Single-polymer particles, active molecular complex, method for producing single-polymer particles, method for measuring tumor size, method for measuring fine structure within tumor, method for imaging biological tissue, drug delivery system, and contrast agent kit

Abstract

The present invention addresses the problem of providing single-polymer particles having a more precisely controlled hydrodynamic diameter, an active molecular complex, a method for producing single-polymer particles, and a method for imaging biological tissue.　In order to solve the aforementioned problem, provided are single-polymer particles characterized in that each particle is formed from a single polymer and in that the molecular weight distribution Mw / Mn is 1.5 or less. This makes it possible to obtain particles having a more precisely controlled hydrodynamic diameter. The single polymer is preferably a structure composed of a single hydrophilic polymer A or a structure in which a side chain of one or more hydrophilic polymers B is bonded to a main chain composed of a single hydrophilic polymer A.

Description

Single polymer particles, active molecular complexes, methods for producing single polymer particles, methods for measuring tumor size, methods for measuring intratumor microstructures, methods for imaging biological tissues, drug delivery systems, and contrast agent kits

[0001] The present invention relates to particles with precisely controlled hydrodynamic diameters. More specifically, the present invention relates to single-polymer particles made of a single polymer, active molecule complexes, methods for producing single-polymer particles, methods for measuring tumor size, methods for measuring intratumoral microstructures, methods for imaging biological tissues, drug delivery systems, and contrast agent kits.

[0002] When administered into the bloodstream, small molecule drugs, such as anticancer drugs, contrast agents, and nucleic acid drugs, are rapidly excreted by the kidneys because their molecular size is 5 nm or less. Furthermore, due to their small molecular size, small molecule drugs are known to leak out of blood vessels and cause damage to surrounding tissues.

[0003] In these cases, by binding or aggregating small molecules with polymers or aggregating them with lipids to increase the apparent molecular weight, it is possible to avoid excretion from the kidneys and vascular leakage and promote delivery to the target site. This technology of controlling the dynamics in the body by controlling the molecular weight of the drug is called passive targeting technology, and research is being conducted on this technology as a field of DDS (drug delivery system) technology.

[0004] For example, Patent Document 1 describes a nuclear magnetic resonance contrast agent using a polymeric micelle containing a hydrophilic polymer chain segment. In this technology, the molecular weight of a hydrophilic low-molecular-weight contrast agent is controlled by using a polymeric micelle, and the contrast agent is effectively delivered to a target site.

[0005] Furthermore, Non-Patent Document 1 describes that micelles composed of block copolymers have great potential as nanomedicines that can control the distribution and function of bioactive agents such as drugs, proteins, or nucleic acids and effectively overcome biological barriers.

[0006] International Publication No. 2006 / 003731

[0007] Horacio Cabral, et al. , Chem. Rev. 2018, 118, 6844-6892

[0008] However, in the technology of Patent Document 1, because polymeric micelles are aggregates formed from a large number of polymers, it is difficult to accurately control the molecular weight of the particles, and the size of each particle varies. As a result, it is difficult to accurately control their dynamics in the body. Similarly, in the technology of Non-Patent Document 1, it is similarly difficult to accurately control the size of each particle. Furthermore, because each polymeric micelle used in the above technology is an aggregate formed from a large number of polymers, its typical hydrodynamic diameter is relatively large, about 30 nm to 80 nm, and it is technically difficult to produce particles with a small hydrodynamic diameter.

[0009] An object of the present invention is to provide a single polymer particle with a more accurately controlled hydrodynamic diameter, an active molecular complex, a method for producing a single polymer particle, a method for measuring tumor size, a method for measuring the microstructure within a tumor, a method for imaging biological tissue, a drug delivery system, and a contrast agent kit.

[0010] As a result of extensive research into the above-mentioned problems, the present inventors have discovered that particles with precisely controlled hydrodynamic diameters can be obtained by using single polymer particles that are made of a single polymer and have a molecular weight distribution of 1.5 or less, and have completed the present invention.

[0011] That is, the present invention provides the following [1] to

[16] . [1] Each particle is formed from a single polymer, and the molecular weight distribution M of the single polymer is ｗ / M ｎ The single polymer particle is characterized in that the hydrodynamic diameter is 1.5 or less. This makes it possible to obtain particles with accurately controlled hydrodynamic diameters.

[0012] [2] The single polymer particle according to claim 1, wherein the single polymer is a structure consisting of a single hydrophilic polymer A, or a structure in which one or more side chains of hydrophilic polymer B are bonded to a main chain consisting of a single hydrophilic polymer A. This makes it possible to obtain particles with more precisely controlled hydrodynamic diameters.

[0013] [3] The single polymer particle according to claim 1 or 2, wherein the hydrophilic polymer A is a polypeptide, a polysaccharide, a vinyl polymer, a polyether polymer, a polyester polymer, or a polyoxazoline. This allows for easy control of the molecular weight of the single polymer, and as a result, allows for control of the hydrodynamic diameter of the single polymer particle.

[0014] [4] The single polymer particle according to any one of claims 1 to 3, wherein the hydrophilic polymer B is polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, poly(2-methoxyethyl acrylate), polyoxazoline, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, or a carboxyvinyl polymer. This allows for easy control of the molecular weight of the single polymer, and as a result, the hydrodynamic diameter of the single polymer particle.

[0015] [5] The single polymer particle according to any one of claims 1 to 4, characterized in that the number of side chains of the hydrophilic polymer B bonded to the hydrophilic polymer A is 1 or more and 100 or less. This makes it possible to obtain particles with more accurately controlled hydrodynamic diameters.

[0016] [6] The single polymer particle according to any one of claims 1 to 5, characterized in that the molecular weight of the single polymer is 10 kDa or more and 1500 kDa or less, thereby making it possible to obtain particles with more accurately controlled hydrodynamic diameters.

[0017] [7] The single polymer particle according to any one of claims 1 to 6, wherein the hydrophilic polymer A is polyaspartic acid, and the hydrophilic polymer B is polyethylene glycol. This makes it possible to obtain particles with more precisely controlled hydrodynamic diameters.

[0018] [8] An active molecule conjugate, characterized in that an active molecule is bound to the single polymer particle according to any one of claims 1 to 7. This makes it possible to obtain an active molecule conjugate whose dynamics in vivo are controlled.

[0019] [9] The active molecule conjugate according to claim 8, wherein the active molecule is an antibody drug, a nucleic acid drug, a small molecule drug, a radioactive drug, a contrast agent, or a chromophore. This makes it possible to obtain a conjugate of a single polymer particle with an antibody drug, a nucleic acid drug, a small molecule drug, a radioactive drug, a contrast agent, or a chromophore, whose dynamics in vivo are controlled.

[0020]

[10] A method for producing single polymer particles, comprising the steps of: (i) synthesizing a linear hydrophilic polymer A by ring-opening polymerization using an α-amino acid-N-carboxyanhydride as a raw material; and (ii) binding a hydrophilic polymer B to the reaction site of the hydrophilic polymer A. This allows for the production of particles with precisely controlled hydrodynamic diameters.

[0021]

[11] A method for measuring tumor size, comprising measuring tumor size using the single polymer particle according to any one of claims 1 to 7 or the active molecule complex according to claim 8 or 9. This makes it possible to measure tumor size.

[0022]

[12] A method for measuring intratumor microstructure, comprising measuring intratumor microstructure using the single polymer particle according to any one of claims 1 to 7 or the active molecular complex according to claim 8 or 9. This makes it possible to measure intratumor microstructure.

[0023]

[13] A method for imaging biological tissue using the single polymer particle according to any one of claims 1 to 7 or the active molecule complex according to claim 8 or 9. This enables more accurate imaging of biological tissue.

[0024]

[14] The method for imaging biological tissue according to claim 13, wherein the biological tissue is a tumor. This allows for more accurate imaging of the tumor.

[0025]

[15] A drug delivery system for transporting the single polymer particle according to any one of claims 1 to 7 or the active molecule complex according to claim 8 or 9 to a target location in a living body, thereby enabling the single polymer particle or the like to be transported to a target location in a living body.

[0026]

[16] A contrast agent kit comprising active molecular conjugates of different sizes among the active molecular conjugates according to claim 8 or 9. This allows for more accurate understanding of the microstructure of biological tissue.

[0027] According to the present invention, it is possible to provide a single polymer particle with a more controlled hydrodynamic diameter, an active molecular complex, a method for producing a single polymer particle, a method for measuring tumor size, a method for measuring the microstructure within a tumor, a method for imaging biological tissue, a drug delivery system, and a contrast agent kit.

[0028] 1 shows the results of molecular weight distribution analysis by GPC of synthesized PBLA. 2 shows the results of GPC analysis of synthesized PAsp-g-PEG2000, PAsp-g-PEG5000, PAsp-g-PEG12000, and PAsp-g-PEG20000. 3 shows the results of synthesis of single polymer particles with controlled hydrodynamic diameter. 4 shows the results of blood retention test of single polymer particles. 5 shows the hydrodynamic diameter of single polymer particles and pharmacokinetics after intravenous administration. 6 shows the results of relaxivity measurement of a contrast agent bound to a single polymer particle of the present invention and a conventional contrast agent. 7 shows an image obtained with the active molecule complex (contrast agent) of the present invention. 8 shows an image obtained with a conventional contrast agent. 9 shows an image obtained with a contrast agent bound to a single polymer particle of the present invention.

[0029] Hereinafter, embodiments of the single polymer particle, active molecular complex, single polymer particle manufacturing method, and biological tissue imaging method according to the present invention will be described in detail. Note that the single polymer particle, active molecular complex, single polymer particle manufacturing method, and biological tissue imaging method described in the embodiments are merely examples for explaining the present invention, and the present invention is not limited thereto.

[0030] [Definition] As used herein, the term "polymer" refers to a molecule with a large molecular weight, whose structure is composed of a large number of repeating units substantially or conceptually derived from molecules with a small molecular weight, and is also referred to as a polymer molecule. Such polymers are broadly classified into biopolymers and synthetic polymers. Specific biopolymers include, for example, polypeptides formed from amino acid units, polysaccharides formed from monosaccharide units, and polynucleotides formed from nucleic acid units. Specific synthetic polymers include polyester-based polymers formed from polycarboxylic acid and polyalcohol units, vinyl-based polymers obtained by polymerizing vinyl groups (CH2=CH-), ether-based polymers having ether bonds (-C-O-C-) in the main chain, and polyamides formed from polycarboxylic acid and polyamine units.

[0031] In this specification, "single polymer" means a single polymer bonded by a covalent bond, not a complex of multiple molecules formed by hydrogen bonds, hydrophobic bonds, van der Waals forces, etc., and "single polymer particle" means a state in which each single polymer is dispersed in an aqueous solvent without secondary intramolecular association, and exhibits particle properties. The single polymer particle of the present invention does not include complexes formed by multiple polymers, such as micelles. Furthermore, unless otherwise specified, the term "single polymer particle" refers to particles formed by a single polymer. The molecular weight distribution M of the single polymer ｗ / M ｎ When the above expression is used, it indicates the molecular weight distribution of a collection of substantially identical single polymer particles obtained by the same manufacturing method or the like.

[0032] As used herein, "biocompatibility" refers to the property of having affinity with biological tissues and organs and not causing foreign body reactions, rejection reactions, etc. The present invention is primarily intended for use in vivo, and the polymers described above are preferably biocompatible polymers.

[0033] In this specification, the term "molecular weight" refers to a molecular weight measured by a TOF-MS method, or a molecular weight calculated and measured from terminal group protons by an NMR method, or a molecular weight calculated from both.

[0034] As used herein, the term "number average molecular weight (M ｎ ")" refers to the molecular weight calculated from the average (arithmetic mean) of the molecular weights of each polymer contained in the aggregate of synthesized polymers. The number average molecular weight is determined by GPC (gel permeation chromatography).

[0035] In this specification, "weight average molecular weight (M ｗ ")" is the average molecular weight calculated based on the weight fraction, and is an average using "weight (molecular weight x number)" as an index, rather than the number of polymers. The weight average molecular weight is determined by the GPC method.

[0036] In this specification, the term "molecular weight distribution" refers to the degree of distribution of the molecular weight and polymerization degree of a polymer, i.e., the degree of variation, and is determined by the GPC method. ｎ Weight average molecular weight M ｗ The ratio (M ｗ / M ｎ When the molecular weight distribution is 1.0 to 1.5, it is judged to be substantially monodisperse. The molecular weight distribution is determined by the GPC method.

[0037] In this specification, "hydrodynamic diameter" refers to the size of a particle estimated from the speed of particle movement. The hydrodynamic diameter d can be calculated by measuring the diffusion time using fluorescence correlation spectroscopy and using the Einstein-Stokes equation (the following mathematical formula (1)). In this specification, "hydrodynamic diameter" is used to mean the same as "particle size." d = kT / 3πη0D (Mathematical formula (1)) (k: Boltzmann constant, T: absolute temperature, η: viscosity of the solvent, D: translational diffusion coefficient)

[0038] [Single Polymer Particles] The single polymer particles of the present invention are formed from a single polymer, and each particle has a molecular weight distribution M ｗ / M ｎ This makes it possible to obtain particles with accurately controlled hydrodynamic diameters.

[0039] The living body has a mechanism for controlling the dynamics of particles in the body depending on the size of the particles taken in, for example, a mechanism that allows particles below a certain size to pass through and blocks particles above a certain size, but by using the single polymer particles of the present invention, the dynamics of particles in the body can be accurately controlled according to the purpose. The single polymer particles of the present invention can be used, for example, as a contrast agent, to prevent single polymers from leaking from blood vessels by adjusting the size of the single polymer particles of the present invention, to cause renal excretion for early excretion from the body, to avoid renal excretion for retention in the body, to serve as single polymer particles that can be delivered deep into tissues, to image biological tissues, to measure tissue size and visualize tissue microstructures, to use single polymer particles or active molecular complexes bound to a drug of an appropriate hydrodynamic diameter or size for therapeutic purposes, and to transport active molecular complexes to a desired location in the body.

[0040] Specific examples of the above-mentioned uses include the following: ・By binding Gd ions, which are used in contrast agents, to particles, it is possible to efficiently deliver them only to the target site, avoiding damage caused by vascular leakage and toxicity to other parts of the body, such as the brain. ・It is possible to create particles of a size (several to several tens of nanometers) that can be delivered to inflamed areas or deep within cancer tissue, which cannot be delivered using conventional micelles and other particles of a size (several tens to several hundred nanometers), while avoiding renal excretion. ・By using particles with precisely controlled size to visualize the microstructure of tissues and then using particles (active molecular complexes) with an appropriate size bound to a drug for treatment based on the results, effective treatment becomes possible.

[0041] The molecular weight distribution of the single polymer particles of the present invention is 1.5 or less. The upper limit is preferably 1.4 or less, more preferably 1.3 or less, and even more preferably 1.2 or less. By setting the molecular weight distribution of the single polymer particles within the above range, particles with more accurately controlled hydrodynamic diameters can be obtained.

[0042] The hydrodynamic diameter of the single polymer particle of the present invention varies depending on the application of the single polymer particle, but is, for example, 1 to 100 nm. The hydrodynamic diameter of the single polymer particle can be controlled by controlling the molecular weight of the single polymer. The molecular weight of the single polymer can be controlled by controlling the molecular weights of hydrophilic polymer A and hydrophilic polymer B, and / or the number of bonds of hydrophilic polymer B, as described below.

[0043] In addition, in the above-mentioned examples of uses of the single polymer particles, when used as a contrast agent, the hydrodynamic diameter of the single polymer particles of the present invention is preferably 0.5 nm or more, more preferably 1 nm or more.

[0044] When used to prevent leakage from blood vessels, the thickness is preferably 2 nm or more, more preferably 3 nm or more.

[0045] When used for renal excretion in order to expel the compound from the body quickly, the particle size is preferably less than 5 nm.

[0046] When used for purposes of avoiding renal excretion in order to retain a single polymer particle in the living body, the particle size is preferably 5 nm or more, more preferably 6 nm or more.

[0047] When used as a single polymer particle that can be delivered deep into tissue, it is preferable that the diameter is smaller, preferably 100 nm or less, more preferably 30 nm or less.

[0048] The single polymer particle can be a single polymer particle composition containing a plurality of single polymer particles. The single polymer particle composition usually contains a collection of single polymer particles obtained by the same manufacturing method, preferably a collection of single polymer particles each of which is substantially identical. The molecular weight distribution M in the single polymer particle composition ｗ / M ｎ represents the molecular weight distribution of the single polymer particle in the single polymer particle composition. The preferred ranges of each component in the single polymer particle composition are the same as the preferred ranges in the single polymer particle.

[0049] First, each component constituting the single polymer particle will be described in detail below.

[0050] (Single Polymer) The single polymer particle of the present invention is formed from a single polymer. The single polymer is preferably a polymer formed mainly from hydrophilic units, more preferably a polymer formed only from hydrophilic units. The single polymer preferably does not have hydrophobic units. Furthermore, the single polymer preferably has the property of existing as a single polymer chain in an aqueous solvent without secondary association between molecules.

[0051] The single polymer is not particularly limited, but may be a structure consisting of only a single hydrophilic polymer A, or a structure in which a side chain of one or more hydrophilic polymers B is bound to a single hydrophilic polymer A. From the viewpoint of accurately setting the molecular size over a wider range, a structure in which a side chain of one or more hydrophilic polymers B is bound to a single hydrophilic polymer A is preferred.

[0052] When the single polymer is a structure in which one or more side chains of hydrophilic polymer B are bonded to a main chain of a single hydrophilic polymer A, it is preferable that the hydrophilic polymer B has a specific molecular weight. The single polymer structure has, for example, a graft copolymer structure, which is a polymer in which one polymer acts as a backbone and other types of polymers are attached as branches.

[0053] The molecular weight of the single polymer is set appropriately depending on the intended use of the single polymer particle, but is preferably 1 to 2000 kDa. The lower limit is more preferably 2 kDa or more, and even more preferably 5 kDa or more. The upper limit is preferably 1750 kDa or less, and more preferably 1500 kDa or less. By controlling the molecular weight of the single polymer, the hydrodynamic diameter of the single polymer particle can be controlled.

[0054] Specific examples of methods for controlling the molecular weight of a single polymer include synthesis by graft copolymerization, obtaining a polymer with a narrow molecular weight distribution by living polymerization or ring-opening polymerization, or obtaining a fraction with an appropriate molecular weight by fractionation after production.

[0055] <Hydrophilic Polymer A> The hydrophilic polymer A forms the main chain of a single polymer. The hydrophilic polymer A has, for example, a linear or branched structure. A linear structure is preferred. The hydrophilic polymer A is not particularly limited, but is preferably a polymer mainly composed of a linear structure, and more preferably a polymer composed only of a linear structure. Specific examples of the hydrophilic polymer A are not particularly limited as long as they are hydrophilic polymers, but biocompatible polymers are preferred. Examples of such biocompatible polymers include polypeptides, polysaccharides, vinyl polymers, polyether polymers, polyester polymers, polyoxazolines, and polynucleotides. Among these, from the viewpoint of ease of molecular weight control, polypeptides or polyamino acids are preferred, and polyaspartic acid, polyglutamic acid, and polylysine are more preferred.

[0056] The molecular weight distribution of the hydrophilic polymer A is not particularly limited, but is preferably 1.5 or less. The upper limit is more preferably 1.4 or less, even more preferably 1.3 or less, and even more preferably 1.2 or less. By setting the molecular weight distribution of the hydrophilic polymer A within the above range, the molecular weight distribution of the single polymer particle can be more controlled. Since the molecular weight distribution of the single polymer particle is greatly affected by the molecular weight distribution of the hydrophilic polymer A, which is the main chain of the single polymer, it is important to control the molecular weight distribution of the hydrophilic polymer A.

[0057] The molecular weight of the hydrophilic polymer A is not particularly limited, but is, for example, 1 to 100 kDa. The lower limit is preferably 1 kDa or more, more preferably 2 kDa or more. The upper limit is preferably 80 kDa or less, and more preferably 50 kDa or less.

[0058] From the viewpoint of ease of molecular weight control, the hydrophilic polymer A is particularly preferably a polypeptide synthesized by ring-opening polymerization reaction using an α-amino acid-N-carboxyanhydride as a raw material.

[0059] <Hydrophilic Polymer B> The hydrophilic polymer B is bonded as a side chain to the main chain consisting of the hydrophilic polymer A. The molecular weight distribution of the hydrophilic polymer B is preferably 1.5 or less, more preferably 1.4 or less, even more preferably 1.3 or less, still more preferably 1.2 or less, and particularly preferably 1.1 or less. The hydrophilic polymer B is bonded to any of the reaction sites present on the hydrophilic polymer A.

[0060] Specific examples of the hydrophilic polymer B include, but are not limited to, hydrophilic polymers, preferably biocompatible hydrophilic polymers. Examples include polyalkylene glycols (e.g., having 2 to 4 carbon atoms), polyvinyl alcohol (PVA), polyvinylpyrrolidone, polyhydroxyethyl methacrylate (PHEMA), poly(2-methoxyethyl acrylate) (PMEA), polyoxazoline, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, and carboxyvinyl polymers. Among these, polyethylene glycol (PEG) is preferred from the viewpoint of ease of molecular weight control. Specific examples of polyethylene glycol include PEG500, PEG2000, PEG5000, PEG10000, PEG20000, PEG40000, and PEG80000.

[0061] The molecular weight of the hydrophilic polymer B is not particularly limited, but is, for example, 0.01 to 1000 kDa. The lower limit is preferably 0.02 kDa or more, more preferably 0.05 kDa or more, and even more preferably 0.1 kDa or more. The upper limit is preferably 500 kDa or less, more preferably 200 kDa or less, and even more preferably 100 kDa or less.

[0062] The number of hydrophilic polymers B bonded to a single hydrophilic polymer A is not particularly limited, but is, for example, 0 to 200. The lower limit is preferably 0 or more, more preferably 1 or more, and even more preferably 3 or more. The upper limit is preferably 150 or less, more preferably 100 or less, and even more preferably 50 or less.

[0063] [Active Molecule Conjugate] The active molecule conjugate of the present invention is obtained by binding an active molecule to the single polymer particle described above. This allows for an active molecule conjugate whose dynamics in vivo are controlled. Examples of the bond between the active molecule include a covalent bond or an ionic bond. This also includes coordination of a metal ion used as a contrast agent to a chelating functional group in a single polymer. A chelating functional group is a functional group that has an element with a lone electron pair (N, O, P, S, etc.) and forms a strong coordinate bond by donating the lone electron pair to a metal ion.

[0064] (Active molecule) The active molecule is not particularly limited, and any active molecule commonly used in the fields of pharmaceuticals, diagnostic agents, etc. can be used, but preferably an antibody drug, a small molecule drug, a nucleic acid drug, a radioactive drug, a contrast agent, or a chromophore.

[0065] Antibody drugs include, but are not limited to, antibodies that specifically bind to disease-related molecules and are linked to drugs such as small molecule drugs, and examples of such drugs include those targeting cancer and autoimmune diseases. Antibodies used in antibody drugs include, but are not limited to, polyclonal antibodies, monoclonal antibodies, and fragments thereof (e.g., Fab, F(ab)2, etc.). The immunoglobulin class and subclass are also not particularly limited. Antibodies selected from antibody libraries using methods such as phage display may also be used, and conventionally known antibodies can be utilized.

[0066] Although there is no particular limitation, small molecule drugs are mainly synthetic chemical substances with a molecular weight of 500 or less, and examples thereof include anticancer drugs.

[0067] Nucleic acid drugs include, but are not limited to, polynucleotides or oligonucleotides formed from DNA, RNA, or derivatives thereof. Polynucleotides can be single-stranded or double-stranded. Polynucleotides can be sequences that code for proteins or sequences that do not code for proteins.

[0068] Radioactive drugs include technetium ( ９９ｍ Tc), thallium (２０１ Tl), gallium ( ６７ Ga) and indium ( １１１ In), fluorine ( １８ When a radiopharmaceutical is used as the active molecule, the active molecule complex can be subjected to imaging by PET (positron emission tomography) or SPECT (single photon emission computed tomography).

[0069] Examples of contrast agents include gadolinium, manganese, iron, iodine, etc. When a contrast agent is used as the active molecule, the active molecule complex can be subjected to imaging by MRI (magnetic resonance imaging).

[0070] Examples of chromophores include fluorescein isothiocyanate (FITC).

[0071] [Method for producing single polymer particles] The method for producing single polymer particles of the present invention is not particularly limited, and examples include synthesis by polycondensation between an amino acid and a carboxylic acid, solid-phase peptide synthesis, etc. Among the synthesis by polycondensation between an amino acid and a carboxylic acid, ring-opening polymerization reaction using an α-amino acid-N-carboxyanhydride as a raw material is preferred.

[0072] The ring-opening polymerization reaction using α-amino acid-N-carboxyanhydride as a raw material is characterized by comprising the following steps: (i) synthesizing a linear hydrophilic polymer A by ring-opening polymerization using α-amino acid-N-carboxyanhydride as a raw material; and (ii) binding a hydrophilic polymer B to the reaction site of the hydrophilic polymer A. The method for producing single polymer particles of the present invention makes it possible to easily produce particles with a more precisely controlled hydrodynamic diameter and a narrow molecular weight distribution. Furthermore, by more strictly controlling each step of the production method, it is possible to adjust the hydrodynamic diameter of the produced particles in 1 nm increments and produce single polymer particles over a wide molecular weight range.

[0073] Step (i) is a step of synthesizing a linear hydrophilic polymer A by ring-opening polymerization using α-amino acid N-carboxyanhydride (also referred to as "α-amino acid NCA") as a raw material. α-amino acid N-carboxyanhydride or a derivative thereof is used as the raw material. Preferably, from the viewpoint of suppressing side reactions, when the amino acid has a highly reactive functional group in its side chain, the functional group is modified with a protecting group. Specific examples of highly reactive functional groups include carboxyl groups. Examples of raw materials having such protecting groups include benzyl esters of α-amino acid N-carboxyanhydride.

[0074] Step (ii) is a step of bonding a side chain of the hydrophilic polymer B to a reactive site of the main chain formed from the hydrophilic polymer A. Such a reactive site is not particularly limited, but is preferably a highly reactive functional group, specifically a carboxyl group or the like.

[0075] [Method for Imaging Biological Tissue] The method for imaging biological tissue of the present invention is characterized by using the single polymer particle or active molecule complex of the present invention described above, which allows imaging of biological tissue to obtain more detailed information on the tissue structure.

[0076] Furthermore, by imaging biological tissue using single polymer particles with precisely controlled size, i.e., hydrodynamic diameter, it is possible to visualize the microstructure of the tissue. At the same time, information on the hydrodynamic diameter or size that can penetrate deep into the tissue can be obtained. Based on the results, more effective treatment can be achieved by using a drug with an appropriate hydrodynamic diameter or size, or by using a single polymer particle or active molecule complex to which a drug is bound.

[0077] [Method for Measuring Tumor Size] In the method for measuring tumor size of the present invention, a plurality of single polymer particles or active molecular complexes with different sizes, i.e., different hydrodynamic diameters, are used. By imaging a tumor using a plurality of single polymer particles controlled to two or more different hydrodynamic diameters, it is possible to visualize the microstructure of tumor tissue and obtain information about the hydrodynamic diameter or size that can penetrate deep into the tissue. Furthermore, by using a drug with an appropriate hydrodynamic diameter or size depending on the results, or by using a single polymer particle or active molecular complex bound to a drug for treatment, more effective tumor treatment is possible.

[0078] [Method for measuring intratumor microstructure] The method for measuring intratumor microstructure of the present invention is characterized by measuring intratumor microstructure using single polymer particles or active molecule complexes. In this method for measuring microstructure, not only can the size of the entire tumor be measured, but the intratumor microstructure can also be measured based on information on whether or not a single polymer can pass through. Furthermore, based on the results, it is possible to select a drug with an appropriate hydrodynamic diameter or size, and provide effective treatment.

[0079] [Drug delivery system] The drug delivery system of the present invention is characterized by transporting single polymer particles or active molecule complexes to a target location in a living body. As described above, living organisms have mechanisms that control the dynamics of particles in the body depending on the size of the particles taken in, for example, mechanisms that allow particles below a certain size to pass and prevent particles above a certain size from passing. The drug delivery system of the present invention can accurately control the dynamics of particles in the body according to the purpose by controlling the size of the single polymer particles of the present invention.

[0080] [Contrast Agent Kit] The contrast agent kit of the present invention is characterized by containing active molecular conjugates of two or more different sizes, i.e., hydrodynamic diameters. By using a plurality of single polymer particles controlled to have two or more different hydrodynamic diameters, it is possible to obtain information about the hydrodynamic diameter or size that can penetrate deep into tissue. Furthermore, based on the results, it becomes possible to select a drug with an appropriate hydrodynamic diameter or size and perform effective treatment.

[0081] Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples, and various modifications are possible within the technical concept of the present invention.

[0082] (Raw Materials) The raw materials used in the following examples are shown in Table 1. Among the raw materials, n-butylamine, N,N-dimethylformamide, and dichloromethane were used after distillation with calcium hydride.

[0083] 1. Experiment 1: Synthesis of a Main Chain Made of Polyaspartic Acid (1) Synthesis of Polyaspartic Acid (PAsp) A main chain made of polyaspartic acid (PAsp) was synthesized using the following procedure. 1,000 mg of β-benzyl-L-aspartic acid N-carboxylic anhydride (NCA-BLA) was weighed into a round-bottom flask and dissolved in 10 mL of N,N-dimethylformamide (DMF). In a separate round-bottom flask, 360 μL of a 100-fold diluted solution of n-butylamine with dichloromethane and 10 mL of DMF were added and mixed. The entire NCA-BLA solution was added to the n-butylamine solution and stirred for 5 days at 35°C. All reactions were carried out under an argon atmosphere, and all organic solvents used were distilled prior to use. After the reaction, the reaction solution was added dropwise to 400 mL of diethyl ether to precipitate polyβ-benzyl aspartate (PBLA). The precipitate was then collected by suction filtration and dried under reduced pressure. The molecular weight distribution of the resulting PBLA was analyzed by GPC, and the molecular weight and degree of polymerization were measured by NMR. The measurement results are shown in Table 2.

[0084]

[0085] Next, 100 mg of PBLA was suspended in 4 mL of acetonitrile, and 4.9 mL of 0.5 M aqueous sodium hydroxide solution was added. The mixture was allowed to react overnight at room temperature to deprotect the benzyl groups from the PBLA. The reaction solution was then placed in a dialysis membrane (molecular weight cutoff: 6,000-8,000) and dialyzed twice with pure water, twice with 0.01 M hydrochloric acid, and twice with pure water (for at least 2 hours each), followed by lyophilization to obtain polyaspartic acid (PAsp).

[0086] (2) Introduction of Azide Groups into the Side Chain Carboxylic Acid Groups of Polyaspartic Acid To measure the hydrodynamic diameter by fluorescence correlation spectroscopy, azide groups were introduced into the side chain carboxylic acid groups of PAsp as follows: 10 mg of polyaspartic acid was weighed out and dissolved in 2.5 mL of 50 mM sodium bicarbonate. 289.6 μL of azido-PEG4-amine prepared in DMF to a concentration of 30 μM was added, followed by 27 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was then placed in a dialysis membrane (molecular weight cutoff: 6,000-8,000) and dialyzed twice with pure water, three times with 0.01 M aqueous sodium hydroxide, three times with 0.01 M hydrochloric acid, and twice with pure water (for at least 2 hours each). The resulting mixture was freeze-dried to obtain polyaspartic acid (PAsp(N)) with azide groups partially introduced into the side chains. ３ )) was obtained. ３ The introduction rate of the azide group in the hydroxybenzoate was calculated by NMR. The experimental results are shown in Table 3.

[0087]

[0088] 2. Experiment 2. Synthesis of a single polymer (1) PAsp(N ３ Introduction of side chains into PAsp(N) (side chains: PEG2000, PEG5000, PEG12000) ３ ) was introduced as a side chain into the main chain of PAsp(N) by the following procedure to produce a single polymer. ３ ) was weighed out and dissolved in 1 mL of DMF. In a separate sample bottle, methoxy PEG2000, methoxy PEG5000, and methoxy PEG12000 were dissolved in DMF at a concentration of 50 mg / mL, and PAsp(N ３ ) so that there would be two equivalents of PAsp(N ３ ) solution. EDC was then added to the PAsp(N ３ ) was added so that the amount was 5 equivalents relative to the carboxy group of PAsp(N) and stirred for 3 hours. ３The number of PEGs introduced into each PAsp was calculated by measuring the decrease in the peak area of ​​each PEG from its initial value using GPC. The GPC results are shown in Table 4. Hereinafter, PAsp with PEG grafted to its side chains will be referred to as PAsp-g-PEGXX (XX is the molecular weight of the PEG in the side chain).

[0089] (2) PAsp(N ３ Introduction of a side chain into PAsp(N) (side chain: PEG20000) ３ PEG20000 was introduced as a side chain into the main chain consisting of PAsp(N) and a single polymer was produced by the following procedure. ３ 2 mg of each was weighed and dissolved in 1 mL of DMF. PEG20000 was dissolved in DMF at a concentration of 50 mg / mL in a separate sample bottle, and PAsp(N ３ ) so that there would be two equivalents of PAsp(N ３ ) solution. Then, NHS was dissolved in DMF at a concentration of 25 mg / mL, and PAsp(N ３ ), and then EDC was added to PAsp(N ３ ) was added in an amount of 0.95 equivalents relative to the carboxy group of PAsp(N) and stirred for 3 hours. ３ The number of PEGs introduced into each of the PEGs was calculated by measuring the decrease in the peak area of ​​each PEG from its initial value using GPC. The measurement results are shown in Table 4.

[0090]

[0091] After synthesis of each PAsp-g-PEG, fractionation was performed by GPC to remove unreacted PEG, and the reaction solution was placed in a dialysis membrane (molecular weight cutoff: 6,000-8,000). The external solution was dialyzed three times against 10 mM phosphate buffer and three times against pure water, followed by freeze-drying to obtain powdered PAsp-g-PEG.

[0092] (3) Introduction of a fluorescent dye into each PAsp-g-PEG (used for subsequent size measurement by fluorescence correlation spectroscopy (and in vivo kinetics measurement), but not required). For size measurement by fluorescence correlation spectroscopy, a fluorescent dye (sulfo-Cy5-DBCO) was introduced into each PAsp-g-PEG using the following procedure. PAsp-g-PEG was dissolved in pure water at a concentration of 25 mg / mL. Sulfo-Cy5-DBCO was dissolved in DMSO at a concentration of 25 mg / mL, and 1 equivalent of sulfo-Cy5-DBCO was added relative to the azide group in the side chain of PAsp-g-PEG. The mixed solution was frozen at -20°C and thawed in a refrigerator at +4°C three times. Subsequently, unreacted sulfo-Cy5-DBCO was purified using a PD-10 column and recovered by lyophilization.

[0093] 3. Experiment 3 Measurement of the physical properties of PAsp-g-PEG (1) Evaluation of the hydrodynamic diameter of each PAsp-g-PEG Each PAsp-g-PEG labeled with Sulfo-Cy5 was diluted with D-PBS(-) to 2 nM, and the diffusion time was measured using fluorescence correlation spectroscopy. The hydrodynamic diameter was calculated using the Einstein-Stokes equation. Cy5 was used as a standard substance when calculating the diffusion coefficient. The results of the fluorescence correlation spectroscopy measurements are shown in Table 5.

[0094] (2) Molecular weight of each PAsp-g-PEG The molecular weight of the entire single polymer was calculated by adding the molecular weight of the main chain measured by NMR measurement to the molecular weight of PEG measured by TOF-MS and the number of side chains introduced. The results are shown in Table 5.

[0095] (3) Measurement of molecular weight distribution of each PAsp-g-PEG The molecular weight distribution (M ｗ / M ｎ The test results are shown in Table 5 and Figure 2.

[0096]

[0097] As a result of these experiments, we synthesized a single polymer with a narrow molecular weight distribution and a controlled hydrodynamic diameter. The molecular weight distribution data and the GPC results shown in Figure 2 also yielded a single peak, indicating that each PAsp-g-PEG had a narrow molecular weight distribution.

[0098] 4. Experiment 4: Evaluation of blood retention Single polymers with molecular weights controlled between 10 kDa and 750 kDa were synthesized, and blood retention was evaluated using single polymer particles composed of the synthesized single polymers. Poly-L-aspartic acid was synthesized, and single polymers were synthesized by binding any number of PEGs with a molecular weight of 2000 to it using the same procedures as in Experiments 1 to 3 above. The molecular weight was controlled by the number of PEGs bound. The hydrodynamic diameter was evaluated, and blood retention was evaluated using fluorescently labeled polymers.

[0099] As shown in FIG. 3, it was demonstrated that by adjusting the molecular weight, a series of polymers with precisely controlled hydrodynamic diameters in the range of 4 to 25 nm with a 1 nm pitch can be obtained.

[0100] As a result of the blood retention test, as shown in Figure 4, it was confirmed that polymers with a hydrodynamic diameter of 6 nm were quickly eliminated from the blood, while polymers with a hydrodynamic diameter of 19 nm or more exhibited long-term blood retention with a blood half-life of 4 days.

[0101] 5. Experiment 5: Evaluation of the Pharmacokinetics of Single Polymer Particles in Muscular Dystrophy Model Mice Fluorescently labeled single polymer particles were administered intravenously to muscular dystrophy model mice, and their biodistribution was evaluated 48 hours later. Single polymer particles were fluorescently labeled in the same manner as in Experiments 1 and 2, using particles with hydrodynamic diameters of 4, 10, 13, 15, and 22 nm synthesized in Experiment 4. In particular, single polymer particles with a hydrodynamic diameter of 4 nm were evaluated as a model for nucleic acid drugs, as this has a hydrodynamic diameter equivalent to that of siRNA. The experimental results are shown in Figure 5 and Table 6.

[0102]

[0103] As shown in Figure 5 and Table 6, polymers of the same size (hydrodynamic diameter) as nucleic acid drugs accumulated in the kidney, which is upstream of renal excretion, at a high level, whereas polymers of 10 nm or larger significantly reduced their accumulation in the kidney. Furthermore, polymers of 10 nm or larger increased their accumulation in the target muscle tissue, confirming the effectiveness of polymer conjugates.

[0104] 6. Experiment 6 Evaluation of cancer contrast effect in a mouse model with subcutaneous colon cancer transplantation. A DOTA complex was bound to the single polymer with a hydrodynamic diameter of 12 nm synthesized in Experiment 4, and Gd was loaded. The relaxivity at 0.47 T was evaluated and compared with that of the clinically used Gd contrast agent Magnescope (Gd-DOTA). It was administered intravenously to a mouse model with subcutaneous colon cancer transplantation. 、 The contrast effect of each contrast agent on cancer was evaluated using 1T-MRI. The experimental results are shown in Table 7 and Figures 7 and 8. Figure 6 also shows the results of investigating the ratio of the contrast effect of each contrast agent on cancer and muscle (Tumor / muscle ratio).

[0105]

[0106] From the results of the relaxivity measurements shown in Table 7, the relaxivity (r １ ), high positive effect (r １ / r ２ ) was obtained. As shown in Figures 7 and 8, the contrast effect of conventional magnescopes was very limited, but the single-molecular-weight contrast agent increased the contrast intensity of the cancer and enhanced the overall contrast of the cancer. Furthermore, a higher contrast effect (tumor / muscle ratio: approximately 1.5) was observed 24 hours after imaging with the single-molecular-weight contrast agent.

[0107] 7. Experiment 7 Evaluation of the number of side chains introduced, the molecular weight of polymer particles, and the contrast effect. Similar to Experiment 2, PEG2000 was reacted at different concentrations with the PAsp main chain to synthesize five polymers with PEG side chains of 2 to 18 introduced. The molecular weights were measured similar to Experiment 3. Next, a DOTA complex was added to these polymers to load Gd. As in Experiment 6, the relaxivity at 0.47 T was evaluated and compared with that of Magnescope (Gd-DOTA), a clinically used Gd contrast agent. The results are shown in Table 8.

[0108]

[0109] From the relaxivity measurement results shown in Table 8, by appropriately selecting the side chains, the number of introduced side chains, and the molecular weight, it is possible to obtain a relaxivity that is significantly higher by 5.9 to 8.5 times than that of the Magnescope currently used clinically, and an extremely high positive effect (r １ / r ２ Such an appropriate molecular design can provide higher contrast during imaging, and also provides the effect of providing the same level of contrast with a smaller dose.

[0110] 8. Experiment 8: Obtaining precise MRI images of cancer microstructures Using the single polymer particles carrying Gd complexes with a hydrodynamic diameter of 12 nm used in Experiment 6, the cancer contrast effect was evaluated in a patient-derived tumor (PDX) model (a mouse model with subcutaneous colon cancer transplants). In the experiment, the particles were intravenously administered to a mouse model with subcutaneous colon cancer transplants of PDX, and the cancer contrast effect was evaluated 24 hours later.

[0111] As shown in Figure 9, MRI imaging using a contrast agent bound to single polymer particles with a hydrodynamic diameter of 12 nm revealed areas where the contrast agent was abundant and areas where it was not. This indicates that there are areas where single polymer particles with a hydrodynamic diameter of 12 nm can penetrate and areas where they cannot, which is thought to reflect the microstructure of the cancer. The arrows indicate high-signal areas (white areas) where the contrast agent is particularly abundant. The above results suggest that by changing the size of the polymer contrast agent, it is possible to use it as a ruler to measure the microstructure (gaps in cancer tissue). This indicates that it is possible to use nanomedicines of different sizes depending on the measured microstructure. The single polymer particle technology of the present invention suggests that it is possible to select nanomedicines of the optimal size for each cancer.

[0112] The compositions of the present invention can provide single polymer particles with more precisely controlled hydrodynamic diameters, active molecule complexes, methods for producing single polymer particles, and methods for imaging biological tissues.

Claims

1. Each particle is formed from a single polymer. The molecular weight distribution Mw / Mn of the single polymer is 1.5 or less. The aforementioned single polymer exists as a single polymer chain in an aqueous solvent without secondary association between molecules. The single polymer is a structure consisting of a single hydrophilic polymer A composed solely of linear structures, or a structure in which one or more side chains of hydrophilic polymer B are bonded to the main chain of a single hydrophilic polymer A, which is a polymer molecule composed of multiple repetitions of units. The hydrophilic polymer A is a polypeptide, polysaccharide, vinyl polymer, polyether polymer, polyester polymer, or polyoxazoline. A single polymer particle characterized in that the hydrophilic polymer B is polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, poly-2-methoxyethyl acrylate, polyoxazoline, carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, or carboxyvinyl polymer.

2. The single polymer particle according to claim 1, wherein the hydrophilic polymer A and the hydrophilic polymer B are of different types.

3. The single polymer particle according to claim 2, wherein the hydrophilic polymer A is a polypeptide, a polysaccharide, a vinyl polymer, a polyester polymer, or a polyoxazoline.

4. The single polymer particle according to claim 1, wherein the hydrophilic polymer A is a polypeptide or a polyamino acid.

5. A single polymer particle according to any one of claims 1 to 4, characterized in that the number of side chains of the hydrophilic polymer B bonded to the hydrophilic polymer A is one or more and 100 or less.

6. The single polymer particle according to any one of claims 1 to 4, characterized in that the molecular weight of the single polymer is 10 kDa or more and 1500 kDa or less.

7. The hydrophilic polymer A is polyaspartic acid. The single polymer particle according to any one of claims 1 to 4, characterized in that the hydrophilic polymer B is polyethylene glycol.

8. The hydrophilic polymer A is polyaspartic acid, The single polymer particle according to claim 5, characterized in that the hydrophilic polymer B is polyethylene glycol.

9. An active molecule complex characterized in that an active molecule is bound to a single polymer particle according to any one of claims 1 to 4.

10. An active molecule complex characterized in that an active molecule is bound to a single polymer particle as described in Claim 5.

11. An active molecule complex characterized in that an active molecule is bound to a single polymer particle as described in Claim 6.

12. An active molecule complex characterized in that an active molecule is bound to a single polymer particle as described in Claim 7.

13. An active molecule complex characterized in that an active molecule is bound to a single polymer particle as described in Claim 8.

14. The active molecule complex according to claim 9, characterized in that the active molecule is an antibody drug, a nucleic acid drug, a small molecule drug, a radiopharmaceutical, a contrast agent, or a chromophore.

15. A method for producing single polymer particles, wherein each particle is formed from a single polymer, characterized by comprising the following steps. (i) A step of synthesizing a linear hydrophilic polymer A by a ring-opening polymerization reaction using α-amino acid-N-carboxy anhydride as a raw material. (ii) The step of bonding hydrophilic polymer B to the reaction site of hydrophilic polymer A. Here, the single polymer exists as a single polymer chain in an aqueous solvent without secondary association between molecules. The single polymer is a structure in which one or more side chains of hydrophilic polymers B are bonded to a main chain made of a single hydrophilic polymer A. The hydrophilic polymer A is a polypeptide or a polyamino acid. The hydrophilic polymer B is polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, poly-2-methoxyethyl acrylate, polyoxazoline, carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, or carboxyvinyl polymer.

16. A method for measuring tumor size, characterized by measuring the size of a tumor using a single polymer particle as described in any one of claims 1 to 4.

17. A method for measuring tumor size, characterized by measuring the size of the tumor using the active molecule complex described in Claim 9.

18. A method for measuring the microstructure within a tumor, characterized by measuring the microstructure within the tumor using a single polymer particle as described in any one of claims 1 to 4.

19. A method for measuring the microstructure within a tumor, characterized by measuring the microstructure within the tumor using the active molecule complex described in Claim 9.

20. A method for imaging biological tissue, characterized by using a single polymer particle as described in any one of claims 1 to 4.

21. A method for imaging biological tissue, characterized by using the active molecule complex described in Claim 9.

22. The method for imaging biological tissue according to claim 20, characterized in that the above-mentioned biological tissue is a tumor.

23. The method for imaging biological tissue according to claim 21, characterized in that the biological tissue is a tumor.

24. A drug delivery system for transporting the active molecule complex described in claim 9 to a target location in a living organism.

25. A contrast agent kit characterized by comprising multiple active molecule complexes of different sizes as described in claim 9.