the surface of the fine particles. During the living radical graft polymerization, a polymerization initiator which is not fixed on the surface of the fine particles may coexist. The compound as the polymerization initiator-containing coupling agent can be selected in consideration of the affinity with the particles and the like. For example, when the particles are made of silicon oxide or metal oxide, the polymerization initiator-containing coupling agent is preferably a silane compound, a titanium compound, a zirconium compound, and an aluminum compound, and a silane compound is particularly preferable. Specifically, (2-bromo-2-methyl) propionyloxypropyltriethoxysilane (BPE), (2-bromo-2-methyl) propionyloxyhexyltriethoxysilane (BHE), ethyl 2-bromoisobutyrate. (EBIB) and the like.

The graft density on the particle surface can be changed by adjusting the ratio of the polymerization initiator-containing coupling agent and the polymerization initiator-free coupling agent (for example, alkylsilane). Specifically, when the particles are silicon oxide particles, the graft density is 0.1 if the ratio of the polymerization initiator-containing coupling agent to the polymerization initiator-free coupling agent is 1: 0. Main chain / nm ² or more. After completion of the polymerization, the target composite particles are obtained from the reaction solution by a single method or a combination of methods usually used in the art, such as extraction, distillation, washing, concentration, precipitation, filtration, drying, adsorption, precipitation, and chromatography. It can be isolated.

Further, when the polymer graft chain is a copolymer (copolymerized type) composed of two or more blocks having different monomer compositions, the blocks may be sequentially produced. At that time, the types and compositions of the monomers used in the production are different for each block.

Further, when the polymer graft chain is composed of three or more blocks, not only the block at the tip portion but also the block adjacent to the core particle may contain a monomer binding to the affinity ligand. Such a polymer graft chain may crosslink the affinity ligand and the binding monomer of the first block after producing the first block and the second block. By cross-linking in this way, the finally obtained composite particle of the present invention has an advantage that the detachment of the graft chain can be suppressed. Diamines such as hexamethylenediamine, dithiols such as 1,2-ethanedithiol and the like can be used for the cross-linking.

Regarding the polymerization conditions for obtaining the desired polymer graft chain length, regarding silica nanoparticles, Patent Document 2 (Japanese Patent Laid-Open No. 2010-280594) and Non-Patent Document 3 (Ohno K. et al., “Macromolecules” , 2012, Vol. 13, pp. 927-936), Patent Document 3 (International Publication No. 2012-073588) and Non-Patent Document 4 (Ohno K. et al., "Macromolecules", regarding iron oxide nanoparticles. , 2013, Vol. 14, pp. 3453-3462).

Finally, the fine particles to which the polymer graft chain having a site that binds to the affinity ligand at the terminal position is bound to the surface is reacted with the affinity ligand. When the affinity ligand is an antibody, protein, or the like, the reaction is carried out, for example, by allowing the affinity ligand to stand in a cool and dark place for 1 hour to 2 days. When the affinity ligand is a nucleic acid or a synthetic compound, the reaction is carried out, for example, by reacting at 4 to 25 ° C. for 1 hour to 2 days.

That is, the present invention is a fine particle having a polymer graft chain bonded to the surface at a position of less than 20%, preferably less than 15%, more preferably less than 10% from the end, for example, a site that binds to an affinity ligand. It is a method for producing a composite particle of the present invention, which comprises a step of reacting with an affinity ligand.
One end of the polymer graft chain is bonded to the surface of the fine particles at a graft density of 0.1 double chain / nm ² or more.

The present invention is also a method for detecting a molecule capable of binding to an affinity ligand.
A step of contacting a molecule capable of binding to the affinity ligand or an object containing the molecule with the composite particle of the present invention is included.

In the above method, the target containing the molecule includes, but is not limited to, cells and tissues.

Examples of the core particles of the composite particles in the detection method include, but are not limited to, fluorescently dyed latex beads and quantum dots, and superparamagnetic iron oxide nanoparticles that can be detected by MRI. Further, a monomer having a fluorescent dye in the side chain may be contained in the polymer graft chain of the composite particle.

The present invention is also a method for purifying a molecule capable of binding to an affinity ligand.
A step of contacting a molecule capable of binding to the affinity ligand or an object containing the molecule with the composite particle of the present invention is included.

In the above method, the target containing the molecule includes, but is not limited to, cells and tissues.

Examples of the core particles of the composite particles in the purification method include, but are not limited to, iron oxide fine particles that can be recovered by a magnet and porous beads that can be used as a chromatographic filler.

The present invention is also a method of inducing a change in a molecule that can bind to an affinity ligand.
A step of contacting a molecule capable of binding to the affinity ligand or an object containing the molecule with the composite particle of the present invention is included.

In the above method, the target containing the molecule includes, but is not limited to, cells and tissues.

In the method of inducing change, by giving the composite particle some medicinal effect, such as by containing a medicinal component in the composite particle, the molecule or the tissue or cell expressing the molecule is stimulated. It is also possible to exert actions such as activation, inhibition, or damage.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.
The following abbreviations are used in the description herein.
THF: tetrahydrofuran BPE: (2-bromo-2-methyl) propionyloxypropyltriethoxysilanePEGMA: polyethylene glycol methacrylate MEONP: nitrophenyloxycarbonyl polyethylene glycol methacrylate EBIB: ethyl 2-bromoisobutyrate GPC: gel filtration chromatography SPR : Surface plasmon resonance NMR: Nuclear magnetic resonance FACS: Fluorescence activated cell sorter

[Manufacturing Example 1-1]
<Manufacturing of fine particles having a polymerization initiator on the surface>
<Manufacture of iron oxide nanoparticles having a polymerization initiator on the surface>
Oleic acid-coated iron oxide nanoparticles having an average particle size (diameter equivalent to a projected area circle) of 20 nm (see Patent Document 3 for the synthesis method) were dispersed in THF to produce a dispersion (1% by mass). Ammonia water (1% by mass) was added to the dispersion, and the mixture was stirred for a while, then BPE (2% by mass) was added, and the mixture was stirred at room temperature for 3 days. During stirring, the solution was periodically ultrasonically irradiated. Then, the solution is centrifuged to recover the particles, the particles are redispersed in THF, and then the centrifugation is repeated, so that the iron oxide nanoparticles having the polymerization initiator group on the surface (projected area equivalent circle diameter). (Average particle size) was 20 nm). See Patent Document 2 for the method of synthesizing BPE. Further, a commercially available iron oxide nanoparticle solution (manufactured by NN-Labs, USA, particle size: 5 nm, 100 mg in 20 mL of toluene) was dispersed in THF (80 g), and then 28% aqueous ammonia (1.65 g) was dispersed. And THF (25 g) is added dropwise, and after stirring for a while, the mixture of BPE (1 g) and THF (25 g) is also added dropwise to produce iron oxide nanoparticles having a polymerization initiator on the surface. I confirmed that.

[Manufacturing Example 1-2]
<Manufacturing of fine particles having a polymerization initiator on the surface>
<Manufacture of silica fine particles having a polymerization initiator on the surface>
As silica fine particles with an average particle size (SEM diameter) of 130 nm (manufactured by Nippon Shokubai Co., Ltd., product name "Seahoster KE-E10"), an ethanol dispersion (7.7% by mass, 30 mL) was added to a 28% ammonia aqueous solution (13). It was added to a mixture of 0.9 g) and ethanol (200 mL). The mixed solution was stirred at 40 ° C. for 2 hours, BPE (2 g) dissolved in ethanol (10 mL) was added dropwise, and the mixture was stirred at 40 ° C. for 18 hours. Then, the silica fine particles recovered by centrifugation were washed with ethanol and then with anisole to obtain silica fine particles having a polymerization initiator group on the surface. The silica fine particles having the polymerization initiator group on the surface were stored in anisole.

[Example 1]
The iron oxide nanoparticles having the polymerization initiator group obtained in Production Example 1-1 on the surface were used as a core, and composite particles in which the polymer graft chains of three blocks and the affinity ligand were bound were produced. The composition of the composite particle is represented in Scheme 1 below. The molar ratio at the time of synthesis preserves a part of the synthesized composite particles, the monomer conversion rate in the polymer graft chain is measured by NMR, and the molecular weight and molecular weight distribution of the free polymer produced from EBIB are measured by gel permeation chromatography (GPC). I asked.

(1) Synthesis of Polymer Graft Chain First Block (Bridged Layer) Ethyl2-bromoisobutyrate (EBIB, 3.9 mg) as a release initiator, polyethylene glycol methyl ether methacrylate (PEGMA, 17 g) as a water-soluble monomer ), Nitrophenyloxycarbonyl polyethylene glycol methacrylate (MEONP, 3 g) as an active ester group-containing monomer, CuBr (28 mg) as a catalyst, 4,4-dinonylbipyridine (dNbipy, 163 mg) as a ligand for a copper complex, and tetrahydrofuran as a solvent ( THF (29 g) and a THF solution (8.4% by mass, 8.3 g) of iron oxide nanoparticles having a polymerization initiator group obtained in Production Example 1-1 on the surface are mixed, degassed, and then sealed. Then, it was polymerized at 60 ° C. for 18 hours. The molar ratio of PEGMA to MEONP in this process was 9: 1. As a result, a free polymer having a number average molecular weight (Mn) of 38,000 and a molecular weight distribution index (Mw / Mn) of 1.23 was formed, and iron oxide nanoparticles in which the first block was bonded to the surface were obtained.

(2) Synthesis of the second block (intermediate layer) of the polymer graft chain Using the iron oxide nanoparticles obtained in (1) to which the polyPEGMA was bonded to the surface as a macroinitiator, the second block (intermediate layer) was used as follows. ) Was manufactured. EBIB (0.6 mg), PEGMA (15 g), CuCl (31 mg), dNbipy (260 mg), THF (14 g), and THF solution of iron oxide nanoparticles obtained in (1) with polyPEGMA bonded to the surface (1). 2.77% by mass (10.8 g) was mixed, degassed, sealed, and polymerized at 60 ° C. for 20 hours. As a result, a free polymer having a number average molecular weight (Mn) of 128,000 and a molecular weight distribution index Mw / Mn of 1.26 was formed, and iron oxide nanoparticles in which the second block and the first block were bonded to the surface were obtained. It was.

(3) Cross-linking with diamine in the first block Iron oxide nanoparticles obtained in (2) in which the second block and the first block are bonded to the surface in order to cross-link the polymer chain using an active ester group derived from MEONP. (684 mg), hexamethylenediamine (6 mg), triethylamine (2.6 mg), and THF (53 g) were mixed and stirred at room temperature for 24 hours. The crosslinked particles were purified by repeating redispersion into THF by centrifugation, and the second block and the first block were bonded to the surface to obtain iron oxide nanoparticles in which the first block was crosslinked.

(4) Synthesis of Polymer Graft Chain Third Block (Immobilization Layer) The iron oxide nanoparticles obtained in (3), in which the second block and the first block are bonded to the surface and the first block is crosslinked, are obtained. The third block was produced as a macroinitiator as follows. Second block obtained with EBIB (1.5 mg), PEGMA (6.8 g), MEONP (1.23 g), CuCl (7.9 mg), dNbipy (66 mg), THF (12 g), and (3). And the first block is bonded to the surface, the first block is crosslinked with a THF solution of iron oxide nanoparticles (2.1 mass t%, 7.1 g), degassed, then sealed, and then at 60 ° C. Polymerized in 24 hours. The molar ratio of PEGMA to MEONP in this process was 9: 1. As a result, a free polymer having a number average molecular weight (Mn) of 25,000 and a molecular weight distribution index Mw / Mn of 1.08 was produced, and the third block, the second block and the first block were bonded to the surface, and the first block was formed. Iron oxide nanoparticles with crosslinked blocks were obtained. The particles were purified by repeated centrifugation into THF, acetone, water, and phosphate buffer (pH = 8.0). The graft density after the third block polymerization was 0.12 strands / nm ² , and the graft density of the entire polymer graft strand was 0.12 strands / nm ² .

(5) Immobilization of antibody on the third block Iron oxide nanoparticles (125 mg) obtained in (4), in which the third block, the second block, and the first block were bonded to the surface and the first block was crosslinked. Was dispersed in phosphate buffer (5 g), and an anti-mouse CD105 antibody (Rat Anti Mouse CD105-UNLB, MJ7 / 17, manufactured by Southern Biotech, 0.5 mg / mL, 1 mL) was added. The reaction solution was allowed to stand in a cool and dark place for 2 days. The particles were purified by repeating redispersion in pure water by centrifugation. As a result, the third block, the second block and the first block were bound to the surface, the first block was crosslinked, and composite particles in which the antibody was bound to the third block were obtained.

[Condition study 1: Study by solution polymerization for the purpose of controlling polymer chain length]
Applying (grafting) a polymer brush to the surface of iron oxide nanoparticles is performed by surface-initiated living radical polymerization. Setting appropriate polymerization conditions is the key to controlling the chain length of graft polymers. First, living radical polymerization of PEGMA (polyethylene glycol methacrylate), which is the main monomer of the polymer brush, was carried out under various conditions using a liberation initiator (ethyl2-bromoisobutyrate) (EBIB).

In addition to EBIB and PEGMA, CuCl as a catalyst, 4,4-dinonylbipyridine (dNbipy) as a ligand for a copper complex, and tetrahydrofuran (THF) as a solvent were mixed at a predetermined concentration, degassed, and then sealed at 50 ° C. Polymerized in. Table 1 summarizes the ratios of various reagents and the specifications of the produced polymers.

As shown in Table 1, by adjusting the polymerization initiator / monomer / catalyst ratio and the polymerization time, polymers having various molecular weights and a small molecular weight distribution index can be synthesized. When producing a desired polymer graft chain, those skilled in the art can find a guideline for polymerization conditions with reference to this.

[Condition study 2: Setting conditions for graft polymerization]
The polymerization conditions found by the solution polymerization of Condition Study 1 were applied to graft polymerization to synthesize composite iron oxide nanoparticles to which polymer brushes of various chain lengths were applied. Specifically, PEGMA, CuCl, dNbipy, and THF were mixed with iron oxide nanoparticles modified with a fixation initiator (BPE) in the same manner as in Condition Study 1, degassed, sealed, and polymerized at 50 ° C. did. EBIB is also mixed with the reaction solution as a liberation initiator for molecular weight analysis of the graft chain. Details of the polymerization conditions are also shown in Table 2.

Table 2 shows the specifications of the synthesized polymer brush-imparted iron oxide nanoparticles. By applying the conditions found in solution polymerization to graft polymerization, composite iron oxide nanoparticles having a desired graft chain length and a graft density of 0.1 strand / nm ² or more could be synthesized.

[Example 2]
Composite particles were obtained in the same manner as in Example 1 except that an anti-mouse VEGFR2 antibody (Rat Anti Mouse VEGF Receptor 2 Antibody (Avas12a1)) was used instead of the anti-mouse CD105 antibody in the step (5). The graft density of the entire polymer graft chain on the composite particles was 0.12 double chain / nm ² .

[Example 3]
The polymerization reaction temperature was set to 50 ° C., and composite particles were obtained in substantially the same manner as in Example 1 except that the polymerization conditions of entry 5 in Table 2 were applied to the second block. In the obtained composite particles, the graft density of the entire polymer graft chain was 0.15 single chain / nm ² .

[Example 4]
Instead of the iron oxide nanoparticles having the polymerization initiator group obtained in Production Example 1-1 on the surface, silica fine particles having the polymerization initiator group obtained in Production Example 1-2 on the surface were used, and (1) and (3). The composite particles were obtained in the same manner as in Example 1 except that the step (5) was omitted and the step (5) was changed as follows.

In the step (5), as the ligand to be immobilized, aHER-Fab, a low molecular weight antibody obtained by fragmenting aHER-IgG into Fab and Fc by papain and separating and purifying only Fab, was used. The method for preparing aHER-Fab is to dissolve in papain digestion buffer (0.1 M AcOH-AcONa, 2 mM EDTA, 1 mM cysteine, pH 5.5) and add papain Agarose from papaya latex papain-immobilized agarose (SIGMA). Then, the mixture was incubated at 37 ° C. for about 8 hours while mixing with a rotator. From the reaction solution separated from the papain-immobilized agarose, aHER-Fab was separated and purified by collecting aHER-Fab by the pass-through fraction by affinity chromatography using a MabSelect SuRe column (GE Healthcare Bioscience).

This aHER-Fab was immobilized on 10 mg (1% by mass) of silica fine particles having the third block and the second block bonded to the surface at a ratio of 2 nmol (1.5 μg). Regarding the quantification of the amount of immobilization, the A280 values (UV absorption derived from protein) of the solution before the reaction and the solution excluding the composite particles after the reaction were compared, and it was confirmed that 0.5 nmol was immobilized. ..

[Example 5]
For the second block, composite particles were obtained in the same manner as in Example 1 except that the polymerization conditions of entry 6 in Table 2 were applied.

[Example 6]
For the second block, composite particles were obtained in the same manner as in Example 1 except that the polymerization conditions of entry 3 in Table 2 were applied.

[Comparative Example 1]
Composite particles were obtained in the same manner as in Example 1 except that the steps (4) and (5) were omitted.

[Comparative Example 2]
For the second block, composite particles were obtained in the same manner as in Example 1 except that the polymerization conditions of entry 2 in Table 2 were applied.

[Quantification of antibody-immobilized amount by ELISA method]
(1) Composite particles of Example 3
A calibration curve of a free anti-HER2 antibody (aHER2-IgG) was prepared using a Total Human IgG ELISA kit (manufactured by AlerCHEK), and the amount of antibody immobilized on the composite particles of Example 3 was quantified. The obtained calibration curve is shown in FIG. 1, and the quantitative results are shown in Table 3. It was confirmed that the antibody was immobilized on the composite particle by the standard ELISA method.

(2) Composite particle of Example 1 Since the antibody immobilized on the composite particle of Example 1 is a rat antibody, it is the same as (1) above except that Rat IgG ELISA (manufactured by Life Diagnostics) is used as a quantification kit. Then, the amount of antibody immobilized on the composite particles of Example 1 was quantified. As a result of the quantification, the amount of immobilization per 1.0 wt% of the particle concentration of the composite particles of Example 1 was calculated to be 0.07 nM (about 0.1 nM). The results are shown in Table 3.

(3) Composite Particles of Example 2 and Comparative Example 1 In the same manner as in (1) above, the amount of antibody immobilized on the composite particles of Example 2 and the composite particles of Comparative Example 1 was quantified. The results are shown in Table 3.

The physical properties of the composite particles of Examples 1 to 6 and Comparative Examples 1 and 2 are summarized in Table 4 below.

[Detection of specific binding to antigen molecule by SPR]
The binding of aHER2-MP to HER2 was confirmed at the molecular level using an intermolecular interaction analyzer Biacore3000 (manufactured by GE Healthcare) based on the SPR principle.

(1) HER2 / Fc chimera (R & D Systems) was immobilized on the sensor chip CM4 as a ligand using an amine coupling kit dedicated to the composite particle apparatus of Example 3. The composite particles of Example 3 and the composite particles of Comparative Example 1 were mixed with a running buffer (20 mM Na-Pi, 150 mM NaCl, 0.005% P20, pH 7.4) at particle concentrations of 1, 2, 4, and 8 mgFe. The solution diluted to / mL was used as an analyze. At a flow rate of 20 μL / min, analyze was added for 120 seconds to observe the bound phase, and the solution was switched to running buffer to observe the dissociated phase for 120 seconds. For repeated measurements, 20 mM aqueous NaOH solution was added for 60 seconds to wash and regenerate the sensor chip. The analysis was performed by fitting analysis using a 1: 1 coupling model using the analysis software attached to the device. As a positive control, aHER2-IgG, which is a free antibody, was also diluted in the order of nM, and the same measurement and analysis were carried out.

The superposition of the binding sensorgrams of the composite particles (negative control) of Comparative Example 1 is shown in FIG. 2A, and the superposition of the binding sensorgrams of the composite particles of Example 3 is shown in FIG. 2B. From FIG. 2A, it was confirmed that the binding response of the composite particle of Example 3 to HER2 / Fc chimera was confirmed, and that the response increased in a concentration-dependent manner. The result of fitting analysis of the binding dissociation constant KD based on the quantitative result in the ELISA and the binding dissociation constant of the antibody immobilized on the composite particle of Example 3 are 2.39 × 10 ^-11 [M]. It was. The binding dissociation constant of the free anti-HER2 antibody (aHER2-IgG) was similarly determined to be 5.45 × 10 ^-11 [M], and the antibody immobilized on the composite particles of Example 3 was It was confirmed that the antibody itself functions without impairing the binding ability.

(2) Composite particle of Example 4 In the same manner as in (1) above, the specific binding of the composite particle of Example 4 to the antigen molecule was detected. The KD of Fab immobilized on the composite particles of Example 4 was 9.0 × 10 ^-9 [M] as a result of calculating from the sensorgram when the Fab concentration equivalent to 15 nM was added. The KD of the free anti-HER2 antibody Fab fragment (aHER-Fab) was separately measured and calculated and found to be 1.8 × 10 ^-9 [M]. Therefore, it can be confirmed that the aHER-Fab immobilized on the composite particles of Example 4 maintains the same order of KD, and that the composite particles also function without significantly impairing the binding ability of the antibody itself. It was. From this result, it was confirmed that the same effect can be obtained even if the technique of the present invention is applied to particles having different sizes and substances, or to ligands having different structures.

(3) Composite particles of Examples 5 and 6 In the same manner as in (1) above, specific binding of the composite particles of Examples 5 and 6 and the composite particles of Comparative Example 2 to the antigen molecule was detected. The solutions of the composite particles of Examples 5 and 6 and the composite particles of Comparative Example 2 were adjusted so that the concentration of the immobilized antibody was equivalent to 40 nM, respectively, and the binding sensorgrams when the solutions were added were superposed. It is shown in FIG. As a result of performing a rough fitting analysis at one concentration point on the composite particles of Example 6, it was found that the bond dissociation constant KD was 1.3 × 10 ^-11 [M], which was the same order as that of Example 1. It was. The composite particles of Example 5 had a small binding response and a reduced binding dissociation constant KD, but the shape of the sensorgram was similar to that of the composite particles of Example 6. On the other hand, the composite particles of Comparative Example 2 had a sensorgram shape that was significantly different from the others. It is presumed that a non-specific binding other than the specific binding to HER2 is visible. That is, by comparing with Comparative Example 2 and Example 5, it was confirmed that the binding ability to a specific molecule was dramatically improved by lengthening the chain length of the graft chain.

[Detection of specific binding to antigen-expressing cells by FACS]
(1) Composite particles of Example 3
The binding of aHER2-MP to HER2-highly expressed cells was confirmed at the cellular level using the FACS device BD FACS Calibur HG (manufactured by Becton Dickinson).

The cells used are cells cultured from SKBR3, which is a HER2 high expression strain derived from human breast cancer cells. The cultured SKBR3 was made into floating cells by trypsin treatment, then replaced with EDTA buffer, and the composite particles of Example 3 having a particle concentration of 6 mgFe / mL were added. The reaction conditions were that the number of reaction cells was 1 × ¹⁰⁶ cells, and the temperature and time were 4 ° C./hour. Here, the one to which only the buffer was added was designated as a negative control, and the one to which a free anti-HER2 antibody (aHER2-IgG, 1 mg / mL) was added was designated as a positive control. Then, after repeating washing three times, FITC-labeled anti-human IgG antibody (Goat anti-human IgG (H + L), manufactured by KPL, 200-fold dilution) was reacted with cells under the same conditions as a secondary antibody, and then the same. Was washed. The number of measured cells was 10,000 cells. The fluorescence intensity distribution of the obtained negative control cells is shown in FIG. 4A, the fluorescence intensity distribution of the positive control cells is shown in FIG. 4B, and the fluorescence intensity distribution of the composite particle cells of Example 3 is shown in FIG. Shown in (c).

As shown in FIG. 4 (c), the fluorescence intensity distribution of the cells reacted with the composite particles of Example 3 shows the same fluorescence intensity shift as in the case of reacting with aHER2-IgG (FIG. 4 (b)). It was. Therefore, it was confirmed that the composite particles of Example 3 exhibited specific binding ability even at the cellular level.

(2) Test substance aCD105-MP. In which Bend3 (mouse cerebral vascular endothelial tumor cell line), which highly expresses both CD105 and VEGFR2 as a composite particle cell line of Examples 1 and 2, is reacted with cells. 1 and aVEGFR-MP. 1 was used with a particle concentration of 1 wt%, and for positive control, 2 μg / mL of the free anti-CD105 antibody (aCD105-IgG) used at the time of immobilization and 2 μg / mL of the free anti-VEGFR2 antibody (aVEGFR-IgG) were used. Was used. For the negative control, rat IgG (antibody mixture produced from rat blood), which is a standard attached to the ELISA kit, used for antibody quantification was used. As the secondary antibody, PE (Phycoerythrin) -labeled anti-rat IgG antibody (diluted by × 50) was used, and the antigen was obtained for the composite particles of Example 1 in the same manner as in (1) above except that the reaction conditions were room temperature for 30 minutes. Specific binding to expressing cells was detected.

The fluorescence intensity distribution of the obtained positive control cells is shown in FIG. 5 (a), and the fluorescence intensity distribution of the cells of the composite particles of Example 1 is shown in FIG. 5 (b). The peak of the fluorescence intensity distribution of the cells reacted with the composite particles of Example 1 was shifted to the right as a whole. In the case of the composite particle of Example 1 (FIG. 5 (b)), the shift value (fluorescence intensity at the peak top) was smaller than that in the case of the positive control (FIG. 5 (a)). These results are considered to be due to the difference in the amount of antibody immobilization and the difference in the expression level of the target molecule on the cell. At the very least, the presence of a significant peak shift indicates that there is specific binding to the cell. Importantly, in the range of the antibody immobilization amount of about 0.1 nM to 10 nM, the specific binding ability as the composite particle of the present invention is similarly exhibited without depending on the antibody immobilization amount. It can be confirmed that it shows.

(3) Except for using BT-474 (human breast cancer cell line) as the cell line and PE (Phycoerythrin) -labeled anti-human IgG antibody as the secondary antibody in order to improve the detection sensitivity of the composite particles of Examples 5 and 6. , Specific binding to antigen-expressing cells was detected for the composite particles of Examples 5 and 6 in the same manner as in (1) above. In this system as well, it was confirmed that binding could be detected by reacting with a positive control-free anti-HER2 antibody (aHER-IgG), and that the detection sensitivity was increased by 10 times or more. The fluorescence intensity distribution showing that a similar fluorescence shift was obtained with aHER-IgG at a lower concentration (20 nM) is shown in FIG.

As a result of evaluating the binding at the cell level in this experimental system, as shown in the graph of FIG. 7, the rate of increase in fluorescence intensity depending on the particle concentration (immobilized antibody concentration) was higher than that of the composite particles of Comparative Example 2. The composite particles of 5 and the composite particles of Example 6 were larger. Together with the evaluation results at the molecular level in the above SPR, it is considered that the composite particles of Example 5 and the composite particles of Example 6 are sufficiently exhibiting specific binding ability. It was also confirmed that the composite particles of Example 6 had higher binding ability than the composite particles of Example 5.

Considering that the lengths of the polymer graft chains of the composite particles of Example 5 and Comparative Example 2 are almost the same, the binding ability of the composite particles of Example 5 is increased due to the difference in the ratio of the antibody immobilization amount. It is considered to have improved.

Claims (7)

Composite particles containing polymer graft chains, microparticles and affinity ligands,
One end of the polymer graft chain is bonded to the surface of the fine particles at a graft density of 0.1 double chain / nm ² or more.
A composite particle in which the affinity ligand is covalently bound within a range of less than 20% of the total length from another end of the polymer graft chain. The living radical of one or more monomers selected from the group consisting of an acrylic acid derivative, a methacrylic acid derivative, an acrylamide derivative, a methacrylamide derivative and a styrene derivative in which the polymer graft chain is based on a polymerization initiator group on the particle surface. Obtained by polymerization and
The composite particle according to claim 1, further comprising a monomer binding to the affinity ligand at a site of less than 20% of the total length of the polymer graft chain. The composite particle according to claim 1 or 2, wherein the number average molecular weight (Mn) of the polymer graft chain is 10,000 or more and 320,000 or less. A method for detecting a molecule, which comprises contacting a molecule capable of binding to the affinity ligand or an object containing the molecule with the composite particle according to any one of claims 1 to 3. A method for purifying a molecule, which comprises contacting a molecule capable of binding to the affinity ligand or an object containing the molecule with the composite particle according to any one of claims 1 to 3. A method for inducing a change in the molecule, comprising contacting the molecule capable of binding the affinity ligand or a subject containing the molecule with the composite particle according to any one of claims 1 to 3. The method for producing a composite particle according to claim 1, further comprising a step of reacting a fine particle having a polymer graft chain having a site that binds to an affinity ligand at a terminal position on the surface and the affinity ligand.
A production method in which one end of the polymer graft chain is bonded to the surface of the fine particles at a graft density of 0.1 single chain / nm ² or more.

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