Targeting Shells for Drug Delivery Systems Using Carbosilane Dendrimers

Abstract

The present invention relates to a targeting shell for a drug delivery system. The purpose of the present invention is to provide a targeting shell for a drug delivery system, which utilizes the reactivity of thiol and alkyl halide, comprises a silole-containing carbosilane dendrimer and a marker protein (such as green fluorescent protein) containing a targeting recognition site, can encapsulate biopolymers or other molecules, and can selectively deliver the above biopolymers and the like into target cells.

Description

Technical Field

[0001] The present invention relates to a targeting shell for a drug delivery system using a carbosilane dendrimer that can be used in a drug delivery system. Background Art

[0002] Biopolymers such as antibodies, peptide aptamers, and nucleic acids are currently attracting attention as next-generation pharmaceuticals. These biopolymers are very different from conventional pharmaceuticals with low-molecular-weight compounds as active ingredients in terms of quality control during the manufacturing process, storage of preparations, and administration methods.

[0003] Generally, when administering a pharmaceutical preparation, the amount of active ingredient that can be delivered to the target site affects the effectiveness, although it depends on the disease to be treated and the properties of the drug. For example, it is known that when a protein or antibody, which is a biopolymer, is used as an active ingredient, antibodies against them are generated depending on the dosage form at the time of administration, and the effectiveness is reduced, thereby causing a problem in which the effectiveness (therapeutic effect) is also reduced.

[0004] Therefore, various drug delivery systems (DDS: drug delivery system) are being actively developed as a means of appropriately delivering such drug preparations to the target site. As carriers for DDS, for example, liposomes, micelles, synthetic resin particles, etc. can be cited. In addition, as preparations using such carriers, for example, there are reports of: preparations in which a ligand that specifically binds to a molecule located at the target site is combined with a liposome, etc. (see patent documents 1 and 2); preparations in which N-acetylglucosamine or other sugars are exposed to the surface of a liposome or synthetic resin particle, etc. (see patent documents 3 and 4); and preparations in which an antibody is combined with a micelle (see patent document 5); and the like.

[0005] In addition, development is also underway for carriers that utilize dendritic polymers that form micelle structures (see Patent Documents 6, 7, and 8). Dendritic polymers are a general term for regularly branched dendritic polymer compounds, which are derived from the Greek word "dendra" (tree). When several molecules of dendritic polymers are associated, they form a sphere, and a nanoscale space is formed inside them. Therefore, molecules can be incorporated in such a way that various functional groups appear in the space, and the degree of association can be increased to tens to hundreds, and the degree of freedom in design is high. Therefore, various new dendritic polymers are currently being actively developed in the field of nanotechnology.

[0006] To date, development of DDS carriers using dendrimers has been conducted for the purpose of preventing and / or treating diseases caused by viral or bacterial infections (see Patent Document 9).

[0007] On the other hand, it is known that silole-containing dendrimers (hereinafter referred to as "carbosilane dendrimers" in this specification) that have the AIE (aggregation-induced emission; a phenomenon in which non-luminescent compounds aggregate with each other and cause efficient luminescence by irradiation with light of a specific wavelength) effect are generally equivalent to forming aggregates when forming micelles, and emit fluorescent light (see Non-Patent Document 1).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: WO2005 / 011632

[0011] Patent Document 2: WO2005 / 011633

[0012] Patent Document 3: Japanese Patent Application Laid-Open No. 2007-1923

[0013] Patent Document 4: Japanese Patent Application Laid-Open No. 2009-46413

[0014] Patent Document 5: Japanese Patent Application Laid-Open No. 2014-73975

[0015] Patent Document 6: Japanese Patent Application Laid-Open No. 2001-206885

[0016] Patent Document 7: Japanese Patent Application Laid-Open No. 2005-120068

[0017] Patent Document 8: Japanese Patent Application Laid-Open No. 2007-238860

[0018] Patent Document 9: Japanese Patent No. 5629888

[0019] Non-patent literature

[0020] Non-patent literature 1: Chem Commun (Camb). 2009 August 7; (29): 4332-53. doi: 10.1039 / b904665h. Epub 2009 May 13 Summary of the invention

[0021] Problems to be solved by the invention

[0022] As described above, although research on carriers for DDS is being actively conducted, it is extremely difficult to efficiently deliver a drug containing a biopolymer as an active ingredient to a target site without impairing its properties.

[0023] In the case of oral administration, unless the carrier can be prevented from being decomposed by strong acid such as gastric acid, the biopolymer as the active ingredient cannot reach the intestine and will not be absorbed. In addition, in the case of intravenous administration (intravenous injection, hereinafter sometimes collectively referred to as "intravenous injection"), although a high concentration of the drug can be temporarily administered to the bloodstream, there are problems other than the above-mentioned gastric acid decomposition, and it is difficult to deliver it to the target site at an effective concentration for the following reasons. That is, the drug concentration in the blood decreases rapidly over time due to excretion into the urine, metabolism in the liver, accumulation in various tissues, etc.; and sometimes it remains in the blood without targeting.

[0024] In addition, sometimes there will be a serious side effect of increasing the amount of medicine delivered to the target site. For example, levodopa, a drug for Parkinson's disease treatment, has a very small amount of blood-brain barrier, so the efficiency cannot be improved. And if the dosage of levodopa is increased, since the decomposition enzyme of levodopa is also present outside the brain as the target site, serious side effects will occur, and the situation that the treatment cannot be continued actually occurs. On the other hand, the multiple administration of small amounts of medicines in one day will cause the problem of increasing the burden on patients and medical personnel.

[0025] In addition to the ability to take in active ingredients with large molecular weight, the carrier for biopolymers also needs to have high compatibility with the organism. This is because if the biocompatibility is low, various reactions will occur on the organism side, and the administration of the carrier itself will have adverse effects on the organism. In addition, it is also necessary to maintain a stable structure before being delivered to the target site, so that the biopolymer as the active ingredient can be quickly released when it is delivered to the target site.

[0026] As described above, biopolymer carriers are required to satisfy various conditions. However, carriers having such characteristics and properties have not been put into practical use so far, and there is a strong social demand for carriers having such characteristics and properties.

[0027] Recently, active research is being conducted on using the targeting and biocompatibility of natural nanoparticles such as exosomes and cells (e.g., red blood cells) for DDS, but the encapsulation technology for drugs has not yet been completed and has not yet reached practical use.

[0028] Therefore, there is a strong social demand for a carrier with high biocompatibility that can encapsulate biopolymers that can serve as active ingredients, can be delivered to target tissues, and can appropriately release the encapsulated biopolymers in the tissues after delivery.

[0029] Means for solving problems

[0030] The present inventors have discovered for the first time that, by utilizing the reactivity of thiols and alkyl halides, a silole-containing carbosilane dendrimer and a labeled protein (e.g., green fluorescent protein) are mixed in an aqueous solvent or a mixed solvent of the aqueous solvent and an organic solvent to form an aggregating molecule that is a complex of the dendrimer and the protein. Here, the aqueous solvent contains physiological saline, phosphate-buffered saline, or other solutes.

[0031] The inventors of the present invention have further found that when the above-mentioned protein is loaded on a dendrimer, the formation of a liposome (hereinafter sometimes referred to as a "vesicle") or micelle in which the protein in the above-mentioned aggregating molecule faces outward can be induced. It has also been found that when the above-mentioned protein, the above-mentioned carbosilane dendrimer and a model drug are mixed in an aqueous solvent, micelles encapsulating the above-mentioned model drug can be formed. Examples of such model drugs include pigments, lectins, antibodies and the like.

[0032] In addition, the inventors of the present invention have also found that the dendrimer containing silole loaded with fluorescent protein emits light in physiological saline, and that the luminescence phenomenon is caused by the resonance energy transfer (FRET) between the silole and the fluorescent protein.

[0033] Furthermore, it was found that the inner diameter of micelles formed by fluorescent protein-loaded silole-containing carbosilane dendrimers is about 50 nm to 500 nm, and micelles having such an inner diameter can encapsulate various drugs including biopolymers.

[0034] In addition, it is known that organisms take up the above micelles into cells by endocytosis and destroy them. The inventors of the present invention have found that by introducing a target tissue binding sequence, specific delivery to the target tissue is possible.

[0035] The present invention was completed under the above circumstances, and its object is to provide a targeting shell for a drug delivery system that can take in compounds or biopolymers of various molecular weights, has high stability in vivo, and can deliver active ingredients to target tissues.

[0036] The present invention includes the following aspects. In one embodiment of the present invention, the present invention relates to a targeting shell for a drug delivery system, which has an aggregating molecule represented by the following formula (I) and can encapsulate any molecule selected from the group consisting of proteins, nucleic acids, and hydrophobic molecules with a molecular weight of less than 200,000.

[0037] [Chemistry 1]

[0038]

[0039] The shell is preferably formed by aggregating the aggregating molecules in an aqueous solvent and has a diameter of 50 nm to 500 nm, because it is the optimal size for high permeability and long retention effect on new blood vessels around cancer cells, particularly in drug delivery of anticancer agents.

[0040] In addition, the targeting sequence presenting part is preferably composed of a protein or peptide having a targeting recognition site for delivery to the target tissue. Furthermore, the peptide preferably specifically binds to any target protein selected from the group consisting of surface antigens, receptors, gates, transporters and channels expressed in the target tissue, and promotes the endocytosis of the combination of the target protein and the peptide into the cell.

[0041] Specifically, the peptide preferably has a sequence described in any one selected from the group consisting of SEQ ID NOs. 1 to 3 in the sequence listing.

[0042] DMPGTVLPGG (SEQ ID NO. 1 in the sequence listing)

[0043] VPTDTDYSGG (SEQ ID NO. 2 in the sequence listing)

[0044] DMPGTVLPGG GGGSEGEWQ QQQHQWAKQE (SEQ ID NO: 3 in the sequence listing)

[0045] Preferably, the peptide has a sequence selected from any one of the group consisting of SEQ ID NOs. 4 to 6 in the sequence listing.

[0046] MASMTGGQQMGR DMPGTVLPGG MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYGKLTLKFISTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF KDDGNYKTRAEVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YITADKQRNG IKANFKTRHN IEDGSVQLADHYQQNTPIGD GPVLLPDNHY LSTQSALLKD PNEKRDHMVL LEFVTAAGSGIT DEVDGT ELYK GGHHHHHH (SEQ ID NO. 4 in the sequence listing)

[0047] MASMTGGQQMGR VPTDTDYSGG MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYGKLTLKFISTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF NDDGNYKTRAEVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YITADKQRNG IKANFKTRHN IEDGSVQLADHYQQNTPIGD GPVLLPDNHY LSTQSALLKD PNDKRDHMVL LEFVTAAGSGIT DEVDGT ELYK GGHHHHHH (SEQ ID NO: 5 in the sequence listing)

[0048] MASMTGGQQMGR DMPGTVLPGG GGGSEGEWQQQQHQWAKQE MSKGEELFTG VVPILVELDGDVNGHKFSVS GEGEGDATYG KLTLKFISTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPEGYVQERTISF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YITADKQRNGIKANFKTRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALLKD PNEKRDHMVLLEFVTAAGSGIT DEVDGTC ELYK GG HHHHHH (sequence number 6 in the sequence list)

[0049] Here, the target tissue is preferably any tissue selected from the group consisting of normal tissue in which inflammation has occurred, tissue containing cells in which undesirable gene expression is observed, tissue composed of cells in which undesirable gene expression is observed, and tissue composed of tumor cells.

[0050] Here, as the above-mentioned normal tissues in which inflammation has occurred, tissues having significant features as autoimmune diseases can be cited. In addition, as tissues containing cells in which undesirable gene expression is observed, tissues in which the association between the onset of disease and gene mutations such as single base substitutions has been clarified through SNP analysis can be cited. As cells in which undesirable gene expression is observed, cells from the above-mentioned tissues in which undesirable gene expression is observed can be cited. In addition, as tissues composed of tumor cells, breast cancer tissues, lung cancer tissues, liver cancer tissues, cervical cancer tissues, etc. can be cited. As the above-mentioned target recognition site, sequence numbers 1 to 3 of the sequence table can be cited, and as the target sequence presentation portion, the amino acid sequences shown in sequence numbers 7 to 11 of the sequence table can be cited.

[0051] In addition, the aggregating molecule represented by the above formula (I) is preferably an aggregating molecule represented by a molecule having any structure selected from the group consisting of the following formulae (II) to (V), wherein TSPP is a fluorescent protein having a target recognition site.

[0052] [Chemistry 2]

[0053]

[0054] [Chemistry 3]

[0055]

[0056] [Chemistry 4]

[0057]

[0058] [Chemistry 5]

[0059]

[0060] Here, it is preferred that the fluorescent protein is any one fluorescent protein selected from the group consisting of red fluorescent protein, yellow fluorescent protein, blue fluorescent protein and green fluorescent protein.

[0061] Effects of the Invention

[0062] According to the present invention, a targeting shell for drug delivery system can be prepared, which has the characteristics required in the manufacture of a carrier capable of delivering drugs, etc. In addition, the targeting shell for drug delivery system can encapsulate various drugs including biopolymers and deliver them to target tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a schematic diagram showing the basic form of a general carbosilane dendrimer.

[0064] Figure 21 is a diagram showing a synthetic scheme for synthesizing dimethylsilane (1)6-Br from dimethylsilane.

[0065] Figure 3 This is a graph showing the particle size distribution of the produced micelles.

[0066] Figure 4 This is a typical electron microscope photograph of the prepared micelles observed using a scanning microscope (SEM).

[0067] Figure 5 The graph shows the results of investigating the luminescence characteristics of products obtained by incubating a complex of GFP and a silole dendrimer represented by formula (X) (hereinafter referred to as "GFP-silole dendrimer complex"), GFP alone, or the silole dendrimer represented by formula (X) alone.

[0068] Figure 6 This is a diagram showing the results of investigating the particle size distribution of micelles produced in the presence of a drug model.

[0069] Figure 7 This is a diagram showing the results of investigating the luminescence characteristics of micelles composed of GFP-silole dendrimer complex encapsulating DiI when excited at 370 nm.

[0070] Figure 8 This is a diagram showing the results of investigating the light emission characteristics of micelles composed of GFP-silole dendrimer complex encapsulating WGA labeled with Alexa594 when excited at 370 nm.

[0071] Fig. 9 This is the electrophoresis image of the PCR product after performing inverse PCR with changing the annealing temperature.

[0072] Fig.10 This is a diagram showing the results of measuring the fluorescence spectrum of fluorescent protein association-driven micelles to which the targeting peptide sequence is bound.

[0073] Fig.11 It is the particle size distribution diagram of the fluorescent protein association-driven micelles of MCF-7 combined with the targeting peptide sequence.

[0074] Fig.12 This is an electron microscope image of the particles formed by observing using a low-temperature low-vacuum scanning electron microscope to determine whether they are micelles or vesicles. In the figure, the structures indicated by white circles and white arrows are believed to be salt crystals. In addition, the structures indicated by black circles and black arrows are believed to be vesicle-like substances.

[0075] Fig.13It is a figure showing the uptake of targeted proteins and micelles containing targeted proteins into MCF cells. (A) shows the uptake of HepG2 targeted type 1 protein (-)(-) and MCF7 targeted type 1 protein (-)(-). (B) shows the uptake of HepG2 targeted type 1 protein (+)(+) and MCF7 targeted type 1 protein (+)(+). (C) shows the uptake of HepG2 targeted type 1 micelles (-)(-) and MCF7 targeted type 1 micelles (-)(-). (D) shows the uptake of HepG2 targeted type 1 protein (+)(+) and MCF7 targeted type 1 protein (+)(+).

[0076] Fig.14 The results of FACS analysis show the difference in micelle uptake 24 hours after A549 cells were exposed to the micelles of the present invention. (A) is the result without micelles, (B) and (C) are the results when non-targeted micelles were used, and (D) and (E) are the results when targeted micelles were used.

[0077] Fig.15 The results of FACS analysis show the difference in micelle uptake 24 hours after MCF7 cells were exposed to the micelles of the present invention. (A) is the result without micelles, (B) and (C) are the results when non-targeted micelles were used, and (D) and (E) are the results when targeted micelles were used.

[0078] Fig.16 These are graphs showing concentration-dependent changes and time-dependent changes of non-targeted micelles. (A) shows the results of the non-targeted micelle (NSS25), and (B) shows the results of the non-targeted micelle (NSS26).

[0079] Fig.17 It is a graph showing the concentration-dependent change and the time-dependent change of the targeted type 1 micelle.

[0080] Fig.18 The graphs show the temporal changes of non-targeted micelles and targeted micelles. (A) shows the results of non-targeted micelles (NSS25), (B) shows the results of non-targeted micelles (NSS26), and (C) shows the results of targeted type 1 micelles.

[0081] Fig.19 The electrophoresis images show the results of gel electrophoresis of proteins constituting the shell. (A) shows the results before staining, and (B) shows the results after staining with Coomassie Brilliant Blue (CBB).

[0082] Fig. 20AThe fluorescence microscope images show the uptake of non-targeted micelles into target cells cultured in collagen-coated culture dishes. The upper layer is a fluorescence microscope image taken 3 hours after the start of the uptake experiment, and the lower layer is a fluorescence microscope image taken 24 hours after the start of the uptake experiment.

[0083] Fig. 20B This is an optical microscope image showing the state when cells are cultured in a poly-d-lysine-coated dish or a collagen-coated dish without micelles.

[0084] Fig. 20C The fluorescence microscope images show the uptake of targeted type 1 micelles into target cells cultured in collagen-coated culture dishes. The upper layer is a fluorescence microscope image taken 3 hours after the start of the uptake experiment, and the lower layer is a fluorescence microscope image taken 24 hours after the start of the uptake experiment. DETAILED DESCRIPTION

[0085] The present invention is described in further detail below. The present invention is a targeting shell for a drug delivery system, which has an aggregating molecule represented by the following formula (I) as described above and can encapsulate any molecule selected from the group consisting of proteins, nucleic acids, and hydrophobic molecules with a molecular weight of 200,000 or less.

[0086] [Chemistry 6]

[0087]

[0088] The carbosilane dendrimer (hereinafter sometimes referred to as "silole dendrimer") used in the present invention is more preferably a compound having the following structure because it can possess both the properties of aggregating and the targeting sequence presenting part described below.

[0089] The structure of a typical carbosilane dendrimer is shown in Figure 1 A~C. Figure 1 The compound represented by A is called fan (0) 3, and the compound has a benzene ring surrounded by a circle as a core. Figure 1 The compound shown in B is called dumbbell (1) 6, in which the core is replaced by silicon with two methyl groups (Me-Si-Me). Figure 1 The 2-molecule sector shown in A (0) is bound to 3 molecules. Figure 1 The compound represented by C is a compound called sphere (1) 6, in which the methyl group of Me-Si-Me of the dumbbell is replaced by a molecule obtained by removing the core of fan (0) 3, forming a sphere.

[0090] The carbosilane dendrimer of the present invention can be prepared according to Figure 2 The synthesis was carried out according to the steps shown (Scheme 1).

[0091] The structure of the carbosilane dendrimer of the present invention is completely different from that of the existing carbosilane dendrimers, and one of the bromine atoms bonded to the molecular terminal participates in the binding with the targeting sequence presenting part. Here, from the aspect of being able to specifically deliver the DDS drug formed by the above-mentioned dendrimer, it is preferred that the targeting sequence presenting part is a protein having a targeting recognition site shown in SEQ ID NOs. 1 to 3 of the sequence listing.

[0092] Furthermore, the protein constituting the targeting sequence has a unique reactive thiol group, and the thiol group is preferably located on the outside of the folded protein, that is, on the surface of the protein.

[0093] This is because the thiol group reacts with the halogen atoms present in the dendrimer to play a role in binding the protein to the dendrimer, and therefore cannot participate in the binding even if it is located inside the folded protein. Here, "protein having a thiol group" includes not only proteins that originally have a thiol group, but also proteins into which a thiol group is newly introduced by genetic engineering methods, proteins containing cysteine ​​in which the cysteine ​​is reduced to a thiol group, and the like.

[0094] The presence position of the sulfhydryl group in the protein is not particularly limited, and sometimes it is preferred that the sulfhydryl group exists in a specific region on the protein. In addition, the luminescent properties of the shell can be controlled sometimes. It should be noted that the cysteine ​​contained in the "protein comprising cysteine" can be introduced into the desired position using a method for replacing any existing amino acid, a method for inserting into the desired position, or other genetic engineering methods. Those skilled in the art can easily use site-directed mutagenesis or other known genetic engineering methods to implement the introduction of cysteine ​​residues into the desired amino acid position of the protein.

[0095] The protein constituting the above-mentioned targeting sequence presenting part is not particularly limited as long as it has the property of association (hereinafter sometimes referred to as "association"), and preferably uses fluorescent proteins including green fluorescent protein (hereinafter sometimes the fluorescent proteins that emit fluorescence other than green are collectively referred to as "GFP") and its mutants. This is because the above-mentioned carbosilane dendrimer bound to such a fluorescent protein emits strong fluorescence when the protein is associated, and the associated state can be easily detected.

[0096] As GFP that can be used in the present invention, for example, in addition to GFP shown in sequence number 7 of the sequence list, GFP shown in sequence number 8, BFP (blue fluorescent protein) shown in sequence number 9 of the sequence list, YFP shown in sequence number 10 of the sequence list, CFP, RFP and other GFP derivatives sold by Clontech can also be cited. In addition, fluorescent proteins derived from sea anemones shown in sequence number 5 of the sequence list can also be used, and GFP shown in sequence number 7 of the sequence list can be preferably used due to the intensity of fluorescence and easy handling. In addition, it is conceivable that Azami-Green or other color mutants sold by MBL can also be preferably used due to structural characteristics.

[0097] Such GFP can be produced using a known method (see, for example, Biochim. Biophys. Acta 1679 (2004) 222-229; Biochem. Biophys. Res. Commun. 330 (2005) 454-460, etc.), or GFP produced by a company that produces such a protein outsourced to use can also be used.

[0098] Using molecules containing functional groups such as silole that cause the AIE effect as the backbone of the dendrimer, aggregating molecules with a targeting sequence presentation part composed of associative proteins such as fluorescent proteins are prepared. When the aggregating molecules are used to form micelles, fluorescence is also generated from the part where the silole group is aggregated, and fluorescence resonance energy transfer (FRET) occurs between the fluorescent protein and the silole, forming strong fluorescence. In addition, when the micelles disintegrate, FRET disappears.

[0099] Therefore, if the micelles prepared using the above-mentioned aggregating molecules are used as carriers for drug delivery, the state of the above-mentioned carrier (micelles) in vivo can be monitored using the change in FRET as an indicator. Furthermore, since such monitoring can be performed, the presence position and state of such carriers can be tracked.

[0100] In order to generate FRET between the fluorescent protein and the silole dendrimer, it is important to determine which position of the fluorescent protein constituting the targeting sequence presentation part is fixed to the above-mentioned silole dendrimer. Those skilled in the art can easily determine the position on the above-mentioned fluorescent protein bound to the dendrimer by preliminary experiments, etc. In the case of using GFP as the fluorescent protein constituting the above-mentioned targeting sequence presentation part, it is preferred that the N-terminal region, the C-terminal region, and the loop region adjacent to any one of the N-terminal region or the C-terminal region are bound to the dendrimer, and it is preferred that amino acids such as cysteine ​​having a thiol group are present in these regions.

[0101] Here, the binding to the "N-terminal region" refers to the case where the portion protruding from the core portion of about 10 amino acids present near the N-terminal is bound to the above-mentioned silole dendrimer. Similarly, the binding to the C-terminal region refers to the case where the portion protruding from the core portion of about 10 amino acids present in the C-terminal region is bound to the above-mentioned silole dendrimer.

[0102] The position of the halogen group supported on the silole dendrimer of the present invention is not particularly limited, but is preferably present in the side chain of the silole dendrimer, and more preferably at the end of the side chain (the position farthest from the silole group) as shown in the following formula (VI).

[0103] As described above, the silole dendrimer used in the method of the present invention is preferably a compound represented by the following formula (VI). This compound can be synthesized from 1,2-diphenylacetylene via a known intermediate 18 to obtain a silole core 2 (1,1-diaryl-2,3,4,5-tetraphenylsilole) as shown in Scheme 2 described below.

[0104] Next, using H2PtCl6·6H2O as a catalyst, 2 is hydrosilylated with trichlorosilane, and a Grignard reaction with arylmagnesium bromide is performed to obtain a silole core dendrimer 3. The obtained dendrimer 3 is treated with dichlorohexylborane and then hydrolyzed with hydrogen peroxide in an alkaline solution to obtain a hexahydroxy derivative 4. Next, the hexahydroxy derivative is O-mesylated and replaced with a bromine anion to obtain 5 (Tetrahedron Lett., 200748: 4365-4368).

[0105] [Chemistry 7]

[0106]

[0107] The protein having a target recognition site constituting the targeting sequence presentation portion is not particularly limited as long as it has the property of association. However, the protein is preferably any fluorescent protein selected from the group consisting of red fluorescent protein, yellow fluorescent protein, blue fluorescent protein and green fluorescent protein.

[0108] This is because it is easy to obtain and because the fluorescence intensity of the fluorescent protein itself is high, strong fluorescence is generated by FRET. Here, the blue fluorescent protein includes any one of fluorescent proteins that exhibit blue or blue-green fluorescence.

[0109] Regarding the aggregating molecule used in the present invention, the above-mentioned protein (targeting sequence presenting part) having a thiol group is mixed with a dendrimer compound (Formula (VI)) having a halogen group in the side chain and incubated to react the thiol group of the protein with the halogen group of the dendrimer compound, thereby binding the protein to the dendrimer compound. At this time, the protein is preferably treated with a reducing agent such as DTT in advance to form a state in which the -SH group is not oxidized. The reaction when one molecule of GFP is introduced through this reaction is shown in Scheme 2.

[0110] [Chemistry 8]

[0111]

[0112] Solution 2

[0113] For example, the reaction shown in Scheme 2 above can be carried out in an appropriate solvent (e.g., a water-soluble solvent such as phosphate buffered saline (PBS) or saline). The reaction conditions at this time vary depending on the protein used, and the reaction temperature can be, for example, 0 to 50°C, preferably 30 to 45°C, and more preferably about 37°C as long as it is lower than the denaturation temperature of the protein used. If GFP is used, the reactivity can be increased temperature-dependently up to 42°C, so from the perspective of preparing aggregating molecules for DDS, GFP is preferably used.

[0114] The reaction time also varies depending on the reaction temperature, and is, for example, about 1 to 24 hours, preferably about 10 to 19 hours, and more preferably about 15 to 18 hours. This is because, with such a reaction time, the protein having a thiol group used is bound to the silole dendrimer to form liposomes or micelles.

[0115] That is, as the protein-dendrimer complex is formed, a liposome or micelle having a structure in which the protein (hydrophilic part) is on the water-soluble solvent side and the core part (hydrophobic part) of the dendrimer is associated between the hydrophobic parts is formed. That is, it is considered that the association of the protein becomes a driving force by binding the above-mentioned associative protein to the side chain of the dendrimer, thereby forming a liposome or micelle.

[0116] Here, at least one protein is bonded to the silole dendrimer by substitution with the halogen atom at the end of the side chain. Therefore, the targeting shell for drug delivery system obtained in the above reaction is obtained in the form of a mixture of the bonds represented by the above chemical formulas (II) to (V).

[0117] When the protein having a thiol group is a fluorescent protein, the mixing ratio (molar ratio) of the protein to the dendrimer can be, for example, 1 to 20, preferably 1:5 to 1:15, and more preferably about 1:10, with the protein being 1:1. However, the optimal value of this ratio varies depending on the concentration.

[0118] In addition, when carrying out the above-mentioned reaction, from the aspect of realizing efficient delivery in DDS described later, it is preferred to introduce the amino acid sequence that will become the targeting recognition site into the protein as the targeting sequence presentation part in advance. The introduction of this targeting recognition site is preferably carried out simply by inverse PCR. This is because, if the plasmid encoding the mutant made in this way is introduced into Escherichia coli for expression, the mutant protein can be easily obtained, and the aggregating molecule for DDS can be obtained.

[0119] By pre-introducing the above-mentioned targeting recognition site into the protein serving as the targeting sequence presenting portion, the desired agent encapsulated in the targeting shell of the drug delivery system of the present invention can be specifically delivered to the above-mentioned normal tissues with inflammation, tissues where undesired gene expression is observed, tissues composed of tumor cells, etc. while maintaining activity.

[0120] The targeting shell for drug delivery system having the above structure is manufactured to encapsulate the desired drug, thereby enabling the drug to be specifically delivered to the target tissue while maintaining the effectiveness of the drug.

[0121] Example

[0122] The following embodiments are merely illustrative and do not limit the scope of the present invention.

[0123] (Example 1) Preparation of Aggregating Molecules and Micelle for DDS

[0124] In this example, GFP as described below was used as the protein that functions as a targeting presentation moiety, and silole dendrimer was used as the dendrimer.

[0125] (1) Preparation of aggregating molecules for DDS

[0126] In this example, a compound represented by the following chemical formula (X) (hereinafter sometimes referred to as "dimethyl dumbbell (1) 6-Br") was used as a dendrimer having a halogen group, and GFP (green fluorescent protein) (SEQ ID NO: 7) was used as a protein having a thiol group.

[0127] [Chemistry 9]

[0128]

[0129] The GFP used here (SEQ ID NO. 7 in the sequence listing) was prepared according to the method reported by the inventors (see Biochim. Biophys. Acta 1679 (2004) 222-229; Biochem. Biophys. Res. Commun. 330 (2005) 454-460). The amino acid at position 251 in the C-terminal region of the amino acid sequence of the GFP shown in SEQ ID NO. 7 was replaced with cysteine, and the original cysteine ​​(positions 48 and 70) was replaced with serine and valine, respectively.

[0130] First, DTT was added to a 20 μM GFP solution (in PBS) to a final concentration of 1 mM, and the mixture was treated at room temperature for 10 minutes to reduce the cysteine ​​on the surface of GFP. 10 μL of a 200 μM silole dendrimer solution (DMSO solvent) represented by the above formula (VI) was added to 400 to 450 μL of a 20 μM GFP solution (in PBS) after cysteine ​​reduction to a final concentration of 10 times the molar equivalent, and mixed using a vortex mixer.

[0131] After mixing, the mixture was allowed to stand at 37°C and incubated overnight to allow GFP to bind to the silole dendrimer and then form micelles. The incubation time was about 16 to 18 hours. The characteristics of the micelle particles in the solution containing the obtained micelles were measured by dynamic light scattering (DLS). The results are shown in Table 1.

[0132] [Table 1]

[0133] Average particle size of the raw material and product measured 5 times in PBS at 25°C [nm]

[0134]

[0135] In addition, the particle size of the reaction solution was measured at 25°C using ZETASIZER NANO-S (manufactured by Malvern) with a laser wavelength of 532 nm. The results are shown in Figure 3 . Figure 3 (A) shows the results of investigating the particle size distribution of the product obtained by incubation with GFP alone. Figure 3 (B) is the result of investigating the particle size distribution of the product obtained by incubating only the silole dendrimer of formula (VI). Figure 3 (C) is the result of investigating the particle size distribution of the product obtained by mixing GFP and the silole dendrimer of formula (VI) and incubating the mixture. The horizontal axis represents the particle size of the obtained product, and the vertical axis represents the percentage of the product of the size indicated by the horizontal axis in the total.

[0136] When GFP and the above-mentioned silole dendrimer were incubated (indicated as "GFP-silole only" in the above-mentioned Table 1), the particle size of the obtained micelle was about 150 nm. In contrast, the particle size observed when only GFP was incubated was about 4 nm, and the particle size observed when only the above-mentioned silole dendrimer was incubated was about 650 nm. It is believed that the increase in the particle size observed when only the above-mentioned silole dendrimer was incubated may be due to the fact that the silole dendrimers aggregated with each other to form large aggregates.

[0137] Next, the obtained micelle particles were observed using a scanning electron microscope (SEM), and a large number of particles with a particle size of about 100 to 500 nm and a small number of particles with a particle size of about 500 nm were confirmed (see Figure 4 ).

[0138] From the above results, it can be seen that the micelles formed using the aggregating molecules for DDS of the present invention have a particle size of about 100 to 500 nm, and the micelles with a particle size of about 100 to 200 nm are more numerous. In addition, it can be confirmed from the electron microscope images using SEM that a spherical micelle structure is formed.

[0139] (2) Confirmation of fluorescence resonance energy transfer

[0140] The silole dendrimer used in this example has the property of emitting light (exhibiting the AIE effect) when the hydrophobic silole core part aggregates, so it was confirmed that it also emits light when forming a micelle structure. Therefore, when preparing micelles composed of GFP-silole dendrimers in which GFP is bound to silole dendrimers, it was studied whether fluorescence resonance energy transfer (FRET) occurs between silole and GFP.

[0141] The experiment of luminescence properties was conducted after removing the unreacted fluorescent protein and dendrimer (free molecules) from the above reaction solution (see Figure 5). The product incubated with silole dendrimer alone was excited at a wavelength of 370 nm (white hollow quadrilateral: □), the product incubated with GFP alone was excited at a wavelength of 488 nm (white hollow triangle: △), and the product incubated with GFP and silole dendrimer (micelle of the present invention) was excited at a wavelength of 370 nm (black circle: ●), and the characteristics of each emission wavelength were investigated. Regarding the micelle composed of the GFP-silole dendrimer complex (the product obtained by incubating GFP and the silole dendrimer of formula (X)), luminescence caused by FRET of silole to GFP was observed at around 510 nm.

[0142] like Figure 5 As shown in FIG. 1 , the peak of the luminescence of the silole dendrimer was observed near 480nm. In addition, no sharp peak was confirmed for the luminescence of the combination of GFP and silole dendrimer, and the maximum value was shown near 510nm. In contrast, a sharp peak was observed near 510nm for the luminescence of GFP, which was believed to be caused by FRET from the above-mentioned silole dendrimer to GFP. In addition, when the micelles disintegrated, the luminescence of GFP, which was believed to be caused by FRET, also disappeared.

[0143] That is, a molecule (GFP-silole dendrimer complex) in which a dendrimer having an AIE effect is bound to an associative protein such as a fluorescent protein is prepared. When micelles are formed using this molecule, FRET occurs between the dendrimer and the fluorescent protein in the micelle state, and FRET disappears due to the disintegration of the micelle.

[0144] As shown above, when the micelle of the present invention is used as an aggregating carrier for DDS, the tissue or organ to be delivered can be confirmed. In addition, even after the micelle delivered to the target tissue or organ disintegrates, the fluorescence of the fluorescent protein can be tracked, thereby also being able to sense the intracellular environment of the fluorescent protein delivered.

[0145] (3) Encapsulation experiments of drugs in micelles composed of GFP-silole dendrimer complexes

[0146] Next, it was confirmed whether micelles encapsulating drugs or the like can be prepared using the micelles of the present invention. In this example, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Promokine PK-CA707-60010, manufactured by PromoCell GmbH), Oil Orange SS (manufactured by Tokyo Chemical Industry Co., Ltd., T0553), goat anti-mouse IgG (manufactured by abcam, ab6708)-Alexa610 (manufactured by Molecular Probes, A30050) and WGA (Wheat Germ Agglutinin: wheat germ agglutinin, manufactured by Molecular Probes) (WGA-Alexa Fluoro (registered trademark) 594 conjugate, W11262) were used as model drugs.

[0147] 10 μL of 200 μM of the silole dendrimer (DMSO solvent) represented by the above formula (X) is added to 20 μM GFP after cysteine ​​reduction to make the final concentration 10 times the molar equivalent, and then any one of DiI (final concentration: 1 μM, fluorescent dye), Oil Orange SS (final concentration: 20 μM, fluorescent dye), goat anti-mouse IgG-Alexa610 (final concentration: 0.1 μM) and WGA (final concentration: 2 μM) is added and mixed using a vortex mixer, and incubated at 37°C overnight (about 16 to 18 hours).

[0148] It was determined whether micelles encapsulating each drug were formed in the sample to which each model drug was added, and when micelles were formed, the particle size of these micelles was measured by the above-mentioned dynamic light scattering method. The results are shown in Table 2. Here, DiI, oil orange SS and goat anti-mouse IgG-Alexa 610 were used as drug models.

[0149] exist Figure 6 A to C show the particle size distribution when the above-mentioned model drug is used. Figure 6 A is the particle size distribution when micelles are prepared in the presence of DiI. Figure 6 B is the particle size distribution when micelles are prepared in the presence of oil orange SS. Figure 6 C is the particle size distribution when micelles are prepared in the presence of goat anti-mouse IgG-Alexa 610. The horizontal axis represents the particle size of the obtained micelles, and the vertical axis represents the percentage of micelles of the size shown on each horizontal axis in the whole. The particle size of the micelles when DiI is added is about 95nm, when Oil Orange SS is added, it is also about 95nm, and when IgG-Alexa610 is added, it is about 180nm.

[0150] [Table 2]

[0151] Average particle size of 5 measurements in PBS at 25°C [nm]

[0152]

[0153] The results of investigating the luminescence properties of micelles encapsulating the fluorescent dye DiI are shown in Figure 7 . Figure 7 B is Figure 7 The spectrum of A around 500 nm is enlarged. The horizontal axis represents wavelength (nm) and the vertical axis represents relative fluorescence intensity (au). First, micelles encapsulating DiI were prepared, and samples were prepared in which the micelles were washed three times with PBS; then, the reaction solution was filtered with a filter with a pore size of 0.45 μm to remove free pigments; and the sample was filtered with a 0.22 μm filter instead of a 0.45 μm filter to remove free pigments, and the fluorescence of these samples was measured.

[0154] like Figure 7 As shown in A and B, when excited at a wavelength of 370 nm, a shoulder peak was confirmed near a wavelength of 480 nm, which is believed to be caused by the luminescence of silole. The peak near 510 nm is believed to be caused by FRET from silole to GFP, and the peak near 570 nm is believed to be caused by FRET from silole to DiI. The above results confirm that DiI is enclosed in micelles.

[0155] Next, the luminescence properties of micelles encapsulating WGA labeled with Alexa594 were investigated (see Figure 8 A and B). Figure 8 A is the result of direct measurement of the reaction solution when Alexa594-labeled WGA was added. Figure 8 B is the result of measurement after removing unreacted proteins (WGA labeled with Alexa594, silole dendrimer, GFP, and other substances not incorporated into micelles) from the reaction solution using an ultrafiltration centrifugal column (manufactured by Millipore). Figure 8 In the figure, the solid line represents the fluorescence characteristics of a product obtained by mixing the GFP-silole dendrimer complex and WGA, and the dotted line represents the fluorescence characteristics of a product obtained by mixing GFP and WGA.

[0156] In the figure, the solid line shows the results of measuring micelles containing Alexa594-labeled WGA in micelles composed of GFP-silole dendrimer complex, and the dotted line shows the results of measuring micelles containing a mixture of GFP and Alexa594-labeled WGA (both excitation wavelengths are 370 nm).

[0157] Figure 8 The results of the direct measurement of the reaction solution without ultrafiltration centrifugal column treatment are shown in A. Figure 8 In A, fluorescence from the silole dendrimer was detected at around 480 nm, fluorescence from GFP was detected at around 510 nm, and fluorescence from WGA was detected at around 610 nm. Figure 8 As shown in B, when ultrafiltration centrifugal column treatment was performed, free silole dendrimer, GFP and WGA were removed (dashed line), and thus almost no luminescence from them was detected. This confirmed that Alexa594-labeled WGA was encapsulated in the prepared micelles.

[0158] (Example 2) Preparation of fluorescent protein combined with targeting peptide sequence

[0159] The fluorescent protein bound to the targeting peptide sequence is prepared as follows: the fluorescent protein is bound to the micelle of the present invention at the C-terminus and is bound to the targeting peptide at the N-terminus, and the targeting peptide binds to a receptor expressed on the surface of cancer cells.

[0160] (1) Reverse PCR

[0161] (1-1) Selection of peptide sequence

[0162] The amino acid sequence of the peptide introduced into the selected target binding site is recorded in Table 3 below. MCF7-1 is a human breast cancer-derived cell MCF7 containing the sequence shown in Table 3 below (hereinafter sometimes referred to as "targeted type 1"), and MCF-2 is a mutant of MCF-1 containing the sequence shown in Table 3 below (hereinafter sometimes referred to as "targeted type 2"). In addition, as shown in Table 3 below, MCF7-1+αstand is another mutant of MCF7-1 having a structure in which a short peptide of an α-helical structure (hereinafter referred to as "αstand") is connected to the above-mentioned MCF7-1 (hereinafter sometimes referred to as "type 1 enhanced").

[0163] [Table 3]

[0164]

[0165] (1-2) Preparation of primers

[0166] Table 4 below lists primers used for inverse PCR of the peptide sequences shown in Table 3. These primers were designed so that, for sequence numbers 1 and 3 in the sequence list, the extension reaction started between DM and PGTVLP of the peptide sequence, and for sequence number 2, the extension reaction started between VP and TDTDYSGG of the peptide sequence. This was done in order to optimally perform inverse PCR.

[0167] [Table 4]

[0168]

[0169] (1-3) Preparation of template plasmid for inverse PCR

[0170] The template plasmid for inverse PCR was prepared by the method described in the following paper.

[0171] "Protease-sensitive signaling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein.-Miho Suzuki, et al. Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression

[0172] Volume 1679, Issue 3, September 17, 2004, Pages 222-229”

[0173] (1-3-1) Plasmid construction of GFPuv5 mutant

[0174] GFPuv5 was obtained from pGFPgcn4 as follows. First, a PCR product with a synonymous mutation for gene manipulation was obtained by using the I167T mutation and a forward primer 5'CATTGAAGATGGCTCCGTTCAA (SEQ ID NO. 18) and a reverse primer 5'TTGTGGCGAGTTTTGAAGTTAG (SEQ ID NO. 19), and then the PCR product was cyclized to produce the construct. The construct obtained as above was named pGFPgcn5.

[0175] Afterwards, the cDNA of GFPuv5 was cloned into pET21a (manufactured by Novagen) to express the protein. The protein expressed here was purified and named GFPuv5tag. For its coding region, primers 5'CTCGACCAT[ATGGCTAGCATGACTGGTGGACAGCAAATGGGT]CGCATGAGTAAAGGAGAAGAACTTTTCA (SEQ ID NO. 20) (attached to the initial 11 amino acid epitope tag used for 10 proteins of the T7 gene in the N-terminal direction of the GFPuv5 series, and the attached tag is indicated by []) and 5'TGACGTGAATTCATTA[GTGATGGTGATGGTGATG]TTTGTAGAGCTCATCCATGC (SEQ ID NO. 21) (His tag is indicated by []) providing a His tag to the C-terminus of the GFPuv5 series were amplified.

[0176] The DNA of the base sequence shown in sequence numbers 1 to 3 was inserted into pET21a and digested with NdeI and EcoRI. The pGFPgcn plasmid was used for gene manipulation, and the pET21a plasmid was used for protein expression under the control of the T7 promoter. The base sequences of the GFPuv5 gene and its mutant genes were confirmed by DNA sequencing (manufactured by ABI PRISM 3100Genetic Analyser). Three other synonymous mutations were found in the experiment (agt of Ser-30 mutated to agc, cat of His-78 mutated to cac, and caa of Gln-183 mutated to cag).

[0177] These mutations are not harmful to fluorescent proteins, so the experiment was continued with these mutations. The fluorescence intensity of the purified GFPuv5tag was about 1.9 times that of the GFPuv4tag. Afterwards, any cysteine ​​residue at position 48 or 70 was replaced with a random amino acid by inverse PCR using pGFPgcn5.

[0178] Oligonucleotides 5'CTTAAATTTATTNNKACTGGAAAAC (SEQ ID NO. 22) and 5'GGTAAGTTTTCCGTATGTTG (SEQ ID NO. 23) were used for mutation of cysteine ​​48, and 5'GTGTTCAANNKTTTTCCCGTTATCCG (SEQ ID NO. 24) and 5'CATACGTCAGAGTAGTGACAAG (SEQ ID NO. 25) were used for mutation of cysteine ​​70. The resulting plasmids were used to transform cultures of E. coli BL21 (DE3), screened for strong fluorescence under daylight excitation, and selected on agar medium. In the case of position 48, several mutants that emitted strong fluorescence were obtained (Ala, Asp, Glu, Gly, Ile, Leu, Asn, Pro, Ser, Thr, Val and Tyr substitutions). However, in the case of mutants in which the amino acid at position 70 was substituted, only the C70V cysteine ​​mutant provided appropriate fluorescence.

[0179] In order to produce a double cysteine ​​mutant GFPuv5 with strong fluorescence intensity, the plasmid with a single mutation was digested with NcoI and EcoRI and ligated to each region again. Single mutants were selected. UV5C0tag (C48S / C70V) showed the highest fluorescence intensity among all recombinants.

[0180] Next, cysteine ​​was introduced into positions 6 and 229 by inverse PCR. For the introduction, a plasmid having a C48S mutation and the following primer sets were used for the respective mutations.

[0181] Glu replacement: 5'TGTCTTTTCACTGGAGTTGTCCC (SEQ ID NO: 26) and 5'TTCTCCTTTACTCATTTTTTC (SEQ ID NO: 27)

[0182] For Ile replacement: 5'TGCACACATGGCATGGATGAGCTC (SEQ ID NO: 28) and 5'CCCAGCAGCAGTTACAAACTC (SEQ ID NO: 29)

[0183] Three protease tags containing a trypsin target sequence (Gln-Gly-Arg) had various spacer sequences (no spacer, Thr spacer, or Gly-Thr spacer) with the necessary cysteine ​​substitution between His-231 and Asp-234. The construct was obtained using puvC48Stag, the resulting plasmid (template), and the primers shown in Table 5 below.

[0184] [Table 5]

[0185]

[0186] (1-3-2) Purification of GFPuv5tag mutant

[0187] Escherichia coli BL 21 (DE3) was transformed with all plasmids. Escherichia coli (12 ml) in the stationary phase after overnight culture was inoculated into LB medium (38 mL) supplemented with ampicillin (50 μg / ml) and IPTG (0.5 mM) and incubated at 37 ° C for 8 hours. Centrifuge at 2,500 x g for 20 minutes, collect the cells, and resuspend them in 10 mL of PBS. The cell mass was dissolved in 10 mL of lysis buffer (pH 8.0) containing 50 mMTris and 8 M urea at room temperature for 15 minutes and vortexed. Centrifuge at 1,200 x g for 15 minutes, and mix the supernatant with Ni2+NTA resin (manufactured by Qiagen Co. Ltd.) suspended in PBS. After washing the resin with PBS and 20 mM imidazole in turn, the bound GFPuv5tag mutant was eluted with a 250 mM imidazole solution.

[0188] To replace the buffer, the eluate was applied to a PD-10 gel electrophoresis filter column (manufactured by Amersham Bioscience Co. Ltd.) equilibrated with 10-fold diluted PBS. The eluted GFPuv5tag mutant proteins were collected and their concentrations were quantified using Coomassie protein detection reagent (manufactured by Pierce). The purified GFPuv5tag mutants were analyzed by 15% SDS-PAGE.

[0189] The base sequence of the template plasmid for inverse PCR is shown in SEQ ID NO: 26.

[0190] (1-4) PCR conditions

[0191] The reaction solutions shown in Table 6 below were prepared, and inverse PCR was performed under the conditions of Table 7 below.

[0192] [Table 6]

[0193]

[0194]

[0195] [Table 7]

[0196]

[0197] (1-5) Confirmation by electrophoresis

[0198] A portion of the PCR reaction solution was taken and subjected to gel electrophoresis using 0.8% PAGE at a voltage of 100 V for 30 minutes, and amplified peptides were confirmed in each sample. The electrophoresis results are shown in FIG. Fig. 9 .

[0199] (2) Removal of independent A sequences and purification of PCR products

[0200] The reaction solution shown in Table 8 below was prepared and reacted at 120°C for 30 minutes to remove the independent A sequence generated by the PCR. The PCR product was then purified using QIAquick (registered trademark) PCR purification kit (manufactured by QIAGEN) according to the instructions attached to the kit.

[0201] [Table 8]

[0202]

[0203] (3) Ligation reaction

[0204] Next, the reaction solution shown in Table 9 below was prepared and reacted at 16°C for 3 hours or more to prepare a circular plasmid for transformation of E. coli DH5α described below. Depending on the mutant, 25°C, 37°C, etc. were used.

[0205] [Table 9]

[0206]

[0207] (4) Transformation of Escherichia coli DH5α

[0208] Thaw 10 μL of competent cells of Escherichia coli DH5α (manufactured by BioDynamics Laboratory) on ice just before use to prepare a competent cell solution. Add 1 μL of the ligation reaction solution to the competent cell solution and place it on ice for 30 minutes. After that, incubate at 42°C for 30 seconds, and then cool on ice for 2 minutes. Add 90 μL of SOC (manufactured by Toyobo Co., Ltd.) culture medium to it and react on an oscillator at 37°C for 1 hour. After that, inoculate it into LB selection medium containing ampicillin (manufactured by Toyobo Co., Ltd.) and let it stand overnight at 37°C.

[0209] (5) Colony PCR

[0210] Colony PCR was performed on colonies obtained by the above transformation to confirm the putative insert.

[0211] (5-1) Preparation of PCR reaction solution

[0212] Prepare the reaction solution for colony PCR shown in Table 10 below.

[0213] [Table 10]

[0214]

[0215] The colony PCR reaction solution was placed in a PCR tube, and the E. coli grown in the ampicillin-containing LB medium was collected and added. PCR was performed according to the schedule shown in Table 11 below.

[0216] [Table 11]

[0217]

[0218]

[0219] (5-2) Electrophoresis

[0220] The PCR reaction solution was subjected to electrophoresis using 1.2% agarose gel at an applied voltage of 100 V for 30 minutes, and colonies confirmed to have amplified were transplanted into a culture bottle containing LB liquid medium (manufactured by Toyobo Co., Ltd.) and incubated at 37°C.

[0221] (6) Plasmid purification

[0222] The plasmid in the E. coli cultured in the LB liquid medium was purified using Wizard Plus SV Minipreps DNA Purification System (produced by Promega) according to the instructions attached to the product. The sequence of the purified plasmid was then analyzed by Eurofin Genomics.

[0223] (7) Transformation of Escherichia coli BL21(DE3)

[0224] 10 μL of E. coli BL21 (DE3) competent cells (manufactured by BioDynamics Laboratory) were thawed on ice just before use to prepare a competent cell solution. 1 μL of the plasmid solution confirmed to contain the target sequence by the above sequencing was added to the competent cell solution and placed on ice for 30 minutes.

[0225] After that, incubate at 42°C for 30 seconds and cool on ice for 2 minutes. Add 90 μL of SOC (manufactured by Toyobo Co., Ltd.) medium to it and react on a shaker at 37°C for 1 hour. Then, inoculate into LB selection medium containing ampicillin and let it stand overnight at 37°C. On the next day, collect the colonies that have been transformed and emit green fluorescence, add them to a culture bottle containing 1 ml of LB liquid medium containing ampicillin, and let it stand overnight at 37°C for pre-culture.

[0226] (8) Purification of fluorescent proteins bound to targeting peptide sequences

[0227] (8-1) Colony culture

[0228] 4 mL of LB liquid medium containing ampicillin was added to a 50 mL tube, and 290 μL of the above pre-culture solution was added thereto as a sample. Four such samples were prepared and cultured on a shaker at 28° C. for 4 hours. Thereafter, 43 μL of 100 mM IPTG (isopropyl-β-thiogalactopyranoside) was added and cultured on a shaker at 28° C. overnight.

[0229] (8-2) Protein recovery

[0230] On the second day, the culture solutions of the above four tubes were pooled into one tube. The three tubes after the transfer of the contents were washed with 1 ml of PBS (-) buffer (manufactured by Wako Pure Chemical Industries, Ltd.), and the washing solution was also added to the pooled tube. The tubes with the culture solutions collected as above were centrifuged at room temperature at 5,000 rpm (centrifuge name: KUBOTA3740, rotor number KUBOTA AF2018, manufactured by Kubota Corporation) for 5 minutes.

[0231] After that, (i) the supernatant was discarded, 3 ml of PBS(-) buffer was added to the precipitated pellet, and (ii) after sufficient stirring, centrifugation was performed at 5,000 rpm for 5 minutes. The above (i) and (ii) were repeated twice. 4 mL of B-PER lysis buffer (manufactured by Reagent) was added to the precipitated pellet, and the pellet was covered with a lid and stirred overnight at room temperature using a shaker.

[0232] (8-3) Purification using His tag

[0233] Take 2 mL of Ni-NTA agarose (manufactured by QlAGEN) into a 15 mL tube, (i) centrifuge at 1,000 rpm for 1 minute, (ii) discard the supernatant, add 1 mL of 1× PBS buffer and stir thoroughly. Repeat the above (i) and (ii) three times to prepare Ni-NTA resin.

[0234] After centrifuging the tube containing the above B-PER lysis buffer at 12,000 rpm for 10 minutes at room temperature, transfer the supernatant to a 15 mL Falcon tube. Add 400 μL of the above Ni-NTA resin that has been stirred well, and stir it at room temperature for 10 minutes using a rotary shaker. Then, centrifuge at 1,000 rpm for 1 minute at room temperature and discard the supernatant. (iii) Add 4 mL of 1×PBS buffer and stir well, (iv) centrifuge at 1,000 rpm for 1 minute at room temperature and discard the supernatant. Repeat (iii) and (iv) twice.

[0235] Then, (v) add 4 mL of 20 mM imidazole (manufactured by Wako Pure Chemical Industries, Ltd.) and stir thoroughly, (vi) centrifuge at 1,000 rpm for 1 minute at room temperature, and discard the supernatant. Repeat (v) and (vi) twice. Add 500 μL of 250 mM imidazole and stir at room temperature for 10 minutes on a rotary shaker. Then centrifuge at 1,000 rpm for 1 minute, and place the supernatant that emits green fluorescence into a new 15 mL Falcon tube as the protein purification solution for the subsequent gel filtration.

[0236] (8-4) Purification by gel filtration

[0237] The imidazole in the protein purification solution was replaced with PBS buffer to purify the fluorescent protein bound to the target peptide sequence. In the above operation, the NAP5 column of GE Heath Care Japan Co., Ltd. was used for purification according to the instructions attached to the product.

[0238] (9) Selection of fluorescent proteins combined with targeting peptide sequences

[0239] According to the conventional method, the absorbance (280nm) of the fluorescent protein concentration combined with the targeting peptide sequence obtained by the above-mentioned purification operation and the absorbance (488nm) of the chromophore concentration (forming ability) were measured, and the fluorescent protein with an A488 / A280 value exceeding 1.5 was selected as the fluorescent protein combined with the targeting peptide sequence of the target. The results are shown in Table 12.

[0240] [Table 12]

[0241]

[0242] (Example 3) Preparation of Shell (Fluorescent Protein Association-Driven Micelle) Incorporating Targeting Peptide Sequence

[0243] As a protein, the fluorescent protein to which the targeting peptide sequence was bound prepared in Example 2 was used instead of GFP, and micelles to which the targeting peptide sequence was bound were prepared in the same manner as in Example 1 (fluorescent protein association-driven micelles).

[0244] (1) Luminescence characteristics

[0245] The luminescence properties of the fluorescent protein association-driven micelles prepared above and combined with the targeting peptide sequence were measured in the same manner as in Example 1 (see Fig.10 ).exist Fig.10In the example of , the reactant shows a complex formed by the combination of a silole dendrimer and a fluorescent protein combined with a targeting peptide sequence, and the unreacted product shows their uncombined state, and each numerical value represents the wavelength (nm) of the excitation light. In the case of micelles combined with a targeting peptide sequence, a luminescent peak is observed near 510nm as in Example 1.

[0246] (2) Particle characteristics

[0247] The particle properties of the liposomes or micelles combined with the targeting peptide sequence were determined by dynamic light scattering in the same manner as in Example 1 (see Fig.11 ). Fig.11 In the graph, the horizontal axis represents the particle size of the obtained micelles, and the vertical axis represents the percentage of micelles of the size indicated by each horizontal axis in the whole. As a result, particles having a particle size of about 100 nm to 200 nm were confirmed in the same manner as in Example 1.

[0248] The above shows that the fluorescent protein-bound micelles with the targeting peptide sequence also form micelles equivalent to those without the targeting sequence. In addition, regarding the micelles obtained in Example 1, it is also speculated that the micelles obtained in this example are all fluorescent protein association-driven micelles formed by the association of fluorescent proteins.

[0249] (Example 4) DDS micelle·liposome (vesicle) morphology determination experiment

[0250] (1) Preparation of basic liposomes or micelles

[0251] Liposomes or micelles were prepared as follows. First, the GFP-containing solution was concentrated by centrifugation at 14,000 x g for 15 minutes using a 10K Amicon filter (manufactured by Amicon), and 1x PBS was added to make the volume to 99 μL. Using a 20 μM GFP-containing solution, 137.5 μL of GFP was concentrated to prepare 50 μM / 50 μL micelles.

[0252] Next, 1 μL of 100 mM DTT was added to the solution and incubated at room temperature for 10 minutes. The entire amount of the incubated solution was added to a NICK column (manufactured by GE Heath Care Japan), and 365 μL of 1x PBS was added. Furthermore, 380 μL of 1x PBS was added to recover almost the entire amount.

[0253] Next, 3.53 μL of 7.78 mM TPS (2,3,4,5-tetraphenyl-1,1-dimethylthiole) was added to the GFP contained in the recovered solution at a molar ratio of 1:10, and the molecules in the solution were aggregated at 37° C. overnight.

[0254] Next, add all the approximately 380 μL of the solution that has been aggregated overnight to a 100K Amicon filter, centrifuge at 14,000 x g for 10 minutes to concentrate, and take approximately 30 μL as the column supernatant. Add 100 μL of 1xPBS to it, and centrifuge at 14,000 x g for 10 minutes to wash. Repeat this washing operation 3 times, and collect approximately 300 μL of the washing solution.

[0255] After centrifugation at 14,000xg for 10 minutes, the column was inverted and the supernatant was recovered using a tabletop centrifuge. An appropriate amount of 1x PBS was added to the empty column and the volume was fixed to 1.2 times the target volume with 1x PBS. The column was placed at 4°C for 1 day for equilibrium and diluted 20 times for particle size measurement. In addition, the fluorescence measurement was performed using Shimadzu RF5300-PC, and the sample diluted 80 times was measured at an excitation wavelength of 370nm and a measurement wavelength of 488nm (range 3x 5).

[0256] In addition, about 350 μL of the solution added to the 100K Amicon filter and centrifuged at 14,000 x g for 10 minutes was used as the column liquid. The above washing solution was combined into 650 μL, diluted 20 times, and the particle size was measured. In addition, the excitation wavelength was 370 nm and the measurement wavelength was 488 nm in the fluorescence measurement, and the sample diluted 25 times was measured (range 3 x 5). The non-targeted liposomes or micelles obtained here were named NSS25 and NSS26.

[0257] (2) Determination results using electron microscopy

[0258] Liposomes or micelles were determined using a low-temperature low-vacuum scanning electron microscope (manufactured by Hitachi High-Technologies, catalog number S-3400N), and the observed molecules were mostly liposomes (vesicles). The sections were observed in a liquid nitrogen atmosphere.

[0259] Using this electron microscope, a reflected electron image of the incident electron beam is obtained as a BSE image, and a secondary electron image is obtained as an SE image. In addition, elemental analysis using X-rays is also performed simultaneously. The BSE image is shown in Fig.12 (A) to (C). In the low-resolution scanning electron microscope image, many vesicle-like molecules were observed. Elemental analysis of some of them also revealed silicon molecules (see Fig.12 ).

[0260] (Example 5) Concentration-dependent stability test of liposomes (vesicles) or micelles

[0261] (1) Preparation of sample micelles (micelles having target binding sites)

[0262] The sample micelles of 50 μM x 50 μL were prepared as follows. As basic micelles, 16.1 μM and 31.6 μM of non-targeted micelles (NSS25 and NSS26) prepared in the above Example (X1) were used, respectively. 11.3 μM of targeted micelles containing the sequence of MCF7 as the target recognition site (sequence numbers 12 and 13 in the sequence list) were used.

[0263] The micelle solutions containing each liposome or micelle were added to Amicon 10K filter, centrifuged at 14,000 x g for 15 minutes, and the supernatants were collected. Each micelle solution was fixed to 99 μL with 1x PBS, 1 μL of 1 mM DTT was added, and allowed to stand at room temperature for 10 minutes.

[0264] Next, each liposome or micelle solution was treated using a NICK column, and 3.21 μL of TPS was added to each of the obtained treated solutions, followed by leaving the solution at 37° C. overnight.

[0265] (2) Confirmation of concentration dependence of liposomes (vesicles) or micelles

[0266] 380 μL of each micelle solution obtained in (1) above was added to an Amicon 100K filter and centrifuged at 14,000 xg for 10 minutes. About 350 μL passed through the filter and fell down, which was referred to as the lower solution, and about 30 μL remaining on the filter was referred to as the upper solution.

[0267] The upper liquid was retained as it was, 100 μL of 1x PBS was added thereto, and the mixture was centrifuged at 14,000×g for 10 minutes to recover about 100 μL of the solution that passed through the filter. This operation was repeated 3 times, and the resulting solution was combined with the lower liquid, diluted 20-fold, and DLS (particle size distribution) was measured in the same manner as for the basic micelles.

[0268] The filter containing the upper liquid was placed in a table centrifuge (manufactured by Kubota Corporation) and centrifuged for 2 to 3 minutes and recovered. Next, an appropriate amount of 1x PBS was added to the empty filter and allowed to stand for 10 minutes. After that, the filter was inverted and placed in a table centrifuge for centrifugation for 2 to 3 minutes, the solution in the filter was recovered, and 1x PBS was added to make a fixed volume of 60 μL to prepare a 50 μM / 50 μL stock solution (1x).

[0269] The stock solution was diluted 5, 10, and 50 times with 1x PBS and left at room temperature for 1 day for equilibrium. Afterwards, the particle size and fluorescence measurements were performed in the same manner as in the case of basic micelles. This showed that micelles prepared at a critical micelle concentration or above could also be stable after 1 month.

[0270] (Example 6) Physical property confirmation test of constituent proteins of DDS micelles

[0271] (1) Protein purification

[0272] In order to confirm the physical properties of the constituent proteins of the micelles, the constituent proteins were purified using Ni NTA Super flow (manufactured by Qiagen) according to the following procedures.

[0273] (1-1) Dissolution of proteins from aggregated molecules

[0274] First, take 50 μL of basic micelles (50 μM / 50 μL) and add them to a 50 mL Falcon tube (manufactured by Falcon), wash with 20 mM imidazole, and then add 250 mM imidazole. Use a rotary shaker to react overnight to dissolve the protein from the micelles. The Falcon tube is directly set in a centrifuge (manufactured by Kubota Shoji Co., Ltd.), centrifuged at 12,000 rpm for 10 minutes, and the resulting supernatant is transferred to a 15 mL Falcon tube.

[0275] (1-2) Preparation of Ni NTA resin slurry

[0276] While the Falcon tube was centrifuged as described above, the Ni NTA resin (included in Ni NTA Superflow) was prepared. First, 2.5 mL of Ni NTA agarose was taken and the preservative was removed. Next, the solution containing the Ni NTA agarose gel was fully shaken, mixed evenly, and transferred to a 15 mL Falcon tube.

[0277] After that, centrifuge at 1,000 rpm for 1 minute in a table centrifuge to precipitate the gel, remove the supernatant with a pipette, add 1 mL of 1x PBS and shake and mix, and vortex mix. Repeat this operation 3 times. After the third centrifugation, remove the supernatant with a pipette to prepare a slurry. Store at 4°C except for the amount used.

[0278] (1-3) Purification of each protein

[0279] 400 μL of the slurry was added dropwise to the structural protein solution in a 15 mL Falcon tube, taking care not to allow the slurry to adhere to the inner wall of the tube. The mixture was then placed on a rotary shaker, the reservoir was adjusted to the fastest setting, and the reaction was allowed to proceed for 10 minutes.

[0280] Next, the tube was removed from the rotary shaker and centrifuged at 1,000 rpm for 1 minute using an animal cell centrifuge (manufactured by TAITEC Co., Ltd.), and the supernatant was discarded. 4 mL of 1x PBS was added to the pellet remaining at the bottom of the tube, vortexed, and centrifuged at 1,000 rpm for 1 minute using the above centrifuge. This operation was repeated twice.

[0281] Add 4 mL of 20 mM imidazole to the obtained pellet and vortex mix, and centrifuge at 1,000 rpm for 1 minute using the above centrifuge. Repeat this operation twice. Next, add 500 μL of 250 mM imidazole to the obtained pellet, set it in a rotary shaker, adjust the storage as fast as possible, and react for 10 minutes. Next, remove the tube from the rotary shaker, centrifuge it at 1,000 rpm for 1 minute using the above animal cell centrifuge, and transfer it to the above new 15 mL Falcon tube.

[0282] 250 mM imidazole was added to the pellet remaining in the old tube again, and the tube was placed on a rotary shaker, and the storage was adjusted to the fastest setting, and the reaction was performed for 5 minutes. Next, the tube was removed from the rotary shaker, and centrifuged at 1,000 rpm for 1 minute using the above-mentioned animal cell centrifuge, and the supernatant was taken and combined with the previous supernatant. This operation was repeated 5 times.

[0283] (1-4) Gel electrophoresis

[0284] For each protein (GFP) purified as above, gel electrophoresis was performed as follows.

[0285] First, prepare about 10 μL of each solution containing 5 μM of each protein and add it to a 1.5 mL capped tube. As a molecular weight marker (ladder band), take 10 μL of Precision Protein Broad Range Prestained Standard (manufactured by BioRad) and add it to a 1.5 mL tube in the same manner. Prepare a separation gel (12%) with the composition shown in Table 13 below and a concentration gel (4%) with the composition shown in Table 14 below.

[0286] [Table 13]

[0287] composition capacity x 4 Separation Gel Buffer 2.5mL 40% acrylamide gel solution (containing 19:1 bisacrylamide-acrylamide) 3.12mL 10% APS 50μL TEMED(Tetramethylethylenediamine) 50μL Distilled water margin Total 10mL

[0288] [Table 14]

[0289] composition capacity x 4 Stacking Gel Buffer 1.25mL 40% acrylamide gel solution (containing 19:1 bisacrylamide-acrylamide) 0.5mL 10% APS 25μL TEMED(Tetramethylethylenediamine) 6μL Distilled water margin Total 5mL

[0290] Each purified protein solution and molecular weight marker were applied, and electrophoresis was performed at 200 V and 20 mA for 60 minutes, and then further for 75 minutes. In addition, a running buffer having the composition shown in Table 15 below was used.

[0291] [Table 15]

[0292] composition capacity 2.5 mM Tris(2-amino-3-hydroxymethyl-1,3-propanediol) 3.03g 192mM Glycine 14.4g 0.1% SDS 1g Distilled water margin Total 1,000L

[0293] The results are shown in Fig.19 . From the gel electrophoresis results, it can be seen that the cage structure composed of carbosilane dendrimers in the targeted type 2 (MCF7-2) is tight, so the fluorescence intensity is strong, but the targeting is low. In addition, it can be seen that in the case of the enhanced type 1 (MCF7-1+αstand), the cage structure is loose, the targeting is high, but the fluorescence is weak. The above shows that the tightness of the cage structure and the structure of the target recognition site are very important.

[0294] (Example 7) Drug Encapsulation Experiment in DDS Liposomes or Micelles

[0295] Orange OT was used as the encapsulated drug. A micellar solution was prepared according to the same procedure as the above-mentioned basic micelle production process, except that 10.28 μL of 7.78 mM TPS was carbonized using an 80 μM GFP-containing solution. Next, 1 μL of Orange OT (8 mM stock solution of Orange OT) was added at a molar ratio (final concentration) of 1:1 to GFP. Next, DiI282 (400 μM stock solution of DiI282) was added at a molar ratio (final concentration) of 20:1 to GFP, and it was taken up overnight at 37°C.

[0296] Next, the column liquid was fixed to volume with 1xPBS to 1.2 times the target volume, and then passed through a PVDF filter (pore size 0.45μm or 0.22μm). Otherwise, it was processed and recovered according to the same steps as the preparation of basic micelles, and the particle size and fluorescence were measured at the same dilution ratio.

[0297] The upper solution was processed in the same manner as the preparation of basic liposomes or micelles, and the particle size and fluorescence were measured at the same dilution ratio. It is believed that both hydrophobic and hydrophilic substances can be used for encapsulation of drugs, but it is believed that the conditions for micelle formation need to be optimized separately.

[0298] (Example 8) Cellular uptake experiment of DDS liposomes or micelles

[0299] (1) Experimental methods

[0300] For MCS7 cells that have reached confluence, 0.25% trypsin solution was used to peel off and prepare a cell suspension (1x10 9 3 mL of the cell suspension was inoculated into 5 collagen-coated culture dishes (manufactured by MatTek) or poly-d-lysine culture dishes (manufactured by MatTek).

[0301] After the cells adhered to the bottom surface of the wells, the medium was removed from each well using a pipette, an appropriate amount of DMEM(+) was added, and the washing operation was repeated three times so that the attached cells did not fall off.

[0302] Add 450 μL of DMEM(+) to each well of the collagen-coated culture dish for 3-hour and 24-hour observations. Next, add 50 μL of 1x PBS to the wells of the negative control. Add 42.7 μL of 1xPBS to 7.3 μL of the type 1 enhanced stock solution to make 50 μL of sample 1, and add this sample 1 to each well of the two culture dishes. In addition, add 41.6 μL of 1x PBS to 8.4 μL of the non-targeted stock solution, and add the resulting sample 2 to the other two culture dishes.

[0303] 50 μL of 1x PBS was also added to the negative control well of the poly-d-lysine culture dish. In addition, in a culture dish for 24-hour observation, sample 2 was added, which was made by adding 41.9 μL of 1x PBS to 8.1 μL of the stock solution of the non-targeted type (NSS26). Immediately after that, the photographs were taken and the cells were cultured at 37°C in the presence of 5% CO2 for 3 hours or 24 hours. After the culture was completed, 1 mL of DMEM (+) was added to wash the cells. This operation was repeated 3 times.

[0304] (2) Observation results using a confocal microscope FV-100 (FV1000D manufactured by Olympus Corporation)

[0305] For DAPI, 405 nm laser was used for excitation, and the luminescence at 460 nm was observed. For the dendrimer part, the luminescence at 515 to 520 nm was observed. The observation results are shown in Figures 20A to 20C . For the 3-hour results ( Fig. 20C Upper layer) and 24-hour observations ( Fig. 20C The results showed that the targeted micelles were more accurately taken into the target cells in the case of 24 hours (see Figures 16 to 18 )middle.

[0306] (3) Analysis methods

[0307] Image J was used as the analysis software. The fluorescence intensity of each region was quantified, and the disintegration of micelles and the remaining fluorescent protein were confirmed by the ratio of the fluorescence intensity of micelles to the fluorescence intensity of fluorescent protein.

[0308] For FACS measurement, FlowJo was used as analysis software. During analysis, data from only cells was used, and the cell population with autofluorescence was selected. Furthermore, the cell population with autofluorescence was subtracted from all data. The results are shown in Figures 13-15The numbers shown in the figure are the number of cells remaining after subtracting the autofluorescence / the total number of measured cells. In the following Table 16, the autofluorescence cell population was determined 5 times, and the average values ​​are shown.

[0309] [Table 16]

[0310]

[0311] The above shows that if the cell is in good condition, it can be targeted; and the long-term contact between micelles and cells will cause non-targeted micelles to be taken in in large quantities through normal endocytosis. However, targeted micelles may be taken in faster than non-targeted micelles. In addition, it is believed that the taken-in micelles may disintegrate in the cells.

[0312] Industrial Applicability

[0313] The present invention is useful in the field of pharmaceutical formulation, particularly in the field of drug delivery.

[0314] Sequence number 1: Targeting recognition sequence peptide introduced into GFP (MCF7-1)

[0315] Sequence number 2: Targeting recognition sequence peptide introduced into GFP (MCF7-2)

[0316] Sequence number 3: Targeting recognition sequence peptide introduced into GFP (MCF7-1+αstand)

[0317] Sequence number 4: GFP introduced into MCF7-1

[0318] Sequence number 5: GFP introduced into MCF7-2

[0319] Sequence number 6: GFP introduced into MCF7-1+αstand

[0320] SEQ ID NO: 7: Amino acid sequence of GFP

[0321] SEQ ID NO: 8: Amino acid sequence of GFP

[0322] SEQ ID NO: 9: Amino acid sequence of BFP

[0323] SEQ ID NO: 10: Amino acid sequence of YFP

[0324] SEQ ID NO. 11: Amino acid sequence of fluorescent protein from the sea anemone

[0325] SEQ ID NO: 12: Forward primer for amplification of MCF7-1

[0326] SEQ ID NO: 13: Reverse primer for amplification of MCF7-1

[0327] SEQ ID NO: 14: Forward primer for MCF7-2 amplification

[0328] SEQ ID NO: 15: Reverse primer for MCF7-2 amplification

[0329] SEQ ID NO: 16: Forward primer for MCF7-1+αstand amplification

[0330] SEQ ID NO: 17: Reverse primer for MCF7-1+αstand amplification

[0331] SEQ ID NO: 18: Forward primer for inverse PCR

[0332] SEQ ID NO: 19: Reverse primer for inverse PCR

[0333] SEQ ID NO: 20: Forward primer for inverse PCR

[0334] SEQ ID NO: 21: Reverse primer for inverse PCR

[0335] SEQ ID NO: 22: Oligonucleotide

[0336] SEQ ID NO: 23: Oligonucleotide

[0337] SEQ ID NO: 24: Oligonucleotide

[0338] SEQ ID NO: 25: Oligonucleotide

[0339] SEQ ID NO: 26: Forward primer for inverse PCR

[0340] SEQ ID NO: 27: Reverse primer for inverse PCR

[0341] SEQ ID NO: 28: Forward primer for inverse PCR

[0342] SEQ ID NO: 29: Reverse primer for inverse PCR

[0343] SEQ ID NO: 30: Forward primer for inverse PCR

[0344] SEQ ID NO: 31: Reverse primer for inverse PCR

[0345] SEQ ID NO: 32: Forward primer for inverse PCR

[0346] SEQ ID NO: 33: Reverse primer for inverse PCR

[0347] Sequence number 34: Forward primer for inverse PCR. Sequence Listing <110> Quarrymen & Co. Inc Saitama University, a national university corporation <120> Endocytosis enhancer by using agglutinate molecule <130> P16SU005PCT <160> 34 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <220> <221> Peptide <222> (1)..(10) <223> MCF7-1 <400> 1 Asp Met Pro Gly Thr Val Leu Pro Gly Gly 1 5 10 <210> 2 <211> 10 <212> PRT <213> Homo sapiens <220> <221> Peptide <222> (1)..(10) <223> MCF7-2 <400> 2 Val Pro Thr Asp Thr Asp Tyr Ser Gly Gly 1 5 10 <210> 3 <211> 29 <212> PRT <213> Homo sapiens <220> <221> Peptide <222> (1)..(29) <223> MCF7-1+alpha stand peptide (MCF7-1+alpha stand PEPTIDE) <400> 3 Asp Met Pro Gly Thr Val Leu Pro Gly Gly Gly Gly Gly Ser Glu Gly 1 5 10 15 Glu Trp Gln Gln Gln Gln His Gln Trp Ala Lys Gln Glu 20 25 <210> 4 <211> 272 <212> PRT <213> Aequorea victoria <220> <221> PEPTIDE <222> (1)..(272) <400> 4 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Met Pro Gly 1 5 10 15 Thr Val Leu Pro Gly Gly Met Ser Lys Gly Glu Glu Leu Phe Thr Gly 20 25 30 Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys 35 40 45 Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu 50 55 60 Thr Leu Lys Phe Ile Ser Thr Thr Gly Lys Leu Pro Val Pro Trp Pro 65 70 75 80 Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr 85 90 95 Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu 100 105 110 Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys Asp Asp Gly Asn Tyr 115 120 125 Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg 130 135 140 Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly 145 150 155 160 His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala 165 170 175 Asp Lys Gln Arg Asn Gly Ile Lys Ala Asn Phe Lys Thr Arg His Asn 180 185 190 Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr 195 200 205 Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser 210 215 220 Thr Gln Ser Ala Leu Leu Lys Asp Pro Asn Glu Lys Arg Asp His Met 225 230 235 240 Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ser Gly Ile Thr Asp Glu 245 250 255 Val Asp Gly Thr Glu Leu Tyr Lys Gly Gly His His His His His His 260 265 270 <210> 5 <211> 272 <212> PRT <213> Aequorea victoria <220> <221> PEPTIDE <222> (1)..(272) <400> 5 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Val Pro Thr Asp 1 5 10 15 Thr Asp Tyr Ser Gly Gly Met Ser Lys Gly Glu Glu Leu Phe Thr Gly 20 25 30 Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys 35 40 45 Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu 50 55 60 Thr Leu Lys Phe Ile Ser Thr Thr Gly Lys Leu Pro Val Pro Trp Pro 65 70 75 80 Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr 85 90 95 Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu 100 105 110 Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe Asn Asp Asp Gly Asn Tyr 115 120 125 Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg 130 135 140 Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly 145 150 155 160 His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala 165 170 175 Asp Lys Gln Arg Asn Gly Ile Lys Ala Asn Phe Lys Thr Arg His Asn 180 185 190 Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr 195 200 205 Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser 210 215 220 Thr Gln Ser Ala Leu Leu Lys Asp Pro Asn Asp Lys Arg Asp His Met 225 230 235 240 Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ser Gly Ile Thr Asp Glu 245 250 255 Val Asp Gly Thr Glu Leu Tyr Lys Gly Gly His His His His His His 260 265 270 <210> 6 <211> 292 <212> PRT <213> Aequorea victoria <220> <221> PEPTIDE <222> (1)..(292) <400> 6 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Met Pro Gly 1 5 10 15 Thr Val Leu Pro Gly Gly Gly Gly Gly Ser Glu Gly Glu Trp Gln Gln 20 25 30 Gln Gln His Gln Trp Ala Lys Gln Glu Met Ser Lys Gly Glu Glu Leu 35 40 45 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 50 55 60 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 65 70 75 80 Gly Lys Leu Thr Leu Lys Phe Ile Ser Thr Thr Gly Lys Leu Pro Val 85 90 95 Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe 100 105 110 Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser Ala 115 120 125 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys Asp Asp 130 135 140 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 145 150 155 160 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 165 170 175 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr 180 185 190 Ile Thr Ala Asp Lys Gln Arg Asn Gly Ile Lys Ala Asn Phe Lys Thr 195 200 205 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 210 215 220 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 225 230 235 240 Tyr Leu Ser Thr Gln Ser Ala Leu Leu Lys Asp Pro Asn Glu Lys Arg 245 250 255 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ser Gly Ile 260 265 270 Thr Asp Glu Val Asp Gly Thr Cys Glu Leu Tyr Lys Gly Gly His His 275 280 285 His His His His 290 <210> 7 <211> 263 <212> PRT <213> Aequorea victoria <220> <221> PEPTIDE <222> (1)..(263) <223> GFP <400> 7 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Met Ser Lys Gly 1 5 10 15 Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly 20 25 30 Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 35 40 45 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Ser Thr Thr Gly Lys 50 55 60 Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val 65 70 75 80 Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 85 90 95 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe 100 105 110 Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly 115 120 125 Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 130 135 140 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His 145 150 155 160 Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn 165 170 175 Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 180 185 190 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro 195 200 205 Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Leu Lys Asp Pro Asn 210 215 220 Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 225 230 235 240 Ser Gly Ile Thr Asp Glu Val Asp Gly Thr Cys Glu Leu Tyr Lys Gly 245 250 255 Gly His His His His His His 260 <210> 8 <211> 258 <212> PRT <213> Aequorea victoria <220> <221> PEPTIDE <222> (1)..(258) <223> GFP <400> 8 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Met Ser Lys Gly 1 5 10 15 Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly 20 25 30 Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 35 40 45 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys 50 55 60 Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val 65 70 75 80 Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 85 90 95 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe 100 105 110 Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly 115 120 125 Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 130 135 140 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His 145 150 155 160 Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn 165 170 175 Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 180 185 190 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro 195 200 205 Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Leu Lys Asp Pro Asn 210 215 220 Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 225 230 235 240 Ile Thr His Gly Met Asp Glu Leu Tyr Lys Gly Gly His His His His 245 250 255 His His <210> 9 <211> 259 <212> PRT <213> Aequorea victoria <220> <221> PEPTIDE <222> (1)..(259) <223> GFP <400> 9 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Met Val Ser Lys 1 5 10 15 Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp 20 25 30 Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly 35 40 45 Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly 50 55 60 Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr His Gly 65 70 75 80 Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe 85 90 95 Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe 100 105 110 Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu 115 120 125 Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys 130 135 140 Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn Ser 145 150 155 160 His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val 165 170 175 Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala 180 185 190 Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu 195 200 205 Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro 210 215 220 Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala 225 230 235 240 Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly His His His 245 250 255 His His His <210> 10 <211> 259 <212> PRT <213> Aequorea victoria <220> <221> PEPTIDE <222> (1)..(259) <400> 10 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Met Val Ser Lys 1 5 10 15 Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp 20 25 30 Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly 35 40 45 Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly 50 55 60 Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly 65 70 75 80 Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe 85 90 95 Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe 100 105 110 Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu 115 120 125 Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys 130 135 140 Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser 145 150 155 160 His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val 165 170 175 Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala 180 185 190 Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu 195 200 205 Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro 210 215 220 Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala 225 230 235 240 Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly His His His 245 250 255 His His His <210> 11 <211> 236 <212> PRT <213> such as Actinia equina <220> <221> Peptide <222> (1)..(236) <220> <221> Peptide <222> (1)..(236) <223> GFP <400> 11 Met Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val 1 5 10 15 Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu 20 25 30 Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val 35 40 45 Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln 50 55 60 Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro 65 70 75 80 Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val 85 90 95 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser 100 105 110 Leu Gln Asp Gly Cys Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn 115 120 125 Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu 130 135 140 Pro Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu 145 150 155 160 Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu 165 170 175 Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr 180 185 190 Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr 195 200 205 Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His His Leu Phe 210 215 220 Leu Gly Thr Cys Gly Gly His His His His His 225 230 235 <210> 12 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(45) <400> 12 cctggtactg ttcttcctgg tggtatgagt aaaggagaag aactt 45 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(27) <223> Primer <400> 13 catatcgcga cccatttgct gtccacc 27 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(45) <223> Primer for MCF7-1 <400> 14 actgatactg attatagtgg aggaatgagt aaaggagaag aactt 45 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(27) <223> Primer for MCF7-1 <400> 15 aggaacgcga cccatttgct gtccacc 27 <210> 16 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(51) <223> Primer for MCF7-2 (primer for MCF7-1) <400> 16 caacaacaac aacatcaatg ggcaaaacaa gaaatgagta aaggagaaga a 51 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(42) <223> Primer for MCF7-2 (primer for MCF7-1) <400> 17 ccattcacct tcactaccac caccaccacc aggaagaaca gt 42 <210> 18 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(22) <223> Primer for MCF7-1+alpha standPEPTIDE <400> 18 cattgaagat ggctccgttc aa 22 <210> 19 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(22) <223> Primer for MCF7-1+alpha standPEPTIDE <400> 19 ttgtggcgag ttttgaagtt ag 22 <210> 20 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Primer for inverse PCR <220> <221> prim_transcript <222> (1)..(70) <223> Primer for inverse PCR <400> 20 ctcgaccata tggctagcat gactggtgga cagcaaatgg gtcgcatgag taaaggagaa 60 gaacttttca 70 <210> twenty one <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(54) <223> Primer for inverse PCR <400> twenty one tgacgtgaat tcattagtga tggtgatggt gatgtttgta gagctcatcc atgc 54 <210> twenty two <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(25) <223> Primer for inverse PCR <220> <221> misc_feature <222> (13)..(14) <223> n is a, c, g, or t <400> twenty two cttaaattta ttnnkactgg aaaac 25 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(20) <223> Primer for inverse PCR <400> twenty three ggtaagtttt ccgtatgttg 20 <210> twenty four <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <220> <221> misc_binding <222> (1)..(26) <220> <221> misc_feature <222> (9)..(10) <223> n is a, c, g or t <400> twenty four gtgttcaann kttttcccgt tatccg 26 <210> 25 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <220> <221> misc_binding <222> (1)..(22) <400> 25 catacgtcag agtagtgaca ag 22 <210> 26 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <220> <221> misc_binding <222> (1)..(23) <400> 26 tgtcttttca ctggagttgt ccc 23 <210> 27 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <220> <221> misc_binding <222> (1)..(21) <400> 27 ttctccttta ctcatttttt c 21 <210> 28 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(24) <223> Primer for inverse PCR <400> 28 tgcacacatg gcatggatga gctc 24 <210> 29 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(21) <223> Primer for inverse PCR <400> 29 cccagcagca gttacaaact c 21 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(33) <223> Primer for inverse PCR <400> 30 cagcgccgtt gtgagctcta caaataatga att 33 <210> 31 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(21) <223> Primer for inverse PCR <400> 31 tgtaatccca gcagcagtta c 21 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(21) <223> Primer for inverse PCR <400> 32 acatgtgagc tctacaaata a 21 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(18) <400> 33 acggccctgt gtaatccc 18 <210> 34 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer <220> <221> prim_transcript <222> (1)..(21) <223> Primer for inverse PCR <400> 34 ggaacatgtg agctctacaa a 21

Claims

1. A targeting shell for a drug delivery system, comprising an aggregating molecule represented by the following formula (I), capable of encapsulating any molecule selected from the group consisting of proteins, nucleic acids, and hydrophobic molecules having a molecular weight of less than 200,000, in, At least one of the bromine atoms bound to the molecular end is bound to the targeting sequence presenting part; here, the targeting sequence presenting part is composed of a fluorescent protein bound to a targeting peptide sequence, and the combination of the target protein and the targeting peptide sequence promotes the incorporation of the aggregating molecule into cells through endocytosis; the protein constituting the above-mentioned targeting sequence presenting part has a unique reactive thiol group, and the protein is bound to the formula (I) through the reaction of the thiol group with the halogen atom present in the above-mentioned formula (I), wherein The fluorescent protein combined with the targeting peptide sequence is a sequence selected from any one of the groups consisting of sequence numbers 4 to 6 in the sequence list, MASMTGGQQMGR DMPGTVLPGG MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYGKLTLKFISTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISFKDDGNYKTRAEVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YITADKQRNG IKANFKTRHNIEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALLKD PNEKRDHMVL LEFVTAAGSGIT DEVDGTELYK GG HHHHHH (sequence number 4 in the sequence list); MASMTGGQQMGR VPTDTDYSGG MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYGKLTLKFISTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISFNDDGNYKTRAEVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YITADKQRNG IKANFKTRHNIEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALLKD PNDKRDHMVL LEFVTAAGSGIT DEVDGTELYK GG HHHHHH (sequence number 5 in the sequence list); MASMTGGQQMGR DMPGTVLPGG GGGSEGEWQQQQHQWAKQE MSKGEELFTG VVPILVELDGDVNGHKFSVS GEGEGDATYG KLTLKFISTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPEGYVQERTISF KDDGNYKTRAEVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YITADKQRNGIKANFKTRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALLKD PNEKRDHMVLLEFVTAAGSGIT DEVDGTC ELYK GG HHHHHH (sequence number 6 of the sequence listing).

2. The drug delivery system targeting shell as claimed in claim 1, wherein The targeting shell for the drug delivery system is formed by aggregating the aggregating molecules in an aqueous solvent and has a diameter of 50nm to 500nm.

3. The targeting shell for drug delivery system according to claim 1, characterized in that: The targeting peptide specifically binds to any target protein selected from the group consisting of surface antigens, receptors, gates, transport proteins and channels expressed in the target tissue, and the combination of the target protein and the targeting peptide sequence promotes the incorporation of the aggregating molecule into cells through endocytosis.

4. The targeting shell for drug delivery system as claimed in claim 3, characterized in that: The target tissue is any one selected from the group consisting of normal tissue in which inflammation occurs, tissue containing cells in which undesirable gene expression is observed, tissue composed of cells in which undesirable gene expression is observed, and tissue composed of tumor cells.

5. The targeting shell for drug delivery system according to claim 1, characterized in that: The aggregating molecule represented by the formula (I) is an aggregating molecule represented by a molecule having any structure selected from the group consisting of the following formulae (II) to (V), Wherein, TSPP is composed of the fluorescent protein combined with the targeting peptide sequence,

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