KR20190108232A - Method for detecting molecular target interactions by conditional and controlled exposure or release of molecules - Google Patents

Abstract

The present invention relates to a method for detecting the interaction of a material by measuring the integrated distribution of a prey material driven to a site as conditionally/controlled exposure/release of bait material, such as drugs, in cells using light and the like. In addition, the present invention provides a method for a material which inhibits or promotes the interaction of the material using the scanning method.

Description

Method for detecting molecular target interactions by conditional and controlled exposure or release of molecules}

The present invention provides a method for detecting the interaction between a bait material, such as a drug, and a prey material, such as a molecular target, based on conditional / controlled exposure / release using light, and a material for controlling the interaction thereof. It relates to a method of detection.

It is known that the development of one new drug using the existing methodology will cost one to two trillions and a period of 10 to 17 years. Representative problems that require such a high cost and duration is that most of the candidates are eliminated during drug development due to side effects such as lack of efficacy or toxicity. The fundamental limitation of this trial and error is that the target of the drug being developed is a molecular target that actually binds to and reacts with them in diseased cells. It is developed by identifying only the disease analysis model or the narrow disease treatment efficacy and spectrum they want to analyze in the human body, without knowing exactly how to bind and react with them and how many such targets are (Chem. Biol. 11: 593, 2004). In fact, almost all commercially available drugs do not know exactly which molecular targets in the human body actually bind and react to show therapeutic efficacy.

Although the importance of natural substances known to have useful physiological activities to the human body through the long history as the fundamental problem of drug development continues to emerge as a drug, their precise molecular targets such as molecular targets are not well understood. (Nature 432: 829, 2004). Identifying the physiological molecular targets of various drugs, including these natural substances, is essential not only to understand their pharmacological effects and side effects, but also to enable the development of more advanced drugs. In particular, the discovery of new molecular targets of drugs in traditional and modern medicines, such as clinically proven natural substances, may provide new therapeutic indications by creating new efficacy and drug repositioning. It is also important that it may be (Nat. Rev. Drug Discov. 3: 673, 2004).

Identifying molecular targets that define the substance of drugs that have such a key meaning is to screen low-molecular compounds with cell-based high-throughput methods with desired efficacy and phenotype-based assays in large compound libraries. This is also important in the field of chemical biology or chemical genomics (Science 300: 294, 2003; Nature 432: 846, 2004). Although screening for low molecular weight compounds affecting specific potency and phenotype, the determination and identification of molecular targets of the bioactive substances found have been spent a great deal of effort and effort, chemical biological methods are not used effectively. In addition, the development of systematic targeting and identification techniques is important in a variety of life sciences and technologies, such as genomics, proteomics, and system biologics, which can be used in various types of cells or organisms, including protein (or low molecular weight) -protein interactions. Effective analysis and detection of the interactions of the various substances in the organism is essential for understanding dynamic biological processes, regulatory networks, and life phenomena.

In identifying and identifying molecular targets as disease factors that bind drugs and the like, affinity chromatography (Microbiol. Rev. 59: 94, 1995; Science 284: 1948, 1999), as a technique for analyzing the interaction of substances, Protein and low molecular weight microarrays, phage display (Chem. Biol. 6: 707, 1999), yeast hybrids (Proc. Natl. Acad. Sci. USA 93: 12817, 1996), with gene expression profiling and heterologous deletions Yeast strain equilibrium analysis (Chem. Biol. 11: 609, 2004) is well known. However, the above technique has a high background, high false positives, low sensitivity, fundamentally inappropriate structural folding, indirectness, post-translational strain control. There are various problems such as the absence of posttranslational modification or limited access to molecular targets such as disease factors present in cells. In addition, experiments and analyzes in an artificial environment other than the natural environment, such as binding conditions in extracellular and in vitro or non-mammalian cell environments, are often misleading. Cause. Therefore, there is a need for a technique for detecting interactions between drugs such as low molecular weight compounds and molecular targets such as disease factors with high sensitivity and accuracy under pharmacologically relevant conditions.

FRET (fluorescence resonance energy transfer) and PCA (protein-fragment complementation assay) are representative methods for analyzing the interaction of substances in pharmacologically suitable human cells. However, this method has a fundamental problem that the interaction cannot be explored if the relative spacing, direction, and position of the bonding materials are not appropriate (Screen 8: 447, 2003). Because PCA technology uses reporter proteins to analyze interactions, it is impossible to ignore the steric hinderance of interactions caused by reporter proteins. Furthermore, it is very difficult due to various technical limitations to analyze the immediate binding of low molecular weight compounds such as drugs and molecular targets such as disease factors in living cells that determine drug efficacy.

In order to overcome all the problems of conventional analysis and search methods, the inventors of the present invention conditionally exposed a bait material, such as a drug, based on light and the like, to mutually interact with a prey material such as a molecular target. We have developed a new analytical method that can easily and conditionally explore the action in human cells.

In other words, when a bait material such as a drug is introduced into a cell by using a transporter, it is entrapped and encapsulated in various small structures such as endosomes, and the small structure and the like by using an exposure inducer activated in response to light. And / or when the bait material is conditionally exposed (released / released) by destroying the transporter, an integrated distribution of the prey material, such as a molecular target in which the label is bound, is concentrated in the site. Measurements in the form of concentrated spots revealed that the interaction of bait and prey could be explored intracellularly.

It has also been found that the search method can be used to detect substances that block / inhibit, activate / induce the interaction of the bait material with the prey material.

Accordingly, the present invention provides a method for exploring the interaction of the bait material with the prey material, which includes measuring the presence of integrated spots and the distribution of labeling materials bound to the prey and conditional / controlled exposure / release. It aims to provide.

In addition, an object of the present invention is to provide a method for detecting a substance that controls the interaction between the bait material and the prey material using the interaction detection method.

According to one aspect of the invention, there is provided a method for exploring the interaction of a bait material with a prey material, the method comprising the following steps:

(a) providing a carrier comprising a bait material and an intracellular introduction material;

(b) providing a cell comprising a prey to which the label is bound;

(c) treating the carrier obtained in step (a) with the cells obtained in step (b) to isolate the small structure in the form of an intracellular pocket;

(d) conditionally destroying the intracellular pocket-shaped microstructure in the cells obtained in step (c) to expose the bait material; And

(e) measuring the distribution of the labeling substance bound to the prey substance and the presence of integrated spots in the cells obtained in step (d).

According to another aspect of the present invention, there is provided a method for detecting a substance that modulates the interaction of a bait material with a prey material, the method comprising the following steps:

(a ') providing a carrier comprising a bait material and an intracellular introduction material;

(b ') providing a cell comprising a prey to which the label is bound;

(c ') treating the carrier obtained in step (a') with the cells obtained in step (b ') to isolate the small structure in the form of an intracellular pocket;

(d ') treating the cells obtained in step (c') with a substance to be detected;

(e ') conditionally destroying the intracellular pocket-shaped small structure in the cells obtained in step (d') to expose the bait material; And

(f ') measuring the distribution of the labeling substance bound to the prey substance and the presence of integrated spots in the cells obtained in step (e').

According to the present invention, conditions such as drugs and molecular targets in the cell by using light, conditionally interacting as if the switch is turned on in the dark (just as stealth fighters are hiding in the radar network conditionally As can be seen, there are several advantages as a basis for screening along with the advantage of clear readout with low background and high S / N ratio. Some examples include:

First, the interaction of physiological modulators in nature can be screened under physiological conditions rather than artificial conditions.

Second, dynamic analysis and screening can be performed in real time according to conditions such as light at the single cell level.

Third, the analysis and screening of interactions between various physiological modulators can be carried out quickly and in large quantities systematically.

Fourth, the interaction between the physiological regulators in vivo may be easily analyzed in a non-invasive state according to the conditions of light and the like described above.

Fifth, structural modifications can be readily applied to screening interactions with various physiological modulators in their original structure.

Therefore, the present invention can be usefully applied to explore the interaction between various physiological modulators such as drugs and molecular targets based on the screening, as well as the search and detection of molecular targets such as new drug candidates and disease factors for drug development. It can be very useful for improving existing drugs or finding new medical uses.

1 is a schematic diagram of a method for analyzing the interaction of a bait material such as a drug and a prey material such as a molecular target according to the present invention.
Figure 2 is a micrograph showing the integration spot caused by the binding of DaMXinib and BMX protein bound to the nanoparticles after irradiation with light according to the method of the present invention.

The method according to the present invention can apply a number of different approaches and approaches related to drug delivery. For example, Annu. Rev. Mater. Res. 41: 1, 2011; US Patent 2012/0253264 A1; Med. Laser Application 21: 239, 2006; J. of Controlled Release 148: 2, 2010; Int. J. Cancer 5: 1, 2015; Nat. Commun. DOI: 10.1038 / ncomms4623, 2014; J. of Biomed. Optics 17: 058001, 2012; Pharm. Research DOI: 10.1007 / s11095-007-9338-9, 2007; Nat. Rev. Drug Discov. 9.8: 615, 2010; Materials Today 8.8: 18, 2005; Material Matters 7.3: 30, 2012; Cancer Treatment InTech. 85, 2013; Drugs 56.5: 747, 1998; Biochimica et Biophysica Acta-Biomembranes 1239.2: 133, 1995; Colloids & Surfaces B: Biointerfaces 75.1: 1, 2010; Asian J. of Pharm. Sci. 8.6: 319, 2013; International J. of Nanomed. Suppl. 1: 51, 2014; Biochemistry 37: 12875, 1998; Antibodies 2.2: 209, 2013; J. of Controlled Release 151.3: 220, 2011; J. of Controlled Release 148.1: 2, 2010; Pharmaceuticals 5.11: 1177, 2012; The FASEB J. 16.10: 1217, 2002; Nat. Med. 10.3: 310, 2004; Photodynamic Therapy-Humana Press 133, 2010; Current Pharmaceutical Biotech. 8.6: 362, 2007; Tumor Biology 23.2: 103, 2002; Pharmaceutical Research 24.11: 2040, 2007; Molecular Pharmaceutics 12.9: 3272, 2015; Theranostics 4.11: 1133., 2014; Acta biomaterialia 24: 279, 2015; Chem Nano Mat. DOI: 10.1002 / cnma. 201500138, 2015; J. of Controlled Release DOI: 10.1016 / j.jconrel.2015.01.032, 2015; Biomaterials 33.6: 1821, 2012; J. of Controlled Release 203: 85, 2015; PloS One 10.3: e0120982, 2015; Nat. Commun. DOI: 10.1038 / ncomms 4546, 2014; Faraday Discussions 161: 515, 2013; Various drug delivery methods disclosed in Biophysical J. 102.5: 1079, 2012) and the like can be applied to the method of the present invention.

When various physiological regulators, such as drugs, are introduced into cells by using various kinds of carriers, they are trapped in various small structures in the form of pockets such as endosomes and are enclustered (clustered entrapping). Conditionally / controlled exposure / release of drugs that are clustered and entrapped by destroying these small structures and / or transporters using exposure inducers under various conditions, such as exposure inducers activated in response to light. In other words, contrary to the general expectation that the drug or the like is exposed and rapidly diffuses and escapes from the cell, the phenomenon of slow exposure with a time delay has been found by the present invention. That is, according to the present invention, after a bait material such as a drug that induces clustered entrapping in a bag-shaped small structure such as an endosome, conditionally / controlled exposure / release, the labeling material is expressed in a cell. Provides a method of exploring the interaction between the bait materials, such as drugs, and the prey materials, such as molecular targets, by measuring the cumulative distribution of the labeled materials, such as bound molecular targets, in the form of concentrated spots. do.

In the present invention, the "physiological control substance" refers to a substance that plays a role of regulating such as to enhance or inhibit the physiological function of the organism. Such physiological modulators may be obtained from natural products such as plants and animals, or may be extracted and purified from microorganisms and metabolites of animal and plant cell lines, and may be obtained biologically or chemically by synthesis. For example, they may be in various forms such as nucleic acids, nucleotides, proteins, peptides, amino acids, sugars, lipids, vitamins or chemical compounds.

In the present invention, "bait" refers to various physiological modulators, including drugs (candidates) used to explore interactions with other physiological regulators.

In the present invention, the term "prey" means a physiological modulator to be searched or analyzed as an interaction partner of the bait material. For example, it may be a molecular target such as a disease factor protein (BMX protein) that interacts with a drug (eg, datinib).

In the present invention, the term "carrier" refers to a bag form such as endosomes, lysosomes, phagosomes, and macropinosomes by introducing a bait material into a cell. Refers to particle media of various sizes, such as nano or micro, that are entrapped and encapsulated in various small structures (vesicles, compartments, etc.). Nanocapsule, nanosphere, nanogel, nanogel, hydrogel, mesoporous silica, liposome, exosome, cyclodextrin, etc. Carriers of various sizes, shapes, and properties can be used, including synthetically prepared particles (particles / crystals, etc.) and polymeric bodies (polymeric complex / capsule / matrix / colloids, etc.). It is also possible to use a multilayer or relatively large sized synthetically prepared carrier so that the conditional / controlled exposure / release of the bait material is slowly sustained release with a time delay.

In the present invention, the carrier includes a bait material as well as an "intracellular introducer". Intracellular introduction material is a substance that induces cell introduction, various foreign substances that are introduced and penetrated into cells such as viruses, various substances such as peptides derived from or parts necessary for introduction and penetration of foreign substances such as viruses into cells And various substances such as proteins related to intracellular introduction and infiltration process and mechanism, including binding of the receptor and the like, sugars, lipids, polymers, and ionic substances such as positive or negative.

In one embodiment, the intracellular introduction material may be a cell permeable / penetrating peptide. The intracellular transduction peptide is also referred to as a protein transduction domain or membrane translocating sequence. There are several examples that have been developed to date, and for example, the intracellularly introduced peptides are not only trans-activating transcriptional activators (TAT) of HIV, and viral protein 22 (VP22) of HSV, but also HP4 of herring protamine. (human protamine P4), Drosophila antennapedia (Antennapedia), mouse Mph-1 (Mutator phenotype protein 1) and the like. That is, the intracellular introduction material may use TAT / HA2 (RRRQRRKKRGGDIMGEWGNEIFGAIAGFLG) or PO72 (GRKKRRQRRRGLFGAIAGFIENGWEGMIDG) derived from the trans-activating transcriptional activator (TAT) of HIV. The intracellular introduction material may include various ionic materials and lipids such as positive or negative. For example, the ionic material and lipids may be commercially available DOTAP, EPC, DPPC, DSPC, DSPE, DMPC, DSPC-PEG, DSPE-PEG, cholesterol, and the like.

In the present invention, the carrier may additionally include not only a bait material and an intracellular introduction material, but also an exposure inducing material reacting to specific conditions such as light, a fluorescent material or a luminescent label, a magnetic material, an antibody, and the like, as necessary. .

In the present invention, a method of loading a bait material or an intracellular introduction material into a delivery vehicle will be described as follows. Direct binding or encapsulation, entrapping, dispersion or colloid / gel, or mixing of various materials directly with a carrier. Synthesis may be made to include in various ways. It is possible to adhere or pack the bait material by hydrophobic interaction according to its characteristics (size, hydrophilicity, hydrophobicity, ionicity, affinity, etc.) without direct coupling such as covalent interaction, It is also possible to include the drug in the delivery vehicle without modification by encapsulation or entrapping, dispersion or colloid, or mixing.

In one embodiment, as a nanoparticle carrier comprising a bait material, such as a drug and an intracellular introduction material, it can be prepared synthetically using a method of directly binding the bait material or intracellular introduction material to the nanoparticles. This direct binding can be accomplished through the interaction of (strept) avidin with biotin. For example, known nanoparticles coated with streptavidin (J. Immunol. Meth. 52: 353, 1982; Anal. Biochem. 60: 149, 1974; J. Biol. Chem. 245: 3059, 1970; USA Patent No. 5,665,582; US Patent Publication No. 2003 / 0092029A1) can be prepared by reacting with a biotin bound material. For example, biotin-bound bait material and intracellular transduction peptides (TAT / HA2, Nature Med. 10: 310, 2004) were applied to Qdot nanoparticles (Thermo Fisher) coated with streptavidin on a commercially available surface. Can be prepared by bonding together. In addition, the nanoparticle transporters to which the bait material and the intracellular transduction peptide are bound may be used in combination with additional intracellular transduction peptides (eg, PO72 (GRKKRRQRRRGLFGAIAGFIENGWEGMIDG, etc.) to further promote intracellular introduction.

A delivery agent comprising a bait material and an intracellular introduction material may be processed into the cell for example for 16 hours, and the delivery agent introduced into the cell over time accumulates in various small structures in the form of intracellular pockets.

Bait materials combined with materials that do not penetrate into cells or do not penetrate cells without transporters such as nanoparticles (for example, biotin, fluorescent materials, etc.) are treated at high concentration to induce introduction, or bait materials are introduced into intracellular materials. Intracellular introduction may be induced by directly binding or by mixing with intracellular introduction materials (peptides such as PO72, lipids such as lipofectamine, etc.) and treating the cells.

In the present invention, the "label" is a substance that is a marker such as fluorescence and luminescence for analyzing the distribution of the prey substance in the cell. If the prey material is a protein like a molecular target of a drug, the labeling material may be a fluorescent material including a fluorescent protein such as GFP, YFP, CFP, TFP, RFP. Prey materials such as molecular target proteins coupled with fluorescent proteins are in the form of DNAs encoding them, and the cells containing the prey materials bound to the labeling substance can be obtained by treating the cells according to a conventional method using lipofectamine or the like. have.

Many attempts have been made to destroy intracellular pocket-like small structures and transporters, such as endosomes, to escape bait materials such as trapped and isolated drugs. For example, light, ultrasound, pH, hyperthermal, electric, magnetic, chemical or small molecular, proteolytic enzymes, viruses, bacteria, plants, etc. Exposure signals, such as proteins or peptides derived from, and substances that are activated in response to these signals (light response exposure substances: AlPcS2a, TPPS2a, Photofrin, Chlorine6, TAT-TPP, TPCS2a, DPc, 5-ALA-induced PpIX, TMPyP) , ZnPc, BPD-MA, mTHPC, Hypericin, etc .; ionizing radiation Reactive exposure inducer: Phosphatidylcholine, etc .; Ultrasonic response induction: PFC, SF6, etc .; pH Reactive exposure induction: Diolein, CHEMS, INF7, etc .; Substances: Specific conditions such as DPPC, DMPC, NH4HCO3, etc. may be appropriately used.

In one embodiment, the conditional exposure can be performed using a photoreactive exposure inducer. The photoreactive exposure inducing substance may be treated with the carrier, the cell, or the carrier and the cell. The photoreactive exposure inducing substance includes a material (photosensitizer, etc.) used in photochemotherapy or photodynamic therapy. For example, the photoreactive exposure inducer may be di-sulphonated aluminum phthalocyanines (e.g., AlPcS2, AlPcS23, etc.) and sulfonated tetraphenylporphyrins (e.g., TPPS23). , TPPS4, TPPSi, TPPS20, etc.), but is not limited thereto. The photoreactive exposure inducing substance destroys a small structure in which the bait material is entrapped and enclosed (in some cases, at the same time as the carrier), thereby conditionally exposing the bait material to the intracellular cell. Induces the integrated distribution of marker material bound to the site.

The cell is not particularly limited, and any cell having a small structure in the form of an intracellular bag may be used, for example, a heLa cell line. For example, the cells to be analyzed are treated with light-responsive exposure inducers (AlPcS2a 5-10 ug / ml or TPPS2a 0.5 ug / ml, etc.), and then red light (670 ± 10 nm) or blue light (420 ± 10 nm). Can irradiate light Conditions such as concentration and treatment time of the exposure-inducing substance and light intensity and irradiation time may be appropriately adjusted according to analytical conditions such as cells, bait materials, and prey materials. After completion of the light irradiation, the distribution of the labeling substance in the cells by time is observed by fluorescence microscopy, either live or fixed in a conventional manner. Measurement of the presence of the integrated spot of the labeling material may be performed using various fluorescence spectrometers such as a conventional fluorescence microscope or a fluorescence sorter (FACS). The interaction of the bait material with the prey material according to the accumulation distribution in the cell can be analyzed more precisely through methods such as fluorescence correlation spectroscopy or photobleaching for fluorescence recovery. Conditional exposure can be analyzed more precisely by treating and immobilizing inhibitors, such as low molecular weight compounds, which inhibit the specific stage of drug-induced endocytosis or intracellular traffiic by introducing from outside. It may be.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example. Analysis of interaction between drug and molecular target

(1) Preparation of Nanoparticle Transporter of Dasatinib

To the streptavidin-coated Qdot nanoparticles (Thermo Fisher), biotin-bound Dasatinib and intracellular transduction peptides (TAT / HA2, RRRQRRKKRGGDIMGEWGNEIFGAIAGFLG) were bound according to the manufacturer's instructions. That is, 100 pmol of biotin-dasatanib was reacted with the nanoparticles (amount capable of binding to 10 pmol of biotinylated molecule) to obtain nanoparticles bound to dasatanib. 100 pmol of biotin-TAT / HA2 was reacted with the obtained nanoparticles to obtain a nanoparticle carrier in which dasatanib and TAT / HA2 were bound together. The obtained nanoparticle transporter of Dasatinib and the intracellularly introduced peptide (PO72, GRKKRRQRRRGLFGAIAGFIENGWEGMIDG) were mixed at the same concentration.

(2) Obtaining BMX-EGFP and EGFP Expression Cells

HeLa cell lines (ATCC No. CCL-2) were incubated at 37 ° C. in DMEM medium containing 10% FBS. According to the manufacturer's instructions, lipofectamine (Invitrogen) was used to express BMX-EGFP or EGFP in which BMX, a molecular target protein of Dasatinib, and EGFP, a fluorescent protein, were combined under CMV promoter. DNA was introduced into HeLa cell lines, respectively. Each obtained HeLa cell line was added with AlPcS2a (Al (III) phthalocyanine chloride disulfonic acid) (about 10 ug / ml, Frontier Scientific), which was a light-reactive exposure inducer, and incubated for about 2 hours.

(3) Treating Cells with Nanoparticle Transporter of Dasatinib

Treating the BMX-EGFP or EGFP-expressing HeLa cell line obtained in the above (2) in DMEM medium, the nanoparticle transporter of dasatinib obtained in the above (1), and endosomes such as endosomes by the hour up to 16 hours Clustered entrapping was confirmed in pocket-shaped microstructures.

(4) interaction analysis

The HeLa cell line expressing BMX-EGFP or EGFP obtained in the above (3) was irradiated with light for about 30 minutes using a red light irradiation device (about 670 nm). After completion of light irradiation, the distribution of fluorescence in the cells was observed by fluorescence microscope at each time. The obtained result is as shown in FIG. As can be seen from the results of FIG. 2, the accumulation spot of fluorescence was observed in the cell line expressing BMX-EGFP, whereas the accumulation spot was not observed in the cell line expressing EGFP.

<110> T & K BioInnovation Inc. <120> Method for detecting molecular target interactions by conditional          and controlled exposure or release of molecules <130> PN0850 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> TAT / HA2 protein transduction domain <400> 1 Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly Gly Asp Ile Met Gly Glu   1 5 10 15 Trp Gly Asn Glu Ile Phe Gly Ala Ile Ala Gly Phe Leu Gly              20 25 30 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> PO72 protein transduction domain <400> 2 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Leu Phe Gly Ala Ile   1 5 10 15 Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly              20 25 30

Claims (11)

A method for exploring the interaction of a bait material with a prey material, the method comprising the following steps:
(a) providing a carrier comprising a bait material and an intracellular introduction material;
(b) providing a cell comprising a prey to which the label is bound;
(c) treating the carrier obtained in step (a) with the cells obtained in step (b) to isolate the small structure in the form of an intracellular pocket;
(d) conditionally destroying the intracellular pocket-shaped microstructure in the cells obtained in step (c) to expose the bait material; And
(e) measuring the distribution of the labeling substance bound to the prey substance and the presence of integrated spots in the cells obtained in step (d). A method of detecting a substance that modulates the interaction of a bait material with a prey material, the method comprising the following steps:
(a ') providing a carrier comprising a bait material and an intracellular introduction material;
(b ') providing a cell comprising a prey to which the label is bound;
(c ') treating the carrier obtained in step (a') with the cells obtained in step (b ') to isolate the small structure in the form of an intracellular pocket;
(d ') treating the cells obtained in step (c') with a substance to be detected;
(e ') conditionally destroying the intracellular pocket-shaped small structure in the cells obtained in step (d') to expose the bait material; And
(f ') measuring the distribution of the labeling substance bound to the prey substance and the presence of integrated spots in the cells obtained in step (e'). The method of claim 1 or 2, wherein said carrier is in the form of nanoparticles. The method according to claim 1 or 2, wherein the intracellular introduction material is an intracellular introduction peptide or an ionic material. The method of claim 4, wherein the intracellularly introduced peptide is TAT / HA2 or PO72. The method of claim 1 or 2, wherein the labeling substance is a fluorescent substance. The method of claim 1 or 2, further comprising treating a photoreactive exposure inducer to said carrier, said cell, or said carrier and cell. The method of claim 7, wherein the photoreactive exposure inducer is AlPcS2a, TPPS2a, Photofrin, Chlorine6, TAT-TPP, TPCS2a, DPc, 5-ALA-induced PpIX, TMPyP, ZnPc, BPD-MA, mTHPC, Hypericin, etc. At least one selected from the group. 8. The method of claim 7, wherein the photoreactive exposure inducer is di-sulfonated aluminum phthalocyanine or sulfonated tetraphenylporphyrin. 8. A method according to claim 7, wherein said conditional destruction of step (d) or step (e ') is carried out by irradiating light of red or blue light. The method according to claim 1 or 2, characterized in that the measurement of the distribution of the labeling substance bound to the prey material in step (e) or (f ') and the presence of integrated spots is carried out using a fluorescence microscope. How to.

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