Three-dimensional cell aggregate introducing agent

Abstract

To provide means for introducing a target material into a three-dimensional cell aggregate.SOLUTION: An agent for introduction into a three-dimensional cell aggregate contains sulfobetaine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a means for introducing a target substance into a three-dimensional cell aggregate.

Background Art

[0002] Biological tissues are formed by three-dimensional cell-cell adhesion of cells. In recent years, it has been revealed that even cultured cells often exhibit functions closer to those of living organisms in terms of protein expression levels when forming three-dimensional structures. In addition, in combination with the establishment of alternative methods for animal experiments, which are highly demanded socially, the development of technologies for forming such three-dimensional structures has been actively carried out (Non-Patent Document 1). As evaluation methods for these cell aggregates, there are the time-course change in the size of cell aggregates, invasion assay, analysis of products and amounts from cell aggregates (to the external solution), flow cytometer analysis by dispersion of cell aggregates, confocal laser microscope observation of fluorescence distribution in cell aggregates, and the like. If the size of the three-dimensional cell aggregate can be increased, the applications as a tissue model can be further expanded, and such applications are also expected. However, as the size of the cell aggregate increases, the diffusion and penetration of reagents into the cells inside the aggregate become difficult, and thus the difficulty of intracellular assays in the aggregated state of living cells and the observation of cells inside the aggregate by confocal laser microscopy also increases significantly. Furthermore, as a fundamental problem, when the cell aggregate becomes larger than 100 μm in diameter, oxygen and nutrients are not sufficiently supplied, and the number of cells undergoing necrosis increases, making it difficult to perform reliable evaluations over a long period. For this reason, the development of a technique capable of rapidly delivering substances and oxygen into the cell aggregate is eagerly desired.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] The problem to be solved by the present invention is to provide a means for introducing a target substance into a three-dimensional cell aggregate.

Means for Solving the Problems

[0005] As a result of intensive research under the above circumstances, the present inventors have found that when sulfobetaine is added to a cell mass in which a large number of cells are aggregated in a three-dimensional direction, it migrates into the inside of the cell mass. Based on the above new findings, the present inventors further examined the structure of the sulfobetaine compound and completed the present invention.

[0006] Therefore, the present invention provides the following items: Item 1. An agent for introducing into a three-dimensional cell aggregate containing sulfobetaine.

[0007] Item 2. The agent for introducing into a three-dimensional cell aggregate according to Item 1, wherein the sulfobetaine has a structure represented by the formula (1):

[0008]

Chemical formula

[0009] [In the formula, R 1 and R 2 are the same or different and each represents a hydrogen atom or a C1-C6 alkyl group. R 3 represents -C(=O)-O- or -C(=O)-NH-. R 4 is

[0010]

Chemical formula

[0011] represents an imidazolium group, a piperidinium group, or a pyrazolium group (wherein R 5 represents, independently or identically, a C1-C6 alkyl group.). n represents an integer from 0 to 2.].

[0012] Item 3. The introduction agent into the three-dimensional cell aggregate according to Item 1 or 2, wherein the number average molecular weight of the sulfobetaine is 3,000 to 90,000.

[0013] Item 4. The introduction agent into the three-dimensional cell aggregate according to any one of Items 1 to 3, wherein the sulfobetaine migrates to the nucleus and / or mitochondria.

[0014] Item 5. The introduction agent into the three-dimensional cell aggregate according to any one of Items 1 to 4, wherein the sulfobetaine shows a migration behavior to liposomes.

[0015] Item 6. A method for transferring a substance into the interior of a three-dimensional cell aggregate, comprising the step of adding a sulfobetaine bound to a target substance to the three-dimensional cell aggregate. A method for transferring a substance into the interior of a three-dimensional cell aggregate.

[0016] Item 7. The introduction agent into the three-dimensional cell aggregate according to Item 1, wherein the sulfobetaine has a structure represented by the formula (1):

[0017] [Chemical formula]

[0018] [wherein R 1 and R 2 represent, independently or identically, a hydrogen atom or a C1-C6 alkyl group. R 3 represents -C(=O)-O- or -C(=O)-NH-. R 4 represents

[0019] [Chemical formula]

[0020] 、 representing an imidazolium group, a piperidinium group, or a pyrazolium group (wherein R 5 represents, independently or identically, a C1-C6 alkyl group.). n represents an integer from 0 to 2.].

Advantages of the Invention

[0021] According to the present invention, a means for introducing a target substance into a three-dimensional cell aggregate can be provided.

Brief Description of the Drawings

[0022]

Fig. 1

Fig. 2

Modes for Carrying Out the Invention

[0023] Agent for introduction into three-dimensional cell aggregates The present invention provides an agent for introducing into a three-dimensional cell aggregate, which contains a compound for introducing into a three-dimensional cell aggregate.

[0024] In the present invention, the agent for introducing into a three-dimensional cell aggregate refers to an agent for introducing a target substance (sometimes referred to as a target molecule in this specification) into a three-dimensional cell aggregate.

[0025] In the present invention, the three-dimensional cell aggregate refers to a mass in which cells aggregate in a three-dimensional direction by cell-cell adhesion, different from single cells, cells grown in a monolayer, etc. When attempting to transfer a target substance into the interior of a three-dimensional cell aggregate, it is necessary for the target substance to permeate not only the cell membranes contained in the three-dimensional cell aggregate but also the locations of cell-cell adhesion. Therefore, generally, even a substance having the ability to transfer into single cells or the interior of cells grown in a monolayer, it is difficult to introduce the substance into the interior of a three-dimensional cell aggregate.

[0026] As the cells constituting the three-dimensional cell aggregate, those derived from mammals are preferred. Examples of mammals include humans, monkeys, dogs, cats, cows, horses, sheep, pigs, mice, rats, hamsters, guinea pigs, rabbits, etc., and humans are preferred. When using mammalian cells, somatic cells, germ cells, etc. can be mentioned, and somatic cells are preferred. Further, examples of somatic cells include fibroblast cells, epithelial cells, muscle cells, liver cells, bone cells, vascular endothelial cells, brain nerve cells, monocytes, granulocytes, lymphocytes, osteoblasts, osteoclasts, pancreatic cells, etc. Also, the three-dimensional cell aggregate may be cells differentiated from pluripotent stem cells such as iPS cells and ES cells. Further, the three-dimensional cell aggregate may be a mixture of two or more of these cells. The size of the three-dimensional cell aggregate is not particularly limited, and for example, those having a diameter of 50 μm to 1000 μm can be mentioned. In the present invention, when the three-dimensional cell aggregate is not substantially spherical, the above diameter means the major axis (the longest diameter).

[0027] In the present invention, typical examples of the compound for introduction into the three-dimensional cell aggregate include sulfobetaine. Sulfobetaine refers to a compound having a positive charge and a negative charge within the same molecule and having no charge as a whole molecule. Further, examples of the compound for introduction into the three-dimensional cell aggregate include a compound having a structure represented by the following formula (1) or a salt thereof:

[0028]

Chemical formula

[0029] [Wherein, R 1 and R 2 are the same or different and each represents a hydrogen atom or a C1-C6 alkyl group. R 3 represents -C(=O)-O- or -C(=O)-NH-. R 4 represents

[0030]

Chemical formula

[0031] , an imidazolium group, a piperidinium group, or a pyrazolium group (wherein, R 5 are the same or different and each represents a C1-C6 alkyl group.). n represents an integer of 0 to 2.]. In the present invention, the compound having the structure represented by the formula (1) includes any of a block copolymer, a random copolymer, and a homopolymer (when y = 0).

[0032] In the present invention, the "C1-6 alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 2-ethylbutyl, and the like.

[0033] In the formula (1), R 1 and R 2 are the same or different and each represents a hydrogen atom or a C1-C6 alkyl group. A plurality of R 1 may be the same or different, and preferably a plurality of R 1 are the same. A plurality of R 2 may be the same or different, and preferably a plurality of R 2 are the same.

[0034] R 1Preferably, it is C1-C6 alkyl, more preferably C1-C3 alkyl, and even more preferably a methyl group. R 2 Preferably, it is C1-C6 alkyl, more preferably C1-C3 alkyl, and even more preferably a methyl group.

[0035] R 3 is * -C(=O)-O- ** , * -O-C(=O)- ** , * -C(=O)-NH- ** , * -NH-C(=O)- ** and indicates * -O-C(=O)- ** , * -NH-C(=O)- ** etc. are preferred (in this specification, among the bonds that R 3 has, the one bonded to the -R 4 -CH2-CH2- side is represented by * , and the one bonded to the =C(-R 1 )- side is represented by ** ).

[0036] R 5 is preferably a C1-C3 alkyl group, and more preferably a methyl group. A plurality of R 5 may be the same or different, and preferably a plurality of R 5 are the same.

[0037] R 3 The imidazolium group represented by means a divalent group formed by removing two hydrogens from

[0038]

Chemical formula

[0039] Also, the piperidinium group represented by R 3 means a piperidinium

[0040]

Chemical formula

[0041] represents a divalent group formed by removing two hydrogens therefrom. Also, R 3 The pyrazolium group represented by is pyrazolium

[0042] [Chemical formula]

[0043] represents a divalent group formed by removing two hydrogens therefrom. As the imidazolium group, piperidinium group, or pyrazolium group, those having a cationic group at a position far from the polymer main chain (for example, a structure in which a ≡NH + or =NH2 + moiety is bonded to an O - -S(=O)2-CH2-(-CH2-) n -CH2-R 4 -C2H4- moiety) are preferred.

[0044] In formula (1), n represents an integer from 0 to 2, preferably 1 to 2, more preferably 1. In formula (1), x is not particularly limited, but for example, represents an integer from 10 to 350, preferably 10 to 80, more preferably 30 to 70. In formula (1), y is not particularly limited, but for example, represents an integer from 0 to 50, preferably 0 to 10, more preferably 0 to 3. In formula (1), z is not particularly limited, but for example, represents an integer from 2 to 110, preferably 2 to 50, more preferably 20 to 50.

[0045] Examples of the compound having the structure represented by formula (1) include a compound represented by the following formula (2) or a salt thereof:

[0046] [Chemical formula]

[0047] In formula (2), R1 , R 2 , R 3 , R 4 , n, x, y, and z are the same as in formula (1). Rα is HOOC-L 1 -,

[0048]

Chemical formula

[0049] or Rα’-L 2 -C(=O)-L 1 - represents (wherein, L 1 and L 2 are the same or different and represent a single bond or a linker group, and Rα’ represents a monovalent group formed by removing one hydrogen from the target substance. R 6 are the same or different and represent a hydrogen atom, a C1-C6 alkyl group (preferably a C1-C4 alkyl group), a cyano group, a phenyl group, a hydroxyl group, an azide group, an acetylcarbonyl group, or an ethoxycarbonyl group.). The group represented by formula (R 6 )3-C-

[0050]

Chemical formula

[0051] Examples of the group represented by

[0052]

Chemical formula

[0053] include, for example, Rω is -L 3 -SH,

[0054]

Chemical formula

[0055] -S-C(=S)-S-R’ or -L3 -S-L 4 -Rω’ represents (wherein L 3 and L 4 are the same or different and represent a single bond or a linker group, and R’ represents, for example, -C 12 H 25 , -C2H5COOH, -CH(CH3)2, -C3H6Si(CH3)3, and Rω’ represents a monovalent group formed by removing one hydrogen from the target substance.).

[0056] L 1 , L 2 , L 3 and L 4 The linker groups represented by are not particularly limited as long as they can bind the target substance to the structure represented by the aforementioned formula (1). For example, when L 1 is a linker group, examples of such linker groups include -C(-R 0 )2-CH2-, -CH2-C(-R 0 )2-, -S-C(=S)-S- etc. (wherein R 0 is the same or different and represents a hydrogen atom or a C1-C6 alkyl group (preferably a C1-C3 alkyl group as the alkyl group, more preferably a methyl group).).

[0057] For example, when L 2 is a linker group, the linker group may have at least one selected from the group consisting of a C1-C6 alkyl group, an oxo group, and a thioxo group as a substituent, and examples thereof include a chain linker group composed of at least one selected from the group consisting of a carbon atom, an oxygen atom, a nitrogen atom, a sulfur atom, and a hydrogen atom. The number of atoms in the main chain portion of such a chain linker group is preferably 1 to 13, more preferably 3 to 11, and even more preferably 5 to 10. Examples of the linker group include -NH-C(=S)-NH-CH2-CH2-NH-, -NH-CH2-CH2-NH-C(=S)-NH-, -S-C(=S)-S- etc.

[0058] For example, when L 3 is a linker group, the linker group is, for example, -(CH2)m -, -(CH2) m -CH2-C(-CN)(-CH3)-, -C(-CN)(-CH3)-CH2-(CH2) m -, -S-C(=S)-S-, etc. (wherein m represents an integer of 1 to 3 (preferably 1 or 2)).

[0059] For example, when L 4 is a linker group, examples of the linker group include -CH2(CH3)-CH2-C(=O)-O-CH2-CH2-NH-C(=S)-NH-, -NH-C(=S)-NH-CH2-CH2-O-C(=O)-CH2-CH2(CH3)-, -S-C(=S)-S-, etc. In the present invention, the compound represented by formula (2) includes any of block copolymers, random copolymers, and homopolymers (when y = 0).

[0060] The target substance is not particularly limited and may be either a high molecule or a low molecule. For example, as high molecular compounds, nucleic acid molecules such as DNA, RNA (siRNA, shRNA, etc.); peptides such as oligopeptides, proteins (antibodies, antibody fragments, enzymes, cytokines, chemokines, receptor polypeptides, etc.), sugar chains, etc. can be mentioned. Examples of low molecules include, for example, antibiotics, anticancer agents, anti-inflammatory agents, fluorocarbons, lipids, fluorescent dyes, etc. Also, the method for binding these target substances to the compound for introduction into three-dimensional cell aggregates is not particularly limited and can be carried out according to known methods.

[0061] In the present invention, the number average molecular weight of the compound for introduction into three-dimensional cell aggregates is not particularly limited, but can be appropriately set, for example, in the range of 3,000 to 90,000, preferably 3,000 to 20,000, etc. The compound for introduction into three-dimensional cell aggregates (including the compound bound with the target substance) can be a known compound or can be produced based on known methods.

[0062] When the compound for introduction into three-dimensional cell aggregates, which is an active ingredient of the present invention, forms a salt, the salt includes acid addition salts and salts with bases. Specific examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, and phosphate; organic acid salts such as oxalate, malonate, succinate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, and trifluoromethanesulfonate; and acidic amino acid salts such as glutamate and aspartate. Specific examples of salts with bases include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt, or calcium salt; salts with organic bases such as pyridine salt and triethylamine salt; and salts with basic amino acids such as lysine and arginine.

[0063] Since the compound, which is an active ingredient of the present invention, may exist in the form of a hydrate or a solvate, these hydrates and solvates are also included in the compound that is an active ingredient of the present invention.

[0064] Examples of the solvent that forms a solvate include alcohols such as ethanol and propanol, organic acids such as acetic acid, esters such as ethyl acetate, ethers such as tetrahydrofuran and diethyl ether, ketones such as acetone, and DMSO.

[0065] In the present invention, either the compound for introduction into three-dimensional cell aggregates, which is an active ingredient of the present invention, or the salt itself can be used as an agent for introduction into three-dimensional cell aggregates, or it can be used as a composition combined with various additives (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizing agents, thickening agents, etc.).

[0066] In an embodiment of the composition, the content of the compound for introduction into three-dimensional cell aggregates or its salt in the composition is not particularly limited, and can be appropriately set from conditions such as, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc., in terms of the content of the compound for introduction into three-dimensional cell aggregates.

[0067] In addition to being used for evaluating three-dimensional cell aggregates, the agent for introducing into three-dimensional cell aggregates can be used for drug delivery system applications. Therefore, by using a compound in which an active ingredient having a therapeutic and / or prophylactic effect on various diseases is bonded to the structure represented by the formula (1) via a linker as needed as the target substance, the active ingredient can be introduced into cells in the patient's body.

[0068] In such an embodiment, the form of the pharmaceutical preparation is not particularly limited, and examples thereof include oral administration agents such as tablets, pills, capsules, powders, granules, and syrups; parenteral administration agents such as injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, drip infusions, external preparations (ointments, creams, patches, inhalants), and suppositories. Among the above preparation forms, preferred examples include oral administration agents (tablets, pills, capsules, powders, granules, syrups, etc.), external preparations (ointments, creams, patches, inhalants), and the like.

[0069] In the present invention, the dosage of the compound for introducing into three-dimensional cell aggregates or a salt thereof varies depending on the administration route, the age, weight, symptoms, etc. of the patient and cannot be generally defined. However, as the dosage of the active ingredient, the daily dosage for adults is usually about 5000 mg or less, preferably about 1000 mg or less, more preferably 500 mg or less. The lower limit of the dosage of the active ingredient is not particularly limited either. For example, as the dosage of the active ingredient, the daily dosage for adults can be appropriately set in the range of usually 1 mg or more, preferably 10 mg or more, more preferably 100 mg or more. When administered once a day, this amount may be included in one preparation. When administered three times a day, one-third of this amount may be included in one preparation.

[0070] The pharmaceutical composition of the present invention is administered to patients such as mammals. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep, etc.

[0071] Method for introducing a target molecule into a three-dimensional cell aggregate The present invention provides a method for transferring a substance into a three-dimensional cell aggregate, which includes a step of adding a compound for introducing into a three-dimensional cell aggregate to which a target substance is bound. For the three-dimensional cell aggregate, target substance, compound for introducing into a three-dimensional cell aggregate, etc. in this embodiment, those similar to those for introduction into a three-dimensional cell aggregate can be appropriately employed. The compound for introducing into a three-dimensional cell aggregate may be added to cells either in vitro or in vivo. In a preferred embodiment, the addition step can be performed by adding the compound for introducing into a three-dimensional cell aggregate to a suspension of the three-dimensional cell aggregate in a medium, buffer solution, etc. The medium, buffer solution, etc. can be appropriately selected from those that can be used for culturing and adjusting the three-dimensional cell aggregate. The medium, buffer solution, etc. may contain up to 10% serum. Further, the method of the present invention may include a step of holding after adding the compound for introducing into a three-dimensional cell aggregate to the three-dimensional cell aggregate. The time of the holding step is not particularly limited, but can be appropriately set, for example, in the range of 0.5 to 24 hours, preferably 0.5 to 4 hours. The temperature in the addition step and the holding step is not limited, but can be appropriately set in the range of 4 to 37°C, preferably 25 to 37°C.

[0072] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Examples

[0073] Example 1 P(DMAPS-random-PEGMA) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The monomers, [2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS) and poly(ethylene glycol) methacrylate (PEGMA, number average molecular weight Mn = 2,080), were dissolved in pure water to a DMAPS concentration of 0.1 M, and 2-(1-isobutyl) sulfanylthiocarbonylsulfanyl-2-methyl propionic acid, a chain transfer agent dissolved in methanol, and the initiator 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] were sequentially added. Here, the molar ratio of monomer:chain transfer agent:initiator was [monomer]:[chain transfer agent]:[initiator]=100:1:0.3, and the final solvent composition was pure water:methanol = 2:1. This solution was subjected to a polymerization reaction in an oil bath at 60 °C for 18 hours. After dialysis in pure water for 5 days for purification and freeze-drying, a white polymer powder with a number average molecular weight of 17,000 was obtained.

[0074] For the modification of rhodamine B at the ω-terminus of P(DMAPS-random-PEGMA), first, P(DMAPS-random-PEGMA) was dissolved in 1 M NaCl to a concentration of 20 mg / ml, 100 molar equivalents of n-buthylamine were added to the polymer, and the mixture was stirred at room temperature for 2 h to substitute the ω-terminus with a thiol group. Then, 20 molar equivalents of 2-aminoethyl methacrylate hydrochloride were added to the polymer and reacted at room temperature for 16 h. The reaction solution was purified by dialysis in pure water using a dialysis membrane for 3 days. After replacing the solvent with phosphate buffer (pH 7.4), 1.5 molar equivalents of rhodamine B isothiocyanate were added to the polymer, and the reaction was carried out by stirring at room temperature in the dark for 16 h. After purification by dialysis in pure water using a dialysis membrane for 7 days and freeze-drying, an ω-terminus rhodamine-modified polymer was obtained.

[0075] [Chemical formula]

[0076] HepG2 cells, a human liver cancer-derived cell line, were seeded at 1250 cells / well in a 96-well plate with low protein adsorption for preparing three-dimensional cell aggregates, and cultured for 4 days in a 37 °C, 5% CO2 incubator in DMEM containing 10% FBS to prepare three-dimensional cell aggregates. After transferring these three-dimensional cell aggregates to a glass-based dish, the above polymer was added to a final concentration of 0.1 mg / mL, and the migration behavior of the polymer into the three-dimensional cell aggregates at 37 °C was observed using slice images obtained with a confocal laser microscope. The results are shown in Fig. 1. As a result of the observation, uptake into the cells existing outside the three-dimensional cell aggregates was recognized simultaneously with the addition, and migration of the polymer was observed from the outer periphery to 30 - 50 μm after 1 hour of addition, whereas it was confirmed that the polymer had reached the center of the three-dimensional cell aggregates 2 hours after the addition. Also, the intracellular distribution of the polymer was considered to avoid the nucleus and be centered on the cytoplasm and mitochondria.

[0077] Example 2 The pyridinium group-containing methacrylamide monomer, [3-(4-(2-methacrylamidoethyl)pyridin-1-ium-1-yl)propane-1-sulfonate], synthesized according to the previously reported (Non-Patent Document 2), was dissolved in an aqueous 1 M sodium chloride solution to a final concentration of 0.1 M, and then adjusted to pH 7 with sodium hydroxide. Further, 4-[(2-carboxyethylsulfanylthiocarbonyl)sulfanyl-4-cyanopentanoic acid as a chain transfer agent was added to an aqueous 1 M sodium chloride solution so as to be 3 molar equivalents to the initiator, and adjusted to pH 7. As an initiator, 2,2’-azobis[2-(2-imidazolin-2-yl)propane] (1 molar equivalent) was added, and reversible addition-fragmentation chain transfer (RAFT) polymerization was carried out under the conditions of 60 °C for 4 hours. Unreacted monomers were removed using a cellulose dialysis membrane and freeze-dried to obtain poly(sulfobetaine methacrylamide), P(PySMAAm) powder with a number average molecular weight of 14,000. Next, doxorubicin, an anticancer agent, was modified to the carboxy group at the α-terminus of P(PySMAAm) by a two-step reaction. First, 1 molar equivalent of P(PySMAAm) was dissolved in 1 M NaCl aq., and 1.5 molar equivalents of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, a condensing agent dissolved in pure water, was added to this polymer solution. Then, 1.2 molar equivalents of doxorubicin and triethylamine were added, and the reaction was carried out overnight under light shielding. After purification using a size exclusion column, the target product was obtained by freeze-drying.

[0078]

Chemical formula

[0079] HepG2 cells, a human liver cancer-derived cell line, were seeded at 1250 cells / well in a low-protein adsorption 96-well plate for preparing three-dimensional cell aggregates and cultured for 4 days in DMEM containing 10% FBS in a 37°C, 5% CO2 incubator to prepare three-dimensional cell aggregates. After transferring these three-dimensional cell aggregates to a glass dish for confocal laser microscopy observation, the above polymer was added to a final concentration of 0.1 mg / mL, and the uptake behavior of this polymer into the three-dimensional cell aggregates at 37°C was observed. The results are shown in Figure 2. As a result of the observation, uptake into the cells existing outside the three-dimensional cell aggregates was observed simultaneously with the addition, and the behavior of migrating to the center of the three-dimensional cell aggregates was confirmed over time.

Claims

1. An agent for introducing into a three-dimensional cell aggregate containing sulfobetaine, wherein the sulfobetaine has a structure represented by the formula (2), and the number average molecular weight of the sulfobetaine is 3,000 to 90,000: An agent for introducing into a three-dimensional cell aggregate: 【Chemical 1】 [Wherein, R １ and R ２ are the same or different and each represents a hydrogen atom or a C1-C6 alkyl group. R ３ represents -C(=O)-O- or -C(=O)-NH-. R ４ is [Chemical 2] , an imidazolium group, a piperidinium group, or a pyrazolium group (wherein R ５ is the same or different and represents a C1-C6 alkyl group). n represents an integer of 0 to 2. X represents an integer of 10 to 350. Y represents an integer of 0 to 50. Z represents an integer of 2 to 110. Rα is HOOC-L1-, 【Chemical Formula 3】 or Rα'-L2-C(=O)-L1- (wherein L1 and L2 are the same or different and represent a single bond or a linker group, and Rα' represents a monovalent group formed by removing one hydrogen from the target substance. R6 are the same or different and represent a hydrogen atom, a C1-C6 alkyl group, a cyano group, a phenyl group, a hydroxyl group, an azide group, an acetylcarbonyl group or an ethoxycarbonyl group). Rω is -L3-SH, 【Chemical Formula 4】 -S-C(=S)-S-R' or -L3-S-L4-Rω' (wherein L3 and L4 are the same or different and represent a single bond or a linker group, R' represents -C12H25, -C2H5COOH, -CH(CH3)2, or -C3H6Si(CH3)3, and Rω' represents a monovalent group formed by removing one hydrogen from the target substance). However, when Rα is HOOC-L1- or 【Chemical Formula 5】 in the case of, Rω represents L3-S-L4-Rω' (wherein L1, R6, L3, L4, Rω' are as described above).].

2. The agent for introducing into a three-dimensional cell aggregate according to claim 1, wherein the sulfobetaine migrates into the nucleus and / or mitochondria.

3. The agent for introducing into a three-dimensional cell aggregate according to claim 1 or 2, wherein the sulfobetaine exhibits migration behavior to liposomes.

4. A method for transferring a substance into a three-dimensional cell aggregate, comprising the step of adding a sulfobetaine bound to a target substance to the three-dimensional cell aggregate, wherein the sulfobetaine has a structure represented by the formula (2), and the number average molecular weight of the sulfobetaine is 3,000 to 90,000 (however, excluding methods for treating humans and methods for operating on humans): 【Chemical Formula 6】 [Wherein, R １ and R ２ are the same or different and each represents a hydrogen atom or a C1-C6 alkyl group. R ３ represents -C(=O)-O- or -C(=O)-NH-. R ４ is 【Chemical Formula 7】 , an imidazolium group, a piperidinium group, or a pyrazolium group (wherein R ５ is the same as or different from, and represents a C1-C6 alkyl group). n represents an integer of 0 to 2. X represents an integer of 10 to 350. Y represents an integer of 0 to 50. Z represents an integer of 2 to 110. Rα is HOOC-L1-, 【Chemical Formula 8】 or Rα’-L2-C(=O)-L1-, wherein L1 and L2 are the same or different and each represents a single bond or a linker group, and Rα’ represents a monovalent group formed by removing one hydrogen atom from the target substance. R6 are the same or different and each represents a hydrogen atom, a C1-C6 alkyl group, a cyano group, a phenyl group, a hydroxyl group, an azide group, an acetylcarbonyl group or an ethoxycarbonyl group.). Rω represents -L3-SH, 【Chemical Formula 9】 -S-C(=S)-S-R' or -L3-S-L4-Rω', wherein L3 and L4 are the same or different and each represents a single bond or a linker group, R' represents -C12H25, -C2H5COOH, -CH(CH3)2, or -C3H6Si(CH3)3, and Rω' represents a monovalent group formed by removing one hydrogen atom from the target substance.), provided that when Rα is HOOC-L1-, or 【Chemical Formula 10】 in the case of, Rω represents L3-S-L4-Rω' (wherein L1, R6, L3, L4, Rω' are as defined above).].

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