JP4834321B2 - Structure - Google Patents

Description

The invention also relates to a porous body, a lid member, a substance held in the porous body by the lid member, the structure and the structure and a connecting member connecting the porous body and the lid member.

  In recent years, stimuli that exert their functions by changing their shape and physical properties in response to input (environmental changes) in response to external stimuli (for example, light irradiation, electric field application, temperature change, pH change, addition of chemical substances, etc.) There is a lot of research on response materials. The stimulus-responsive material is expected to be applied in many fields in that the function expression can be controlled from the outside using the above characteristics. As one of the application fields of the stimulus responsive material, for example, application to drug delivery technology can be mentioned.

  Conventionally, a general drug is administered into the body by a method such as injection, administration, or application, and reaches a target site while circulating through the body. However, according to these methods, the amount of the drug compound that can reach the target site on the way, is widely diffused in tissues other than the target site, absorbed or decomposed from the digestive tract, etc., and finally reaches the target site The (concentration) is generally much lower than the dose. Therefore, it is necessary to administer more than the necessary amount of drug.

  For such a problem, a method for modifying a site related to absorption / decomposition of a drug compound to a structure different from the chemical structure to be absorbed / degraded without lowering the drug effect, or a method using a drug carrier There is. In particular, a technique for intensively delivering a drug to a target affected area or target substance (for example, an organ, tissue, cell, pathogen, etc.) using a drug carrier is called a drug delivery system (hereinafter referred to as DDS). By using the DDS technique, it is expected that side effects can be reduced by increasing the therapeutic effect of the drug and reducing the dose. Examples of drug carriers currently under investigation include liposomes and lipid microspheres.

  Liposomes are composed of lipid aggregates composed of hydrophilic groups and hydrophobic groups, and can exist stably by forming a spherical shape with a double layer placed inside the hydrophobic group against the external water environment. is there. For example, naturally occurring lipids such as lecithin and cholesterol are dissolved in an organic solvent and diffused in water by sonication or the like to form a liposome having a stable bilayer structure. The drug can be encapsulated in the liposome. It is considered that when the drug is a highly hydrophobic compound, it is held inside the double membrane, and when the drug is highly hydrophilic, it is held in the inner aqueous phase of the liposome double membrane.

  The lipid microsphere is obtained by suspending soybean oil containing a drug in water together with lecithin. Lecithin is present on the surface of the lipid microsphere, and soybean oil containing the drug is encapsulated therein. Preparations in which various anti-inflammatory drugs are encapsulated in such lipid microspheres have been clinically applied. Japanese Patent Application Laid-Open No. 05-221852 discloses a method for dissolving lipid microspheres by dissolving a fat-soluble anticancer agent in a fatty acid ester, and adding a surfactant such as phospholipid and homogenizing to coat the surface of fine particles made of the fatty acid ester. A method of forming is disclosed. However, the structure of the liposome is easily destroyed by contact with a lipid component or protein component present in the blood, and it cannot be said that the liposome has sufficient long-term stability in the body.

  Polyethylene glycol (PEG) -modified liposomes for the purpose of improving stability, polysaccharide-modified liposomes, and the like have been studied, but sufficient stability is often not obtained. Moreover, in order to employ lipid microspheres, the drug needs to be a fat-soluble substance. Furthermore, as in the case of liposomes, there are indications such as low stability in blood and easy migration to the reticuloendothelial system, and further improvements are required as drug carriers.

  Regarding the stability of the drug carrier as described above in blood, a block copolymer composed of polyethylene glycol (hydrophilic site) and polyamino acid (hydrophobic site) is a micelle-like in aqueous solution or phosphate buffer. There is a report that the stability in blood is improved by the technology of encapsulating a drug such as an anticancer drug in the structure by utilizing the formation of a stable structure (Drug Delivery System Vol. 6 (Drug Delivery System Vol. 6). 2), pp77, 1991).

  U.S. Pat. No. 3,854,480 also discloses a drug delivery device that releases a drug at a controlled rate over an extended period of time. This specification describes a core of a matrix (for example, polydimethylsiloxane) in which a drug is dispersed in a film made of a material insoluble in body fluids such as blood (for example, polyethylene and a copolymer of ethylene and vinyl acetate). A technique is disclosed in which a drug is permeated and released from the core part and the membrane by diffusion through the provided structure.

  However, these documents do not disclose a technique for intensively releasing the drug to the affected area. In order to enhance the therapeutic effect of drugs and reduce side effects, in addition to the stability of the body in the blood and the body after administration, the drug can be released where necessary. Development of a drug carrier having a release control function is also desired.

  In response to such a demand, a technique is disclosed in which a compound such as a drug is encapsulated in a silica-based porous material that is stable even in body fluids such as blood, and the release of the compound can be controlled by external stimulation (J. AM). CHEM.Soc., Vol.125, pp4451, 2003). This document discloses mesoporous silica (MCM-41) modified with 2- (propyldisulfyl) ethylamine (average particle size: 200 nm, average pore size: 2.3 nm, hereinafter, silica structure) as a material encapsulating a drug. Has been. Here, the amino group arranged on the surface of the silica structure is obtained by immersing the silica structure in an aqueous solution of compound (ATP, vancomycin) and further adding CdS (average particle diameter: 2.0 nm) modified with thiol acetate thiol. The silica structure is capped with CdS by cross-linking by chemical bonding between the CdS surface and the carboxyl group on the surface of the CdS, and the structure containing the drug is produced. Moreover, as a method for releasing the compound encapsulated in the silica structure, by treating with a reducing agent such as DTT or mercaptoethanol, the SS bond of the aforementioned disulfanyl group is cleaved, and CdS is separated from mesoporous silica. A method is disclosed in which the encapsulated compound can be released to the outside.

  In addition, J.H. Material. In Biomed Mater Res, 51, pp293, (2000), nanoparticles coated with gold using a hydrogel of N-isopropylacrylamide-acrylamide copolymer (NIPAAm-AAm) as a core are in the range of 800 to 1200 nm. It discloses a technique that absorbs infrared light, swells the core NIPAAm-AAm in a photothermal conversion reaction, and breaks the fine particle structure. Furthermore, the possibility of drug release by photoresponse when the drug is suspended or dissolved in NIPAAm-AAm is shown. Near-infrared light having a wavelength of 800 to 1200 nm is useful because it passes through human tissues and is harmless. However, it is difficult to say that NIPAAm-AAm, which is the core material, is sufficient in terms of safety for tissues such as the human body.

According to the above-mentioned method, it is considered that the release of the drug can be controlled by coexisting the silica structure containing the drug in the affected part and the reducing agent, or by using the gold-coated NIPAAm-AAm fine particles. There are still technical problems to be solved regarding the method of making the reducing agent exist only in the affected area and the safety thereof.
JP 05-221852 A US Pat. No. 3,854,480 Drug Delivery System Vol. 6 (2), pp77, 1991 J. et al. AM. CHEM. Soc. Vol. 125, pp4451, 2003 J. et al. Material. Biomed Mater Res, 51, pp293, (2000)

  The present invention has been made to satisfy the demand for the prior art related to a structure capable of controlling the release of a substance such as a drug, and is capable of reliably controlling the release of a substance such as a drug at a predetermined site. Is to provide.

As a result of diligent research, the present inventors have found that the following structures are suitable for solving the above-described problems.
That is, the first form of the structure of the present invention is a structure that holds a compound therein,
The structure is
A porous body, a lid member, and a connecting member for connecting the lid member and the porous body;
The connection part with the lid member of the connection member includes a capturing body that specifically recognizes and captures the lid member,
At least a part of the lid member is made of gold,
The capture body that specifically recognizes and captures the lid member is a capture body having an antibody variable region that specifically recognizes and captures gold .
The second form of the structure of the present invention is a structure that holds a compound therein,
The structure is
A porous body, a lid member, and a connecting member for connecting the lid member and the porous body;
The connection part with the lid member of the connection member includes a capturing body that specifically recognizes and captures the lid member,
At least a part of the lid member is made of silicon oxide;
The capture body that specifically recognizes and captures the lid member is a capture body having an antibody variable region that specifically recognizes and captures silicon oxide .

  One of the effects exhibited by the structure of the present invention is that when holding one or more substances, the surface of the porous body (including the outer surface and the inner surface of the pore), preferably at least the inner surface of the pore and the opening thereof By holding the release substance at the peripheral edge, it is possible to make the most of the characteristics of the porous body having a large surface area per unit volume. Moreover, it becomes possible by using a cover member to suppress that the substance hold | maintained at the porous body diffuses naturally. Further, the stability of the lid member can be further improved by being physically or chemically connected to the surface of the porous body.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the best embodiment according to the present invention will be described in detail with reference to the accompanying drawings. In addition, the essential point regarding unembodiment is mentioned later.
(Structure)
The structure of the present invention holds one or more substances in a releasable manner, and is a biopolymer that physically or chemically connects a porous body, a lid member, and the porous body and the lid member. And a joining member made of an organic compound containing a compound (hereinafter also referred to as “connecting member”).

  The size of the structure is defined according to the size of a porous body, which will be described later, serving as a base material, and can be selected and designed depending on the application. For example, when used in DDS applications, the maximum particle size or particle size is preferably 200 nm or less, and more preferably 50 nm or less for the purpose of transporting to finer capillaries. The lower limit is not particularly limited, but in consideration of handleability, the particle size or the maximum length of the particles is preferably 5 nm or more. The substance is held on at least one selected from the group consisting of the outer surface of the porous body, the pore internal space, and the inner surface of the porous body, ie, the inner wall of the pore. The substance is preferably held on the outer surface or the inner surface, and more preferably at least held on the inner surface and the peripheral edge of the opening of the pore.

(Porous body)
The porous body used for the structure of the present invention has a large surface area, that is, a specific surface area in a unit volume in order to exhibit the effects of the present invention. Furthermore, the pores provided in the porous body are in contact with the environment outside the structure in order to achieve the effects of the present invention, and communicate with the outside from the surface of the structure. The shape of the pores is selected according to the properties of the substance releasably retained in the pores and the external environment, that is, the dispersion or solution in which the substance is suspended or dissolved. It is preferable that the pores penetrate the porous body. The pore diameter is preferably 1 nm to 10 μm, more preferably 50 nm to 1000 nm.

The material of the porous body used in the present invention can be appropriately selected from conventionally known materials as long as it can form the structure of the present invention. Specifically, it comprises any one or more of metals, metal oxides, inorganic semiconductors, organic semiconductors, natural polymer compounds, synthetic polymer compounds, plastics, pulps, (non) woven fabrics, or a composite thereof. be able to. Specific examples of materials include gold, silver, platinum, aluminum, copper and the like. Examples of the metal oxide include silicon oxide, alumina, titanium oxide, zinc oxide, magnetite, ferrite, NbTa composite oxide, WO ₃ , In ₂ O ₃ , InSnO, MoO ₃ , V ₂ O ₅ , SnO ₂ , etc., hydroxy Examples thereof include insoluble inorganic salts such as apatite and calcium phosphate gel. Examples of organic polymer compounds include conventionally known styrene polymerizable monomers such as styrene, α-methylstyrene, β-methylstyrene, conventionally known methacrylic polymerizable monomers such as methyl acrylate and ethyl acrylate, and methylene aliphatic monomers. From conventionally known vinyl esters such as carboxylic acid esters, vinyl acetate and vinyl propionate, conventionally known vinyl ethers such as vinyl methyl ether, vinyl polymerizable monomers such as conventionally known vinyl ketones such as vinyl methyl ketone, etc. An organic polymer compound produced by polymerizing or copolymerizing a polymerizable monomer selected from the group can be exemplified.

  Other films made of plastics such as polyethylene terephthalate (PET), diacetate, tricetate, cellophane, celluloid, polycarbonate, polyimide, polyvinyl chloride, polyvinylidene chloride, polyacrylate, polyethylene, polypropylene, polyester, polyvinyl chloride, polyvinyl alcohol, acetyl Porous polymer film made of cellulose, polycarbonate, nylon, polypropylene, polyethylene, Teflon, wood board, glass board, silicon substrate, cotton, rayon, acrylic, silk, polyester, cloth, fine paper, medium paper, art paper , Bond paper, recycled paper, baryta paper, cast coated paper, cardboard paper, resin coated paper, etc. , But it is not limited thereto.

  Among the above materials, considering the application as a drug carrier such as drug delivery, metal oxidation takes into account the stability in body fluids such as blood, biocompatibility, and the production cost of porous materials. Things are preferred. Examples thereof include silicon oxide, aluminum oxide, iron oxide, and ferrite.

  Among them, when controlling the release of substances held by a structure using light / magnetism, the constituent material of the porous body is a material that absorbs light as a signal or is affected by magnetism. Not desirable. From such a viewpoint, silicon oxide is the most preferable porous material constituting material.

On the other hand, as the shape of the porous body, one having the following structure can be used.
Hollow columnar structure: a porous body in which a large number of cylinders having an arbitrary shape are arranged (FIG. 14 (d))
-Porous structure: a porous body in which a lot of holes of an arbitrary shape are randomly opened (FIG. 14 (a))
-Opal structure: a porous body in which spherical objects are densely deposited (FIG. 14B)
Inverse opal structure: porous body in which the substance / pores are reversed in the opal structure (FIG. 14 (c))
Concave structure: porous body having a plurality of concave holes in the substrate (FIG. 14 (f))
Furthermore, a convex structure (FIG. 14E), a protruding structure (FIG. 14G), a fibrous structure (FIG. 14H), and the like can be given.

  The porous body may be provided on a suitable support substrate or may be only a porous body. In the structure of the present invention, when a supporting base material is used, the supporting base material can be selected from conventionally known materials according to the use. For example, it can be selected from metals, metal oxides, plastics, and composites thereof.

  As a preferable method for forming the porous body, it is preferable to use a material selected from the above and a conventionally known processing method corresponding to the material. Examples of the forming method include photolithography, etching, sand blasting, FIB processing, and the like. Moreover, when providing a porous body in an aluminum material, it is also possible to form by electrochemical methods, such as an anodic oxidation method.

  Further, when the material is silicon oxide, a forming method using a sol-gel method is known. According to this method, it is also possible to produce a porous body having an average pore size: about 2 to 20 nm and an average particle size: about 200 nm. An example of this method is described below. Under acidic conditions, a coating layer is formed using an alkoxysilane solution to which a surfactant is added, reacted at 35 ° C. for 20 hours, and then heated at 80 ° C. for 48 hours. A silicon oxide layer mixed in a network is formed. Next, by removing the surfactant phase mixed in the layer (for example, by heating at 500 ° C. for 6 hours), the region occupied by the surfactant phase has a pore diameter of 1 nm to 1000 nm. A technique for obtaining a porous material layer left as a pore structure can be used. As a method for removing the surfactant from the silicon oxide-surfactant complex produced by the above steps, there may be mentioned, for example, a method of eluting and removing the surfactant by treating with an organic solvent in addition to the heating method described above. Which method is adopted is appropriately selected according to the physical properties of the substrate to be used, such as heat resistance and solvent resistance.

  According to this method, it is also possible to produce a porous body having an average pore size: about 2 to 20 nm and an average particle size: about 200 nm.

(Cover member)
The lid member prevents the compound held by the structure of the present invention from disappearing due to natural diffusion or the like, and receives an input signal such as external light or magnetic field change, so that the lid member is By releasing from the compound, the compound held in the structure is released to the outside of the structure. Furthermore, it is desirable for the lid member to change its temperature at its periphery by being given a light or magnetic field change.

Suitable materials for such a lid member include metals such as gold, silver, copper, platinum, zinc, aluminum, aluminum, silicon, magnetic metals such as iron, cobalt, nickel, and oxides thereof. Is mentioned. Preferably, gold, silver, copper, platinum, or the like that easily converts the absorbed light or magnetic field into heat can be mentioned. In the present invention, gold or silicon oxide is used for at least a part of the lid member .

  The size of the lid member is not particularly limited, but if the pore size of the porous body is extremely small, the substance held in the porous body of the present invention may diffuse from the gap between the pore wall surface and the lid member. is there. Further, when the lid member is significantly larger than the porous body, the lid member cannot sufficiently function as the lid member because the lid member covers the openings of the plurality of pores incompletely. Considering the above-mentioned preferable pore diameter of 1 nm to 10 μm, or considering the diameter of the porous body, the preferable diameter of the lid member is 1 to 100 nm, more preferably 2 to 50 nm.

  Among these, it is most preferable to use gold as a part of the cover material surface layer. For example, it is known that fine particles (particle size: 1 nm to 100 nm) coated with gold using silica as a core member absorb light of 800 nm to 1200 nm and generate heat. By this heat generation, it is possible to remove the bond between the biopolymer compound described later and the lid member.

(Biopolymer compounds)
The biopolymer compound that constitutes the connecting member physically / chemically connects the porous body and the lid member. The biopolymer compound preferably has a binding site to the surface of the porous body and a binding site to the lid member, and more preferably, at least one of these binding sites includes at least a part of the variable region of the antibody. It is configured.

Here, the “antibody” in the present invention is an antibody produced in all vertebrate lymphoid cells and a deletion of amino acids constituting the antibody within a range capable of maintaining a desired function as an antibody. It is a protein that has a substituted or added amino acid sequence and maintains a relationship in structure / function with the original antibody. The binding site of the connecting member, which is a part having binding properties to the porous body surface and / or the lid member surface, may be an antibody fragment or domain and a part thereof (hereinafter referred to as “antibody part”). . Examples of antibody moieties that can be binding sites include variable regions V _H or V _L , or complexes thereof such as single chain Fv in which V _H and V _H and V _L are linked via a peptide consisting of several amino acids. ScFv (ie, single chain Fv), and some of them.

When gold is contained in at least a part of the surface of the lid member and the connecting member has a binding site that binds to the gold portion, the connecting member includes a gold-binding protein. Good. The gold-binding protein may include an antibody portion, and may particularly include and include Fab ′, (Fab ′) ² , Fd and Fv, or a part thereof.

When scFv is used for the construction of the connecting member, it is desirable to provide a linker composed of one or more amino acids between V _H and V _L (in random order) forming scFv. It is important that the residue length of the amino acid linker is designed so as not to prevent formation of a structure necessary for binding of V _H or V _L to the antigen. The amino acid linker length is generally 5 to 18 residues, and 15 residues are most frequently used and studied.

  The components of the connecting member as described above can be obtained by genetic engineering techniques.

  Further, the binding site of the biopolymer compound to the surface of the porous body and the binding site to the surface of the lid member may be such that both binding sites are antibody portions, one is an antibody portion and the other is 5 or more amino acids. The peptide chain may be a peptide chain consisting of 5 or more amino acids. Hereinafter, the antibody domain that constitutes the antibody portion that is the binding site to the lid member surface is referred to as a first domain, and the antibody domain that constitutes the antibody portion that is the binding site to the porous body surface is referred to as a second domain.

The case of an antibody domain is schematically shown in FIGS. 4A to 4D. In each figure, the first domain is 1, and the second domain is 2. 4A shows the case where both the first and second domains are V _H , FIG. 4B shows the case where the first domain is V _H and the second domain is _VL , and FIG. 4C shows that the first domain is _VL and the second domain is In the case of _VH , and FIG. 4D is the case where both the first and second domains are _VL . It is desirable that the first domain and the second domain do not form a complementary binding site. Even if the connecting member is composed of a constituent part other than the polypeptide chain between the first domain and the second domain, both domains are connected continuously or directly with an intervening peptide chain. Also good. A structure in which the polypeptide chains are continuously connected by a polypeptide chain is preferable in terms of expression of each function and simplification of the production process. The intervening peptide chain may be a linker composed of one or more amino acids. The linker is preferably 1 to 10 amino acids. More preferably, it consists of 1 to 5 amino acids. When the amino acid length of the linker is 11 to 15, both domains increase the degree of freedom of arrangement, and therefore, a complementary bond is formed (scFv) between the domains, and the lid member and the porous body may not be bonded. is there. Further, as for the form, it may have a secondary structure that is designed so as to exhibit a shape that allows the target substances to which each domain binds to easily bind to each other.

The biopolymer compound composed of the first and second domains further has another domain including at least a part of V _H and / or at least a part of V _L , that is, a third domain and / or a fourth domain. It may be a thing. The third domain forms a complex with the first domain, and the fourth domain forms a complex with the second domain. For example, when the first domain is V _H , the third domain is V _L that can form Fv with the first domain, and the formed complex is associated with the first domain part and the third domain part. To form a binding site with the surface of the lid member. When the second domain is V _L , the fourth domain is V _H that can form Fv with the second domain, and the formed complex is associated with the second domain part and the fourth domain part. To form a binding site with the surface of the porous body.

  5, 6, 7, 11, 12 and 13 show that the first domain 1 and the third domain 3 form a complex, and the second domain 4 and the fourth domain 4 form a complex. The situation is shown schematically in FIGS. 8, 9, 10, 11, 12, and 13, respectively. By forming a complex, it can be expected to be structurally more stable and suppress functional deterioration due to structural changes. More preferably, such composites are combined to form a binding site with the lid member surface or the porous body surface. This is because, by forming a binding site in association with each other, it can be expected to improve the binding ability such as improvement of the binding rate and suppression of the dissociation rate. As described above, the configuration can be appropriately determined so that the binding ability of each domain and domain complex is exhibited on the surface of the porous material and the surface of the lid member.

  The schematic of FIG. 7, comprising a polypeptide chain consisting of a first domain and a second domain and a polypeptide chain consisting of a first domain and a third domain, wherein the first domain and the third domain form a complex. A configuration as shown in the figure is also possible. In this configuration, the Fv or Fv-like complex formed from the first domain and the third domain binds to the surface of the lid member, and the two second domains bind to the porous body, thereby anchoring the lid member ( anchor biting the porous body). Similarly, a polypeptide chain composed of a first domain and a second domain and a polypeptide chain composed of a first domain and a fourth domain, wherein the second domain and the fourth domain form a complex, A configuration as shown in FIG. 10 is also possible. In this configuration, the Fv or Fv-like complex formed from the second domain and the fourth domain is bonded to the surface of the porous body, and the two first domains are bonded to the lid member, thereby fixing the porous body anchor. It functions as (anchor biting the lid member).

  The pair of domains forming the complex may be provided as independent polypeptide chains, or may be a polypeptide chain that is linked directly or by being linked by a linker. The linker that connects the domains of the complex has 1 to 10, more preferably 1 to 5 amino acids for the same reason as the above-described linker that connects both domains having a binding site to the lid member and the porous body. A continuous one is preferable, but the length is not limited as long as the structure is necessary. For example, in the case of FIG. 13 described below, there are 1 to 5 amino acids between the first and second domains and between the third and fourth domains, and between the domains of the second and third amino acids. May be 15 to 25 amino acids. FIG. 6 schematically shows a state in which a first domain and a third domain are formed, and FIGS. 9 and 13 schematically show a polypeptide chain in which a second domain and a fourth domain are connected by a linker. In FIGS. 6, 9 and 13, the first domain and the second domain are also connected by a linker, and in FIG. 13, the third domain and the fourth domain are also connected by a linker. Once, a series of polypeptide chains consisting of main-third domain, first domain-second domain-fourth domain in FIG. 9, first domain-second domain-fourth domain-third domain in FIG. ing. The sequence of each domain in these single-chain polypeptides can be selected and determined as appropriate according to desired properties such as binding to gold or target and long-term stability of the gold-binding protein. .

  Further, in a biopolymer compound composed of antibody fragments, a combination of the domain complexes forming a binding site as described above (for example, the first domain and the third domain, or the second domain and the fourth domain). It is also possible to genetically modify a part of the domain that does not significantly affect the desired binding ability for the purpose of improving the binding property and stability. For example, a method for allowing a disulfide bond to be formed at the junction interface between the domains described above includes, for example, the above-described domain complex (for example, the first domain and the third domain, or the second domain and the second domain). A method of introducing a cysteine residue into a desired site at the junction interface of (four domains) can be used. In addition, by providing two cysteines in the linker, it is also possible to use a method for assisting the formation of a domain complex that forms the same binding site and for improving stability.

The antibody variable region used in the connecting member preferably has at least one of the amino acid sequences of SEQ ID NOs: 1 to 57. The variable region has one or more amino acid sequences selected from the group consisting of amino acid sequences in which one or several amino acids are deleted, substituted or added from any of these 57 amino acid sequences. However, it does not matter as long as the connecting member can exert its function. The amino acid sequences of antibody variable regions having the amino acid sequences of SEQ ID NOs: 1 to 57 are exemplified in SEQ ID NOs: 58 to 77. The codes of SEQ ID NOS: 58 to 77 are set as follows (for example, the code 7s1 of SEQ ID NO: 58 is expressed as “58: 7s1”):
58: 7s1, 59: 7s2, 60: 7s4, 61: 7s7, 62: 7s8, 63: 7p2, 64: 7p3, 65: 7p4, 66: 7p7, 67: 7p8, 68: 10s1, 69: 10s2, 70: 10s3, 71: 10s4, 72: 10s5, 73: 10p1, 74: 10p2, 75: No. 4, 76: No. 4; 7, and 77: No. 10. .

  Such a biopolymer compound constituting the connecting member can be produced as a protein by genetic engineering. For example, a biopolymer compound can be obtained as a fusion protein by introducing a nucleic acid encoding a biopolymer compound into various expression vectors, and expressing and purifying it. It is also possible to produce it as a single polypeptide chain.

  When formed as a single chain, for example, by inserting a nucleic acid encoding an affinity peptide into the 5 ′ end (or 3 ′ end) of a nucleic acid encoding an antibody variable region, the N terminus (or C terminus) of the variable region is inserted. ) To a nucleic acid encoding a protein fused with a substrate affinity peptide. In this case, it is possible to insert or insert a nucleic acid encoding the linker sequence described above between the nucleic acid encoding the antibody fragment and the nucleic acid encoding the affinity peptide. Furthermore, even if it is produced so as to be constituted by a complex of a plurality of polypeptide chains, its form is not a problem as long as it does not impair the function required for the connecting member.

  For example, when one of the binding sites of the connecting member of the present invention to the porous body and the lid member is composed of an antibody fragment and the other is composed of an affinity peptide, the antibody variable region or the complex thereof has the above-mentioned at the N-terminus or C-terminus. It is also possible to form a protein fused with an affinity peptide. These lid member affinity peptide chains can be selected in terms of their binding properties based on a peptide display method represented by a conventionally known phage display method, a design using computational chemistry, or the like, and a suitable one can be used. It is also possible to prepare a candidate library with reference to the conventionally known amino acid sequences of peptides (Nature Materials, Vol. 2, pp577, 2003) and perform screening. When a silicon oxide-based material, particularly SBA-15, is used as the porous body, it is preferable to use SEQ ID NO: 80 or 81.

  Further, in the combination of scFvs that form a binding site (for example, the first domain and the third domain, or the second domain and the fourth domain), the binding property is improved and the stability is improved. For the purpose of improving, it is also possible to genetically modify a part of the portion that does not greatly affect the desired binding ability. For example, there is a method of enabling formation of a disulfide bond at the junction interface between the scFv, for example, scFv (for example, the first domain and the third domain, or the second domain and the second domain). A cysteine residue is introduced at a desired site at the junction interface of the fourth domain). Also, by providing two cysteines in the linker, scFv formation assistance for forming the same binding site and stabilization methods are possible.

  The biopolymer compound can be obtained as a fusion protein by introducing a nucleic acid encoding the biopolymer compound as described above into various expression vectors, and expressing and purifying it.

  A protein expression vector can be designed and constructed by incorporating it into a configuration necessary for expressing a transgene encoding a biopolymer compound such as a known promoter according to a known host cell to be selected. it can. When using Escherichia coli or the like as a host cell, it is possible to reduce degradation by protease by quickly excluding foreign gene product proteins or their components from the cytoplasm. In addition, even when this foreign gene product is toxic to the microbial cells, it is known that the influence can be reduced by secreting it outside the microbial cells. Usually, many of the proteins secreted through the known cytoplasmic membrane or inner membrane have a signal peptide at the N-terminus of the precursor, and are cleaved by a signal peptidase in the secretion process to become a mature protein. Many signal peptides are arranged at the N-terminal with a basic amino acid, a hydrophobic amino acid, and a cleavage site by a signal peptidase.

  Secretion expression can be achieved by arranging a nucleic acid encoding a conventionally known signal peptide represented by pelB, which is one of the signal peptides, on the 5 ′ side of the nucleic acid encoding the target protein.

  Also, when the biopolymer compound is formed as a complex of a plurality of polypeptide chains. Individual polypeptides can be produced by separate vectors, but a plurality of polypeptide chains can be produced in one vector. In this case, a nucleic acid encoding pelB can be arranged on the 5 ′ side of the nucleic acid encoding each domain or polypeptide chain to promote secretion. Furthermore, when expressing as a polypeptide chain consisting of one or more domains, secretion can be promoted by similarly placing a nucleic acid encoding pelB at the 5 ′ end of the polypeptide chain. Thus, the protein in which the signal peptide is fused to the N-terminus can be purified from the periplasma fraction and the culture supernatant.

  In consideration of the convenience of purification of the expressed protein, the N or C terminal of the antibody molecule, or a polypeptide chain formed by consecutively combining individual antibody fragments or a plurality of antibody fragments. It is possible to arrange a purification tag in genetic engineering. Examples of the purification tag include a histidine tag in which 6 residues of histidine are continuous (hereinafter, His × 6), a glutathione-binding site of glutathione-S-transferase (GST), and the like. Examples of the method for introducing the tag include a method of inserting a nucleic acid encoding a purification tag into the 5 ′ or 3 ′ end of a nucleic acid encoding a gold-binding protein in the expression vector, or a commercially available purification. For example, a tag introduction vector may be used.

  Hereinafter, an example of a method for producing a biopolymer compound using the above expression vector will be described.

  A protein that is a biopolymer compound, or a polypeptide chain that constitutes a component thereof, is transformed into a host cell for protein expression that is conventionally known by transforming the above-described protein expression vector designed according to the host cell. It is synthesized in a host cell using a protein synthesis system of Thereafter, the target protein accumulated in the host cell or secreted outside the cytoplasm can be obtained by purifying from the cell internal fraction or the cell culture supernatant, respectively. For example, when Escherichia coli is used as a host cell, a nucleic acid encoding a conventionally known signal peptide typified by pelB is placed on the 5 ′ side of a nucleic acid encoding a gold-binding protein so that it is secreted and expressed outside the cytoplasm. Can try.

  It is also possible to encode a plurality of polypeptide chains that constitute a gold-binding protein as a constituent element in one expression vector. In that case, a nucleic acid encoding pelB can be arranged on the 5 ′ side of the nucleic acid encoding each polypeptide chain serving as a component, and secretion outside the cytoplasm can be promoted during expression.

Thus, the gold-binding protein in which the signal peptide is fused to the N-terminus can be purified from the periplasma fraction and the medium supernatant fraction.
As a purification method, the protein component is concentrated from the above fraction with ammonium sulfate or the like and then suspended again in an appropriate buffer. For example, when the purification tag is a His tag, it is a nickel chelate column, and when the purification tag is GST, glutathione is immobilized. The target protein can be purified by using a crystallization column.

  It is also possible to obtain gold-binding protein expressed in bacterial cells as insoluble granules. In this case, the insoluble granules can be centrifuged from a cell lysate obtained by pulverizing bacterial cells obtained from the culture solution with a French press or ultrasonic waves. The obtained insoluble granule fraction is solubilized with a buffer solution containing a conventionally known denaturing agent containing urea and guanidine hydrochloride, and then subjected to column purification as described above under denaturing conditions. The resulting column elution fraction can be subjected to refolding work to remove the denaturant and rebuild the active structure. As a refolding method, a conventionally known method can be appropriately used. For example, a conventionally known method such as a step dialysis method or a dilution method can be used according to the target protein.

  Each domain or polypeptide chain of the gold-binding protein can be expressed in the same host cell, or it can be coexisted after being expressed using a different host cell. is there.

  Furthermore, it is known that the above-described vector containing a nucleic acid encoding a gold-binding protein can be used to express a target protein in vitro using a cell extract. Suitable cells include Escherichia coli, wheat germ, rabbit reticulocyte and the like. However, protein synthesis using the cell-free extract is generally performed under reducing conditions. Therefore, it is more preferable to perform a conventionally known refolding treatment in order to form a disulfide bond in the antibody fragment.

Furthermore, any one of the binding sites that bind to the porous body surface or the lid member surface of the aforementioned biopolymer compound can introduce the biopolymer compound chemical modification group.
For example, when gold is exposed on at least a part of the surface of the lid member, introduction of an SH group at the end other than the binding site with the porous body surface of the biopolymer compound having binding properties on the porous body surface By introducing a group, the biopolymer compound of the present invention can be obtained. It is also possible to introduce a functional group having a silanol group or alkoxysilane into a biopolymer compound having a binding site for gold.

The connection site | part with the porous body of a connection member can be formed as what comprises the part which consists of a peptide which has the binding property with respect to a porous body. When at least a part of the surface of the porous body contains at least one of a metal and a metal oxide, the connecting part of the connecting member is bonded to the part containing at least one of the metal and the metal oxide. Can be configured. Furthermore, it is also possible to use a configuration in which at least a part of the surface of the porous body contains silicon oxide, and the connecting portion of the connecting member is bonded to a portion including at least one of a metal and a metal oxide. In addition, as a binding site for the porous body of the connection member,
(1) Maintains binding to silicon oxide in which one or several amino acids are deleted, substituted or added to the amino acid sequence of SEQ ID NO: 80 and (2) amino acid sequence of SEQ ID NO: 81 Those having two or more amino acid sequences selected from the group consisting of amino acid sequences can be used.
(Substance for release)
The substance retained in the structure used in the present invention is selected according to the use of the structure, and can be selected from a wide variety of compounds such as various compounds. For example, water-soluble drugs and fat-soluble drugs can be used.
(Compound release control)
The release control of the substance from the structure according to the present invention is a technique for controlling the holding in the structure and the release of the substance from the structure by an external stimulus, that is, an external signal load.

  Examples of the external signal include light, magnetism, and electricity. Considering the use of DDS, light and magnetism (change in magnetic field) are preferable. In particular, it is desirable that it is harmless to the administration side for administering the drug. From such a viewpoint, light and magnetism are preferable. In the case of light, the wavelength and the like are appropriately selected in view of the effect on the lid used and the administration side. For example, as described above, silica or an acrylamide polymer is used as a core (core, Fine particles (particle size: 1 nm to 100 nm) coated with gold (particle size: 1 nm to 100 nm) as a particle size: 5 nm to 50 nm) absorb light of 800 nm to 1200 nm, which hardly harms tissues and cells in the animal body. Fever. By this heat generation, it is possible to release the compound part from the structure by releasing the bond between the biopolymer compound and the cover member and releasing the cover member from the porous body.

  Further, when the lid member is made of gold fine particles, for example, it is known that heat is generated when a high frequency of 10 MHz to 2 GHz in the microwave region is applied from a short wave. It is also possible to apply to the compound release control of the present invention by detaching the lid member from the structure by such a change in the thermal characteristics of the lid member due to such a magnetic field change. The irradiation time can be appropriately determined according to the wave number used for irradiation.

  The signal input device used for the release control means of the present invention is not limited as long as it can change the temperature of the periphery of the lid member bonded to the porous body holding the substance of the present invention. For example, a lamp capable of irradiating light with a wavelength of 800 to 1200 nm or a YAG laser may be used. Furthermore, it is possible to use various microwave generators.

  An example of the drug release (diffusion) process is schematically shown in FIG. The drug 12 is held in the pores 11 of the porous body (13). A gold-coated silica fine particle 14 in which gold is coated on a silica fine particle having a diameter of 30 μm to a thickness t = 3 nm is prepared, and a silica-affinity peptide-fused gold-binding Fv15 is bonded to the surface thereof. By binding Fv to the surface of the porous body constituting the opening of the hole 11, the particle is held in the opening so as to close the hole with the fine particles 14 combined with Fv (16). When the porous body is placed in an optical magnetic field, the fine particles absorb energy (17) and generate heat. As a result, Fv breaks the bond, the fine particles blocking the pores are released from the openings, and the drug 12 held in the pores is released by diffusing out of the pores (18). It should be noted that the drug 12 may be held in the vicinity of the pores, not within the pores, as long as the diffusion can be suppressed by the fine particles.

  According to the structure of the present invention, the following second to seventh effects can be further obtained.

  As a second effect of the present invention, an organic compound containing a biopolymer compound capable of binding to the porous body surface and the lid member surface as a connecting member, and particularly an antibody or a fragment thereof, and a peptide are used. A highly specific connection to the surface of the porous body and the surface of the lid member is possible. Thereby, it becomes possible to reduce the interaction which a connection member couple | bonds with the substance which is not a joint object with connection members, such as a substance for discharge | release, and has a bad influence. Therefore, if the biopolymer compound is a material comprising an antibody variable region having a binding property to the surface of the porous body and / or an antibody variable region having a binding property to the surface of the lid member, higher recognition specificity can be obtained. I can expect.

  As a third effect of the present invention, at least a part of the porous body is made of metal or metal oxide, so that it can exist stably even in a body fluid environment such as blood. Furthermore, biocompatibility can be enhanced by using silicon oxide as the porous body and gold as the lid member.

  The fourth effect of the present invention is that the lid member is detached from the surface of the porous body by an external stimulus input (substance release signal), and the structure is based on the signal input by the substance held by the structure. In this way, the substance to be released at the target time and site can be more reliably released from the porous body.

  The fifth effect of the present invention is that the physical or chemical connection between the surface of the porous body and the surface of the lid member is performed via a biopolymer compound, so that the porous body and the porous body constituting the structure It is possible to release the substance based on the detachment of the lid member by a slight temperature change with little influence on the part of the patient to be treated to which the structure is to be applied or the periphery thereof.

  As a sixth effect of the present invention, a temperature change can be induced on at least the surface of the lid member by using at least one of light and a magnetic field change as a signal for inducing the release of the substance. It is also possible to select a light and magnetic field in a region that is substantially harmless to the treatment subject when it is introduced into the treatment subject animal or person. Thereby, in the substance release control, it is possible to expand the application application without requiring a special expensive facility / equipment.

  As a seventh effect of the present invention, by arranging gold on at least a part of the surface of the lid member, it is possible to select a light or magnetic change region that is harmless to the living body and to stabilize the surface of the lid member. And can provide long-term stability of the release control function.

In the following examples, mesoporous silica (SBA-15) is used as the porous body, gold-coated silica beads (gold layer: 1 nm, silica: 10 nmφ) as the lid member, and gold-binding scFv as the biopolymer compound that connects them. A structure consisting of a protein in which an SBA-15 affinity peptide is fused to the C-terminus of is prepared.
(Example 1) Production of porous body As a porous body, mesoporous silica (SBA-15) is produced by the following method.

Poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide); <block copolymer comprising 20 units of ethylene oxide, 70 units of propylene oxide, 20 units of ethylene oxide, hereinafter referred to as EO ₂₀ -PO ₇₀ -EO ₂₀ A silica reaction solution consisting of> 4 g, 0.041 mol tetraethoxysilane (TEOS) /0.24 mol HCl, 6.67 mol H ₂ O is prepared.

This silica reaction solution was reacted at 35 ° C. for 20 hours, and further reacted at 80 ° C. for 48 hours. Subsequently, by heating for 6 hours at 500 ° C., by burning the block copolymer resin EO ₂₀ -PO ₇₀ -EO ₂₀ included, obtaining a porous silica.

  In the obtained porous silica, the average pore diameter is 7.9 nm, and the average thickness of the silica wall surface between the pores is 3 nm.

(Example 2)
Preparation of gold-binding scFv-SBA-15 affinity peptide fusion protein A protein in which the SBA-15 affinity peptide IPHVHHKHPHV (SEQ ID NO: 80) is fused to the C-terminus of gold scFv is prepared by the following steps.
(1) Preparation of expression vector Vector pUT-XX, which was changed in advance by cleaving the multicloning site of pET-15b (Novagen) with NheI / SacII and NotI / SacII, as shown in FIGS. 2A and 2B Prepare VL (clone name: VL # No, 7, SEQ ID NO: 76) and VH (clone name: 7s4, SEQ ID NO: 60) of pET-15b (Novagen), each of which is a component of gold-binding scFv The multicloning site is inserted into a vector modified as shown in Fig. 2 (a) and (b), and the resulting vectors are designated as pUT-VLNo, 7, and pUT-7s4. Linker (GGGGGS) × 3, VH coding gene, SBA-15 affinity peptide (hereinafter also referred to as “Si tag” as shown in FIG. 3), His × 6 (hereinafter referred to as “Si tag”) , "His-tag") is translated continuously, and the expression vector pUT-scFv as expressed as a fusion protein prepared as follows (Figure 3).

PCR is performed using pUT-7s4 obtained above as a template and the following primers.
SiscFv-B (SEQ ID NO: 78)
5′-NNNNNCCATGGCCCCAGGTGCAGTTGGTGGAGT-3 ′
SiscFv-F (SEQ ID NO: 79)
5'-NNNNNCCGCGGCACGGTGGGGGTGCTTGTGTGCACGTGCATGGGGATAACCATTCAGATCCCTCTTCT-3 '
PCR is performed using a commercially available PCR kit (Takara Bio LA-Taq kit) according to the protocol recommended by the manufacturer.

  The PCR product obtained as a result is subjected to 2% agarose electrophoresis. Next, using a gel extraction kit (Promega), crude purification is performed from the gel to obtain a PCR fragment of about 400 bp. As a result of sequencing, it is confirmed that it has the target base sequence. pUT-VL No. 7 and the PCR fragment obtained above are cleaved using NotI / SacII. Subsequently, agarose electrophoresis is performed, and the target fragments are purified on the Vector side and the Insert side, respectively. Si tag and His-tag are bonded to both ends of the PCR fragment to form an Insert piece.

  The purified nucleic acid fragments obtained above are mixed so that Vector: Insert = 1: 5, and a ligation reaction is performed in the same manner as in Example 1.

  Thereafter, 40 μL of JM109 competent cells are transformed with the ligation reaction solution. Transformation is performed under conditions of heat shock in ice → 42 ° C. × 90 sec → ice. 750 μL of LB medium is added to the BL21 solution transformed by heat shock, and shaking culture is performed at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 6000 rpm for 5 minutes, 650 μL of the culture supernatant was discarded, the remaining culture supernatant and the precipitated cell fraction were stirred, and LB / amp. Spread on a plate and let stand at 37 ° C overnight.

A colony is randomly selected from the plate, shake-cultured in 3 mL of LB / amp. Liquid medium, and a plasmid is extracted using a commercially available MiniPrep kit (manufactured by Promega) according to the method recommended by the manufacturer. The obtained plasmid is cleaved using NotI / SacII. Next, agarose electrophoresis is performed to confirm that the target gene fragment has been inserted.
This plasmid is designated as pUT-scFvSp.

Hereinafter, 40 μL of BL21 (DE3) competent cells are transformed with the plasmid pUT-scFvSp obtained in the above operation. Transformation is performed under conditions of heat shock in ice → 42 ° C. × 90 sec → ice.
750 μL of LB medium is added to the BL21 solution transformed by heat shock, and shaking culture is performed at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 6000 rpm for 5 minutes, 650 μL of the culture supernatant was discarded, the remaining culture supernatant and the precipitated cell fraction were stirred, and LB / amp. Spread on a plate and let stand at 37 ° C overnight.
(2) Preculture A colony on the plate was randomly selected and 3.0 mL LB / amp. Shake culture overnight at 28 ° C. in the medium.
(3) Main culture The above preculture solution is inoculated into 750 mL of 2 × YT medium, and the culture is further continued at 28 ° C. When the OD600 exceeds 0.8, IPTG is added so that the final concentration is 1 mM, and the culture is further performed overnight at 28 ° C.
(4) Purification The target polypeptide chain is purified from the insoluble granule fraction by the following steps.
(A) Recovery of insoluble granules The culture solution obtained in (3) above is centrifuged at 6000 rpm x 30 min to obtain a precipitate as a cell fraction. The obtained cells are suspended in 15 ml of Tris solution (20 mM Tris / 500 mM NaCl) in ice. The obtained suspension is crushed with a French press to obtain a bacterial crushing solution. Next, the bacterial disruption solution is centrifuged at 12,000 rpm × 15 min, the supernatant is removed, and the precipitate is obtained as an insoluble granule fraction.
(B) Solubilization of insoluble granule fraction Add 10 mL of 6M guanidine hydrochloride / Tris solution to the insoluble fraction obtained in (A) and soak overnight. Next, it is centrifuged at 12,000 rpm × 10 min to obtain a supernatant as a solubilized solution.
(C) Metal chelate column As a metal chelate column carrier, His-Bind (manufactured by Novagen) is used. Column adjustment, sample loading, and washing steps are performed at room temperature (20 ° C.) in accordance with the recommended method of the supplier. The target His tag fusion polypeptide is eluted with a 60 mM imidazole / Tris solution. As a result of SDS-PAGE (acrylamide 15%) of the eluate, it is confirmed that it is a single band and is purified.
(D) Dialysis The above eluate is dialyzed at 4 ° C. using a 6M guanidine hydrochloride / Tris solution as an external solution to remove imidazole in the eluate to obtain the respective polypeptide chain solutions.
(E) Refolding In the same manner as described above, while scFv-Sp polypeptide chain solution fused with gold-binding Fv and the above peptide was separately separated by the following steps, guanidine hydrochloride was performed by dialysis (4 ° C.). Perform protein refolding.

a) Using a 6M guanidine hydrochloride / Tris solution, the express coefficient and ΔO. D. A sample with a concentration of 7.5 μM (volume after dilution: 10 ml) is prepared from the (280 nm-320 nm) value. Next, β-mercaptoethanol (reducing agent) is added to a final concentration of 375 μM (protein concentration 50 times), and reduction is performed at room temperature in the dark for 4 hours. This sample solution is put into a dialysis bag (MWCO: 14,000) to obtain a sample for dialysis.
b) The dialysis sample is immersed in 6M guanidine hydrochloride / Tris solution as an external solution for dialysis, and dialyzed for 6 hours with gentle stirring.
c) Decrease the concentration of guanidine hydrochloride in the external solution stepwise to 3M and 2M. Dialyse for 6 hours at each external concentration.
d) Add oxidized glutathione (GSSG) to the Tris solution to a final concentration of 375 μM and L-Arg to a final concentration of 0.4 M), add the 2M dialysis solution from 3) above to a guanidine hydrochloride concentration of 1M. Then, dialyze with a solution adjusted to pH 8.0 (4 ° C.) with NaOH for 12 hours with gentle stirring.
e) Prepare an L-Arg Tris solution containing 0.5 M guanidine hydrochloride in the same manner as in d) above, and dialyze for 12 hours.
f) Finally, dialyze against Tris solution for 12 hours. 7) After completion of dialysis, centrifuge at 10,000 rpm for about 20 minutes to separate the aggregate and supernatant. For the solution obtained above, the external solution is further replaced with a phosphate buffer (hereinafter referred to as PBS), and SPR measurement is performed using the solution. Gold bondability is confirmed.

Example 3 Production of Structure (1) 200 mg of SBA-15 produced in Example 1 is immersed overnight in 3 μM ATP / phosphate buffer: PBS (pH 7.4).
(2) Gold fine particles (20 nm, Tanaka Kikinzoku, 0.15 mmol) are suspended in 0.01 mmol ATP / PBS.
(3) Next, 1.5 μM scFv / PBS fused with the silica-affinity peptide prepared in Example 2 was mixed with the suspension of SBA-15 and (2) subjected to the immersion treatment in (1) above. Stir for 24 hours.
(4) Subsequently, centrifugation is performed at 12,000 rpm × 5 min, the supernatant is removed, and a precipitate is obtained. This precipitate is vacuum-dried to obtain a structure.

Example 4 Control of Compound Release from Structure-1
(1) 20 mg of the structure obtained in Example 3 is suspended in PBS (pH 7.4) and dispersed in the solution using ultrasonic waves. This operation is repeated three times to wash ATP adsorbed on the structure.
(2) The structure is left to stand for 12 hours in a state suspended in PBS. After standing, 0, 4, 8, 12 hours later, a part of the solution is taken out and measured by HPLC (C18, reverse phase column) (detection wavelength: 275 nm). It is confirmed that the amount of ATP decreases with time.
(3) Next, YAG laser (1064 nm, 164 mJ / pulse, 7 nsec, 10 Hz) is irradiated for 1 hour.
(4) After laser irradiation, the solution is sampled every 10 minutes and analyzed by HPLC. It is confirmed that ATP is released over time.

Example 5 Control of Compound Release from Structure-2
(1) 20 mg of the structure obtained in Example 3 is suspended in PBS (pH 7.4) and dispersed in the solution using ultrasonic waves. This operation is repeated three times to wash ATP adsorbed on the structure.
(2) The structure is left to stand for 12 hours in a state suspended in PBS. After standing, 0, 4, 8, 12 hours later, a part of the solution is taken out and measured by HPLC (C18, reverse phase column) (detection wavelength: 275 nm). It is confirmed that the amount of ATP decreases with time.
(3) Next, with a synthesized signal generator (manufactured by HP), 0.5 GHz light is repeated 5 times in a cycle of irradiation for 10 seconds / pause for 50 seconds.
(4) The solution after signal irradiation is sampled and analyzed by HPLC. It is confirmed that the amount of ATP after signal irradiation increases with respect to the amount of ATP in the solution of (2) above, and ATP is released by the signal.

(Example 6)
In Example 2, VH (VH clone name A14P-) represented by SEQ ID NO: 82 in which alanine at position 14 of VH (VH clone name: 7s4) of the gold-binding scFv-SBA-15 affinity peptide protein was changed to proline was used. 7s4, SEQ ID NO: 83).
-Preparation of expression plasmid A mutation is introduced into a target site using pUT-scFvSp obtained in Example 2 as a template. Mutagenesis is performed according to the method recommended by the manufacturer using a QuickChange kit (manufactured by STRATAGENE). Primers used at that time are as follows.
A14P-f (SEQ ID NO: 84)
GAGCAGAGGTGAAAAAGCCCAGGGGAGTCTCTGAAG
A14P-r (SEQ ID NO: 85)
CTTCAGAGACTCCCCTGGCTTTTCACCTCTCGCTC
It is confirmed by sequencing that the obtained plasmid has inserted the DNA represented by SEQ ID NO: 80.

  Using the plasmid pUT-scFv2Sp obtained in the above operation, 40 μL of BL21 (DE3) competent cells are transformed. Transformation is performed under conditions of heat shock in ice → 42 ° C. × 90 sec → ice. 750 μL of LB medium is added to the BL21 solution transformed by heat shock, and shaking culture is performed at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 6000 rpm for 5 minutes, 650 μL of the culture supernatant was discarded, the remaining culture supernatant and the precipitated cell fraction were stirred, and LB / amp. Spread on a plate and let stand at 37 ° C overnight.

  Thereafter, the target protein is obtained by the same operations as in (2) to (4) of Example 2. Moreover, a structure is obtained by the same operation as in Example 3 using the obtained protein. Further, the same evaluation as in Example 4 is performed, and it is confirmed that ATP is released as in Example 4.

(Example 7)
In Example 6, the gold-binding scFv-SBA-15 affinity peptide protein VH (VH clone name: A14P) was changed from 34th valine to phenylalanine, 44th glutamine to glutamic acid, and 45th leucine to arginine. A protein is prepared so as to be VH represented by 86 (VH clone name PFER-7s4, SEQ ID NO: 87).
-Preparation of expression plasmid A mutation is introduced into a target site using pUT-scFv2Sp obtained in Example 6 as a template. Mutagenesis is performed according to the method recommended by the manufacturer in the same manner as described above using the QuickChange kit (manufactured by STRATAGENE). The target plasmid is obtained by three operations.
The primers used are as follows.

PCR primer V37F-f (SEQ ID NO: 88) for introducing the mutation at the first site
TTACTGGATCAACTGGTTCCCGCCAGATGCCCGG
V37F-r (SEQ ID NO: 89)
CCGGGGCATCTGGCGGAACCAGTTGATCCCAGTAA
PCR primer G44E-f (SEQ ID NO: 90) for introducing mutation at the second site
CAGATGCCCGGCAAGAACTACTGAAGATGGAGGGG
G44E-r (SEQ ID NO: 91)
CCCCATCCATTCCAGTTCTTTGCCGGGCATCTG
PCR primer L45F-f (SEQ ID NO: 92) for introducing the mutation at the third position
GCCCGGCAAAGAAAAGGAGATGGATGGGGATG
L45F-r (SEQ ID NO: 93)
CATCCCCATCCATTCCCTGCCTTTGCCCGGGC
It is confirmed by sequencing that the obtained plasmid has inserted the DNA represented by SEQ ID NO: 82.

  Using the plasmid pUT-scFv2Sp obtained in the above operation, 40 μL of BL21 (DE3) competent cells are transformed. Transformation is performed under conditions of heat shock in ice → 42 ° C. × 90 sec → ice. 750 μL of LB medium is added to the BL21 solution transformed by heat shock, and shaking culture is performed at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 6000 rpm for 5 minutes, 650 μL of the culture supernatant was discarded, the remaining culture supernatant and the precipitated cell fraction were stirred, and LB / amp. Spread on a plate and let stand at 37 ° C overnight.

  Thereafter, the target protein is obtained by the same operations as in (2) to (4) of Example 2. Moreover, a structure is obtained by the same operation as in Example 3 using the obtained protein. Further, the same evaluation as in Example 4 is performed, and it is confirmed that ATP is released as in Example 4.

The present invention is a structure comprising at least a porous body holding one or more compounds,
(1) A lid member for holding the compound on the surface of the porous body / or the periphery thereof, (2) at least a material for physically or chemically connecting the lid member and the surface of the porous body Provided is a structure comprising a part of an organic material made of a biopolymer compound. By applying the present invention, a structure capable of stably holding a compound in a porous body can be obtained. Furthermore, the present invention includes means for controlling compound release from the structure. By applying the present invention, it is possible to control the release of the compound held by the structure at a desired timing from the structure of the present invention.

  That is, the present invention can appropriately supply a target substance in various chemical reactions. Moreover, the target substance can be released by applying an external signal only to the affected area by applying to the DDS technique. This makes it possible to minimize the dose to be administered to the patient, and to suppress undesirable effects such as side effects.

1 is a schematic diagram of the structure and compound release of the present invention. FIG. (A) And (b) is a figure which shows an example of the vector preparation figure for biopolymer compound manufacture. It is a figure which shows an example of the vector preparation figure for biopolymer compound manufacture. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows typically the structure of an example of gold | metal binding protein. It is a figure which shows various microstructures. (a) Porous structure, (b) Opal structure, (c) Inverse opal structure, (d) Columnar structure, (e) Convex structure, (f) Concave structure, (g) Projection A structure and (h) a fibrous structure are shown, respectively.

Explanation of symbols

1: First domain 2: Second domain 3: Third domain 4: Fourth domain 5, 6: Linker

Claims (6)

A structure that holds a compound therein,
The structure is
A porous body, a lid member, and a connecting member for connecting the lid member and the porous body;
The connection part with the lid member of the connection member includes a capturing body that specifically recognizes and captures the lid member,
At least a part of the lid member is made of gold,
Structure characterized by capturing body that captures specifically recognize the lid member is a capturing body having an antibody variable region to capture specifically recognize gold. The structure according to claim 1, wherein the antibody variable region that specifically recognizes and captures gold has one or more amino acid sequences represented by SEQ ID NOs: 1 to 57. In the antibody variable region that specifically recognizes and captures gold, one or several amino acids of one of the amino acid sequences shown in SEQ ID NOs: 1 to 57 are deleted, substituted, or added. The structure according to claim 1, which has one or more amino acid sequences. A structure that holds a compound therein,
The structure is
A porous body, a lid member, and a connecting member for connecting the lid member and the porous body;
The connection part with the lid member of the connection member includes a capturing body that specifically recognizes and captures the lid member,
At least a part of the lid member is made of silicon oxide;
Structure characterized by capturing body that captures specifically recognize the lid member is a capturing body having an antibody variable region to capture specifically recognize the silicon oxide. The antibody variable region that specifically recognizes and captures silicon oxide has the amino acid sequence represented by SEQ ID NO: 80 or one or several amino acids of any one of the amino acid sequences represented by SEQ ID NO: 81 The structure according to claim 4 , which has two or more amino acid sequences deleted, substituted or added. The structure according to any one of claims 1 to 5, wherein the lid member is detached from the connection member by input of light or a magnetic field as an external signal.

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