Polymer chain modified nanoparticles and methods for producing the same, and polymerization initiation group modified nanoparticles and methods for producing the same

Abstract

The present invention provides a group of polymer chain-modified nanoparticles in which the number of polymer chains is controlled, and a method for producing the same, as well as a group of polymerization initiator-modified nanoparticles in which the number of polymerization initiators is controlled, and a method for producing the same. [Solution] The polymer chain modified nanoparticle group according to the present invention is composed of a plurality of polymer chain modified nanoparticles, each polymer chain modified nanoparticle having a nanoparticle, 1 to 5 single-stranded nucleic acids with one end immobilized on the surface of the nanoparticle, a polymerization initiator bound to the other end of each single-stranded nucleic acid, and a polymer chain formed by polymerizing monomer components starting from each polymerization initiator. When the plurality of polymer chain modified nanoparticles are classified into one or more groups according to the number of single-stranded nucleic acids that each polymer chain modified nanoparticle has, the number of polymer chain modified nanoparticles constituting the group with the largest number of particles is 80% or more of the total number of the plurality of polymer chain modified nanoparticles.

Description

Technical Field

[0001] The present invention relates to a polymer chain-modified nanoparticle group and a method for producing the same, a polymerization initiation group-modified nanoparticle group and a method for producing the same.

Background Art

[0002] Metal nanoparticles such as gold nanoparticles have characteristic optical properties based on localized surface plasmon resonance, and thus are used in applications such as biosensing and bioimaging. In recent years, research on using metal nanoparticles as carriers for drugs and genes has also been advanced. Metal nanoparticles used in these applications are often modified on their surfaces with polymer chains for the purpose of improving dispersion stability, imparting biocompatibility or functionality, etc.

[0003] Conventionally, as methods for producing metal nanoparticles modified with polymer chains, a method of mixing a polymer chain having a functional group such as a thiol group at its end with metal nanoparticles and adsorbing the polymer chain on the surface of the metal nanoparticles ("grafting to" method); a method of synthesizing metal nanoparticles from a precursor compound (such as chloroauric acid) in the presence of a polymer chain having a functional group such as a thiol group at its end and adsorbing the polymer chain on the surface of the metal nanoparticles ("in situ" method); a method of previously modifying the surface of metal nanoparticles with a polymerization initiation group and synthesizing a polymer chain starting from this polymerization initiation group ("grafting from" method); etc. are known (for example, see Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the future, when utilizing polymer-modified metal nanoparticles for various applications, it will be important to precisely control the number of polymer chains. For example, when forming micelles in which metal nanoparticles are arranged on the outside and polymer chains on the inside, and using these micelles as carriers for drugs or genes, it will be necessary to control the number of polymer chains immobilized on the metal nanoparticles to a small number (e.g., one). However, in principle, it is difficult to control the number of polymer chains using conventional manufacturing methods disclosed in Non-Patent Document 1, etc.

[0006] The present invention has been proposed in view of the above, and aims to provide a group of polymer chain-modified nanoparticles in which the number of polymer chains is controlled and a method for producing the same, as well as a group of polymerization initiator-modified nanoparticles in which the number of polymerization initiators is controlled and a method for producing the same. [Means for solving the problem]

[0007] The following embodiments are specific means for solving the above problems. <1> A group of polymer chain-modified nanoparticles composed of multiple polymer chain-modified nanoparticles, The polymer chain-modified nanoparticle comprises a nanoparticle, 1 to 5 single-stranded nucleic acids with one end immobilized on the surface of the nanoparticle, a polymerization initiator bound to the other end of each single-stranded nucleic acid, and a polymer chain formed by polymerizing monomer components starting from each polymerization initiator. A group of polymer chain-modified nanoparticles in which, when the plurality of polymer chain-modified nanoparticles are classified into one or more groups based on the number of single-stranded nucleic acids each polymer chain-modified nanoparticle has, the number of polymer chain-modified nanoparticles constituting the group with the largest number of particles is 80% or more of the total number of the plurality of polymer chain-modified nanoparticles.

[0008] <2> The polymer chain-modified nanoparticles are defined by the following formula (1): NP(L1-NA-L2-PI-Pol) n (1) (In the formula, NP represents nanoparticles, NA represents single-stranded nucleic acid, PI represents a polymerization initiator, Pol represents a polymer chain, L1 is absent or represents a linker having a functional group adsorbable to the nanoparticles, L2 is absent or represents a linker, and n represents an integer from 1 to 5.) Having a structure represented by, <1> The polymer chain-modified nanoparticle group described above.

[0009] <3> The aforementioned nanoparticles are metal nanoparticles. <1> or <2> The polymer chain-modified nanoparticle group described above.

[0010] <4> The polymerization initiator is a living polymerization initiator. <1> ~ <3> A group of polymer chain-modified nanoparticles as described in any one of the items.

[0011] <5> A group of polymerization initiation group modified nanoparticles composed of multiple polymerization initiation group modified nanoparticles, The polymerization initiator-modified nanoparticle comprises a nanoparticle, 1 to 5 single-stranded nucleic acids with one end immobilized on the surface of the nanoparticle, and a polymerization initiator bound to the other end of each single-stranded nucleic acid. A group of polymerization initiator-modified nanoparticles in which, when the plurality of polymerization initiator-modified nanoparticles are classified into one or more groups according to the number of single-stranded nucleic acids that each polymerization initiator-modified nanoparticle has, the number of polymerization initiator-modified nanoparticles constituting the group with the largest number of particles is 80% or more of the total number of the plurality of polymerization initiator-modified nanoparticles.

[0012] <6> The polymerization initiator-modified nanoparticles are defined by the following formula (2): NP(L1-NA-L2-PI) n (2) (In the formula, NP represents nanoparticles, NA represents single-stranded nucleic acid, PI represents a polymerization initiator, L1 is absent or represents a linker having a functional group adsorbable to the nanoparticles, L2 is absent or represents a linker, and n represents an integer from 1 to 5.) Having a structure represented by, <5> The group of polymerization initiator-modified nanoparticles described above.

[0013] <7> The polymerizable initiator-modified nanoparticle group according to <5> or <6>, wherein the nanoparticle is a metal nanoparticle.

[0014] <8> The polymerizable initiator-modified nanoparticle group according to any one of <5> to <7>, wherein the polymerizable initiator is a living polymerization initiator.

[0015] <9> A method for producing a polymer chain-modified nanoparticle group, comprising a step of polymerizing a monomer component starting from each polymerizable initiator possessed by the polymerizable initiator-modified nanoparticle group according to any one of <5> to <8> to obtain a polymer chain.

[0016] <10> A method for producing a polymerizable initiator-modified nanoparticle group according to any one of <5> to <8>, comprising: using, as a modifying molecule, a first molecule which is a single-stranded nucleic acid having a polymerizable initiator bonded to one end side, or a second molecule which is a single-stranded nucleic acid, and immobilizing the other end side of the first molecule or one end side of the second molecule on the surface of the nanoparticle in a surface modification step; a sorting step of sorting the nanoparticles to which a desired number of the modifying molecules are immobilized by electrophoresis; When the second molecule is used in the surface modification step, the method for producing a polymerizable initiator-modified nanoparticle group further includes a polymerizable initiator bonding step of bonding a polymerizable initiator to the other end side of the second molecule after the sorting step.

Advantages of the Invention

[0017] According to the present invention, it is possible to provide a polymer chain-modified nanoparticle group in which the number of polymer chains is controlled and a method for producing the same, and a polymerizable initiator-modified nanoparticle group in which the number of polymerizable initiators is controlled and a method for producing the same.

Brief Description of the Drawings

[0018] [Figure 1] It is a diagram showing the results of gel electrophoresis of a DNA-AuNP conjugate (Init-DNA-AuNP) having a polymerizable initiator at the end. [Figure 2]It is a diagram showing the absorption spectrum of a DNA-AuNP monoc conjugate (Init-monoDNA-AuNP) having a polymerization initiation group at the end. [Figure 3] It is a diagram showing the absorption spectrum of a poly(N-isopropylacrylamide)-AuNP monoc conjugate (monoPNIPAM-AuNP) obtained by polymerizing N-isopropylacrylamide starting from the polymerization initiation group possessed by Init-monoDNA-AuNP.

Embodiments for Carrying out the Invention

[0019] Hereinafter, specific embodiments to which the present invention is applied will be described in detail. In this specification, the term "(meth)acrylic acid" means both "acrylic acid" and "methacrylic acid". The same applies to other terms such as "(meth)acrylate" and "(meth)acrylamide".

[0020] <Nanoparticle group modified with polymerization initiation group> The nanoparticle group modified with a polymerization initiation group according to this embodiment is composed of a plurality of nanoparticle groups modified with a polymerization initiation group. Each nanoparticle modified with a polymerization initiation group has a nanoparticle, 1 to 5 single-stranded nucleic acids whose one end is immobilized on the surface of the nanoparticle, and a polymerization initiation group bonded to the other end of each single-stranded nucleic acid. When the plurality of nanoparticle groups modified with a polymerization initiation group are classified into one or more groups according to the number of single-stranded nucleic acids possessed by each nanoparticle group modified with a polymerization initiation group, the number of nanoparticle groups modified with a polymerization initiation group constituting the group with the largest number of particles is 80% or more of the total number of the plurality of nanoparticle groups modified with a polymerization initiation group.

[0021] The number of nanoparticle groups modified with a polymerization initiation group constituting the group with the largest number of particles is preferably 90% or more, more preferably 95% or more, of the total number of the plurality of nanoparticle groups modified with a polymerization initiation group, and the upper limit may be 100%. That is, the nanoparticle group modified with a polymerization initiation group according to this embodiment is characterized in that the number of single-stranded nucleic acids immobilized on each nanoparticle and the number of polymerization initiation groups bonded to the single-stranded nucleic acids are precisely controlled.

[0022] Each polymerization initiator-modified nanoparticle is, for example, represented by the following formula (2): NP(L1-NA-L2-PI) n (2) (In the formula, NP represents nanoparticles, NA represents single-stranded nucleic acid, PI represents a polymerization initiator, L1 represents a linker that is absent or has a functional group adsorbable to nanoparticles, L2 represents a linker that is absent or has a functional group adsorbable to nanoparticles, and n represents an integer from 1 to 5.) It has a structure represented by the following:

[0023] In equation (2) above, n may be an integer from 1 to 3, or it may be 1 or 2, or it may be 1.

[0024] The nanoparticles NP in formula (2) above may be either inorganic nanoparticles or organic nanoparticles. Examples of inorganic nanoparticles include metal nanoparticles made of metals such as gold, silver, copper, platinum, aluminum, tin, zinc, and tungsten. Examples of organic nanoparticles include latex particles made of polymers such as polystyrene, polymethyl methacrylate, and styrene-(meth)acrylic acid copolymer. Among these nanoparticles, metal nanoparticles are preferred, and gold nanoparticles, which have high oxidation stability, are more preferred from the viewpoint of long-term storage.

[0025] The volume-average particle size of the nanoparticles NP is preferably 1 nm to 500 nm, and more preferably 2 nm to 100 nm. The volume-average particle size of the nanoparticles NP is measured, for example, by dynamic light scattering.

[0026] In formula (2) above, the single-stranded nucleic acid NA may be a DNA strand or an RNA strand. Furthermore, the phosphodiester bond in the single-stranded nucleic acid may be replaced with other bonds such as phosphotriester bonds, phosphorothioate bonds, phosphorodithioate bonds, H-phosphonate bonds, or alkylphosphonate bonds, as needed. The base length of the single-stranded nucleic acid NA is preferably 5 to 500 bases, and more preferably 10 to 100 bases.

[0027] Single-stranded nucleic acid NA can be immobilized on nanoparticles NP via a linker L1 having a functional group adsorbable to the nanoparticle NP. Here, "adsorption" refers to physical adsorption by van der Waals forces, or chemical adsorption by covalent bonds, ionic bonds, coordination bonds, hydrogen bonds, etc. The linker L1 may have one or more functional groups adsorbable to the nanoparticle NP. That is, there may be one or more adsorption sites between the nanoparticle NP and the linker L1.

[0028] Examples of functional groups that can be adsorbed onto nanoparticles (NP) include thiol groups (-SH), methylthio groups (-SCH3), mercaptothio groups (-S-SH), dithiocarbamate groups (-N-CS2), xanthanthate groups (-O-CS2), thiocarbonyl groups (>C=S), thiocarboxyl groups (-C(O)SH), acetylthio groups (-SC(O)CH3), sulfide groups (-S-), disulfide groups (-SS-), sulfo groups (-SO3H), sulfino groups (-SOOH), and carboxyl groups (-COOH).

[0029] Among the linker L1 structures, structures other than functional groups that can be adsorbed onto nanoparticles NP can be adopted without particular limitation, as long as they can be introduced into single-stranded nucleic acid molecules by known methods.

[0030] Linker L1 may be located at the 3' end or the 5' end of the single-stranded nucleic acid NA. For example, linker L1 may be bound to the 3' end of the single-stranded nucleic acid NA via an ether bond or ester bond with the hydroxyl group at the 3' end of the single-stranded nucleic acid NA, or via a phosphodiester bond with a phosphate residue, or it may be bound to the 5' end of the single-stranded nucleic acid NA via an ether bond or ester bond with the -CH2OH group at the 5' end of the single-stranded nucleic acid NA, or via a phosphodiester bond with a phosphate residue.

[0031] An example of linker L1 is represented by the following formulas (L1-1) or (L1-2). In the formulas, * indicates a binding site to single-stranded nucleic acid NA, and it binds to the 3' or 5' end of single-stranded nucleic acid NA via a phosphodiester bond between the phosphate residue at the 3' or 5' end of the single-stranded nucleic acid NA. a represents an integer from 1 to 20.

[0032] [ka]

[0033] Furthermore, if linker L1 has the structure represented by the above formula (L1-2), linker L1 may be adsorbed onto nanoparticle NP in a state where the disulfide bond has been reductively cleaved to form two thiol groups.

[0034] Alternatively, the single-stranded nucleic acid NA may be directly immobilized on the nanoparticle NP without the linker L1. For example, if the nanoparticle NP is a latex particle, the single-stranded nucleic acid NA can be immobilized on the nanoparticle NP by reacting the functional groups such as carboxyl groups and epoxy groups present on the surface of the latex particle with the functional groups such as amino groups present on the single-stranded nucleic acid NA. The single-stranded nucleic acid NA may be directly immobilized on the nanoparticle NP at its 5' end or at its 3' end.

[0035] The polymerization initiator PI in formula (2) above can be any group capable of polymerizing the monomer starting from the polymerization initiator. A living polymerization initiator capable of living polymerization is preferred as the polymerization initiator PI, a living radical polymerization initiator capable of living radical polymerization is more preferred, and an atom transfer radical polymerization initiator capable of atom transfer radical polymerization (ATRP) is even more preferred.

[0036] Specific examples of polymerization initiator groups (PI) include α-haloacyloxy groups, α-haloacyl groups, and halosulfonyl groups. Examples of halogen atoms included in α-haloacyloxy groups, α-haloacyl groups, and halosulfonyl groups include chlorine atoms, bromine atoms, and iodine atoms, with chlorine atoms and bromine atoms being preferred from the viewpoint of reactivity.

[0037] Examples of α-haloacyloxy groups include 2-chloroisobutylyloxy group, 2-bromoisobutylyloxy group, 2-iodoisobutylyloxy group, 2-chlorobutylyloxy group, 2-bromobutylyloxy group, 2-iodobutylyloxy group, 2-chloropropionyloxy group, 2-bromopropionyloxy group, 2-iodopropionyloxy group, chlorophenylacetoxy group, bromophenylacetoxy group, and iodophenylacetoxy group.

[0038] Examples of α-haloacyl groups include 2-chloroisobutyryl group, 2-bromoisobutyryl group, 2-iodoisobutyryl group, 2-chlorobutyryl group, 2-bromobutyryl group, 2-iodobutyryl group, 2-chloropropionyl group, 2-bromopropionyl group, 2-iodopropionyl group, dichloroacetyl group, dibromoacetyl group, and diiodoacetyl group.

[0039] Examples of halosulfonyl groups include chlorosulfonyl groups, bromosulfonyl groups, and iodosulfonyl groups.

[0040] The polymerization initiator PI can be immobilized on the single-stranded nucleic acid NA via the linker L2. The structure of the linker L2 can be any known structure that can be introduced into a single-stranded nucleic acid molecule without any particular limitations.

[0041] Linker L2 may be located at the 5' end or the 3' end of the single-stranded nucleic acid NA. For example, linker L2 may be bound to the 5' end of the single-stranded nucleic acid NA via an ether bond or ester bond with the -CH2OH group at the 5' end of the single-stranded nucleic acid NA, or via a phosphodiester bond with a phosphate residue, or it may be bound to the 3' end of the single-stranded nucleic acid NA via an ether bond or ester bond with the hydroxyl group at the 3' end of the single-stranded nucleic acid NA, or via a phosphodiester bond with a phosphate residue.

[0042] An example of linker L2 is represented by the following formula (L2-1). In the formula, * indicates a binding site with single-stranded nucleic acid NA, and it binds to the 5' or 3' end of single-stranded nucleic acid NA via a phosphodiester bond between the phosphate residue at the 5' or 3' end of the single-stranded nucleic acid NA. ** indicates a binding site with polymerization initiator PI. b is an integer from 1 to 20.

[0043] [ka]

[0044] Alternatively, the polymerization initiator PI may be directly immobilized on the single-stranded nucleic acid NA without the linker L2. For example, the polymerization initiator PI can be immobilized on the single-stranded nucleic acid NA via functional groups such as amino groups present on the single-stranded nucleic acid NA. The polymerization initiator PI may be directly immobilized on the 5' end of the single-stranded nucleic acid NA, or it may be directly immobilized on the 3' end of the single-stranded nucleic acid NA.

[0045] <Method for producing polymerization-initiated group modified nanoparticles> [First aspect] A first aspect of the method for producing a group of polymerization initiator-modified nanoparticles according to this embodiment includes a surface modification step in which a first molecule, which is a single-stranded nucleic acid with a polymerization initiator group attached to one end, is used as a modification molecule, and the other end of the first molecule is immobilized on the surface of a nanoparticle, and a selection step in which nanoparticles on which a desired number of first molecules are immobilized are selected by electrophoresis.

[0046] First, in the surface modification step, a first molecule, which is a single-stranded nucleic acid with a polymerization initiator group attached to one end, is used as the modification molecule, and the other end of the first molecule is immobilized on the surface of the nanoparticle. As the first molecule, for example, one represented by formula: L1-NA-L2-PI is used. In the formula, L1, NA, L2, and PI are the same as in formula (2) above.

[0047] The first molecule can be produced, for example, by reacting a single-stranded nucleic acid having an amino group-containing functional group introduced at one end with an active ester of a polymerization initiator. An example of an active ester of a polymerization initiator is one represented by the following formula.

[0048] [ka]

[0049] Alternatively, the first molecule can be produced by an automated nucleic acid solid-phase synthesis method using a polymerization initiator having a phosphoramidite group. An example of a polymerization initiator having a phosphoramidite group is represented by the following formula.

[0050] [ka]

[0051] The first molecule can be immobilized on the surface of the nanoparticles by allowing a solution containing nanoparticles and the first molecule to stand at 1 to 80°C for 0.5 to 24 hours. The molar ratio of nanoparticles to the first molecule (first molecule / nanoparticle) is preferably 0.1 to 10.

[0052] Furthermore, if the nanoparticles are metal nanoparticles such as gold nanoparticles, it is preferable to treat the surface of the nanoparticles with an organophosphorus compound such as bis(p-sulfonatophenyl)phenylphosphine (BSPP) to make it negatively charged before immobilizing the first molecule on the surface of the nanoparticles. This prevents aggregation of the nanoparticles.

[0053] For example, BSPP can be bonded to the surface of nanoparticles by mixing nanoparticles and BSPP in a solvent such as water or dimethyl sulfoxide at a temperature of 0°C to 80°C (preferably 10°C to 60°C). The concentration of BSPP is not particularly limited, but it is preferably 10 mg / mL or less.

[0054] Next, in the sorting step, nanoparticles with a desired number of immobilized first molecules are sorted by electrophoresis. When groups of nanoparticles with various numbers of immobilized first molecules are subjected to gel electrophoresis, nanoparticles with fewer first molecules migrate a longer distance, while nanoparticles with more first molecules migrate a shorter distance. Therefore, the nanoparticle groups can be separated into one or more groups based on the number of first molecules. By then cutting out the gel from the desired region, nanoparticles with the desired number of immobilized first molecules can be recovered.

[0055] Furthermore, if the base length of the nucleic acid contained in the first molecule is less than 80 bases, it is preferable to anneal the first molecule with an extension nucleic acid to lengthen its base length in order to improve the separation efficiency by electrophoresis. As the extension nucleic acid, a nucleic acid of 80 to 300 bases that has the complementary sequence of the nucleic acid contained in the first molecule at one end can be used.

[0056] When using extension nucleic acids, the selection process involves subjecting the first molecule, which has been annealed with the extension nucleic acid, to electrophoresis. Then, by cutting out the gel from the desired region as described above, nanoparticles with the desired number of immobilized first molecules are recovered. After recovering the nanoparticles, complementary nucleic acids that are complementary to the extension nucleic acid are added, and the extension nucleic acid and complementary nucleic acids are annealed. By centrifugation, nanoparticles from which the extension nucleic acid has been removed can be obtained.

[0057] [Second aspect] A second aspect of the method for producing a group of polymerization initiator-modified nanoparticles according to this embodiment includes a surface modification step in which a second molecule, which is a single-stranded nucleic acid, is used as a modification molecule, and one end of the second molecule is immobilized on the surface of the nanoparticle; a selection step in which nanoparticles on which a desired number of second molecules are immobilized are selected by electrophoresis; and a polymerization initiator-binding step in which a polymerization initiator is attached to the other end of the second molecule.

[0058] First, in the surface modification step, a second molecule, which is a single-stranded nucleic acid, is used as the modification molecule, and one end of the second molecule is immobilized on the surface of the nanoparticle. As the second molecule, for example, one represented by formula: L1-NA is used. In the formula, L1 and NA are the same as in formula (2) above. However, it is preferable to introduce an amino group-containing functional group to the end of the single-stranded nucleic acid NA opposite to L1 in order to attach a polymerization initiator in the subsequent polymerization initiator attachment step.

[0059] The second molecule can be immobilized on the surface of the nanoparticles by allowing a solution containing nanoparticles and the second molecule to stand at 1 to 80°C for 0.5 to 24 hours. The molar ratio of nanoparticles to the second molecule (second molecule / nanoparticle) is preferably 0.1 to 10.

[0060] Next, in the sorting step, nanoparticles with a desired number of immobilized secondary molecules are sorted by electrophoresis. When groups of nanoparticles with varying numbers of immobilized secondary molecules are subjected to gel electrophoresis, nanoparticles with fewer secondary molecules migrate a longer distance, while nanoparticles with more secondary molecules migrate a shorter distance. This allows the nanoparticle groups to be separated into one or more groups based on the number of secondary molecules. By then cutting out the gel from the desired region, nanoparticles with the desired number of immobilized secondary molecules can be recovered.

[0061] Next, in the polymerization initiator bonding step, polymerization initiators are bonded to the other end of the second molecule of the nanoparticles selected in the selection step. If an amino group-containing functional group is introduced to the other end of the second molecule, the polymerization initiator can be bonded to the other end of the second molecule by reacting the second molecule with the active ester of the polymerization initiator.

[0062] Furthermore, if the base length of the nucleic acid contained in the second molecule is less than 80 bases, it is preferable to anneal the second molecule with the extension nucleic acid before the selection process to extend its base length. Examples of the extension nucleic acid include the same nucleic acids as in the first embodiment.

[0063] <Polymer chain-modified nanoparticle group> The polymer chain-modified nanoparticle group according to this embodiment is composed of a plurality of polymer chain-modified nanoparticles, each polymer chain-modified nanoparticle having a nanoparticle, 1 to 5 single-stranded nucleic acids with one end immobilized on the surface of the nanoparticle, a polymerization initiator bound to the other end of each single-stranded nucleic acid, and a polymer chain formed by polymerizing monomer components starting from each polymerization initiator. When the plurality of polymer chain-modified nanoparticles are classified into one or more groups according to the number of single-stranded nucleic acids in each polymer chain-modified nanoparticle, the number of polymer chain-modified nanoparticles constituting the group with the largest number of particles is 80% or more of the total number of polymer chain-modified nanoparticles.

[0064] The number of polymer chain-modified nanoparticles constituting the group with the largest number of particles is preferably 90% or more, more preferably 95% or more, and may have an upper limit of 100% of the total number of polymer chain-modified nanoparticles. In other words, the polymer chain-modified nanoparticle group according to this embodiment is characterized in that the number of single-stranded nucleic acids immobilized on each nanoparticle and the number of polymer chains extending from the ends of the single-stranded nucleic acids are precisely controlled.

[0065] Polymer chain-modified nanoparticles are, for example, those of the following formula (1): NP(L1-NA-L2-PI-Pol) n (1) (In the formula, NP represents nanoparticles, NA represents single-stranded nucleic acid, PI represents a polymerization initiator, Pol represents a polymer chain, L1 is absent or represents a linker having a functional group adsorbable to nanoparticles, L2 is absent or represents a linker, and n represents an integer from 1 to 5.) It has a structure represented by the following:

[0066] Since the nanoparticle NP, linker L1, single-stranded nucleic acid NA, linker L2, polymerization initiator PI, and n in formula (1) above are the same as in formula (2) above, a detailed explanation is omitted.

[0067] The polymer chain Pol in formula (1) above is not particularly limited as long as it is obtained by polymerizing monomer components starting from a polymerization initiator PI. Examples of monomer components include unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid and its anhydride, itaconic acid and its anhydride; (meth)acrylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, chloroethyl (meth)acrylate, polyethylene glycol (meth)acrylate; (meth)acrylamides such as (meth)acrylamide, N-methyl (meth)acrylamide, N-isopropyl (meth)acrylamide, N-phenyl (meth)acrylamide; allyl compounds such as allyl acetate, allyl caproate, allyl caprylate; vinyl ethers such as hexyl vinyl ether, octyl vinyl ether, methoxyethyl vinyl ether; vinyl esters such as vinyl butyrate, vinyl isobutyrate, vinyl benzoate; and styrenes such as styrene, methylstyrene, and chlorostyrene. These monomer components may be used individually or in combination of two or more. When there are two or more monomer components, the polymer chain Pol may be a block copolymer or a random copolymer.

[0068] The average degree of polymerization of polymer chains Pol is said to be, for example, 10 to 10000.

[0069] <Method for producing polymer chain-modified nanoparticles> The method for producing the polymer chain-modified nanoparticle group according to this embodiment includes the step of using the polymerization initiator group-modified nanoparticle group according to this embodiment described above, and polymerizing monomer components starting from each polymerization initiator group possessed by the polymerization initiator group-modified nanoparticle group to obtain a polymer chain.

[0070] As the monomer component, any monomer component for obtaining the polymer chain Pol in formula (1) above can be used without particular limitation.

[0071] Polymer chain-modified nanoparticles can be produced by adding polymerization initiator-modified nanoparticles and monomer components to a solvent, adding a catalyst as needed, and reacting for a predetermined time.

[0072] Examples of solvents include hydrocarbon solvents such as benzene and toluene; ether solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole, and dimethoxybenzene; halogenated hydrocarbon solvents such as methylene chloride, chloroform, and chlorobenzene; ketone solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; alcohol solvents such as methanol, ethanol, propanol, isopropanol, n-butyl alcohol, and tert-butyl alcohol; nitrile solvents such as acetonitrile, propionitrile, and benzonitrile; ester solvents such as ethyl acetate and butyl acetate; carbonate solvents such as ethylene carbonate and propylene carbonate; and water. These solvents may be used individually or in combination of two or more.

[0073] When the polymerization initiator group of the polymerization initiator-modified nanoparticles is an atom transfer radical polymerization initiator, a metal complex is used as a catalyst. Examples of metal complexes include complexes of 0-valent copper, 1-valent copper, 2-valent copper, 2-valent ruthenium, 2-valent iron, or 2-valent nickel, with 1-valent copper complexes being preferred. Examples of copper compounds constituting the 1-valent copper complex include cuprous chloride, cuprous bromide, cuprous iodide, cuprous cyanide, cuprous oxide, and cuprous perchlorate. Examples of ligands constituting the 1-valent copper complex include 2,2'-bipyridyl and its derivatives; 1,10-phenanthroline and its derivatives; and aliphatic amines such as trialkylamines, tetramethylethylenediamine, pentamethyldiethylenetriamine, and hexamethyl(2-aminoethyl)amine.

[0074] The reaction temperature for producing polymer chain-modified nanoparticles is preferably, for example, 10°C to 60°C. The reaction time is also preferably, for example, 1 hour to 24 hours. [Examples]

[0075] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples.

[0076] <Experimental Example 1: Synthesis of DNA derivatives with polymerization initiation groups at their terminals (Init-DNA) (1)> 2-bromoisobutyrate N-hydroxysuccinimide (0.156 g, 0.59 mmol), the active ester of the polymerization initiator for atom transfer radical polymerization (ATRP), was dissolved in 15 mL of anhydrous dimethylformamide. Meanwhile, a 16-base DNA strand was prepared, having a 6-aminohexyl group at the 5' end and modified with dithiothreitol (DTT) at the 3' end. The base sequence of the DNA strand was 5'-TTTTTTTTTTTTTTTC-3' (SEQ ID NO: 1). 35 μL of an aqueous solution of the DNA strand (pH 8.5, 100 μM) was added to the solution of the active ester of the polymerization initiator, and the mixture was incubated at 4°C for 2 hours, then left to stand overnight at room temperature. After the reaction, the DNA derivative (Init-DNA) with polymerization initiators at its ends was purified by ethanol precipitation. Finally, a 100 μM aqueous solution of Init-DNA (containing 100 mM NaCl) was prepared as a stock solution. The structure of the obtained Init-DNA is as follows.

[0077] [ka]

[0078] <Experimental Example 2: Synthesis of DNA derivatives with polymerization initiators at their terminals (Init-DNA) (2)> First, following a previously reported study (SE Averick et al., Angew. Chem. Int. Ed., 53 (2014) 2740), a polymerization initiator having a phosphoramidite group was synthesized using the following scheme.

[0079] [ka]

[0080] Specifically, 4-amino-1-butanol (1) (2.70 mL, 0.029 mmol) and triethylamine (4.42 mL, 0.0318 mmol) were added to anhydrous dichloromethane (20 mL), and then α-bromoisobutyrate bromide (2) (3.44 mL, 0.0279 mmol) was slowly added dropwise under ice cooling. The mixed solution was stirred at 0°C for 12 hours. Then, 5% NaOH aqueous solution (20 mL) was added and the mixture was stirred for 4 hours. The reaction solution was washed twice with 1 M NaOH aqueous solution (25 mL), twice with 1 M HCl aqueous solution (25 mL), and once with saturated brine (25 mL). The organic layer was collected and dehydrated with anhydrous sodium sulfate. After drying under reduced pressure, intermediate (3) was obtained as a pale pink liquid.

[0081] Next, intermediate (3) (0.34 g, 1.43 mmol) was added to pyridine (3 mL) and dissolved. This solution was concentrated under reduced pressure at 50 °C and dried for 30 minutes. This procedure was repeated three times. Anhydrous dichloromethane (10 mL) and N,N-diisopropylethylamine (1.24 mL, 7.14 mmol) were added to intermediate (3). Then, under a nitrogen atmosphere, 2-cyanoethyl=N,N-diisopropylchlorophosphoramidite (4) (478 μL, 2.14 mmol) was slowly added dropwise at 0 °C. This mixed solution was stirred at room temperature for 2 hours. The reaction solution was extracted by adding ethyl acetate and saturated sodium bicarbonate aqueous solution. The organic layer was recovered and dehydrated with anhydrous sodium sulfate. The compound concentrated using an evaporator was purified by silica gel chromatography (developing solvent: ethyl acetate / hexane / triethylamine = 100 / 100 / 2 (mL)). The recovered eluent was concentrated and dried under reduced pressure to obtain a colorless liquid amidite compound (5).

[0082] Using the obtained amidite compound (5), a DNA derivative (Init-DNA) having polymerization initiators at its terminals was synthesized by automated solid-phase nucleic acid synthesis. The base sequence of the synthesized DNA strand is 5'-TAGCTCCAACTACCAC-3' (SEQ ID NO: 2). Synthesis and deprotection conditions followed the standard protocol. The obtained Init-DNA was purified by high-performance liquid chromatography (eluent: acetonitrile-ammonium acetate mixed solution) and recovered by lyophilization. The structure of the obtained Init-DNA is as follows.

[0083] [ka]

[0084] <Experimental Example 3: Synthesis of DNA-AuNP monoconjugates with polymerization initiators at their termini (Init-monoDNA-AuNP)> First, organophosphorus-immobilized gold nanoparticles (BSPP-AuNP) were prepared according to a previously reported method (Y. Akiyama et al., Small, 11 (2015) 3153). Specifically, bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP; final concentration: 1 mg / mL) was added to an aqueous solution of 2 nM AuNP (particle size: 5 nm) and allowed to stand at 50°C for 1 hour. This BSPP-AuNP was centrifuged twice (18700 × g, 10°C, 90 minutes), and a buffer solution (0.5 × TBE, 100 mM NaCl, 1 mg / mL BSPP) was added.

[0085] Next, the 100 μM Init-DNA aqueous solution (containing 100 mM NaCl) prepared in Experimental Example 1 was mixed with a buffer solution of BSPP-AuNP (4 μM) so that the molar ratio of AuNP to DNA varied (DNA / AuNP = 0.5 to 1.5). The resulting mixed solution was then allowed to stand at 20 to 25°C for 12 to 48 hours. To this mixed solution, a 94-base extension DNA strand was added in a molar amount four times the amount of BSPP-AuNP, and the mixture was allowed to stand at 50°C for 5 minutes, followed by 5°C for 15 minutes. The base sequence of the extension DNA strand is 5'-GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (Sequence ID 3). Next, BSPP-AuNPs (Init-DNA-AuNPs) immobilized with various numbers of Init-DNA were subjected to electrophoresis using a 3% agarose gel (100V, 30 minutes, electrophoresis buffer: 0.5×TBE).

[0086] An example of electrophoresis results is shown in Figure 1. Lane 1 in Figure 1 shows the electrophoresis of BSPP-AuNP as a control, while lanes 2 and 3 show the electrophoresis of Init-DNA-AuNP. In lane 2, the molar ratio of AuNP to DNA was DNA / AuNP = 1.5, and the standing time after mixing was 12 hours. In lane 3, the molar ratio of AuNP to DNA was DNA / AuNP = 1.2, and the standing time after mixing was 48 hours. As can be seen from Figure 1, the electrophoresis distance changed depending on the number of immobilized Init-DNA molecules.

[0087] After electrophoresis, DNA-AuNP monoconjugates (Init-monoDNA-AuNPs), in which one molecule of Init-DNA was immobilized per AuNP, were recovered by excising the desired region of the gel. The number of Init-monoDNA-AuNPs immobilized with one molecule of Init-DNA was 100% of the total number of AuNPs.

[0088] Since the Init-monoDNA-AuNP obtained above has an extension DNA strand bound to it, the extension DNA strand was removed as follows. First, a 94-base complementary DNA strand, complementary to the extension DNA strand, was added to an aqueous solution of Init-monoDNA-AuNP in a 4:4 molar amount relative to the AuNP, and the mixture was incubated at 60°C for 10 minutes, followed by standing at 20-25°C for 1 hour. The base sequence of the complementary DNA strand is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTC-3' (SEQ ID NO: 4). Next, centrifugation (18700×g, 10°C, 90 minutes) was performed twice to recover the Init-monoDNA-AuNP from which the extension DNA strand had been removed (yield: 5-15%).

[0089] A dispersion of Init-monoDNA-AuNP (0.5×TBE, 1 mg / mL BSPP) was prepared by adding 1% Tween20 (final concentration: 0.02% (vol / vol)). This dispersion (final concentration of Init-monoDNA-AuNP: 200 nM) was added to a measurement cell, and its physical properties were evaluated. The zeta potential and absorption spectrum of the Init-monoDNA-AuNP dispersion were measured at 25°C and 20-25°C, respectively. BSPP-AuNP was used as a control sample.

[0090] The measurement results showed that the zeta potential of the Init-monoDNA-AuNP dispersion was -14.90 mV, and the zeta potential of the BSPP-AuNP dispersion was -16.89 mV. Furthermore, as shown in Figure 2, the maximum absorption wavelength of the Init-monoDNA-AuNP dispersion was 519.6 nm, and the maximum absorption wavelength of the BSPP-AuNP dispersion was 519.8 nm. Thus, there were no significant changes in the zeta potential or absorption spectrum before and after immobilization of Init-DNA. This indicates that Init-monoDNA-AuNP with high dispersibility was obtained.

[0091] <Experimental Example 4: Preparation of Poly(N-Isopropylacrylamide)-AuNP Monoconjugate (monoPNIPAM-AuNP)> Based on the synthesis conditions reported previously (Kim DJ et al., Macromol. Chem. Phys., 206 (2005) 1941), an aqueous solution (400 μL) containing N-isopropylacrylamide (NIPAM) (11.3 mg, 100 μmol), CuBr(I) (3.8 mg, 26.5 μmol), and N,N,N',N”,N”-pentamethyldiethylenetriamine (11.1 μL, 53.0 μmol) was prepared and degassed by bubbling with nitrogen gas. A dispersion of 200 nM Init-monoDNA-AuNP was also prepared and degassed by bubbling with nitrogen gas. After mixing both solutions, sonication was performed at 25°C for 30 seconds. Subsequently, the mixture was stirred at 25°C for 1 hour to obtain monoPNIPAM-AuNP.

[0092] A dispersion of monoPNIPAM-AuNP (0.5 × TBE, 1 mg / mL BSPP) was prepared by adding 1% Tween20 (final concentration: 0.02% (vol / vol)). This dispersion (final concentration of monoPNIPAM-AuNP: 200 nM) was added to a measurement cell, and the absorption spectrum was measured at 20-25°C. For comparison, poly(N-isopropylacrylamide) synthesized using monoDNA-AuNP without polymerization initiators and a polymerization initiator, and poly(N-isopropylacrylamide) synthesized using BSPP-AuNP and a polymerization initiator were prepared.

[0093] The absorption spectra of each dispersion are shown in Figure 3. As shown in Figure 3, a maximum absorption (wavelength: approximately 520 nm) characteristic of dispersed AuNP was observed in all cases, but the absorbance was highest when using Init-monoDNA-AuNP. This result suggests that monoPNIPAM-AuNP, in which a single poly(N-isopropylacrylamide) chain is immobilized on AuNP, was obtained.

Claims

1. A group of polymer chain-modified nanoparticles composed of multiple polymer chain-modified nanoparticles, The polymer chain-modified nanoparticle comprises a nanoparticle, one to five single-stranded nucleic acids with one end immobilized on the surface of the nanoparticle, a polymerization initiator bound to the other end of each single-stranded nucleic acid, and a polymer chain formed by polymerizing monomer components starting from each polymerization initiator. A group of polymer chain-modified nanoparticles in which, when the plurality of polymer chain-modified nanoparticles are classified into one or more groups according to the number of single-stranded nucleic acids that each polymer chain-modified nanoparticle has, the number of polymer chain-modified nanoparticles constituting the group with the largest number of particles is 80% or more of the total number of the plurality of polymer chain-modified nanoparticles.

2. The polymer chain-modified nanoparticles are defined by the following formula (1): NP(L) １ -NA-L ２ -PI-Pol) ｎ (1) (In the formula, NP represents nanoparticles, NA represents single-stranded nucleic acid, PI represents polymerization initiator, Pol represents polymer chain, L １ It either does not exist or indicates a linker having a functional group that can be adsorbed onto the nanoparticles, L ２ (This indicates that it does not exist or indicates a linker, and n is an integer between 1 and 5.) A group of polymer chain-modified nanoparticles according to claim 1, having a structure represented by the given name.

3. The polymer chain-modified nanoparticle group according to claim 1 or 2, wherein the nanoparticles are metal nanoparticles.

4. The polymer chain-modified nanoparticle group according to claim 1 or 2, wherein the polymerization initiator is a living polymerization initiator.

5. A group of polymerization initiation group modified nanoparticles composed of multiple polymerization initiation group modified nanoparticles, The polymerization initiator-modified nanoparticle comprises a nanoparticle, 1 to 5 single-stranded nucleic acids with one end immobilized on the surface of the nanoparticle, and a polymerization initiator bound to the other end of each single-stranded nucleic acid. A group of polymerization initiator-modified nanoparticles in which, when the plurality of polymerization initiator-modified nanoparticles are classified into one or more groups according to the number of single-stranded nucleic acids that each polymerization initiator-modified nanoparticle has, the number of polymerization initiator-modified nanoparticles constituting the group with the largest number of particles is 80% or more of the total number of the plurality of polymerization initiator-modified nanoparticles.

6. The polymerization initiator-modified nanoparticles are defined by the following formula (2): @(L １ --A-L ２ --I) ｎ (2) (In the formula, NP represents a nanoparticle, NA represents a single-stranded nucleic acid, PI represents a polymerization initiation group, and L １ either does not exist or represents a linker having a functional group adsorbable to the nanoparticle, and L ２ either does not exist or represents a linker, and n represents an integer of 1 to 5.) The group of polymerization initiator-modified nanoparticles according to claim 5, having a structure represented by .

7. The group of polymerization initiator-modified nanoparticles according to claim 5 or 6, wherein the nanoparticles are metal nanoparticles.

8. The group of polymerization initiator-modified nanoparticles according to claim 5 or 6, wherein the polymerization initiator is a living polymerization initiator.

9. A method for producing polymer chain-modified nanoparticles, comprising the step of using the polymerization initiator group-modified nanoparticles described in claim 5 or 6, and polymerizing monomer components starting from each polymerization initiator group possessed by the polymerization initiator group-modified nanoparticles to obtain a polymer chain.

10. A method for producing a group of polymerization initiator-modified nanoparticles according to claim 5 or 6, A surface modification step involves using a first molecule, which is a single-stranded nucleic acid with a polymerization initiator group attached to one end, or a second molecule, which is a single-stranded nucleic acid, as a modification molecule, and immobilizing the other end of the first molecule or one end of the second molecule on the surface of a nanoparticle. The process includes a selection step of selecting nanoparticles on which a desired number of the modified molecules are immobilized by electrophoresis, A method for producing a group of polymerization initiator-modified nanoparticles, wherein, when the second molecule is used in the surface modification step, the method further includes a polymerization initiator bonding step after the selection step, in which a polymerization initiator is bonded to the other end of the second molecule.

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