JP2020068749A - Agent for introduction into three-dimensional cell aggregate - Google Patents

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

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

  Biological tissues are formed by three-dimensionally intercellularly adhering cells, but in recent years, it has become clear that even in cultured cells, the formation of three-dimensional structures often exhibits functions closer to those of living organisms in terms of protein expression level. Along with the establishment of alternative methods for animal experiments, which have been socially demanded, and technological development for forming this three-dimensional structure is competing (Non-Patent Document 1). As a method for evaluating these cell aggregates, changes over time in the size of cell aggregates and invasion assays, analysis of products / amounts from the cell aggregates (to the external fluid), flow cytometer analysis by dispersion of cell aggregates , Confocal laser microscopy observation of fluorescence drying in a cell aggregate. If the size of the three-dimensional cell aggregate can be increased, its application as a tissue model can be further expanded, and such application is expected. However, as the size of the cell aggregate increases, it becomes more difficult for the reagent to diffuse and permeate into the cells inside the aggregate. The level of difficulty of observation is much higher. 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 that cause necrosis increases. Therefore, the reliability of the evaluation and the long-term evaluation should be improved. Hard to do. Therefore, development of a method capable of rapidly delivering a substance or oxygen into the inside of a cell aggregate is eagerly desired.

E. Fennema et al. Trends Biotechno l. 20th, 31, 108-115.J Proceedings of the 66th Symposium on Macromolecules, Title: Creation of new sulfobetaine polymers with upper critical solution temperature (UCST), Presenters: Yoshifumi Oishi, Nobuyuki Morimoto, Masaya Yamamoto

  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.

  The present inventor, as a result of earnest research under the above circumstances, found that when sulfobetaine was added to a cell aggregate in which a large number of cells were aggregated in a three-dimensional direction, the cells migrate to the inside of the cell aggregate. The present inventors have further studied the structure and the like of the sulfobetaine compound based on the above new findings and completed the present invention.

Accordingly, the present invention provides the following sections:
Item 1. An agent for introducing into a three-dimensional cell aggregate containing sulfobetaine.

  Item 2. Item 3. The agent for introducing into a three-dimensional cell aggregate according to Item 1, wherein the sulfobetaine has a structure represented by Formula (1):

[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 is, -C (= O) -O- or -C (= O) shows the -NH-. R ⁴ is

, An imidazolium group, a piperidinium group, or a pyrazolium group (in the formula, R ⁵ is the same or different and represents a C1-C6 alkyl group).
n shows the integer of 0-2. ].

  Item 3. Item 3. The three-dimensional cell aggregate mass introduction agent according to Item 1 or 2, wherein the sulfobetaine has a number average molecular weight of 3,000 to 90,000.

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

  Item 5. Item 3. The agent for introducing into a three-dimensional cell aggregate according to any one of Items 1 to 4, wherein the sulfobetaine exhibits a transfer behavior to liposomes.

Item 6. Including a step of adding sulfobetaine having a target substance bound to a three-dimensional cell aggregate,
A method for transferring a substance into a three-dimensional cell aggregate.

  Item 7. Item 3. The agent for introducing into a three-dimensional cell aggregate according to Item 1, wherein the sulfobetaine has a structure represented by Formula (1):

[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 is, -C (= O) -O- or -C (= O) shows the -NH-. R ⁴ is

, An imidazolium group, a piperidinium group, or a pyrazolium group (in the formula, R ⁵ is the same or different and represents a C1-C6 alkyl group).
n shows the integer of 0-2. ].

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

Confocal laser microscope image of ω-terminal rhodamine-modified P (DMAPS-ran-PEGMA) added to HepG2 cell aggregate in Example 1. (Left) 1 hour after addition (Right) 2 hours after addition Confocal laser microscope image of α-terminal doxorubicin-modified P (PySMAAm) added to HepG2 cell aggregates in Example 2. (Left) 1 hour after addition (Middle) 2 hours after addition (Right) 4 hours after addition

The agent for introducing into a three-dimensional cell aggregate The present invention provides an agent for introducing into a three-dimensional cell aggregate including a compound for introducing into a three-dimensional cell aggregate.

  In the present invention, the agent for introducing into a three-dimensional cell aggregate refers to an agent for introducing a target substance (which may be referred to as a target molecule in the present specification) into the three-dimensional cell aggregate.

  In the present invention, the three-dimensional cell aggregate refers to an aggregate in which cells are aggregated in three-dimensional directions by cell-cell adhesion, unlike single cells, cells proliferating in a monolayer, and the like. When the target substance is transferred to the inside of the three-dimensional cell aggregate, the target substance needs to permeate not only the cell membrane contained in the three-dimensional cell aggregate but also the site of cell-cell adhesion. Therefore, it is generally difficult to introduce the substance into the three-dimensional cell aggregate even if it is a single cell or a substance having the ability to migrate to the inside of cells grown in a monolayer. ..

  The cells constituting the three-dimensional cell aggregate are preferably derived from mammals. Examples of mammals include humans, monkeys, dogs, cats, cows, horses, sheep, pigs, mice, rats, hamsters, guinea pigs and rabbits, with humans being preferred. When mammalian cells are used, somatic cells, germ cells and the like can be mentioned, with somatic cells being preferred. As somatic cells, for example, fibroblasts, epithelial cells, muscle cells, hepatocytes, osteocytes, vascular endothelial cells, cranial nerve cells, mononuclear cells, granulocytes, lymphocytes, osteoblasts, osteoclasts, Examples thereof include pancreatic cells. Further, the three-dimensional cell aggregate may be cells obtained by inducing differentiation of pluripotent stem cells such as iPS cells and ES cells. Further, the three-dimensional cell aggregate may be a mixture of two or more kinds of these cells. The size of the three-dimensional cell aggregate is not particularly limited, and examples thereof include those having a diameter of 50 μm to 1000 μm. In the present invention, when the three-dimensional cell aggregate is not substantially spherical, the above diameter means the longest diameter (longest diameter).

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

[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 is, -C (= O) -O- or -C (= O) shows the -NH-. R ⁴ is

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

  In the present invention, the “C1-6 alkyl group” refers to a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, and examples thereof include methyl, ethyl, n-propyl, isopropyl and n-butyl. , Isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-ethylflorpyr, n-hexyl, isohexyl, 2-ethylputyl and the like.

In 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.

As R ¹ , C1-C6 alkyl is preferable, C1-C3 alkyl is more preferable, and methyl group is more preferable. As R ² , C1-C6 alkyl is preferable, C1-C3 alkyl is more preferable, and methyl group is more preferable.

R ³ is ^* -C (= O) -O- ^** , ^* -OC (= O)- ^** , ^* -C (= O) -NH- ^** , ^* -NH-C (=. O)- ^** , ^* -OC (= O)- ^** , ^* -NH-C (= O)- ^**, etc. are preferable (in the present specification, among the bonds possessed by R ³ The one bonded to the —R ⁴ —CH ₂ —CH ₂ — side is represented by ^* , and the one bonded to the = C (—R ¹ ) — side is represented by ^** ).

As R ⁵ , a C1-C3 alkyl group is preferable, and a methyl group is more preferable. Plural R ^{5 s} may be the same or different, and preferably plural R ^{5 s} are the same.

The imidazolium group represented by R ³ is imidazolium group.

Represents a divalent group obtained by removing 2 hydrogens from. The piperidinium group represented by R ³ is piperidinium.

Represents a divalent group obtained by removing 2 hydrogens from. Further, the pyrazolium group represented by R ³ means pyrazolium

Represents a divalent group obtained by removing 2 hydrogens from. The imidazolium group, piperidinium group, or pyrazolium group has a cation group at a position distant from the polymer main chain (for example, ≡NH ⁺ or = NH ₂ ⁺ moiety is O ⁻ —S (═O) ₂ —CH _{2 - (- CH 2 -) n} -CH 2 -R 4 -C 2 H 4 - moiety and those having a structure of binding) are preferred.

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

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

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

Or Rα′-L ² —C (═O) -L ¹ — (in the formula, L ¹ and L ² are the same or different and each represents a single bond or a linker group, and Rα ′ represents one bond from the target substance). R ⁶ is a monovalent group formed by removing hydrogen, and R ⁶ is the same or different and is a hydrogen atom, a C1-C6 alkyl group (preferably a C1-C4 alkyl group), a cyano group, a phenyl group, a hydroxyl group, an azido group, Indicates an acetylcarbonyl group or an ethoxycarbonyl group.). Formula ^(R ₆₎ 3 -C-

As the group represented by, for example,

And the like (in the formula, Ph represents a phenyl group and Et represents an ethyl group).
Rω is −L ³ −SH,

-S-C (= S) shows the -S-R 'or ^{-L 3 -S-L 4 -Rω'} ( wherein, ^{L 3} and ^{L 4} are the same or different and each represents a single bond or a linker group, R 'is, for example _{-C 12 H 25, -C 2 H} 5 COOH, -CH (CH 3) 2, shows a _{-C 3 H 6 Si (CH 3} ) 3, Rω' is one of hydrogen from the target substance Represents a monovalent group obtained by removing).

The linker group represented by L ¹ , L ² , L ³ and L ⁴ is not particularly limited as long as it can bond the target substance to the structure represented by the above formula (1). For example, when L ¹ is a linker group, as the linker group, —C (—R ⁰ ) ₂ —CH ₂ —, —CH ₂ —C (—R ⁰ ) ₂ —, and —S—C (= S ) —S— and the like (in the formula, R ⁰ is the same or different, and is a hydrogen atom or a C1-C6 alkyl group (the alkyl group is preferably a C1-C3 alkyl group, more preferably a methyl group)). Show.).

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 a carbon atom. A chain linker group composed of at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a hydrogen atom. The number of atoms in the main chain portion of the chain linker group is preferably 1 to 13, more preferably 3 to 11, and most preferably 5 to 10. The linker group, for _{example, -NH-C (= S)} -NH-CH 2 -CH 2 -NH -, - NH-CH 2 -CH 2 -NH-C (= S) -NH -, - S- C (= S) -S- etc. are mentioned.

For example, when ^{L 3} is a linker group, Examples of the linker _{group, - (CH 2) m -} , - (CH 2) m -CH 2 -C (-CN) (- CH 3) -, - C ( _{-CN) (- CH 3) -CH} 2 - (CH 2) m -, - S-C (= S) in -S-, and the like (wherein, m is an integer of 1 to 3 (preferably 1 or 2) is shown).

For example, when ^{L 4} is a linker group, Examples of the linker _{group, -CH 2 (CH 3) -CH} 2 -C (= O) -O-CH 2 -CH 2 -NH-C (= S) - _{NH -, - NH-C (} = S) -NH-CH 2 -CH 2 -O-C (= O) -CH 2 -CH 2 (CH 3) -, - S-C (= S) -S- Etc.
In the present invention, the compound represented by the formula (2) includes any of a block copolymer, a random copolymer and a homopolymer (when y = 0).

  The target substance is not particularly limited, and may be a high molecule or a low molecule. For example, high molecular compounds include nucleic acid molecules such as DNA and RNA (siRNA, shRNA); peptides such as oligopeptides and proteins (antibodies, antibody fragments, enzymes, cytokines, chemokines, receptor polypeptides, etc.), sugar chains Etc. Examples of the low molecule include antibiotics, anticancer agents, antiinflammatory agents, fluorocarbons, lipids, fluorescent dyes and the like. Further, the method for binding these target substances to the compound for introducing into the three-dimensional cell aggregate is not particularly limited, and it can be performed according to a known method.

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

  When the compound for introducing into a three-dimensional cell aggregate, which is the active ingredient of the present invention, forms a salt, the salt includes a salt of an acid addition salt and a base. Specific examples of the acid addition salt include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate and phosphate, oxalate, malonate and succinic acid. Salt, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethane Examples thereof include organic acid salts such as sulfonates, and acidic amino acid salts such as glutamate and aspartate. Specific examples of the salt with a base 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, bases such as lysine and arginine. A salt with a soluble amino acid can be mentioned.

  Since the compound which is the 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 which is the active ingredient of the present invention. ..

  Examples of the solvent forming the solvate include ethanol, alcohols such as propanol, organic acids such as acetic acid, esters such as ethyl acetate, ethers such as tetrahydrofuran and diethyl ether, ketones such as acetone, DMSO and the like. It

  In the present invention, even if the compound for introducing into a three-dimensional cell aggregate, which is the active ingredient of the present invention, or a salt thereof is used as an agent for introducing into a three-dimensional cell aggregate, various additives (for example, an isotonic agent , A chelating agent, a stabilizer, a pH adjuster, an antiseptic, an antioxidant, a solubilizing agent, a thickening agent, etc.).

  In the embodiment of the composition, the content of the compound for introducing into the three-dimensional cell aggregate in the composition or a salt thereof is not particularly limited, and is, for example, 90 in terms of the content of the compound for introducing into the three-dimensional cell aggregate in the composition. It can be appropriately set from conditions such as mass% or more, 70 mass% or more, 50 mass% or more, 30 mass% or more, 10 mass% or more, 5 mass% or more, 1 mass% or more.

  The agent for introducing into a three-dimensional cell aggregate can be used for drug delivery system applications, in addition to evaluation for a three-dimensional cell aggregate. Therefore, by using an active ingredient having a therapeutic and / or prophylactic effect for various diseases as a target substance and using a compound bound to the structure represented by the formula (1) through a linker as the case requires, It can be introduced intracellularly in the patient's body.

  In such an embodiment, the form of the pharmaceutical preparation is not particularly limited and, for example, orally administered agents such as tablets, pills, capsules, powders, granules and syrups; injections (intravenous injection, intramuscular injection, local injection, etc.) ), Gargles, drops, external preparations (ointments, creams, patches, inhalants), parenteral administration agents such as suppositories, and the like. Of the above-mentioned formulation forms, preferred are, for example, orally administered agents (tablets, pills, capsules, powders, granules, syrups, etc.), external preparations (ointments, creams, patches, inhalants), etc. Can be mentioned.

  In the present invention, the dose of the compound for introducing into the three-dimensional cell aggregate or the salt thereof varies depending on the route of administration, age, body weight, symptom of the patient and the like and cannot be specified unconditionally. The daily dose is usually about 5000 mg or less, preferably about 1000 mg or less, more preferably 500 mg or less. The lower limit of the dose of the active ingredient is not particularly limited, and for example, the dose of the active ingredient can be appropriately set in the range of a daily dose for an adult of usually 1 mg or more, preferably 10 mg or more, more preferably 100 mg or more. .. When administered once a day, this amount should be contained in one preparation, and when administered three times a day, one third of this should be contained in one preparation.

  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 and the like.

Method of introducing a target molecule into a three-dimensional cell aggregate, the present invention includes a step of adding a compound for introducing into the three-dimensional cell aggregate into a three-dimensional cell aggregate, the compound for introducing into the three-dimensional cell aggregate. A method of migrating the substance of Regarding the three-dimensional cell aggregate, the target substance, the compound for introducing into the three-dimensional cell aggregate, etc. in the present embodiment, the same ones as those introduced into the three-dimensional cell aggregate can be appropriately adopted. In the method, the compound for introducing into the three-dimensional cell aggregate may be added to cells either in vitro or in vivo. In a preferred embodiment, the adding step can be performed by adding the compound for introducing into the three-dimensional cell aggregate to a suspension of the three-dimensional cell aggregate in a medium, a buffer solution or the like. As the medium, buffer, etc., those which can be used for culturing and adjusting the three-dimensional cell aggregate can be appropriately adopted. Further, the medium, the buffer solution and the like may contain up to 10% serum. In addition, the method of the present invention may include a step of adding the compound for introducing into the three-dimensional cell aggregate to the three-dimensional cell aggregate and then holding the compound. 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 adding 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.

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

Example 1
P (DMAPS-ran-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 purified to a DMAPS concentration of 0.1M. 2- (1-isobutyl) sulfanylthiocarbonylsulfanyl-2-methyl propionic acid which is a chain transfer agent dissolved in water and methanol and an initiator 2-2'-Azobis [2- (2-imidazolin-2-yl) propane ] Were sequentially added. Here, the molar ratio of the monomer to the chain transfer agent and the initiator is [monomer]: [chain transfer agent]: [initiator] = 100: 1: 0.3, and the final solvent composition is pure water: methanol = 2: I set it to 1. The solution was subjected to a polymerization reaction in an oil bath at 60 ° C for 18 hours. It was dialyzed in pure water for 5 days, purified, and lyophilized to obtain a white polymer powder having a number average molecular weight of 17,000.

  P (DMAPS-ran-PEGMA) ω-terminal Rhodamine B modification was carried out by first dissolving P (DMAPS-ran-PEGMA) in 1 M NaCl to 20 mg / ml and adding 100 molar equivalents to the polymer. After n-buthylamine was added and the mixture was stirred at room temperature for 2 h to replace the ω-terminal with a thiol group, 20 mol equivalent of 2-aminoethyl methacrylate hydrochloride was added to the polymer and reacted for 16 h at room temperature. The reaction solution was purified by dialysis in pure water for 3 days using a dialysis membrane. After replacing the solvent with phosphate buffer (pH 7.4), add 1.5 molar equivalents of rhodamine B isothiocyanate to the polymer, and stir at room temperature for 16 h in the dark to allow the reaction to occur and use a dialysis membrane. It was purified by dialysis in pure water for 7 days, and freeze-dried to obtain a ω-terminal rhodamine-modified polymer.

HepG2 cells, a human hepatoma-derived cell line, were seeded at 1250 cells / well in a low protein adsorption 96-well plate for the preparation of three-dimensional cell aggregates at 37 ° C in 5% CO ₂ incubator in DMEM containing 10% FBS. Three-dimensional cell aggregates were prepared by culturing for 4 days. After transferring this three-dimensional cell aggregate to a glass-based dish, the above polymer was added to a final concentration of 0.1 mg / mL, and the transfer behavior of the polymer to the three-dimensional cell aggregate at 37 ° C was measured by a confocal laser scanning microscope. The slice image at was observed. The results are shown in Figure 1. As a result of observation, uptake into the cells existing outside the three-dimensional cell aggregate was observed at the same time as the addition, and migration of the polymer from the outer periphery to 30-50 μm was observed 1 hour after the addition, whereas addition 2 It was confirmed that the polymer reached the center of the three-dimensional cell aggregate after a lapse of time. In addition, the intracellular distribution of the polymer was considered to be distributed mainly in the cytoplasm and mitochondria, avoiding the nucleus.

Example 2
A pyridinium group-containing methacrylamide monomer, [3- (4- (2-methacrylamidoethyl) pyridin-1-ium-1-yl) propane-1-sulfonate], which was synthesized according to a previous report (Non-patent Document 2), was used at a final concentration of 0.1 M. After being dissolved in a 1 M sodium chloride aqueous solution, the pH was adjusted to 7 with sodium hydroxide. Further, 4-[(2-carboxyethylsulfanylthiocarbonyl) sulfanyl-4-cyanopentanoic acid as a chain transfer agent was added to 1 M sodium chloride aqueous solution so as to be 3 mol equivalent to the initiator, and the pH was adjusted to 7. 2,2'-azobis [2- (2-imidazolin-2-yl) propane] (1 molar equivalent) was added as an initiator and reversible addition-fragmentation chain transfer (RAFT) polymerization was carried out at 60 ° C for 4 hours. went. Unreacted monomers were removed using a cellulose dialysis membrane and freeze-dried to obtain poly (sulfobetaine methacrylamide) and P (PySMAAm) powder having a number average molecular weight of 14,000. Next, doxorubicin, which is an anticancer drug, was modified into a carboxy group which is the α-terminal of P (PySMAAm) by a two-step reaction. First, 1 molar equivalent of P (PySMAAm) was dissolved in 1 M NaCl aq., And the condensing agent 4- (4,6-dimethoxy-1,3,5-triazin-2-) was dissolved in pure water in this polymer solution. After adding yl) -4-methylmorpholinium chloride in an amount of 1.5 mol equivalents, 1.2 mol equivalents of doxorubicin and triethylamine were added, and the reaction was carried out overnight in the dark. After purification using a size exclusion column, the desired product was obtained by freeze-drying.

HepG2 cells, a human hepatoma-derived cell line, were seeded at 1250 cells / well in a low protein adsorption 96-well plate for the preparation of three-dimensional cell aggregates at 37 ° C in 5% CO ₂ incubator in DMEM containing 10% FBS. Three-dimensional cell aggregates were prepared by culturing for 4 days. After transferring this three-dimensional cell aggregate to a glass dish for confocal laser scanning microscope observation, the above polymer was added to a final concentration of 0.1 mg / mL, and the polymer was added to the three-dimensional cell aggregate at 37 ° C. The entrapment behavior was observed. The results are shown in Figure 2. As a result of the observation, the uptake into the cells existing outside the three-dimensional cell aggregate was observed at the same time as the addition, and the behavior of shifting to the center of the three-dimensional cell aggregate with time was confirmed.

Claims (7)

An agent for introducing into a three-dimensional cell aggregate containing sulfobetaine. The agent for introducing into a three-dimensional cell aggregate according to claim 1, wherein the sulfobetaine has a structure represented by the formula (1):
[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 is, -C (= O) -O- or -C (= O) shows the -NH-. R ⁴ is
, An imidazolium group, a piperidinium group, or a pyrazolium group (in the formula, R ⁵ is the same or different and represents a C1-C6 alkyl group).
n shows the integer of 0-2. ]. The agent for introducing into a three-dimensional cell aggregate according to claim 1 or 2, wherein the sulfobetaine has a number average molecular weight of 3,000 to 90,000. The agent for introducing into a three-dimensional cell aggregate according to claim 1, wherein the sulfobetaine is transferred to the nucleus and / or mitochondria. The agent for introducing into a three-dimensional cell aggregate according to any one of claims 1 to 4, wherein the sulfobetaine exhibits a transfer behavior to a liposome. Including a step of adding sulfobetaine having a target substance bound to a three-dimensional cell aggregate,
A method for transferring a substance into a three-dimensional cell aggregate. The agent for introducing into a three-dimensional cell aggregate according to claim 1, wherein the sulfobetaine has a structure represented by the formula (1):
[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 is, -C (= O) -O- or -C (= O) shows the -NH-. R ⁴ is
, An imidazolium group, a piperidinium group, or a pyrazolium group (in the formula, R ⁵ is the same or different and represents a C1-C6 alkyl group).
n shows the integer of 0-2. ].