Composition for detecting choroid plexus in brain in active state using low-molecular-weight fluorescent material and use thereof

Abstract

The present invention relates to a composition for detecting the choroid plexus in the brain in an active state using a low-molecular-weight fluorescent material, and use thereof. Particularly, the present invention relates to: a composition for detecting the choroid plexus in the brain, comprising a fluorescent probe compound represented by chemical formula 1 as an active ingredient; a kit for detecting the choroid plexus in the brain, comprising the composition as an active ingredient; and a method for imaging the choroid plexus in the brain. In addition, the present invention relates to: a method for analyzing the choroid plexus in the brain in vitro, comprising a step of treating brain tissue with a brain choroid plexus composition of the present invention and detecting fluorescence generated from a fluorescent probe compound; and a method for screening for a therapeutic agent for a disease caused by an abnormality in the choroid plexus in the brain, comprising a step of treating isolated brain tissue with a candidate compound and a step of detecting fluorescence generated from a fluorescent probe compound after treating the brain tissue with a brain choroid plexus composition of the present invention.

Description

Composition for detecting choroid plexus of active brain using low-molecular-weight fluorescent substance and use thereof

[0001] The present invention relates to a composition for detecting the choroid plexus of an active brain using a low-molecular-weight fluorescent substance and its use.

[0002] The choroid plexus is located in the lateral, third, and fourth ventricles and is a tissue that produces cerebrospinal fluid and is formed by numerous villi composed of tough epithelial cells surrounding a highly vascularized stromal core containing immune cells and fibroblasts.

[0003] The choroid plexus is composed of a layer of cuboid epithelial cells surrounding the core of the capillaries and loose connective tissue. The epithelium of the choroid plexus is continuous with the ependymal cell layer (ventricular layer) that lines the ventricle, and the precursor ependymal cells are monociliated but differentiate into multiciliated ependymal cells. Unlike the ependyma, the choroid plexus epithelium has tight junctions between the cells on the side facing the ventricles, and these tight junctions prevent most substances from passing through the layer into the cerebrospinal fluid (CSF). Thus, the choroid plexus acts as a blood-CSF barrier. The choroid plexus is folded into numerous villi around each capillary, forming frond-like projections that protrude into the ventricles. The villi, together with the brush border of microvilli, greatly increase the surface area of ​​the choroid plexus. Cerebrospinal fluid is formed when plasma is filtered from the blood through epithelial cells, and as cerebrospinal fluid is formed, substances move actively in and out of the cerebrospinal fluid.

[0004] In addition, the choroid plexus has been found to secrete various biologically active molecules in addition to cerebrospinal fluid, and the system of the choroid plexus and the blood-cerebrospinal fluid barrier is known to be involved in learning and memory, depression, brain network plasticity, and neurogenesis.

[0005] Meanwhile, research on the developmental process of the structure of the choroid plexus and its impact on brain function is still insufficient, and there is no technology that can specifically detect and analyze the choroid plexus visually.

[0006] Accordingly, the purpose of the present invention is to provide a composition for detecting the choroid plexus of the brain, which comprises a low-molecular-weight fluorescent probe compound as an active ingredient.

[0007] In addition, another object of the present invention is to provide a brain choroid plexus detection kit comprising the composition for detecting brain choroid plexus of the present invention as an active ingredient.

[0008] In addition, another object of the present invention is to provide a method for imaging the choroid plexus of the brain using the composition for detecting the choroid plexus of the brain of the present invention.

[0009] Another object of the present invention is to provide a method for analyzing the choroid plexus of the brain in vitro, comprising the step of treating brain tissue with the composition for detecting the choroid plexus of the brain of the present invention and detecting fluorescence generated from the fluorescent probe compound.

[0010] Another object of the present invention is to provide a method for screening a therapeutic agent for a disease caused by a choroid plexus abnormality of the brain, comprising the steps of treating a candidate compound to isolated brain tissue; and treating the brain tissue with a composition for detecting choroid plexus of the brain of the present invention, and then detecting fluorescence generated from the fluorescent probe compound.

[0011] To achieve the above purpose, the present invention provides a composition for detecting the choroid plexus of the brain, comprising a fluorescent probe compound represented by the following chemical formula 1 as an active ingredient.

[0012] <Chemical Formula 1>

[0013]

[0014] In one embodiment of the present invention, the composition may target epithelial cells of the cerebral choroid plexus.

[0015] In one embodiment of the present invention, the fluorescent probe compound may exhibit SLCO1B3 (Solute Carrier Organic Anion Transporter Family Member 1B3)-dependent fluorescence.

[0016] In one embodiment of the present invention, the fluorescent probe compound may not exhibit fluorescence when the expression or activity of SLCO1B3 is reduced or inhibited, and may exhibit fluorescence when SLCO1B3 is expressed or active.

[0017] In addition, the present invention provides a brain choroid plexus detection kit comprising the brain choroid plexus detection composition of the present invention as an active ingredient.

[0018] The present invention also provides a method for imaging the choroid plexus of the brain using the composition for detecting the choroid plexus of the brain of the present invention.

[0019] The present invention also provides a method for analyzing the choroid plexus of the brain in vitro, comprising the step of treating brain tissue with a composition for detecting the choroid plexus of the brain of the present invention and detecting fluorescence generated from the fluorescent probe compound.

[0020] In one embodiment of the present invention, the fluorescent probe compound may target epithelial cells of the choroid plexus of the brain.

[0021] Furthermore, the present invention provides a method for screening a therapeutic agent for a disease caused by a choroid plexus abnormality of the brain, comprising the steps of treating a candidate compound to isolated brain tissue; and treating the brain tissue with a composition for detecting the choroid plexus of the brain of the present invention, and then confirming fluorescence generated from the fluorescent probe compound.

[0022] In one embodiment of the present invention, the disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), prion disease, motor neuron disease, and choroid plexus cyst.

[0023] The composition containing the fluorescent probe compound provided in the present invention has the effect of rapidly and accurately detecting the choroid plexus of the brain, and in particular, can specifically target and fluorescently stain epithelial cells in the choroid plexus of the brain, so that it can be usefully used to study the structure and function of the choroid plexus using a visualized imaging method, and further has the effect of being usefully used to discover the pathogenesis of choroid plexus-related brain diseases and treatments.

[0024] Figure 1 is a schematic diagram showing screening of brain cell fluorescent substances using a brain cell mixed culture solution according to one embodiment of the present invention.

[0025] Figure 2 shows the results of fluorescent staining confirmed by treatment with a low-molecular-weight fluorescent substance (LC-B7) of the present invention in the cerebral cortex and choroid plexus regions isolated from mice.

[0026] Figure 3 shows the level of fluorescence-positive cells according to the treatment of the small molecule fluorescent substance (LC-B7) of the present invention in the brain tissues of mice and rats, as confirmed by a flow cytometer. The Y-axis of each plot represents cell morphology, the X-axis represents fluorescence intensity, and the trapezoidal partitioned area represents the morphology-fluorescence intensity distribution of detection-positive cells.

[0027] Figure 4 shows a photograph showing the staining pattern observed in human stem cell-derived choroid plexus organoids (ChP organoids) and cerebral organoids following treatment with a small molecule fluorescent substance (LC-B7).

[0028] Figure 5 shows the results of fluorescence imaging without a fixation process after treating organoids of different brain regions derived from human stem cells with a small-molecule fluorescent substance (LC-B7).

[0029] Figure 6 shows a photograph of a stained area of ​​the choroid plexus derived from human stem cells after treating it with a low-molecular-weight fluorescent substance (LC-B7).

[0030] Figure 7 shows the results of comparing the staining patterns of markers for epithelial cells, endothelial cells, and mesenchymal cells, which are constituent cells of the choroid plexus, with the staining patterns of LC-B7, in order to confirm whether the low-molecular-weight fluorescent substance (LC-B7) of the present invention stains specific cells in the choroid plexus of the mouse-derived (upper) and human stem cell-derived (lower) brain.

[0031] Figure 8 shows the results of comparing the fluorescent staining by treatment with the low-molecular-weight fluorescent substance (LC-B7) of the present invention in mouse and human stem cell-derived choroid plexus and the fluorescent staining using an antibody against TTR, a choroid plexus stem cell marker.

[0032] Figure 9 illustrates a process for screening a target protein of a CRISPRa-based LC-B7 fluorescent material in one embodiment of the present invention.

[0033] Figure 10 shows the results of screening as a CRISPRa-based LC-B7 dye target protein of the present invention.

[0034] Figure 11 shows the results of confirming the change in intensity of the fluorescent staining signal after treating cells showing a positive fluorescent reaction to the small molecule fluorescent substance (LC-B7) of the present invention with SLCO1B3 inhibitor (Prednisolone phosphate) at various concentrations (left figure) and the results of analyzing the level of cells killed through SYTOX (right figure).

[0035] Figure 12 shows the results of analyzing the level of fluorescence-positive cells in a group treated with SLCO1B3 siRNA as an SLCO1B3 inhibitor and a control group in cells showing a fluorescence-positive reaction to the low-molecular-weight fluorescent substance (LC-B7) of the present invention.

[0036] Figure 13 shows the results of confirming fluorescent staining according to LC-B7 treatment in SLCO1B3 overexpressing cells after treating HEK293T cells with SLCO1B3 lenti virus vector (HG13013-ACGLN). As a control group, a group of HEK293T cells that were not treated with SLCO1B3 lenti virus vector but were treated with LC-B7 was used.

[0037] The present invention is characterized by providing a novel low-molecular-weight fluorescent substance capable of specifically detecting and analyzing the choroid plexus of an active brain and a method for detecting the choroid plexus of the brain using the same.

[0038] As previously described in the prior art, the choroid plexus of the brain plays an important role in all brain functions, including not only producing cerebrospinal fluid but also immune function and neural network formation.

[0039] However, research on the developmental process of the structure of the choroid plexus and its influence on brain function is still insufficient, and there is no technology to visually detect the choroid plexus.

[0040] Accordingly, the inventors of the present invention, while conducting research to develop a fluorescent marker capable of specifically visualizing and detecting the choroid plexus of the brain, discovered for the first time that a low-molecular compound of the following chemical formula 1 specifically fluorescently stains the choroid plexus of the brain.

[0041] Accordingly, the present invention provides a composition for detecting the choroid plexus of the brain, comprising a fluorescent probe compound represented by the following chemical formula 1 as an active ingredient.

[0042] <Chemical Formula 1>

[0043]

[0044] The fluorescent probe compound represented by the chemical formula 1 according to the present invention is a new derivative compound of Cy3, which is known as a conventional fluorescent probe, and is a Glucose-2N-Cy3 derivative compound. The inventors of the present invention named the compound of the chemical formula 1 'LC-B7'.

[0045] The present inventors confirmed that among various derivative compounds based on Cy3, the derivative compound of the above chemical formula 1 has the characteristic of specifically fluorescently staining the choroid plexus of the brain.

[0046] According to one embodiment of the present invention, it was investigated whether the low-molecular-weight fluorescent probe compound of the above chemical formula 1 could target a certain region of the brain and cause fluorescent staining. As a result of the investigation, it was confirmed that a high fluorescent positive reaction was specific to the choroid plexus region, while a low fluorescent positive reaction was found in the entire brain tissue region and the cerebral cortex region.

[0047] These results were similar to those observed in cerebral organoids and choroid plexus organoids prepared using human stem cells, with a significantly higher staining area observed in choroid plexus organoids compared to cerebral organoids.

[0048] In addition, when a fluorescent probe compound of chemical formula 1 was injected into a living mouse via tail vein, it was confirmed that it passed through the blood-cerebrospinal fluid barrier (BCSFB) and specifically labeled and fluorescently stained the choroid plexus of the brain.

[0049] Through these results, the inventors of the present invention were able to determine that the LC-B7 fluorescent probe compound of the present invention can specifically label and detect the choroid plexus region in brain tissue.

[0050] Furthermore, in another embodiment of the present invention, it was investigated whether the LC-B7 fluorescent probe compound of the present invention can target and detect specific cells within the choroid plexus of the brain.

[0051] The choroid plexus is composed of three cell layers: epithelial cells, endothelial cells, and mesenchymal cells. The expression pattern of marker proteins for each cell type and the fluorescent staining pattern for the fluorescent probe compound of the present invention were compared and analyzed.

[0052] As a result, the staining patterns of AQP1 (aquaporin1) and TTR (transthyretin), which are markers for epithelial cells, were found to have the highest similarity to the staining patterns of the fluorescent probe compound of the present invention, while the staining patterns of COL1A1 (Collagen alpha-1 chain), which is a marker for mesenchymal cells, were found to have the lowest similarity.

[0053] Through this, the inventors of the present invention were able to find out that the fluorescent probe compound of the present invention specifically targets and fluorescently stains epithelial cells within the choroid plexus of the brain.

[0054] In addition to these results, the inventors of the present invention investigated which molecules the fluorescent probe compound of the present invention targets or interacts with in epithelial cells in the choroid plexus using a CRISPRa-based screening method.

[0055] As a result, it was confirmed that the fluorescent probe compound of the present invention targets SLCO1B3 (Solute Carrier Organic Anion Transporter Family Member 1B3) and exhibits SLCO1B3-dependent fluorescence.

[0056] In order to elucidate the interaction between the fluorescent probe compound of the present invention and SLCO1B3, cells showing a positive fluorescence response when treated with the fluorescent probe compound were treated with an SLCO1B3 inhibitor. As a result, it was found that the intensity of the fluorescent staining signal by the fluorescent probe compound of the present invention decreased in a concentration-dependent manner of the SLCO1B3 inhibitor treatment. On the other hand, in cells overexpressing SLCO1B3, the fluorescent staining signal was detected and increased.

[0057] Through these results, the inventors of the present invention were able to find out that the fluorescent probe compound of the present invention has the characteristic of not exhibiting fluorescence when the expression or activity of SLCO1B3 is reduced or inhibited, and exhibiting fluorescence when SLCO1B3 is expressed or active.

[0058] Therefore, the present invention can provide a composition for detecting choroid plexus of the brain, which comprises a fluorescent probe compound represented by the above chemical formula 1 as an active ingredient, and can also provide a kit for detecting choroid plexus of the brain, which comprises the composition as an active ingredient.

[0059] The composition of the present invention may include a mixed solution in addition to the fluorescent probe compound of the present invention. The mixed solution may preferably be water, an organic solvent, or a mixed solution thereof, but is not limited thereto, and any buffer solution known in the art used for fluorescence detection may be used without limitation.

[0060] The above detection kit may further include a substance that can be selectively bound to or accumulated in the choroid plexus of the brain together with the fluorescent probe compound of the present invention.

[0061] In addition, the present invention can provide a method for imaging the choroid plexus of the brain using the composition of the present invention.

[0062] The fluorescent probe compound of the present invention can specifically fluorescently stain the choroid plexus of the brain, specifically, the epithelial cells within the choroid plexus of the brain, so that the fluorescence generated from the fluorescent probe compound can be visualized and imaged using equipment such as a confocal fluorescence microscope, a two-photon fluorescence microscope, an optical coherence tomography, a two-photon fluorescence microscope, or an optical coherence tomography.

[0063] In addition, in the brain choroid plexus imaging method of the present invention, the content of the fluorescent probe compound of the present invention, which is an effective ingredient in the composition, can be appropriately adjusted depending on the form and purpose of use, the condition of the target subject, etc.

[0064] The method for imaging the choroid plexus of the brain according to the present invention can be administered to mammals, including humans, via various routes. The administration method can be any conventionally used method, and for example, it can be administered orally, rectally, intravenously, intramuscularly, subcutaneously, intrauterinely, or intracerebroventricularly. The composition of the present invention can be formulated and used in oral formulations such as powders, tablets, capsules, suspensions, and emulsions, or in parenteral formulations such as transdermal preparations, suppositories, and sterile injectable solutions, according to conventional methods.

[0065] The present invention also provides a method for analyzing the choroid plexus of the brain in vitro, comprising the step of treating brain tissue with a composition for detecting the choroid plexus of the brain of the present invention and detecting fluorescence generated from the fluorescent probe compound.

[0066] In particular, the fluorescent probe compound of the present invention can target epithelial cells of the cerebral choroid plexus, so that epithelial cells in the cerebral choroid plexus can be visually observed and analyzed.

[0067] Furthermore, the present invention can provide a method for screening a therapeutic agent for a disease caused by a choroid plexus abnormality, comprising the steps of treating a candidate compound to isolated brain tissue; and treating the brain tissue with the composition for detecting choroid plexus abnormalities of the present invention, and then confirming fluorescence generated from the fluorescent probe compound.

[0068] The above disease may include any disease caused by an abnormality of the choroid plexus of the brain, but is not limited thereto, and the disease may be a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), prion disease, motor neuron disease, and choroid plexus cyst.

[0069] That is, when the fluorescent probe compound of the present invention is used, the choroid plexus region of the brain, which was difficult to observe and analyze in the past, can be visually imaged and analyzed, so that not only can the presence or absence of structural or functional abnormalities in the choroid plexus of the brain be confirmed, but also, by confirming whether the candidate drug has the activity of restoring the structure or function of the choroid plexus when treated, it is possible to screen whether the candidate drug can be used as a treatment for a disease caused by an abnormality in the choroid plexus of the brain.

[0070] Furthermore, the composition including the fluorescent probe compound provided in the present invention can provide a diagnostic method capable of diagnosing a disease caused by an abnormality in the choroid plexus of the brain by visually analyzing the choroid plexus of the brain.

[0071] In this way, the fluorescent probe compound of the present invention can be usefully used for research on the choroid plexus, which performs various important functions in complex brain tissue, and for discovering treatments for related brain diseases.

[0072] The present invention will now be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0073]

[0074] <Example 1>

[0075] Screening of small-molecule fluorescent substances with fluorescent staining activity against brain cells

[0076] The present inventors performed the following experiments to screen for low-molecular-weight fluorescent substances capable of fluorescently staining brain cells among various derivative compounds having a specific functional group attached to the Cy3 compound structure.

[0077] Specifically, mouse mixed-brain cells were cultured in a 96-well plate, and treated with a library of various fluorescent substances (LC-library) in which a specific functional group was attached to the Cy3 compound structure at a concentration of 1 μM for 1 hour at 37°C in a CO2 incubator. Afterwards, the cells were washed once with PBS, and each cell treated with the fluorescent substance library was screened for substances with strong staining signals using an ImageXpress Micro confocal High-content imaging system (Molecular Devices), a fluorescence image mass analysis device. At this time, 10 random locations in each well were selected, and the fluorescence intensity at each location was evaluated.

[0078] In addition, the presence of staining specificity of cultured cells was investigated through flow cytometry. After removing cells from the culture medium, an AttuneTM Nxt flow cytometer (Thermofisher) was used, and the FSC-A / SSC-A values ​​were adjusted as the initial cell sorting setting value (Gating) to first exclude dead cell debris, and the SSC-A / SSC-H values ​​of live cells were adjusted again so that only single cells could be analyzed (overlapping cells were excluded). Approximately 10,000 single cells were examined, the fluorescence values ​​were recorded, and the patterns were analyzed.

[0079]

[0080] As a result, it was confirmed that the LC-B7 low-molecular-weight fluorescent substance having the above chemical structure showed the strongest fluorescence positivity for brain cells, and a fluorescence-positive cell cluster was confirmed in the group treated with the LC-B7 low-molecular-weight fluorescent substance (see Fig. 1).

[0081]

[0082] <Example 2>

[0083] Identification of staining sites of LC-B7 small-molecule fluorescent substance targeting rodent brain regions

[0084] As confirmed through the above <Example 1> that among various derivative compounds using Cy3 as a backbone, the LC-B7 low-molecular-weight fluorescent substance stains some cells in a mixed culture of mouse brain glial cells, the inventors of the present invention performed an experiment to determine in which part of the mouse brain the brain cells stained by the LC-B7 low-molecular-weight fluorescent substance exist.

[0085] Specifically, we treated mouse brain sections with LC-B7 to investigate whether there were specific areas that were stained in actual mouse brain tissue. That is, after euthanizing the mice with CO2, the upper neck area was cut, the skull was opened, the brain was extracted, the extracted brain was placed in a dissection solution, and sections with a thickness of approximately 300 μm were prepared using a brain slicer (vibratome). Then, the brain sections were placed in aCSF containing LC-B7 at a concentration of 400 nM and incubated at room temperature for 1 hour, after which the tissues were taken out and photographed in fluorescence. In addition, the above aCSF solution (in g per 1 L, NaCl 7.25, KCl 0.373, NaH2PO4 0.1697, NaHCO3 2.184) was first prepared for storage, and immediately before the experiment, 0.27 g of D-glucose, 150 μl of 3 mM MgCl2, and 300 μl of 1 mM CaCl2 were added to 150 mL of aCSF and continuously bubbled with 95% O2 gas. In addition, a 2x concentrated stripping solution (in g per 1 L, NaCl 10.168, KCl 0.372, NaH2PO4 0.344, NaHCO3 4.2) was first prepared for storage, diluted with distilled water immediately before the experiment, and then 18.2 g of sucrose, 0.45 g of D-glucose, 0.056 g of ascorbic acid, 750 μl of 3 mM MgCl2, and 250 μl of 1 mM CaCl2 were added per 250 mL, bubbled with 95% O2 gas for about 10 minutes, and then kept as cold as possible before use.

[0086] In addition, to confirm whether the stained area was the choroid plexus, 10-week-old mice were euthanized with CO2, the upper neck area was cut, the skull was opened, the brain was extracted, the choroid plexus was obtained from the ventricle area, and then placed in DMEM containing 500 nM of LC-B7 small molecule fluorescent material, incubated for 1 hour at room temperature, and then photographed with a Nikon fluorescence microscope.

[0087]

[0088] As a result, as shown in Fig. 2, it was confirmed that cells in each ventricle of the brain were fluorescently stained by LC-B7, as seen in the brain coronal section of the mouse (left figure), and it was confirmed that the choroid plexus of the purely isolated mouse was fluorescently stained by LC-B7 (right figure).

[0089]

[0090] Furthermore, the inventors of the present invention extracted brain tissues from different types of rodents, mice and rats, on the first day after birth, separated them into individual cells, treated them with LC-B7, and then performed flow cytometry using the same method as described above to analyze the areas showing fluorescence positivity.

[0091]

[0092] As a result, as shown in Fig. 3, in the case of mice, 2.79% of the entire brain tissue showed a positive fluorescence reaction, and only 0.63% of the cerebral cortex showed a positive fluorescence reaction for LC-B7, but the choroid plexus showed a high positive fluorescence reaction of 25.8%. In addition, experiments performed on house mice showed similar positive fluorescence reactions, and in particular, a very high positive fluorescence reaction of 41.3% was confirmed in the choroid plexus.

[0093]

[0094] Through these results, the inventors of the present invention were able to find out that the LC-B7 low-molecular-weight fluorescent substance of the present invention can specifically stain the choroid plexus of the brain.

[0095]

[0096] <Example 3>

[0097] Identification of staining sites with LC-B7 small-molecule fluorescent agent in human embryonic stem cell-derived brain organoids.

[0098] As previously confirmed in <Example 2> that the LC-B7 small molecule fluorescent substance can specifically stain the choroid plexus of the brain in rodent brains, the inventors of the present invention prepared human choroid plexus organoids (ChP organoids) using human stem cells to confirm whether the LC-B7 small molecule fluorescent substance also works in human cells, and then performed fluorescent staining by treating them with LC-B7. At this time, not only choroid plexus organoids but also cerebral organoids were prepared and compared to see whether fluorescent staining was performed.

[0099] The human stem cell-derived choroid plexus organoids (ChP organoids) and cerebral organoids used in the above experiments were prepared according to the instructions for use of the STEMdiff™ Choroid Plexus Organoid Differentiation Kit (#100-0824) and STEMdiff™ Cerebral Organoid Kit (#08570) from STEMCELL Technologies. First, for the preparation of choroid plexus organoids, human stem cells in good condition were cultured, and when they became 70-80% confluent, the EB generation process was performed as day 0 of cell growth. The EB generation culture medium and EB seeding culture medium (solution containing Y-27632) were prepared according to the manufacturer's instructions. Human stem cells were dissociated from culture wells (6 wells) using Gentle Cell Dissociation (100-0485, STEMCELL Technologies) solution and transferred to EB seeding medium. 9,000 cells were seeded per well of a 96-well round-bottom ultra-low attachment plate (Corning 7007). The cells were cultured in a 37°C incubator until day 2 to ensure maximum stability. On days 2 and 4, 100 μl of EB seeding medium was added. From day 5, the cells were incubated for 48 hours in a 37°C incubator, and three-dimensional expansion was initiated using Matrigel (Corning 354277) on day 7. EBs were placed on UV-sterilized parafilm, 15 μl of Matrigel was sprayed per EB, and the parafilm was placed in a 100Φ cell culture plate and incubated in a 37°C cell incubator for 30 minutes to ensure sufficient polymerization of Matrigel.Afterwards, EBs were seeded on 6-well ultra-low adherent plates (100-0083, STEMCELL Techonologies) and cultured in expansion medium for 3 days. From day 10 onwards, the manufacturer's differentiation medium was used, and rotational culture was performed at 65 rpm using an orbital shaker (INFORS HT Celltron orbital shaker). On day 13, the differentiation medium was completely replaced, and the cells were cultured for an additional 2 days. From day 15 onwards, the manufacturer's maturation medium was used, and 3 mL of the medium per 6 well was completely replaced every 3 to 4 days for culture.

[0100] In addition, the production of cerebral organoids was similar to the production of the choroid plexus organoids described above, and was produced according to the method in the manual using a unique composition culture medium for the production of choroid plexus organoids.

[0101] Additionally, the culture medium for growing each generated organoid was treated with LC-B7 small molecule fluorescent substance at a concentration of 400 nM and cultured in a 37°C cell incubator for 1 hour. Afterwards, fluorescence images were taken using a CELENA X high-content imaging system (Logos Biosystems) with a 1.25x objective lens, and image analysis was performed using CELENA X cell analysis software.

[0102]

[0103] As a result, as shown in Figs. 4 and 5, when LC-B7 small molecule fluorescent substance was treated in cerebral organoids, staining occurred only in specific areas, whereas in choroid plexus organoids, a much wider area was stained compared to cerebral organoids, and in particular, choroid plexus-related cells were stained in cerebral organoids. In addition, as shown in Figs. 5 and 6, the basal part of the choroid plexus pouch was well stained by LC-B7 small molecule fluorescent substance, and it was confirmed that there was a difference in the intensity of fluorescent staining in the choroid plexus area as well.

[0104] These results allowed the inventors to demonstrate that the LC-B7 small molecule fluorescent substance can specifically stain the choroid plexus of the human brain, and to anticipate that the LC-B7 small molecule fluorescent substance has detection specificity depending on the degree of maturity of the differentiated region or the level of expression of a specific protein in each organoid.

[0105]

[0106] <Example 4>

[0107] Identification of specific cells within the choroid plexus stained with LC-B7, a small molecule fluorescent substance.

[0108] The present inventors, noting that staining with the LC-B7 small molecule fluorescent substance differs depending on the region of the choroid plexus, used specific markers of cells distributed within the choroid plexus to identify the actual cell types being stained. The choroid plexus is composed of epithelial cells, endothelial cells, and mesenchymal cells. The similarity of the staining patterns was compared by treating together epithelial cell markers AQP1 and TTR, endothelial cell markers CD31 and OTX2, and mesenchymal cell marker COL1A1.

[0109] Specifically, the choroid plexus of the mouse was extracted using the same method as the previous screening method, and then the extracted choroid plexus was washed with HBSS (LB003-02, WELGENE) to remove any blood, etc. for choroid plexus tissue culture. The choroid plexus culture medium was made by adding HEPES, Glutamax, Pen / strep, 1 μg / mL EGF, and 1:200 B27 to DMEM containing 5% FBS. The choroid plexus was finely ground together with the culture medium using an insulin syringe (1 cc / 30 G 8 mm, YCNC103008, Yongchang). 200 μl of matrigel was added to 200 μl of the culture medium and choroid plexus cell mixture, and mixed. 20 to 50 μl of the matrigel-cell mixture was dispensed into the center of each well of a 24-well plate. The Matrigel dome was cultured in a 37°C cell incubator for approximately 30 minutes to solidify, and then choroid plexus culture medium was added and cultured in a 37°C cell incubator, with 50% of the culture medium replaced every other day. On the second day of culture, 20 μM cytosine arabinoside (Ara-C) was treated to inhibit fibroblast growth.

[0110] Choroid plexus cells cultured with LC-B7 were treated at a concentration of 400 nM on day 12 or 17 and cultured for 1 hour in a 37℃ cell incubator. Hoechst was added in between, and fluorescence microscopy was performed in a living state immediately after incubation. After fluorescence microscopy, the cells were fixed with 4% PFA and subjected to antibody tissue staining. After blocking with 5% BSA for 1 hour, TTR antibody (PA5-20742, Invitrogen) 1:500, AQP antibody (32737, Santacruz) 1:200, and COL1A1 antibody (PA5-29569, Invitrogen) were treated, and the reaction was allowed to proceed overnight. The next day, after washing three times with PBS, secondary antibodies were treated at a 1:400 dilution ratio, incubated for 1 hour, washed three times with PBS again, and fluorescence photographs were taken with the same microscope as when photographing in a living state.

[0111]

[0112] In addition, the pulverization of human choroid plexus organoids was performed according to the STEMCELL Technologies method, that is, 100 mg of papain was diluted in an activation solution (1.1 mM EDTA, 0.067 mM mercaptoethanol, 5.5 mM L-cysteine ​​HCL) to make 250 units / mL, and the dilution volume was prepared according to the CoA (certificate of analysis) for each papain product lot. The papain stock solution was mixed with DNase I solution in HBSS to make a concentration of 30 units / mL papapin and 125 units / mL DNase I to prepare a separation solution. In addition, an ovomucoid protease inhibitor solution was also prepared (10 mg / mL, HBSS solution). Once all the necessary solutions were prepared, the organoids were transferred to a 24-well plate, washed once with PBS, and then the PBS was removed. 500 μl of the separation solution was added and incubated in an orbital shaker at 90 rpm for 30 minutes in a 37°C cell incubator. The organoids were then disrupted 5 to 6 times with a 1-mL pipette at room temperature for the first time. The cells were then incubated again in an orbital shaker at 90 rpm for 10 minutes in a 37°C cell incubator, and then disrupted 5 to 6 times with a 1-mL pipette at room temperature for the second time. All cells were collected and placed in a 15-mL tube, followed by treatment with approximately 1 to 2 mL of an ovomucoid protease inhibitor solution. After centrifugation at 300 × g for 5 minutes, the supernatant was removed and the cells were diluted with HBSS. The debris was removed with a 37 μm strainer, dispensed into 96-well plates, and cultured in choroid plexus organoid maturation medium for approximately 2–3 weeks. Afterwards, LC-B7 staining and antibody fluorescent staining were performed in the same manner as in the choroid plexus culture experiment, and fluorescence photography was performed.

[0113] Since LC-B7 is a probe suitable for live shooting, a tissue staining technique was performed to fix the cells after live shooting and then attach the TTR antibody again to compare them with the TTR antibody staining image. In addition, the live image and the antibody tissue staining image were created separately, and since deformation occurred during the tissue fixation process, software (ImageJ plugin BigWarp) was used for alignment.

[0114]

[0115] As a result, as shown in Fig. 7, when compared with the staining patterns of the marker proteins of each cell, the staining patterns according to the LC-B7 fluorescent material treatment were found to have the highest similarity to the staining patterns of AQP1 (aquaporin 1) and TTR (transthyretin), which are markers of epithelial cells, while the staining pattern similarity to COL1A1 (Collagen alpha-1 chain), which is a marker of mesenchymal cells, was found to be low.

[0116] In addition, as shown in Fig. 8, it was confirmed that the staining form according to the LC-B7 fluorescent material treatment of the present invention exhibits a staining form most similar to TTR (transthyretin), a marker of epithelial cells of the choroid plexus.

[0117] Through this, the inventors of the present invention were able to find out that the LC-B7 low-molecular-weight fluorescent substance of the present invention can more effectively and specifically fluorescently stain epithelial cells of the choroid plexus.

[0118]

[0119] <Example 5>

[0120] LC-B7 staining target protein screening

[0121] The present inventors have confirmed that the LC-B7 low-molecular-weight fluorescent substance of the present invention can specifically fluorescently stain the choroid plexus of the brain, and in particular, that it can fluorescently stain choroid plexus epithelial cells as specific target cells. Accordingly, experiments were conducted to determine which proteins in the choroid plexus epithelial cells are targeted by the LC-B7 fluorescent substance.

[0122] For this purpose, the Cas9-VPR transcriptional activation system (VPR: VP64-p65-Rta) was used, and the system can increase the expression of the corresponding gene when the sgRNA is attached to the gene promoter region. In addition, the present inventors constructed a SLC (solute carrier) transporter sgRNA library. The SLC transporter acts as a cellular metabolite channel and is known to transport a wide range of essential nutrients, metabolites such as glucose, amino acids, vitamins, neurotransmitters, inorganic substances, or metal ions. Choroid epithelial cells are specialized for the transport of water, ions, proteins, and vitamins, and are equipped with glucose, fructose, and urate transporters, so the present inventors screened which proteins LC-B7 targets as target molecules in choroid epithelial cells. In particular, since LC-B7 originated from a synthetic library containing glucose transporter-targeting fluorescent molecules, the Cas9-VPR transcriptional activation system was used to analyze the target protein (transporter) of LC-B7 using an sgRNA library targeting 480 SLC transporters, which is broader than the glucose transporter, to determine which protein (transporter) LC-B7 interacts with.

[0123] Specifically, the CRISPRa experiment using the SLC transporter gene library involved sorting only cells that showed a fluorescence-positive reaction to LC-B7 through fluorescence-based flow cytometry, further culturing them, repeating the flow cytometry, and base-sequencing the sgRNA in the genome of the cleanly separated fifth-round sorted cells. This process is illustrated in Figure 9.

[0124]

[0125] As a result, as shown in Fig. 10, each cell has a unique sgRNA base sequence for each SLC transporter, so the amount of cells can be estimated using a next-generation sequencing system. Among the cells that showed a positive fluorescence reaction, the proportion of cells with SLCO1B3 gene sgRNA was found to be the highest at 45%.

[0126] Therefore, the inventors of the present invention were able to find out that the LC-B7 low-molecular-weight fluorescent substance of the present invention targets SLCO1B3 in the epithelial cells of the choroid plexus of the brain.

[0127]

[0128] <Example 6>

[0129] Validation of LC-B7 staining target protein through SLCO1B3 inhibitor treatment

[0130] To verify whether SLCO1B3 is the target protein of the LC-B7 small molecule fluorescent substance, the fluorescence-positive cells accumulated through SLC-CRISPRa were treated with Prednisolone phosphate, an SLCO1B3 inhibitor, and the fluorescent staining signal was analyzed.

[0131]

[0132] As a result, as shown in Fig. 11, it was found that as the concentration of predinosolone phosphate increased, the intensity of the fluorescent staining signal by LC-B7 decreased (Fig. 11 left drawing). Considering that this decrease in fluorescent staining signal may occur due to a decrease in cell number due to drug toxicity, the degree of dead cells was determined using SYTOX, but it was found that there was no correlation between the number of dead cells and the intensity of the fluorescent staining signal (Fig. 11 right drawing).

[0133] These results showed that the intensity of the fluorescent staining signal of LC-B7 was determined by the activity of SLCO1B3, and confirmed once again that the target protein of LC-B7 was SLCO1B3.

[0134]

[0135] <Example 7>

[0136] Validation of LC-B7 staining target protein through SLCO1B3 inhibitory siRNA treatment

[0137] To reconfirm that the LC-B7 staining target protein is SLCO1B3, this time, SLCO1B3 siRNA (ThermoFisher Scientific, Cat#AM16708) was treated in fluorescence-positive cells, and the fluorescence staining signal was analyzed. The control siRNA treatment group was used as a control.

[0138]

[0139] As a result, as shown in Fig. 12, it was found that the number of fluorescent-positive cells was significantly reduced in the group treated with SLCO1B3 siRNA compared to the control group.

[0140] Through this, the inventors were able to confirm that the LC-B7 staining target protein is SLCO1B3.

[0141]

[0142] <Example 8>

[0143] Validation of LC-B7 staining target protein through SLCO1B3 overexpression

[0144] Furthermore, the inventors of the present invention assumed that SLCO1B3 is a target molecule of LC-B7, and overexpressed SLCO1B3 in HEK293T cells rather than in the choroid plexus, and analyzed the degree of fluorescence staining according to LC-B7 treatment.

[0145] To this end, first, a SLCO1B3 expression lentivirus was constructed using the following method. That is, after purchasing the SLCO1B3 lentivirus vector (HG13013-ACGLN, SinoBiological), the LipfectamineTM 3000 (L3000001, Thermofisher) was used according to the instructions, and the packaging plasmid used was the third-generation plasmid system (LV053, Applied Biological Material). Briefly, lenti virus packaging medium (47.4 mL Opti-MEM reduced serum medium, 2.5 mL FBS, 0.1 mL Sodium Pyruvate (100 mM), 0.5 mL Glutamax for 50 mL volume) and HEK293T cell culture medium (44.5 mL DMEM, 5 mL FBS, 0.5 mL Glutamax for 50 mL volume) were prepared before the experiment, and HEK239T cells were cultured in 6-well dishes. On the first day of the experiment, the cultured HEK293T cells were transferred to the lenti virus packaging medium and cultured overnight. Transfection was performed on the morning of the second day of the experiment (cell confluent 95% or more), and after 6 hours of culture, the medium was replaced with the lenti virus packaging medium and cultured again overnight. On the third day of the experiment, 2 mL of the culture medium in which the cells were growing was obtained, and new lenti virus packaging medium was added again. On the fourth day of the experiment (around 52 hours after transfection), 2 mL of the culture medium in which the cells were growing was obtained again, and a total of 4 mL of supernatant was secured. The supernatant was centrifuged (2000 rpm, 10 minutes), the cell debris was discarded, and only the supernatant was collected again. The cell debris was filtered once more using a 45 μm filter.

[0146] Additionally, microscopic imaging of LC-B7 staining levels in SLCO1B3 overexpressing cells was performed using the following method.

[0147] HEK293T cells were seeded in 96-well plates, and when they reached 50% confluency, they were treated with the constructed SLCO1B3 lentivirus. After 72 hours, the fluorescent protein was confirmed to be expressed well, and then LC-B7 was treated at 400 nM and stained for 1 hour at 37°C in a CO2 incubator. Afterwards, live images were taken at 20x magnification using an ImageXpress Micro confocal high-content imaging system (Molecular Devices).

[0148] In addition, flow cytometry analysis was performed using the following method: HEK293T was dispensed into a 96-well plate, and when it exceeded 50% confluent, the prepared SLCO1B3 lenti virus was treated, and after 72 hours, it was confirmed that the fluorescent protein was well expressed, and then LC-B7 was treated at 400 nM and stained for 1 hour at 37℃ in a CO2 incubator. The fluorescent staining status of the cells was analyzed using an Attune NxT flow cytometer (Thermofisher).

[0149]

[0150] As a result, as shown in Fig. 13, no fluorescent staining was observed in the group treated with LC-B7 alone in HEK293T cells other than the choroid plexus, whereas fluorescent staining was observed in the group treated with LC-B7 in HEK293T cells in which SLCO1B3 was overexpressed.

[0151] Through this, the inventors were able to reconfirm that the specific target protein of LC-B7 is SLCO1B3.

[0152]

[0153] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. A composition for detecting the choroid plexus of the brain, comprising a fluorescent probe compound represented by the following chemical formula 1 as an active ingredient. <Chemical Formula 1> 2. In paragraph 1, A composition for detecting the choroid plexus of the brain, characterized in that the composition targets epithelial cells of the choroid plexus of the brain.

3. In paragraph 1, A composition for detecting the choroid plexus of the brain, characterized in that the fluorescent probe compound exhibits SLCO1B3 (Solute Carrier Organic Anion Transporter Family Member 1B3)-dependent fluorescence.

4. In paragraph 3, A composition for detecting the choroid plexus of the brain, characterized in that the fluorescent probe compound does not exhibit fluorescence when the expression or activity of SLCO1B3 is reduced or inhibited, and exhibits fluorescence when SLCO1B3 is expressed or active.

5. A kit for detecting choroid plexus of the brain, comprising the composition of paragraph 1 as an active ingredient.

6. A method for imaging the choroid plexus of the brain using the composition of paragraph 1.

7. A step of treating the composition of claim 1 to brain tissue and confirming fluorescence generated from the fluorescent probe compound, A method for analyzing the choroid plexus of the brain in vitro.

8. In paragraph 7, A method for analyzing the choroid plexus of the brain in vitro, characterized in that the fluorescent probe compound targets epithelial cells of the choroid plexus of the brain.

9. A step of treating the candidate compound to the isolated brain tissue; and Comprising a step of confirming fluorescence generated from a fluorescent probe compound after treating the brain tissue with the composition of the first clause. A method for screening for therapeutic agents for diseases caused by abnormalities in the choroid plexus of the brain.

10. In paragraph 9, A method for screening a therapeutic agent for a disease caused by a choroid plexus abnormality in the brain, wherein the disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), prion disease, motor neuron disease, and choroid plexus cyst.

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