JP2003327545A - Carrier for captured substance, complex of carrier and captured substance, method of introducing captured substance and adsorbent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a carrier for captured substance having a high safety, capable of producing in an industrial scale, effectively passing the captured substance through a membrane in a simple operation, and to provide a complex of the carrier and the captured substance obtained by linking the captured substance to the carrier for captured substance, a method of introduction for the captured substance using the carrier for the captured substance, and an adsorbent having adsorbing function to component of the membrane. <P>SOLUTION: The carrier forms the complex with the captured substance to assist the captured substance to pass through the membrane, comprises more than one cyclic molecule having functional groups as substituents bonding with the captured substance, a linear molecule holding the more than one cyclic molecules in a penetrating state and bulky substituents capping both ends of the linear molecules. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a trapped substance carrier that assists the trapped substance to pass through a membrane, particularly a trapped substance carrier suitable for gene transfer into cells, and a trapped substance bound to the trapped substance carrier. The present invention also relates to a trapped substance carrier complex, a trapped substance introduction method using the trapped substance carrier, and an adsorbent having a function of adsorbing a membrane component and the like.

[0002]

2. Description of the Related Art Recently, attempts have been actively made to effectively use macromolecules such as genes as drugs. But,
Even if these macromolecules are directly administered to a living body or a cell, they are decomposed by an enzyme or the like before being taken up. Therefore, a carrier is required in order to efficiently release the administered macromolecule into the cytoplasm and, in some cases, further deliver it to the nucleus, and various gene carriers have been developed especially in gene transfer. . Currently used gene carriers are classified into viral vectors and non-viral vectors. Adenovirus, retrovirus, Sendai virus and the like are typical examples of viral vectors. The method using a viral vector has merits such as high gene transfer efficiency and enabling constant gene expression, but there is a problem that it cannot be said that safety has been sufficiently studied. On the other hand, as a method using a non-viral vector, in addition to the classical calcium phosphate method and the DEAE dextran method, recently, a method using a polycation and a lipofection method using a cationic lipid have been widely used. The methods using these non-viral vectors are currently widely used because of their high safety, large-scale preparation, and simple procedure. However, it has a problem that the gene transfer efficiency is inferior to that of a viral vector. Therefore, there is a demand for the development of a technique with a higher gene transfer efficiency, which uses a non-viral vector excellent in safety. In addition to the genes, there is a demand for a method of efficiently introducing proteins into cells as drugs.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to solve the various problems in the prior art and achieve the following objects. That is, according to the present invention, a trapped substance carrier that has high safety, can be prepared in a large amount, has a simple procedure, and allows the trapped substance to efficiently pass through the membrane, and the trapped substance is bound to the trapped substance carrier An object of the present invention is to provide a trapped substance carrier complex, a trapped substance introducing method using the trapped substance carrier, and an adsorbent having an adsorption function for a membrane component and the like.

[0004]

Means for Solving the Problems The present inventors have found that a capture substance can be bound by introducing a substituent into a cyclic molecule of a molecular assembly typified by polyrotaxane, and the capture substance to which the capture substance is bound is bound. By administering the capture substance carrier to the living body or cells, the trapped substance can be passed through the membrane. Particularly, when the trapper substance carrier is administered to the cell, the trapped substance enters the cell and is released into the cytoplasm to effectively capture the substance. We obtained the knowledge that it functions as a product carrier. The present invention is based on these findings, and means for solving the above problems are as follows. That is, <1> a plurality of cyclic molecules which are a carrier for a trapping substance that binds to a trapping substance to form a complex and assists the passage of the trapping substance through a membrane, and has a functional group capable of binding to the trapping substance as a substituent. And a linear molecule that can be retained while penetrating the plurality of cyclic molecules, and a bulky substituent that caps both ends of the linear molecule. <2> The trapped substance carrier according to <1>, wherein the membrane is a biological membrane. <3> The capture product carrier according to <2>, wherein the biological membrane is a cell membrane. <4> The capture carrier according to any one of <1> to <3>, which is a polyrotaxane. <5> The capture product is the capture product carrier according to any one of <1> to <4>, which is a nucleic acid. <6> The trapped substance carrier according to any one of <1> to <5>, wherein the functional group is a cationic group under physiological conditions. <7> The capture substance carrier according to <6>, wherein the functional group is a group containing an amino group at the terminal. <8> The trapped substance carrier according to any one of <1 to 7 above, wherein the bulky substituent is bound to a linear molecule so as to be degradable in vivo. <9> The captured substance carrier according to any one of <1> to <8>, wherein a plurality of cyclic molecules can be desorbed, and the desorbed cyclic molecules have an adsorption function. <10> The plurality of cyclic molecules is the trapped substance carrier according to any one of <1> to <9>, which is cyclodextrin. <11> The capture carrier according to <10>, wherein the cyclodextrin is any one of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. <12> The capture molecule carrier according to any one of <1> to <11>, wherein the linear molecule is desorbable, and the desorbed linear molecule interacts with a substance. <13> In any one of <1> to <12>, wherein the linear molecule is polyalkylene glycol, poly (epsilon-lysine), polylactic acid, polyglycolic acid, or a block or random copolymer thereof. It is the capture carrier described. <14> A captured substance carrier complex comprising the captured substance carrier according to any one of <1> to <13> and a captured substance captured by the captured substance carrier. <15> The capture product is bound to the capture product carrier according to any one of <1> to <13>, and the capture product is administered to any of living organisms (excluding humans) and cultured cells. It is a method of introducing a trapped substance, which comprises passing through a membrane in any of cells. <16> A plurality of cyclic molecules, a linear molecule that can be held in a state of penetrating the plurality of cyclic molecules, and a bulky substituent that caps both ends of the linear molecule, Is capable of desorbing in the reaction field, and the desorbed plurality of cyclic molecules have an adsorption function. <17> The desorbed plurality of cyclic molecules are the adsorbent of <16>, which adsorbs to the membrane component.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION (Captured substance carrier) The captured substance carrier of the present invention holds a plurality of cyclic molecules each having a functional group capable of binding to a captured substance as a substituent, and a state of penetrating the plurality of cyclic molecules. There is no particular limitation except that it has a possible linear molecule and a bulky substituent that caps both ends of the linear molecule, and can bind to a cyclic molecule, a linear molecule, a bulky substituent and a trapped substance. The functional group can be appropriately selected from known materials.

The trapped substance carrier of the present invention has a function of assisting the trapped substance to pass through the membrane.
Constituting the trapped substance carrier of the present invention, a plurality of cyclic molecules having a functional group capable of binding to the trapped substance as a substituent, and a linear molecule that can be held in a state of penetrating the plurality of cyclic molecules,
It is considered that any one or some of the following various functions of the structure having a bulky substituent that caps both ends of the linear molecule (hereinafter referred to as a polyrotaxane structure) are overlapped. That is, when the polyrotaxane structure is decomposed and the cyclic molecule is released from the linear molecule and released, the hole of the cyclic molecule blocked by the linear molecule is exposed to adsorb the membrane component and improve the permeability of the membrane. Increase. When the linear molecule is amphipathic and has the property of a surfactant, the linear molecule is exposed by the elimination of the cyclic molecule, and the action of the surfactant enhances the permeability of the membrane. Further, the polyrotaxane structure, which was one molecule, is decomposed into many cyclic molecules, so that the osmotic pressure of the reaction field sharply rises and the reaction field is destabilized. At the same time, the binding property between the functional group of the cyclic molecule and the trapped substance is changed from the high polyvalent polyvalent bond in the polyrotaxane structure to the low binding property of the cyclic molecule alone, and the trapped substance is easily released from the cyclic molecule. . In one aspect thereof, the trapped substance carrier of the present invention binds to the trapped substance and enters the cell, and assists the trapped substance to be released into the cytoplasm. In the conventional non-viral gene transfer agents using lipids having polycations, the low transfer efficiency is considered to be due to enzymatic degradation in endosomes. It is considered that one of the issues is to release it. The principle by which the trapped substance carrier of the present invention takes up the trapped substance into cells and reaches the nucleus is not exactly understood, but the trapped substance carrier taken into the endosome together with the trapped substance by endocytosis is It is considered that the functions of the polyrotaxane structure enhance the permeability of the endosome membrane, thereby assisting the release of the trapped substance into the cytoplasm. Since the various functions are considered to be caused by the decomposition of the polyrotaxane structure, the functions are suppressed at the time of administration, and the damage to the cells etc. is suppressed locally and at the same time reaches the cells etc. It does not fill the pores of the cyclic molecule with other lipids before, and exerts a high effect of adsorbing the membrane component when the polyrotaxane structure is decomposed.

Any linear molecule may be used as long as it can hold a plurality of cyclic molecules in a penetrating state, and those which are harmless to cells to be introduced are preferable.
Examples of hydrophilic / hydrophobic polymers are polyalkylene glycols such as polyethylene glycol (PEG) and polypropylene glycol (PPG) and block copolymers thereof, polyamino acids such as poly (epsilon-lysine), polysaccharides, polylactic acid, polyglycol. Examples thereof include fatty acids such as acids and their block or random copolymers. Among these, the point that a bulky substituent can be introduced, the point that it is harmless, and the property as a surfactant,
When the polyrotaxane structure is decomposed and the linear molecule is exposed, it interacts with a substance and contributes to the destabilization of membranes such as endosomes as described above.
G and PPG are particularly preferred.

The bulky substituent may be any as long as it caps both ends of the linear molecule to prevent elimination of the plurality of cyclic molecules, and may be an amino acid, an oligopeptide, or a single molecule. Examples include sugars, oligosaccharides, nucleic acids, fluorescent molecules and the like. Specifically, N-benzyloxycarbonyl-L-phenylalanine, alanine, valine,
Leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, aspartic acid, glutamic acid, glycine, serine, threonine, tyrosine,
Examples thereof include any unit of cysteine, lysine, arginine, and histidine, derivatives thereof, oligopeptides composed of a plurality of units, glucose, galactose, mannose, maltose, and the like, but are not particularly limited.

Of these, bulky substituents include
A biodegradable group that is degradable in vivo to a linear molecule is preferable from the viewpoint of controlling the escape of the cyclic molecule from the linear molecule. That is, as described above, the binding property between the functional group of the cyclic molecule represented by cyclodextrin and the trapped substance is decreased by the decomposition of the polyrotaxane structure and the cyclic molecule becoming a simple substance, and thus the cyclic molecule is linear. At the same time as leaving the molecule, the bond between the cyclic molecule and the trapped substance is weakened, and the trapped substance is easily released. Further, at the same time when the cyclic molecule is released from the linear molecule, the pores of the cyclic molecule are exposed and the linear molecule is exposed to enhance the permeability of the membrane. Further, the effect of osmotic pressure promotes destabilization of the membrane. Therefore,
By controlling the decomposition of bulky substituents in time and space, these functions can be controlled in time and space.

The bulky substituent can be cleaved by a specific protease in the endosome by introducing an enzymatically or non-enzymatically hydrolyzable bond, for example, a protease recognition sequence, or , A method of designing to bind in a pH-dependent manner, and a method of designing to be cleaved by utilizing a redox reaction by using a thiol bond. When the bulky substituent is biodegraded, cyclic molecules are released from the polyrotaxane structure. The analysis includes gel filtration chromatography, reverse phase chromatography, NM
It can be performed by R or the like.

Since the carrier for trapping substances has low flexibility and association property of the molecular chain, when such a bulky substituent that can be decomposed in vivo is used, the hydrophobic association in the molecule prevents access of the enzyme and the like. Enzymes and the like can easily approach the ends without being sterically hindered, and decomposition proceeds.

The plurality of cyclic molecules are not particularly limited as long as they have a functional group capable of binding to a trapped substance, and examples thereof include cyclodextrin (CD), crown ether, cyclofructan and the like. .
Among these, it is easy to introduce a functional group as a substituent, and it has been well investigated that it is nontoxic alone when introduced into a cell, preferably cyclodextrin, and as the cyclodextrin, Examples include cyclodextrins having different glucose unit numbers such as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. Further, as the cyclic molecule, one having a function of adsorbing a lipid membrane by itself (in a state of being desorbed from a linear molecule having a polyrotaxane structure) is preferable. Among these, α-cyclodextrin adsorbs an alkyl chain of a lipid membrane in the ring of cyclodextrin in a simple substance, and β-cyclodextrin adsorbs cholesterol among membrane components in a simple substance. It is known to increase permeability and is particularly preferable as a cyclic molecule.

In the cyclodextrin, the functional group can be introduced by utilizing the hydroxyl group of cyclodextrin. These functional groups may be directly bonded to the hydroxyl group or may be bonded via another functional group. The number of cyclic molecules is not particularly limited as long as it is plural, but it is more than half of the stoichiometric number of cyclic molecules calculated from the molecular weight of the linear molecule PEG, stability of the bond due to polyvalent bond. It is preferable from the viewpoints of obtaining the compound and of having a great influence on the osmotic pressure when decomposed.

The functional group of the trapped substance carrier of the present invention is not particularly limited as long as it can bind the trapped substance.
It can be appropriately selected according to the captured substance. For example,
When the captured substance is a nucleic acid or a gene, a group that is cationic under physiological conditions is used as the functional group, and a group containing an amino group at the terminal is particularly preferable. The group containing an amino group at the terminal includes a primary amino group,
Secondary amino groups, tertiary amino groups, and quaternary ammonium salts, such as aminoethylcarbamoyl (AEC) groups,
Diethylaminoethyl group, histidylethylcarbamoyl group and the like are preferable. Specific examples of the capture carrier having a terminal group containing an amino group as a substituent include a polyrotaxane structure having 175 aminoethylcarbamoyl (AEC) groups in one molecule (175AEC-α / E4-
TYR-Z), a polyrotaxane structure having 11 AECs in the same molecule (11AEC6).
0HEC-α / E4-TYR-Z) and the like. An example of the above-mentioned capture substance carrier having an amino group is schematically shown in FIG. Since the trapped substance carrier of the present invention has a polyrotaxane structure having low flexibility in the skeleton, even if a large number of functional groups are introduced, the intramolecular association property is low, and the trapped substance can be effectively and stably bound. It is excellent as a carrier for captured substances.

The trapped material is not particularly limited,
Examples include virus inhibitors, nucleic acids such as genes, saccharides, contrast agents, and various other drugs.

The trapped substance carrier of the present invention can be administered to, for example, a living body or a cell, and can be mixed with the trapped substance at the time of administration, bound to the trapped substance, and administered. The trapped substance carrier can be suitably used for various tests and tests that require administration of the trapped substance having a large molecular weight to the cytoplasm from the outside by administering to the cultured cells. In addition, the entrapped substance carrier can be suitably used for administration to living organisms including humans, for assisting the drug to pass through various membrane components, and for transporting the drug into cells. The administration method is not particularly limited and can be appropriately selected depending on the purpose. When administered to cultured cells, it is sufficient to administer a trapped substance carrier bound to the trapped substance in the medium, and when administered to a living body, for example,
It can be administered orally, intravenously, intraperitoneally, intranasally, intracutaneously, subcutaneously, intramuscularly, etc. The dosage form, effective dose to be administered depends on the type and composition of entrapped substance, administration method, age of patient and It can be appropriately determined in consideration of weight and the like. Further, it may be prepared by mixing with other carrier for formulation.

(Captured substance carrier complex) The captured substance carrier complex of the present invention is not particularly limited except that it comprises the captured substance carrier of the present invention and the captured substance captured by the captured substance carrier. As described above, the capture agent carrier of the present invention is mixed with the capture agent upon administration to bind to the capture agent,
Although it can be administered, it can also be bound in advance with a capture substance to prepare a capture substance-carrier complex.

(Method for introducing trapped substance) In the method for introducing trapped substance of the present invention, the trapped substance is bound to the carrier for trapped substance of the present invention, and the substance is administered to either living body (except human) or cultured cells. There is no particular limitation except that the trapped substance is passed through the membrane in any of the living body and the cultured cells, preferably the trapped substance is passed through the cell membrane to convey the trapped substance to the cytoplasm, and particularly preferably the gene is transferred. It can be used as a gene transfer method in which a capture product is bound to a capture product carrier and introduced into cells. By the method of introducing a trapped substance of the present invention, a gene released into the cytoplasm can enter the nucleus and be expressed.

(Adsorbent) The adsorbent of the present invention has a bulky structure in which a plurality of cyclic molecules, a linear molecule that can be retained while penetrating the plurality of cyclic molecules, and both ends of the linear molecule are capped. There is no particular limitation except that it has a substituent, the plurality of cyclic molecules can be desorbed in the reaction field, and the plurality of desorbed cyclic molecules have an adsorption function, and the cyclic molecule, the linear molecule The bulky substituent and the bulky substituent can be appropriately selected from known materials, and as the linear molecule and the bulky substituent, those exemplified for the trapped substance carrier can be preferably used. The plurality of cyclic molecules in the adsorbent have an adsorbing function in a desorbed state, and those adsorbing to the membrane component are preferable. Examples of such a cyclic molecule include cyclodextrin, and in particular, α-cyclodextrin that adsorbs an alkyl chain of a lipid membrane in the ring of cyclodextrin in a simple substance, and β that adsorbs cholesterol among membrane components in a simple substance. -Cyclodextrin is preferred. These cyclic molecules may have a functional group as a substituent, and the functional group is not particularly limited, but a substituent capable of binding to various drugs is also preferable. The adsorbent can be suitably used for administration to a living body, cells or the like together with a drug or a drug bound thereto to adsorb a membrane component or the like in a reaction field and increase its permeability.

[0020]

Example 1 As a specific example of the trapped substance carrier of the present invention, the polyrotaxanes shown in Table 1 were synthesized.

[0021]

[Table 1] AEC: aminoethylcarbamoyl HEC: hydroxyethylcarbamoyl
amoyl)

175AEC-α / E4-TYR in Table 1
-Z represents AEC-polyrotaxane, but before the slash (/) in the symbol, information on the penetrating cyclic molecule,
That is, in this example 175 amino ethylcarbamoyl (AEC) group _{(-C (= O) NH 2} CH 2 CH 2 N
H ₂ ) as a functional group on the α-cyclodextrin (α-CD) in the polyrotaxane. Further, after the slash, there is information about the penetrating linear molecule and the bulky substituent to be capped, that is, polyethylene glycol (PE having an average molecular weight of 4,000) capped with benzyloxycarbonyl-L-tyrosine (Z-TYR).
G) is represented. Similarly, 11AEC60HEC-α / E4-
TYR-Z represents 11 AEC groups and 60 hydroxyethylcarbamoyl (HEC) groups (-C (= O) NH ₂
CH ₂ CH ₂ OH) in the α-CD in the polyrotaxane and capped by Z-TYR average molecular weight 4,0.
00 represents PEG.

AEC-polyrotaxanes such as 175AEC-α / E4-TYR-Z were synthesized by the following method.
α-CD is benzyloxycarbonyl-L-tyrosine (Z
The polyrotaxane pierced by the polyethylene glycol (PEG) chain capped with -L-TYR) was prepared by known methods. Briefly, α-CD
The inclusion complex of (Yokohama Bio Research Corporation) and PEG is a saturated aqueous solution of α-CD and α- (3-aminopropyl) -ω- (3-aminopropyl) polyoxyethylene (PEO-BA: Mn = 4. 000 (provided by Sanyo Kasei Co., Ltd.) was prepared by simply mixing the aqueous solutions. Then, Z-L-TYR (Kokusan Kagaku Co., Ltd.) was used as a terminal amino group of the inclusion complex in a DMF solvent with benzotriazole-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide salt (BOP reagent nova biochem). The polyrotaxane skeleton was synthesized by a condensation reaction. The chemical structure is 750M
FT-NMR spectrometer using ¹ H-NMR of Hz (Varian FT-NMR Gemini 750, Palo Alto, USA)
Specified by The number of α-CDs was 4.75 (C ₁ H of α-CDs) and 3.49 (PE by ¹ H-NMR.
G CH ₂ CH ₂ O) was determined by comparing the integrated values of the signals.

Next, in order to introduce the aminoethylcarbamoyl molecule into the polyrotaxane skeleton, the hydroxyl group of α-CDs in the polyrotaxane was converted to 1,1′-carbonyldiimidazole (CDI: Ald) in a DMSO solvent.
A CDI-activated polyrotaxane solution activated by the above method was prepared. This CDI-activated polyrotaxane solution was added dropwise to an excess of ethylenediamine (Wako Pure Chemical Industries, Ltd.) solvent to synthesize AEC-polyrotaxane.

[0025]

[Chemical 1]

The number of AEC groups introduced is the CDI which is the activator.
Adjusted by the amount of. The chemical structure is 750MHz¹
For FT-NMR spectrometer using H-NMR
More specific. AEC number is¹2.7 by H-NMR
0 (CH of ethylenediamine _Two) And 3.49 (PEG
CH_TwoCH_TwoO) to compare the integrated value of the signal
Determined by

11AEC60HEC-α / E4-T
YR-Z was prepared by adding the above CDI-activated polyrotaxane solution dropwise to a mixed solvent (9: 1) of ethylenediamine and aminoethanol (Wako Pure Chemical Industries, Ltd.) to form an AEC group and hydroxyethylcarbamoyl (HEC).
The group was introduced. The chemical structure is 750 MHz ¹ H-NM
Specified by FT-NMR spectrometer using R. AEC numbers were 2.70 (CH _{2 of} ethylenediamine) and 3.49 (CH ₂ C of PEG) by ¹ H-NMR.
H ₂ O) signal and HEC number are 3.01 (CH _{2 of} aminoethanol) and 3.49 (CH ₂ CH of PEG).
₂ O) was determined by comparing the integrated values of the signals.

Example 2 Conditions for forming a complex between the polyrotaxane shown in Table 1 and the plasmid DNA (pDNA) were tested. As pDNA, a plasmid pLuci having luciferase gene (manufactured by pGL3 Promega)
Was used. pLuci (1.2 μg) and 175AEC-
α / E4-TYR-Z was mixed at various charge ratios. That is, mixing was performed by changing the ratio of the number of positive charges of NH ₂ groups of 175AEC-α / E4-TYR-Z to the number of negative charges of pLuci. After culturing for 15 minutes, 0.1 μg / ml EtBr was added, and 1% agarose gel (buffer solution was 0.04 M Tris-ace) was added.
Electrophoresis was performed for 30 minutes with TAE buffer containing 0.001 M EDTA in the tate). 210 AEC-α / E4-
Also for TYR-Z, 175 AEC-α / E4-TY
Tested as RZ. The results of electrophoresis are shown in FIG. It was confirmed that both AEC-polyrotaxane complexes formed a complex with the plasmid DNA pLuci at a charge ratio of 2 or more. Also, 360 AEC-α / E
4-TYR-Z and 505AEC-α / E4-TYR
Similarly, with -Z, the formation of a complex with pLuci was confirmed at a charge ratio of 2 or more (not shown). Furthermore, 11AEC60HEC-α / E4-TY
The same test was performed for RZ. The results are shown in Figure 3. Formation of a complex with pLuci was confirmed at a charge ratio of 1 or more. In addition, all the trapped substance carriers shown in Table 1 were completely dissolved in phosphate buffered saline (PBS).

Example 3 Transfection was carried out using the pDNA / captured substance carrier complex. Add 3T3 cells to 96-well tissue culture plates, 1 per well
0000 seeds were sown. After 4 hours, the medium was replaced with 100 μl / well of medium with or without serum. Next, 10 μl / well of a buffer solution containing 0.32 μg of the complex of the plasmid pLuci having the luciferase gene and the capture carrier (175AEC-α / E4-TYR-Z) was added. As a control, a capture substance carrier that does not form a complex with pLuci was added. After incubating for 4 hours, the cells were washed with phosphate buffered saline, 100 μl / well of serum-containing medium was added, and the cells were further cultured for 20 hours. Then, the cells were solubilized and the luciferase activity was measured using a commercially available kit. In addition, in the case of the above-mentioned medium with serum, DNA-
The cell viability when the capture carrier complex was added was measured. Cell viability was measured by absorbance at 415 nm with the WST cell counting kit (Dojindo).

As a comparison, transfection was performed in the same manner with LipofectAMINE, which is a commercially available transfection reagent, and cell viability was measured. Li
The results of transfection and the measurement of cell viability by pofectAMINE are shown in FIGS.
The results of transfection with -α / E4-TYR-Z and measurement of cell viability are shown in FIGS. 6 and 7.

As shown in FIG. 6, the highest gene transfer efficiency was obtained when 175AEC-α / E4-TYR-Z was used as a trapped carrier and a charge ratio of 2 was used. Further, the presence of serum is desirable as a condition for cell culture, but it is known that transfection efficiency generally decreases in the presence of serum during transfection. However, under the above-mentioned conditions, gene transfer efficiency was not significantly reduced even in the presence of serum. In addition, cell viability was lower than that of polylysine (PLL), which is an existing polycationic polymer (FIG. 7).

An ethidium bromide binding test was carried out in order to further investigate the binding state of the trapped substance carrier of the present invention and DNA. Salmon sperm DNA (manufactured by Nissan Kagaku) was used as the DNA. Salmon sperm DNA and 175AEC-α / E
Mix 4-TYR-Z with various charge ratios
NA-175AEC-α / E4-TYR-Z complex was obtained. 10 μg / ml DNA-175AEC in PBS
-0.5 μg in a solution containing α / E4-TYR-Z complex
/ Ml EtBr addition, fluorescence spectrophotometer FP650
0 (manufactured by JASCO Corporation), relative fluorescence intensity (relative i
ntensity) was measured. The measurement result is shown in FIG. Figure 8
N / P is salmon sperm DNA and 175AEC-α / E
It represents the charge ratio when mixed with 4-TYR-Z. That is,
175AEC-α / E4-TYR-Z against the number (P) of negative charges of the phosphate group of salmon sperm DNA
Represents the number (N) of positive charges of NH ₂ groups in This test result is very similar to the case of a general polycation, and it can be seen that the globularization occurs in which the DNA has a more spherical and compact shape at a charge ratio of about 1. That is, when the DNA is in a coil shape in which the DNA is relatively loosely spread, EtBr can enter between the structures of the DNA, but when the DNA is in a concentrated globule shape, EtBr
It is known that the ethidium bromide binding test correlates with the structure of DNA because tBr cannot penetrate into the structure of DNA. DNA that was coiled with DNA alone
However, it can be seen that when the complex is formed with the trapped substance carrier, the state is close to that of concentrated globules due to the effect that the charge is erased. By taking such a compact shape, it is less susceptible to decomposition by enzymes,
It is thought that the transfection efficiency will be improved.

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, a trapped substance carrier which is highly safe, can be prepared in a large amount, has a simple procedure, and can pass the trapped substance efficiently through the membrane, and the trapped substance on the trapped substance carrier It is possible to provide a trapped substance carrier complex having bound thereto, a method for introducing a trapped substance using the trapped substance carrier, and an adsorbent having a function of adsorbing a membrane component or the like.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of a trapped substance carrier of the present invention having an amino group as a functional group.

FIG. 2 is a diagram showing analysis results by agarose gel electrophoresis in a test for forming a complex of AEC-α / E4-TYR-Z and pLuci.

FIG. 3 11AEC60HEC-α / E4-TYR-Z
FIG. 6 is a diagram showing an analysis result by agarose gel electrophoresis in a formation test of a complex of pLuci with pLuci.

FIG. 4 is a diagram showing the transfection efficiency by LipofectAMINE.

FIG. 5 shows the survival rate after transfection with LipofectAMINE.

FIG. 6 is a diagram showing the transfection efficiency of 175AEC-α / E4-TYR-Z.

FIG. 7 shows the survival rate after transfection with 175AEC-α / E4-TYR-Z.

FIG. 8 is a diagram showing the result of an ethidium bromide binding test.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61K 48/00 B01J 20/26 B01J 20/26 H C12N 15/09 C12N 15/00 A (72) Invention Nobuhiko Yui 4-103 Matsugaoka, Tatsunokuchi-cho, Nomi-gun, Ishikawa Prefecture (72) Inventor Toru Otani 108-1 Yu, Akishima-cho, Tsurugira-cho, Ishikawa-gun, Ishikawa Prefecture 4B024 AA20 CA01 CA09 EA04 GA11 HA17 HA20 4C076 AA95 AA99 BB01 BB13 BB15 BB16 BB21 BB25 CC35 CC47 CC50 EE23 EE24 EE26 EE39 EE48 EE49 FF34 FF63 4C084 AA13 MA52 MA56 MA59 MA65 NA03 ZB332 ZC802 4C086 AA01 AA02 EA16 MA01 MA20 MA04 NA20 ZB33 ZC80 4G066 AC01B AD20B20

Claims (17)

[Claims] 1. A trapping substance carrier that binds to a trapping substance to form a complex and assists passage of the trapping substance through a membrane, and has a plurality of cyclic molecules having as a substituent a functional group capable of binding to the trapping substance. And a linear molecule that can be retained while penetrating the plurality of cyclic molecules, and a bulky substituent that caps both ends of the linear molecule. 2. The entrapped carrier according to claim 1, wherein the membrane is a biological membrane. 3. The captured substance carrier according to claim 2, wherein the biological membrane is a cell membrane. 4. The trapped substance carrier according to claim 1, which is a polyrotaxane. 5. The captured substance carrier according to any one of claims 1 to 4, wherein the captured substance is a nucleic acid. 6. The entrapment carrier according to claim 1, wherein the functional group is a cationic group under physiological conditions. 7. The trapped substance carrier according to claim 6, wherein the functional group is a group containing an amino group at a terminal. 8. The trapped substance carrier according to claim 1, wherein the bulky substituent is bound to a linear molecule so as to be decomposed in vivo. 9. The trapped substance carrier according to claim 1, wherein a plurality of cyclic molecules can be desorbed, and the desorbed cyclic molecules have an adsorption function. 10. The capture carrier according to claim 1, wherein the plurality of cyclic molecules are cyclodextrins. 11. The capture carrier according to claim 10, wherein the cyclodextrin is any one of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. 12. The capture carrier according to claim 1, wherein the linear molecule is desorbable, and the desorbed linear molecule interacts with a substance. 13. The linear molecule according to claim 1, wherein the linear molecule is polyalkylene glycol, poly (epsilon-lysine), polylactic acid, polyglycolic acid, or a block or random copolymer thereof. Capture carrier. 14. A captured substance carrier complex comprising the captured substance carrier according to any one of claims 1 to 13 and a captured substance captured by the captured substance carrier. 15. The captured substance carrier according to any one of claims 1 to 13 is bound with a captured substance and administered to either a living body (excluding human) or a cultured cell, and the living body and the cultured cell. A method for introducing a trapped substance, which comprises passing the membrane through any one of the above. 16. A method comprising: a plurality of cyclic molecules; a linear molecule that can be retained in a state of penetrating the plurality of cyclic molecules; and a bulky substituent that caps both ends of the linear molecule. An adsorbent characterized in that the plurality of cyclic molecules can be desorbed in a reaction field, and the desorbed plurality of cyclic molecules have an adsorption function. 17. The adsorbent according to claim 16, wherein the plurality of desorbed cyclic molecules are adsorbed on the membrane component.