KR20200064971A - Fucoidan-based Theragnostic Composition - Google Patents

Abstract

The present invention relates to a fucoidan-based theragnostic composition and, more particularly, to preparation of a theragnostic composition and a use thereof, which may use an assembly of fucoidan with a fluorescent dye or a photosensitizer, thereby not only diagnosing cancer or vascular disease lesion through fluorescence imaging, but also obtaining a therapeutic effect at the same time. According to the present invention, the assembly in which the fluorescent dye or the photosensitizer is covalently linked to fucoidan can be useful in diagnosis of tumor tissues and ophthalmic vascular diseases through fluorescence imaging, and obtain a therapeutic effect on cancer cells and coronary artery smooth muscle cells as well as an inhibitory effect on angiogenesis in ophthalmic diseases. In addition, when additionally carrying out a photodynamic treatment with the assembly according to the present invention, it is possible to effectively treat cancer and vascular diseases with less side effects.

Description

Fucoidan-based Theragnostic Composition

The present invention relates to a fucoidan-based seragnosis composition, and more specifically, the present invention can only diagnose a cancer or vascular disease lesion by fluorescence imaging by using a covalently bound conjugate of a fucoidan and a fluorescent dye or photosensitizer. In addition, it relates to the production and use of the seragnosis composition that can also obtain a therapeutic effect at the same time.

Diagnosis and treatment are two main categories in the clinical application of diseases, and recently the concept of theragnosis, a technique that simultaneously performs diagnostics and therapy using imaging functions, has been introduced.

The photosensitizer, which is used in photodynamic therapy (PDT), absorbs light of a specific wavelength and generates a fluorescence signal. Although it is possible to selectively kill only target cells using singlet oxygen or free radicals, which are reactive oxygen species that occur only at the target site, there are advantages to minimize side effects seen in anticancer drugs. Photodynamic therapy has been actively researched since the early 20th century and has been used for diagnosis and treatment of cancer, treatment of ophthalmic diseases, treatment of vascular diseases such as arteriosclerosis, acne and dental diseases, autologous bone marrow transplantation, antibiotics , AIDS treatment, skin transplant surgery or arthritis, etc., it is used to increase immunity, and its application range is gradually expanding. In recent years, by combining photodynamic therapy with immunotherapy, the possibility of treating metastatic cancer existing in an unlit area has been opened. With the development of technology, recently, by using a photosensitizer as a therapeutic agent for sonodynamic therapy, it is possible to treat tumors located deep within the human body, which have been difficult to treat with conventional photodynamic therapy. In addition, selective fluorescence imaging technology for various disease lesions has been developed using near infrared fluorescent dyes, and it is applied to clinical trials. Since the photosensitizer generates a strong fluorescence signal when irradiated with light of a specific wavelength, these characteristics are used. Efforts are also being made to apply to the diagnosis of fluorescence images of diseased lesions such as cancer.

In the case of a photosensitive agent for photodynamic diagnosis or treatment used in the related art, it is hydrophobic, and after administration to a patient through intravenous injection, non-specific accumulation occurs in normal tissues including skin as well as cancer tissues. This not only makes it difficult to diagnose the tumor region by lowering the target-to-background ratio, but also has the risk of damaging important surrounding normal tissues during photodynamic therapy. In addition, when a patient undergoing photodynamic therapy is exposed to bright light such as sunlight, reactive oxygen production is activated from the photosensitizer accumulated in the skin, causing side effects of skin photosensitivity. For this reason, after photodynamic therapy, the patient is recommended to stay in a dark room for at least 6 weeks until the photosensitizer accumulated in normal tissue such as skin disappears, which causes inconvenience to the patient. There is an attempt to solve the skin photosensitivity problem by increasing the hydrophilicity of the photosensitizer, but in this case, a large amount of the photosensitizer is sufficient for treatment because the large amount of the photosensitizer administered intravenously is rapidly discharged through the urine. In order to accumulate in the tissue, there is a disadvantage that a high dose of a photosensitizer must be administered.

Coupling the photosensitizer to a hydrophilic polymer such as chitosan, glycol chitosan, poly(ethylene glycol), poly-L-lysine, and carboxymethyl dextran In this case, it is possible to stably disperse the photosensitizer in an aqueous solution, while improving the accumulation efficiency for the tumor. However, these hydrophilic polymer-photosensitizers and conjugates help improve the hydrophilicity of photosensitizers, but because they do not have target specificity for cells associated with cancer cells or other diseases, specificity for target cells is not achieved. To have it, a target ligand such as folic acid, an antibody, or an aptamer had to be additionally conjugated to the polymer. In this case, steps and costs for manufacturing increase and mass production of the binder becomes difficult. In addition, even if the hydrophilic polymer accounts for more than 70% of the mass of the hydrophilic polymer and the photosensitizer combination, the therapeutic effect can be obtained only by the photosensitizer, and the hydrophilic polymer itself has no therapeutic effect on cancer cells, etc. Not only is the therapeutic effect that can be low, but there is a big disadvantage that the photodynamic treatment effect is not obtained when light does not reach the site where the polymer-photosensitizer conjugate is delivered.

In addition, attempts have been made to image lesions such as cancer by using a conjugate of a fluorescent dye and a hydrophilic polymer (ie, a ligand-fluorescent dye-hydrophilic polymer conjugate) in which a ligand capable of targeting a specific cell is conjugated. However, in this case, only the imaging function for the target site can be obtained, and in order to obtain a therapeutic effect, a complex process of additionally conjugation or loading of the drug to the polymer is required.

Republic of Korea Patent No. 10-2017-0048202 Republic of Korea Patent No. 10-2008-0095182

In order to overcome the above problems, the present inventors not only enable the conjugate to simultaneously achieve fluorescence imaging and photodynamic therapy by preparing a conjugate covalently coupled to a photosensitizer or a fluorescent dye using fucoidan. , By making it possible to obtain the therapeutic effect and target specificity effect of fucoidan, it was confirmed that an improved therapeutic effect that could not be expected before was obtained and the present invention was completed.

Accordingly, it is an object of the present invention to provide a conjugate in which fucoidan and a fluorescent dye or fucoidan and a photosensitizer are bonded through a covalent bond.

Another object of the present invention is to treat cancer and vascular-related diseases, such as arteriosclerotic plaque or senile macular degeneration and glaucoma, using the above-mentioned conjugates, as well as real-time fluorescence imaging of the site, as well as therapeutic effects on these diseases It is to provide a composition for the diagnosis of fluorescence imaging and a composition for photodynamic therapy that may have.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

In order to achieve the technical problem as described above, the present invention provides a conjugate in which fucoidan and a photosensitizer or a fluorescent dye are covalently linked, and a composition for fluorescence imaging or a composition for photodynamic therapy comprising the same.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention relates to a conjugate in which a photosensitizer or fluorescent dye is covalently bonded to fucoidan. Here, the conjugate includes the same meaning as the conjugate or conjugate.

In one aspect, the present invention relates to a fluorescent dye-fucoidan conjugate in which fucoidan and a fluorescent dye are covalently linked.

In the present invention, it may be characterized in that the carboxyl group of the fucoidan and the amine group of the fluorescent dye are covalently bonded using a coupling agent.

In the present invention, the fluorescent dye is cyanine (cyanine), rhodamine (rhodamine), coumarin (cumarine), EvoBlue (EvoBlue), oxazine (oxazine), vodpi (BODIPY), carbopyronine, naphthalene, b It may be a fluorescent dye selected from the group consisting of phenyl, anthracene, phenanthrene, pyrene, carbazole or derivatives based on the dye.

In one embodiment of the present invention, the fluorescent dye is fluorescein (Fluorescein), CR110: Carboxyrodamine 110: Rhodamine Green (trade name), TAMRA: Carboxytetramethylrodamine: TMR, Carboxyrodamine 6G: CR6G , BODIPY FL (trade name): 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 493/503 (trade name): 4 ,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, BODIPY R6G (trade name): 4,4-difluoro Rho-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 558/568 (trade name): 4,4- Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 564/570 (trade name): 4,4-difluoro-5 -Styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 576/589 (trade name): 4,4-difluoro-5-(2-pyrrolyl)-4 -Bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-difluoro-5-(4-phenyl-1,3-butadienyl) -4-Bora-3a,4a-diaza-s-indacene-3-propionic acid, EvoBlue10 (trade name), EvoBlue30 (trade name), MR121, ATTO 655 (trade name), ATTO 680 (trade name), ATTO 700 (trade name) , ATTO MB2 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name), Alexa Fluor 633 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Flamma 496 (trade name), Flamma 507 (trade name), Flamma 530 (trade name), Flamma 552 (trade name), Flamma 560 (trade name), Flamma 575 (trade name), Flamma 581 (trade name), Flamma 648 (trade name), Flamma 675 (trade name), Flamma 749 (trade name), Flamma 774 (trade name), Flamma 775 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name) ), 5(6)-TAMRA-X (trade name), 5TAMRA (trade name), Cy5 ^TM , Cy5.5 ^TM , Cy7 ^TM or Licor NIR ^TM ,IRDye38 ^TM ,IRDye78 ^TM , IRDye80 ^TM , LaJolla Blue ^TM , Licor NIR ^TM , Indocyanine green (ICG) and may be selected from the group consisting of ZW800-1C.

In the present invention, the binding ratio of the fucoidan to the fluorescent dye is preferably 1:2 to 1:4, and when more fluorescent dyes are combined, fucoidan and fucoidan through a connector capable of decomposing in the target cell It is desirable to combine fluorescent dyes.

The fluorescent dye-fucoidan conjugate according to the present invention is, but is not limited to, dissolving fucoidan in a buffer solution (first step); Adding and stirring a coupling agent to the first step melt (second step); Separating the reaction mixture of the second step and adding a near infrared fluorescent dye and stirring (third step); And after dialysis the reaction mixture of the third step in distilled water, freeze-drying to obtain a fluorescent dye-fucoidan conjugate in which the near-infrared fluorescent dye is covalently bound (fourth step).

Since the fluorescent dye-fucoidan conjugate according to the present invention can obtain high binding specificity to P-selectin and vascular endothelial growth factor even after formation of the conjugate, in cancer cells, atherosclerotic plaques and ophthalmic diseases It is possible to diagnose a fluorescence image of angiogenesis sites and platelet-rich thrombus.

Accordingly, the present invention relates to a composition for diagnosing fluorescent images comprising the fluorescent dye-fucoidan conjugate.

In one aspect, the present invention relates to a fucoidan-fucoidan conjugate, wherein the fucoidan and the photosensitizer are covalently linked.

In the present invention, the photosensitive agent and the fucoidan are bound to the carboxyl group of the fucoidan by using a linker containing a disulfur bond or a diselenium bond.

Such photosensitizers include, but are not limited to, hematoporphyrins, porphycenes, pheophorbides, purpurins, chlorins, protoporphyrins, phthalocyanines a porphyrin-based compound selected from the group consisting of (phthalocyanines); And hypericin, rhodamine, atto, ATTO, rose Bengal, psoralen, phenothiazinium-based dyes and merocyanine It may be selected from the group consisting of non-porphyrin-based compounds selected from the group.

The photosensitizer-fucoidan conjugate according to the present invention, but is not limited to, dissolving fucoidan in a buffer solution (first step); Adding and stirring a coupling agent to the first step melt (second step); Adding and stirring a linker (linker) containing a disulfur or diselenium bond to the second step reaction mixture (step 3); Dialysis the reaction mixture of the third step in distilled water, followed by freeze drying to obtain a fucoidan derivative having an amine group (fourth step); And dissolving the photosensitizer in the organic solvent, adding a coupling agent thereto and stirring (step 5); Mixing and reacting the fucoidan derivative having the amine group with the fifth step reaction solution (step 6); After dialysis the reaction mixture of the sixth step in a phosphate buffer solution and distilled water, freeze-drying to obtain a photosensitizer-fucoidan conjugate in which the photosensitizer and the fucoidan are covalently bonded through a connector (step 7). It can be produced by a method.

In the photosensitizer-fucoidan conjugate according to the present invention, the fucoidan polymer itself exhibits direct cytotoxicity against cancer cells or smooth muscle cells, or a combination of vascular endothelial growth factor (VEGF) and fucoidan ( By inhibiting cancer growth by binding) or by further inhibiting the formation of angiogenesis in ophthalmic diseases, it is possible to simultaneously obtain a therapeutic effect by fucoidan itself and a photodynamic treatment effect using a photosensitizer.

The photosensitizer-fucoidan conjugate according to the present invention can also obtain fluorescence imaging efficacy for endothelial cells of cancer cells, arteriosclerotic plaques, and new blood vessels, and smooth muscle, which is a major factor causing vascular restenosis Cell proliferation can be effectively suppressed.

Accordingly, the present invention relates to a composition for diagnosing a fluorescence image or a composition for photodynamic treatment comprising the photosensitive agent-fucoidan conjugate.

In the present invention, the coupling agent refers to a reagent capable of promoting or forming a bond between two or more functional groups present in a molecule, between molecules, or both. In the present invention, the coupling agent may preferably use N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS).

In preparing the fluorescent dye-fucoidan and photosensitizer-fucoidan conjugate, after converting the carboxyl group of the fucoidan to a functional group such as amine, thiol, azide, or alkyne using a coupling agent, the fluorescent dye or the photosensitizer and A conjugate can be formed. For example, when an alkyne or azide group is introduced into fucoidan, a fluorescent dye or a photosensitizer may be combined through click chemistry, which is a reaction between azide and alkyne.

In the present invention, a photosensitizer used in combination for photodynamic diagnosis or treatment may be applied with a photosensitizer that can be applied by a person skilled in the art. For example, a porphyrin-based compound selected from the group consisting of porphyrins, chlorins, pheophorbides, bacteriochlorins, porphycenes, phthalocyanines; Or biporphyrin selected from the group consisting of hypericin, rhodamine, rose bengal, psoralen, phenothiazinum-based dyes and merocyanine It may be used in the group consisting of compounds, but is not limited thereto. More specifically, hematoporphyrins, porphycenes, pheophorbides, purpurins, chlorins, protoporphyrins, phthalocyanines (hematoporphyrins) in the form of free bases or metal complexes phthalocyanines) porphyrin compounds selected from the group consisting of; Or consisting of hypericin, rhodamine, atto, ATTO, rose Bengal, psoralen, phenothiazinium dyes and merocyanine Any one selected from the group consisting of non-porphyrin-based compounds selected from the group can be used alone or in combination.

In the present invention, the linker connecting the photosensitizer or the fluorescent dye and the fucoidan with a covalent bond is a zero-length connector, that is, an amide bond to which the amine group of the fluorescent dye or photosensitizer and the carboxyl group of the fucoidan are connected. , Carbon bonds, disulfur or diselenium bonds. The linker according to the present invention may have amine groups at both ends. According to the present invention, the amine group of the linker is covalently bonded to the carboxyl group of the fucoidan to form a fucoidan conjugate in which the linker is covalently bonded.

In one embodiment of the present invention, the linker is a coupling agent such as EDC-NHS, selenocystamine, diselenodipropionic acid, selenocystine, cystine , Cystamine and may be selected from the group consisting of mixtures thereof.

In one embodiment of the present invention, the photosensitizer may be chlorin e6, a chlorin-based photosensitizer represented by the following. The carboxyl group of chlorin e6 is covalently bonded to the amine group of the fucoidan conjugate in which the linker is covalently bound to form a photosensitizer-fucoidan conjugate.

According to the present invention, the fucoidan conjugate in which the linker is covalently linked is taken into the target cell, and disulfide or iselenium bond is broken by the glutathione, a reducing agent present in excess in the cell, and the fucoidan is bound. The photosensitizer or fluorescent dye that has been released may be released in the target cell.

The present invention also relates to a composition for photodynamic therapy containing the photosensitizer-fucoidan conjugate.

The fluorescent dye or photosensitizer fucoidan conjugate of the present invention can exert a therapeutic effect by specifically targeting P-selectin overexpressing cells.

Diseases that can be diagnosed/treated with the fluorescent dye or the photosensitizer fucoidan complex according to the present invention include terminal black melanoma, hypokeratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenocarcinoma, adenocarcinoma, astrocytic ( astrocytic) tumor, glucagonoma, hemangioblastoma, hemangioma, hemangioma, hepatic adenoma, hepatoma, hepatocellular carcinoma, insulinoma, epithelial neoplasm, epithelial squamous cell neoplasm, invasive squamous cell carcinoma, large cell carcinoma, smooth muscle Tumor diseases such as sarcoma, malignant melanoma, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloblastoma, pituitary adenoma, glioma, brain tumor, pharyngeal cancer, larynx cancer, thymoma, mesothelioma, breast cancer, lung cancer, stomach cancer, esophageal cancer , Colon cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureteral cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasmacytoplastic tumor, leukemia, childhood cancer, Cancer diseases such as skin cancer, ovarian cancer, and cervical cancer, but are not limited thereto. Other diseases that can be treated with the fluorescent dye or photosensitizer fucoidan complex according to the present invention include sickle cell disease, arterial thrombosis, rheumatoid arthritis, ischemia and reperfusion, arteriosclerotic plaque, vascular stenosis after stent procedure, senile macular degeneration Ophthalmic diseases such as sex and glaucoma, acne and dental diseases.

The composition for diagnosis of fluorescence imaging or the composition for photodynamic therapy according to the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. The composition for photodynamic treatment of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

Fluorescent imaging diagnostic compositions or photodynamic therapy compositions according to the present invention can be formulated using methods known in the art. Formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

In addition, the composition for diagnosis of fluorescence imaging or the composition for photodynamic therapy according to the present invention may be administered orally or parenterally according to a desired method, and the dosage is the patient's weight, age, sex, health status, diet, and administration time. , Administration method, excretion rate and disease severity may be appropriately changed.

The fucoidan-fluorescent dye complex or fucoidan-photosensitive agent combination according to the present invention allows the photosensitizer or the fluorescent dye to dissolve well in water, and allows the fluorescence imaging and photodynamic treatment to be effectively performed, as well as the fucoidan polymer itself The therapeutic effect or target specificity effect on P-selectin overexpressing cells can be obtained. According to the present invention, the fucoidan polymer itself shows direct cytotoxicity to cancer cells or smooth muscle cells, or inhibits cancer growth by binding of fucoidan with vascular endothelial growth factor (VEGF). Alternatively, by additionally obtaining an inhibitory effect on angiogenesis in an ophthalmic disease, it is possible to obtain an improved therapeutic effect that could not be expected in a conventional photosensitizer or fluorescent dye and hydrophilic polymer.

The photosensitizer-fucoidan conjugate according to the present invention can not only obtain fluorescence imaging efficacy for endothelial cells of cancer cells, arteriosclerotic plaques and new blood vessels, but also provide therapeutic effects and photosensitizers by fucoidan itself. The used photodynamic treatment effect can be obtained at the same time. The site where the stent is mounted for the expansion of blood vessels causes restenosis of blood vessels due to the proliferation of smooth muscle cells over time, and when the photosensitizer-fucoidan complex according to the present invention is used It can effectively suppress the proliferation of smooth muscle cells, the main factor causing stenosis.

Since the fluorescent dye-fucoidan conjugate according to the present invention can obtain high binding specificity to P-selectin and vascular endothelial growth factor even after formation of the conjugate, cancer cells, atherosclerotic plaques, and ophthalmic diseases In addition, it is possible to diagnose a fluorescence image of an angiogenesis site in, as well as to obtain a therapeutic effect by fucoidan.

The fluorescent dye-fucoidan conjugate and the photosensitizer-fucoidan conjugate according to the present invention are not only useful for fluorescence imaging of tumor tissue and ophthalmic vascular disease, but also therapeutic effect on cancer cells and coronary artery smooth muscle cells and emerging in ophthalmic diseases Angiogenesis inhibitory effect can be obtained. In addition, when photodynamic therapy is additionally performed in the photosensitizer-fucoidan conjugate, cancer can be effectively treated with low side effects.

1 is a schematic diagram of a fucoidan-based fluorescent dye or photosensitizer conjugate. The photosensitizer or fluorescent dye is covalently bonded to the fucoidan through a linker, and the linker may include an amide bond, a carbon bond, a disulfide bond, or an selenium bond.
2 is a schematic diagram for synthesizing a conjugate of a fluorescent dye having an amine group and a fucoidan. Fluorescent dye-fucoidan conjugates were prepared using EDS and NHS, which are coupling agents.
3 is a data confirming the optical properties of the prepared Flamma774-Fucoidan conjugate through (A) UV-Vis absorbance and (B) fluorescence spectrum.
FIG. 4 is data confirming (A) fucoidan and (B) Flamma774-Fucodian conjugate prepared through FT-IR analysis.
FIG. 5 is data confirming (A) fucoidan and (B) Flamma774-Fucoidan conjugate prepared through ¹ H-NMR analysis.
6 is a data confirming the optical properties of the prepared ATTO655-Fucoidan conjugate through (A) UV-Vis absorption and (B) fluorescence spectrum.
7 is a schematic diagram of a method of synthesizing a ZW800-Fucoidan conjugate by binding a near infrared fluorescent dye ZW800 dye to fucoidan.
8A is data confirming the optical properties of the ZW800-Fucoidan conjugate in a 1:2 ratio according to the ratio through UV-Vis absorption and fluorescence spectra.
8B is data confirming the optical properties of the ZW800-Fucoidan conjugate in a ratio of 1:4 according to the ratio through UV-Vis absorption and fluorescence spectra.
9 is a data confirming the binding affinity of the prepared ZW800-Fucoidan conjugates and fucoidan itself and a vascular epithelial growth factor (VEGF165) ligand through surface plasmon resonance (SPR) analysis.
10 is a data confirming the binding affinity of the prepared ZW800-Fucoidan conjugate and fucoidan itself and P-selectin through surface plasmon resonance (SPR) analysis.
11 is a data confirming the optical properties of the prepared FSD750-Fucoidan conjugate through (A) UV-Vis absorption and (B) fluorescence spectrum.
FIG. 12 shows Ce6-Fucoidan conjugates in which chlorin e6 (Ce6), a photosensitive agent, is combined with fucoidan through a connector containing a disulfide bond (-SS-), and this material is self-assembled through self assembly. It is a synthetic schematic showing nanoparticle sized nanoparticles for photodynamic diagnosis and treatment.
13A is a graph showing the aqueous phase distribution (hydrodynamic size) of the prepared Ce6-Fucoidan (fucoidan molecular weight 18 kDa) conjugate.
13B is a UV-Vis absorption spectrum according to the solvent of the prepared Ce6-Fucoidan (18 kDa) conjugate.
13C is a fluorescence spectrum according to the solvent of the prepared Ce6-Fucoidan (18 kDa) conjugate.
14A is a graph showing the aqueous phase distribution (hydrodynamic size) of the prepared Ce6-Fucoidan (fucoidan molecular weight 100 kDa) conjugate.
Figure 14B is a photograph of the shape of the prepared Ce6-Fucoidan (100 kDa) material analyzed with a transmission electron microscope.
15A is a UV-Vis absorption spectrum according to the solvent of the prepared Ce6-Fucoidan (100 kDa) conjugate.
15B is a fluorescence spectrum according to the solvent of the prepared Ce6-Fucoidan (100 kDa) conjugate.
15C is a fluorescence spectrum obtained by treating glutathione (GSH) with Ce6-Fucoidan (100 kDa) at a concentration of 0 μM, 5 μM, and 5 mM, respectively.
15D is a data obtained by treating glutathione (GSH) with 0 μM, 5 μM, and 5 mM concentration for 4 hours in Ce6-Fucoidan (100 kDa), and measuring the amount of monooxygen production while irradiating 670 nm light. The fluorescence of the single-oxygen-detecting reagent (SOSG) increases as monooxygen occurs.
16 is a result of ¹ H-NMR analysis of a free photosensitive agent (free Ce6) and the prepared Ce6-Fucoidan conjugate.
17 is a confocal fluorescence micrograph obtained after treating the photosensitive agent-fucoidan conjugate Ce6-Fucoidan and the free photosensitive agent (free Ce6) at the same concentration in cancer cells. It was confirmed that Ce6-Fucoidan conjugates can be ingested much better into cancer cells.
18A is a result of analyzing cell survival rates by treating Ce6-Fucoidan conjugates and free photosensitizer cancer cells at various concentrations. It can be seen that the therapeutic effect by the conjugate itself can be obtained even without light irradiation.
18B is a result of analyzing the cell survival rate when a photodynamic therapy was performed using a 670 nm laser after treatment of a cancer cell with a Ce6-Fucoidan conjugate and a free photosensitizer. It was possible to freeze the highly improved cancer treatment effect by light irradiation.
19A is a result of near infrared fluorescence imaging obtained 5 minutes and 24 hours after intravenous injection of Ce6-Fucoidan conjugate. It can be seen that when the Ce6-Fucoidan conjugate is administered, the position of cancer tissue can be diagnosed from a fluorescence image.
19B is a result of quantitative analysis of tumor-to-background signal ratio values.
FIG. 19C shows the results of fluorescence imaging by collecting tumors and major organs 24 hours after administration of the Ce6-Fucoidan conjugate. It can be seen that Ce6-Fucoidan conjugates accumulated in the tumor tissue.
FIG. 19D shows the results of fluorescence imaging by confocal microscopy after 24 hours of Ce6-Fucoidan conjugate administration, incision of tumors and production of frozen sections. It can be seen that the concentration of the photosensitizer is high in the tumor administered with the Ce6-Fucoidan conjugate.
Figure 20A is a result showing the anti-tumor effect using a photosensitizer-fucoidan conjugate Ce6-Fucoidan conjugate. In the case of intravenous administration of the Ce6-Fucoidan conjugate, it can be seen that a significant anticancer effect can be obtained even without light irradiation, and a very high cancer treatment effect can be obtained when light is irradiated to the tumor.
Fig. 20B shows the distribution of tumors in tumor tissues by CD31 staining after incision of tumor tissue 24 hours after the combined treatment of Ce6-Fucoidan conjugates and photodynamic therapy, and cell apoptosis effect by TUNEL staining. It is the result of checking.
Figure 21A is the result of cutting the major organs of mice in each experimental group on the 10th day and confirming whether they are toxic through H&E staining.
21B is a result of measuring changes in mouse weight for each experimental group.
22A is a result of analyzing the cell survival rate by treating Ce6-Fucoidan conjugates and free photosensitizers in coronary artery smooth muscle cells at various concentrations.
FIG. 22B shows the results of analyzing cell viability after treating Ce6-Fucoidan conjugates and free photosensitizers at various concentrations in coronary artery smooth muscle cells and performing photodynamic therapy using a 670 nm laser.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings in order to describe the present invention in more detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[Example 1] Preparation of Flamma774-Fucoidan conjugate

A covalent bond was synthesized using a coupling agent between the amine group of Flamma774, a near infrared fluorescent dye of Bioacts, and the carboxyl group of fucoidan. Flamma774-amine is a fluorescent substance having a molar mass of 971.15 g/mol, a maximum excitation wavelength of 774 nm, and a maximum emission wavelength of 806 nm. Since the near-infrared fluorescent dye complex has high permeability to biological tissues, it can be used for bioimaging in drug delivery and tumor research. Fucoidan was used as a Sigma Aldrich product with a molecular weight of 18,000 Da extracted from Fucus vesiculosus.

Various coupling agents can be used to bind the fluorescent dye to fucoidan, where N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) are used to dye. Activated to obtain a fucoidan-NHS ester complex, and was subjected to Flamma774-amine binding thereto. To explain the process in more detail, 10 mg of fucoidan was dissolved in 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, EDC 19.7 mg, sulfo-NHS 2.17 mg was added thereto, and the mixture was stirred for about 30 minutes. . Then, after separation using a PD-10 column, 1.02 mg of Flamma774-amine fluorescence was added and stirred for one day. Then, dialysis was performed in distilled water for one day to remove unreacted products and by-products, and freeze-dried to obtain a powder to obtain a fluorescent dye-fucoidan conjugate in which Flamma774 was covalently bound.

Through the optical measurement analysis of Figure 3, it was confirmed that about 3.8 Flamma774 per molecule of fucoidan was bound.

[Example 2] Preparation of ATTO655-Fucoidan conjugate

The amine group of the fluorescent dye ATTO655 and the carboxyl group of fucoidan were formed using a coupling agent to form a covalent bond. ATTO655-amine is a fluorescent substance having a molar mass of 798 g/mol, a maximum excitation wavelength of 663 nm, and a maximum emission wavelength of 680 nm. Fucoidan was used as a Sigma Aldrich product with a molecular weight of 18,000 Da extracted from Fucus vesiculosus.

Fucoidan is activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) to obtain fucoidan-NHS ester conjugates and the process of combining ATTO655-amine with it. It went through. To explain the process in more detail, 10 mg of fucoidan was dissolved in 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, EDC 19.7 mg, sulfo-NHS 2.17 mg was added thereto, and the mixture was stirred for about 30 minutes. . Then, after separation using a PD-10 column, 0.399 mg of ATTO655-amine fluorescence was added and stirred for one day. Then, dialyzed against distilled water for one day, freeze-dried to obtain a powder to obtain a fluorescent dye-fucoidan conjugate covalently bound to ATTO655.

Through the optical measurement analysis of Figure 6, it was confirmed that about 1.3 ATTO dyes are bound per molecule of fucoidan.

[Example 3] Preparation of ZW800-Fucoidan conjugate

The amine group of ZW800, a near-infrared fluorescent dye developed by Professor Hak-soo Choi's research team at Harvard University, formed a covalent bond using a coupling agent. ZW800-amine is a near infrared fluorescent substance having a molar mass of 887 g/mol, a maximum excitation wavelength of 753 nm, and a maximum emission wavelength of 772 nm. Fucoidan was used as a Sigma Aldrich product with a molecular weight of 18,000 Da extracted from Fucus vesiculosus.

Fucoidan is activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) to obtain fucoidan-NHS ester conjugates and ZW800-amine binding process. It went through. The reaction ratio of fucoidan to ZW800 fluorescent dye was reacted at 1:2 and 1:4, respectively. To explain the process in more detail, 20 mg of fucoidan was dissolved in 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, EDC 38.34 mg, sulfo-NHS 4.34 mg was added thereto, and the mixture was stirred for about 30 minutes. . Then, after separation using a PD-10 column, if the reaction ratio of the fucoidan to ZW800 fluorescent dye is 1:2, 1.97 mg of ZW800-amine fluorescence is added, and the reaction ratio of the fucoidan to ZW800 fluorescent dye is 1:4 days. In the case, 3.94 mg of ZW800-amine fluorescence was added and stirred for one day. Then, by dialysis against distilled water for one day, and freeze-dried to obtain a powder, a fluorescent dye-fucoidan conjugate in which ZW800 was covalently bound was obtained.

Through the optical measurement analysis of FIG. 8, when the reaction ratio of the fucoidan to ZW800 fluorescent dye was 1:2, it was confirmed that about 1.84 ZW800 fluorescent dyes were combined per molecule of the fucoidan. When the reaction ratio of fucoidan to ZW800 fluorescent dye was 1:4, it was confirmed that about 2.88 ZW800 fluorescent dyes were bound per molecule of fucoidan.

[Example 4] Binding affinity of ZW800-Fucoidan conjugate and VEGF165 ligand

The binding affinity between the synthesized ZW800-Fucoidan conjugate and the human VEGF165 ligand was analyzed using surface plasmon resonance (SPR). SPR sensor technology uses a phenomenon that causes a signal change when a biomaterial such as a protein is bound to a sensor surface.Relationship between biomolecules in real time without specific labels (fluorescence, radioactivity, etc.) using SPR optical principles (Kinetics Affinity , Ka, Kd, KD). As the SPR analysis equipment, Biacore T200 equipment and CM5 chip were used for analysis, and then data were processed through Biaevaluation software.

Equilibrium binding of the SPR assay to the binding affinity of the human VEGF165 ligand of fucoidan with nothing labeled, and in the case of ZW800-Fucoidan conjugates (1) and (2) with a reaction ratio of fucoidan to ZW800 fluorescent dye of 1:2 and 1:4 The results of analysis through the dissociation rate constant (KD) value are shown in FIG. 9. It was measured to be 178.7 nM for the ZW800-Fucoidan conjugate reacted at 1:2, 72.42 nM for the ZW800-Fucoidan conjugate reacted at 1:4, and 4.053 nM for Fucoidan. It can be seen that these results show strong binding of 10 ^-9 M units to VEGF even after formation of the conjugate of fucoidan and fluorescent dye. Therefore, the fluorescent dye-fucoidan conjugate can obtain not only the fluorescence imaging diagnosis of vascular disease, but also the effect of inhibiting angiogenesis in ophthalmic diseases through binding of the fluorescent dye-fucoidan conjugate with VEGF.

[Example 5] Binding affinity of ZW800-Fucoidan conjugate and P-selectin

In Figure 10, the reaction ratio of the fucoidan to ZW800 fluorescent dye is 1:2 and 1:4 in the case of ZW800-Fucoidan conjugates (1) and (2), and the binding affinity of P-selectin of fucoidan with nothing labeled It shows the results of analysis through the equilibrium dissociation rate constant (KD) value of the SPR assay. It was measured to be 387.8 nM for the ZW800-Fucoidan conjugate reacted at 1:2, 218.8 nM for the ZW800-Fucoidan conjugate reacted at 1:4, and 2.981 nM for Fucoidan. It can be seen that these results show strong binding of 10 ^-9 M units to P-selectin even after the formation of the conjugate of fucoidan and fluorescent dye. These results indicate that P-selectin can be diagnosed with metastatic cancer cells overexpressing on the cell surface or vascular disease lesions such as atherosclerosis using a fluorescent dye-fucoidan conjugate.

[Example 6] Preparation of FSD750-Fucoidan conjugate

A covalent bond can be formed by covalently bonding the amine group of the FSD750, a bioinfrared fluorescent material, of Fact750, and the carboxyl group of fucoidan. FSD750-amine is a fluorescent substance having a molar mass of 1252.42 g/mol, a maximum excitation wavelength of 749 nm, and a maximum emission wavelength of 774 nm. Fucoidan was used as a Sigma Aldrich product with a molecular weight of 18,000 Da extracted from Fucus vesiculosus.

Fucoidan is activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) to obtain fucoidan-NHS ester conjugates and FSD750-amine binding process. It went through. To explain the process in more detail, 5 mg of fucoidan was dissolved in 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, and 9.585 mg of EDC and 1.085 mg of sulfo-NHS were added thereto and stirred for about 30 minutes. . After separation using a PD-10 column, 0.695 mg of FSD750-amine fluorescence was added and stirred for one day. Then dialyzed against distilled water for one day, freeze-dried to obtain a powder to obtain a fluorescent dye-fucoidan conjugate in which FSD750 is covalently bound.

Through the optical measurement analysis of FIG. 11, it was confirmed that about 1.2 FSD750 fluorescent dyes were combined per molecule of fucoidan.

[Example 7] Preparation of photosensitizer-fucoidan (18 kDa) conjugate

The photosensitizer-fucoidan conjugate was synthesized by covalently bonding the photosensitive agent chlorin e6 (Ce6) with the carboxyl group of fucoidan through a linker. Fucoidan was used as a Sigma Aldrich product with a molecular weight of 18,000 Da extracted from Fucus vesiculosus.

First, in order to synthesize fucoidan in which an amine group was introduced, cystamine dihydrochloride, a linker containing a disulfide bond, was covalently bonded to the carboxyl group of fucoidan using EDC and sulfo-NHS. To explain the process in more detail, 54.36 mg of fucoidan was dissolved in 18 mL of 10 mM PBS buffer, 23.0 mg (0.5 mL) of EDC, and 27.1 mg (0.5 mL) of sulfo-NHS were added and stirred for about 30 minutes. Then, 27 mg (1 mL) of cystamine dihydrochloride, a linker containing a disulfide bond, was added and stirred for one day. Then, dialyzed against distilled water for one day, freeze-dried to obtain a powder to obtain a fucoidan derivative having an amine group.

After activating the carboxyl group of Ce6 with EDC and sulfo-NHS, it was subjected to a process of combining fucoidan with an amine group introduced therein. 5 mg of Ce6 was dissolved in 2.5 mL of dimethyl sulfoxide (DMSO), to which 16.3 mg of EDC and 19 mg of sulfo-NHS were added and stirred for 1 hour. Then, 30.15 mg of fucoidan into which the amine group was introduced was dissolved in 2.5 mL of DMF:H2O co-solvent (1:1 v/v), mixed with Ce6 reaction solution, and stirred for one day. Then dialyzed for one day using phosphate buffer (pH7.4) and distilled water, and freeze-dried to obtain a powder to obtain a photosensitizer-fucoidan conjugate. 13A, it can be seen that the prepared photosensitizer-fucoidan conjugate forms nanoparticles in an aqueous solution, and according to FIGS. 13B and C, it can be seen that the fluorescence properties of the prepared conjugate are suppressed.

[Example 8] Preparation of fucoidan (100 kDa)-photosensitive agent conjugate

The photosensitizer-fucoidan conjugate was synthesized by covalently bonding the photosensitive agent chlorin e6 (Ce6) with the carboxyl group of fucoidan through a linker. Fucoidan used in the preparation of the conjugate was a fucoidan having a molecular weight of 100,000 Da extracted from Undaria pinnatifida, and was used by Harim Bio.

First, in order to synthesize fucoidan having an amine group introduced therein, cystamine dihydrochloride was covalently bound to fucoidan by a linker containing a disulfide bond using carboxyl groups of fucoidan, EDC, and sulfo-NHS. To explain the process in more detail, 405 mg of fucoidan was dissolved in 18 mL of 10 mM PBS buffer, to which 23.0 mg (0.5 mL) of EDC, 27.1 mg of sulfo-NHS (0.5 mL) was added and stirred for about 30 minutes. Then cystamine dihydrochloride was added 27 mg (1 mL) and stirred for one day. Then, dialyzed against distilled water for one day, freeze-dried to obtain a powder to obtain a fucoidan derivative having an amine group.

After activating the carboxyl group of Ce6 with EDC and sulfo-NHS, it was subjected to a process of combining fucoidan with an amine group introduced therein. 20 mg of Ce6 was dissolved in 10 mL of DMSO, to which 65.2 mg of EDC and 76 mg of sulfo-NHS were added and stirred for 1 hour. Then, 101 mg of fucoidan in which the amine group was introduced was dissolved in 5 mL of DMF:H2O cosolvent (1:1 v/v), mixed with the Ce6 reaction solution, and stirred for one day. Then dialyzed for one day using phosphate buffer (pH7.4) and distilled water, and freeze-dried to obtain a powder to obtain a photosensitizer-fucoidan conjugate.

14A is a result of measuring the average size of particles by a particle size analyzer using the photosensitizer-fucoidan conjugate prepared above, and analyzed to 259 nm. 14B is a result of analyzing the photosensitizer-fucoidan complex with a transmission electron microscope. As shown, it can be seen that nanoparticles having a size of about 85 nm were obtained.

After dissolving the photosensitizer-fucoidan conjugate prepared in a NaOH/SDS mixed solution serving as a surfactant and a phosphate buffered saline (PBS) solution, 0.1 M, pH7.4, the amount of Ce6 bound through absorbance 15A and 15B are measured UV-vis absorption and fluorescence spectra.

In addition, as the treated Ce6-Fucoidan (100 kDa) was treated with glutathione (GSH) concentrations of 0 μM, 5 μM, and 5 mM, the disulfide bonds were broken and the Ce6 fluorescence intensity of the quenched derivative changed. Fluorescence intensity change according to the concentration of glutathione was measured for viewing, and FIG. 15C is a fluorescence spectrum observing this. There is no change in the intensity of fluorescence when Ce6-Fucoidan (100 kDa) is treated with a concentration of 5 μM, which is the concentration of glutathione present in the extracellular or blood, but the intensity of fluorescence when treated with 5 mM, the concentration of glutathione in cancer cells It was confirmed that increases by about 5 times.

15D is treated with the prepared Ce6-Fucoidan (100 kDa) glutathione (glutathione; GSH) 0 μM, 5 μM, 5 mM concentration for 4 hours, mono-oxygen at 30 second intervals while irradiating light with a 670 nm laser This is the result of analyzing the production. When the glutathione concentration in cancer cells was treated with 5 mM, it was confirmed that the production of monooxygen increased by about 2 times. These results show that when a connector containing a disulfide bond is used, when the disulfide bond is decomposed by glutathione, a reducing agent that is ingested into the cancer cells targeted by the conjugate and present at a high concentration in the cancer cells, the fluorescence and photodynamic treatment effects Indicates that cancer cells can be recovered specifically.

Figure 16A is a light feeling of claim ¹ H-NMR analysis of the Ce6, FIG. 16B is the optical sense - a ¹ H-NMR analysis of the fucoidan conjugate. ^{Through 1} H-NMR analysis, it was calculated that about 20 Ce6 per molecule of fucoidan is bound.

[Example 9] Confirmation of intake of photosensitizer-fucoidan conjugate to cancer cells

The degree to which the photosensitizer-fucoidan conjugate and the free photosensitizer (free Ce6) were ingested into HT1080 cells, cancer cells, was compared through confocal fluorescence microscopy.

1) Cell culture

The human fibrosarcoma cell line HT1080 was obtained from the American Type Culture Collection (ATCC, USA). HT1080 cells were cultured at 37°C, 5% carbon dioxide and standard humidity conditions in MEM (Eagle's Minimum Essential Media) medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.

2) Cell intake confirmation experiment

HT1080 cells were placed in 5×10 ⁴ cells for each well of LabTek II Chambered Coverglass, and cultured for 24 hours so that the cells adhered well. The photosensitizer-fucoidan conjugate Ce6-Fucoidan and the free photosensitizer (free Ce6) were treated with cancer cells for 6 hours at a concentration of 2 μ based on Ce6. Thereafter, the drug not ingested into the cells was removed by washing, and after adding a new cell culture solution, the amount taken into the cells was compared through a confocal fluorescence microscope (observation condition = excitation: 633 nm, emission: 650 nm long-pass filter). According to FIG. 17, the fluorescence intensity of Ce6-Fucoidan-treated cancer cells was about 18 times higher than that of Ce6-treated cancer cells, and this resulted in the fact that the photosensitizer-fucoidan conjugate was effectively ingested into the cancer cells compared to the photosensitizer. In addition, it can be seen that the optical properties that were quenched are restored in the cancer cells.

[Example 10] Analysis of therapeutic performance of photosensitizer-fucoidan conjugate against cancer cells

18A is a result of measuring the cell viability according to the concentration of the photosensitive agent-fucoidan conjugate and the free photosensitive agent treated on cancer cells. It can be seen that fucoidan has an anti-cancer effect by fucoidan itself, unlike other biocompatible polymers.

Figure 18B shows the photodynamic therapy effect of Ce6-Fucoidan, a photosensitizer-fucoidan conjugate, on HT1080 cancer cells. The wavelength used for photodynamic therapy was 670 nm, and the light intensity was 10 J/cm ² . After treating HT1080 cancer cells with Ce6 as a control and Ce6-Fucoidan conjugate according to the present invention for 6 hours at various concentrations, they were exchanged with fresh MEM medium and irradiated with light at 10 J/cm ² using a 670 nm laser. After further incubation for 24 hours, the survival rate of the cells was analyzed using a CCK-8 assay assay kit. As shown, it was found that Ce6-Fucoidan conjugate has much better phototoxicity to HT1080 cancer cells than the existing Ce6, and the concentration (IC ₅₀ ) value required to kill half of the cells is 2.73 μM. Analysis.

[Example 11] Tumor target effect of photosensitizer-fucoidan conjugate

In Example 11, in order to analyze the tumor target effect of the photosensitizer-fucoidan conjugate, an experiment was conducted in which the conjugate was administered intravenously to a laboratory animal and a fluorescent image was taken.

HT1080 human fibrosarcoma transplanted animal models were injected intravenously with phosphate buffer (negative control), free photosensitizer (free Ce6) or photosensitizer-fucoidan conjugate (Ce6-Fucoidan), respectively, at 5 minutes and 24 hours. Fluorescence images were taken with (λ _ex 660/20 nm, λ _em 710/40 nm). According to the results of FIG. 19A, it was confirmed that the free photosensitizer (free Ce6) had a short residence time in vivo and was discharged out of the body within a short period of time, and remained almost in the body for 24 hours, and there was no fluorescent image signal in the tumor tissue. . On the other hand, Ce6-Fucoidan conjugate stayed in the body for a long time through the bloodstream and was continuously accumulated in the tumor tissue, and it was confirmed that a clear fluorescent image signal appeared in the tumor tissue at 24 hours. In FIG. 19B, by quantifying the fluorescence image signal value of the tumor tissue, the amount of the photosensitizer accumulated in the tumor tissue is compared, so that the photosensitizer-fucoidan conjugate is capable of targeting the tumor and selectively delivered to the tumor, and is also optical in the tumor. It was suggested that the improved properties can lead to an improved therapeutic effect. 19C is a result of confirming ex vivo fluorescence images by obtaining tumor tissue and spleen, kidney, liver, and lung tissue after euthanizing the mouse. As can be seen from the results, the photosensitizer-fucoidan conjugate remains for a longer time in each tissue in the body including the tumor compared to when the photosensitizer is administered, and the amount of the photosensitizer delivered to the tumor is also free photosensitive agent. Compared to (free Ce6), it can be seen that it is a suitable targeted therapy that can increase the effect of photodynamic therapy. 19D is a result of confirming the degree of penetration of the photosensitizer into the tumor tissue by confocal microscopy after freezing the obtained tumor tissue to produce a frozen section. In the control group (PBS), the free photosensitizer (free Ce6) treatment group showed little fluorescence signal by the photosensitizer in the tumor tissue, whereas in the photosensitizer-fucoidan conjugate treatment group, the photosensitizer in the tumor tissue Through the presence of a strong fluorescence signal, it was confirmed that the conjugate was well delivered to the tumor tissue and penetrated into the tumor tissue, and the results consistent with FIGS. 19A, B, and C were confirmed. In addition, it has been suggested that the position of a tumor can be detected from a fluorescence image by using an optical characteristic that is recovered from a tumor when a photosensitizer-fucoidan conjugate is used.

[Example 12] Animal test for the effect of enhancing the photodynamic treatment of the photosensitizer-fucoidan conjugate

The effect of photodynamic therapy with a photosensitizer-fucoidan conjugate was evaluated in an oncology animal model. A HT1080 cancer cell line was injected xenograft subcutaneously into a tumor model, intravenously injected with a free photosensitizer (Ce6) or Ce6-Fucoidan, and irradiated (PDT) using a 670 nm laser to the tumor site. To evaluate the anti-tumor effect, the tumor size was measured daily for 10 days to analyze differences between groups. In the case of the control experimental animals, a phosphate buffer solution without a photosensitizer was injected intravenously.

1) Tumor model construction and tumor growth inhibitory effect analysis

The human fibrosarcoma cell line HT1080 was obtained from the American Type Culture Collection (ATCC, USA). HT1080 cells were cultured at 37° C., 5% carbon dioxide and standard humidity conditions in MEM (Eagle's Minimum Essential Media) medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. It was confirmed that subcutaneous tumors were generated 5-7 days after injection of HT1080 cancer cells 5×10 ⁶ cells/100 μL subcutaneously into a nude mouse (Balb/c nude). When the tumors were about 70-80 mm ³ in size, they were divided into four groups (negative control, free Ce6 + PDT, Ce6-Fucoidan, and Ce6-Fucoidan + PDT) to perform each experiment. On day 1, free Ce6 or Ce6-Fucoidan was administered at 5 mg Ce6 equivalent/kg body weight through the tail vein of the mouse, and the negative control group was administered intravenously with phosphate buffer (PBS). On the second day, the PDT group irradiated the laser locally on the tumor site to perform photodynamic therapy (PDT), and the PDT irradiated light with a 670 nm wavelength laser at 50 mW/cm ² and 20 J/cm ² . Did. Tumor growth graphs were prepared by measuring the size of the tumor until day 10, and the tumor size between each group was compared and analyzed. As can be seen from the results of FIG. 20A, it was confirmed that the anti-tumor effect in the Ce6-Fucoidan + PDT group was the best, and the tumor size of the Ce6-Fucoidan or Ce6-Fucoidan + PDT group on the 10th day was 76.0 of the negative control, respectively. % ( P <0.01) or 0% ( P <0.001) was confirmed to inhibit tumor growth. The Free Ce6 + PDT group was found to have no statistically significant tumor growth inhibitory effect compared to the control group. In particular, when the photosensitizer-fucoidan conjugate was administered, a statistically significant anti-cancer effect was obtained without light irradiation (Ce6-Fucoidan), and when light was irradiated (Ce6-Fucoidan + PDT), very good anti-cancer The effect was obtained.

2) Observation of the effect of inhibiting angiogenesis in tumor tissue through tissue staining

In order to confirm the distribution of new blood vessels in the tumor tissue, on the third day of the experiment, the mice were euthanized to obtain tumor tissue, and paraffin blocks and tissue slides were prepared. Then, CD31 staining was performed using tissue slides to confirm vascular distribution. For CD31 staining, anti-CD31 antibody (abcam) was used for 2 hours at room temperature, and secondary antibody (anti-rabbit IgG-HRP) was reacted for 1 hour at room temperature, followed by diaminobenzidine (DAB) chromogen substrate (DAKO, Carpinteria, CA) ), and counterstain with Meyer's hematoxylin, dehydration with ethanol, and mounting. The stained tissue slide sample was imaged with a tissue microscope. As a result, as shown in FIG. 20B, the expression level of CD31 was similar in the negative control group, free Ce6 + PDT, and Ce6-Fucoidan treatment group, but the Ce6-Fucoidan + PDT treatment group had a marked staining of CD31 compared to the other three groups. It was confirmed that it was reduced. This result is similar to the results of Figure 9 of Example 4, Fucoidan was combined with VEGF to suppress the angiogenesis by suppressing the signaling system of VEGF, or the possibility that most of the neovascularization in the tumor was destroyed by the effect of photodynamic therapy. present.

3) Observation of cell death-induced changes in tumor tissue through tissue staining

In order to confirm changes in apoptosis and cell death in tumor tissues, mice were euthanized on the third day of the antitumor effect experiment to obtain tumor tissues, and paraffin blocks and tissue slides were prepared. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using tissue slides. TUNEL stained tissue slide samples were imaged with a tissue microscope. As a result, as shown in Figure 20B, compared to other groups, Ce6-Fucoidan + PDT treatment group was confirmed that the most occurred cell death.

[Example 13] In vivo safety evaluation of the photosensitizer-fucoidan conjugate

In order to evaluate the safety of the photosensitizer-fucoidan conjugate in vivo, an experiment was conducted to confirm histological changes for each organ. At the same time as the experiment for confirming the tumor growth inhibitory effect of Example 12 was conducted, as well as the size of the tumor as well as the body weight until the 10th day, the mice were euthanized on the 10th day, and then the heart, lung, liver, spleen and kidney of the mouse were collected. To prepare a paraffin block and tissue slide, H&E staining was performed.

According to FIG. 21A, it was found that no significant histological change occurred with the control group in all treatment groups. In addition, according to FIG. 21B, the photosensitizer-fucoidan conjugate treatment group (Ce6-Fucoidan) or the photosensitizer-fucoidan conjugate and photodynamic therapy combination treatment group (Ce6-Fucoidan + PDT) was compared with the control group for 10 days. It can be seen that no significant weight loss occurred during the period. Through these results, it was proved that the photosensitizer-fucoidan conjugate is biocompatible and safe.

[Example 14] Analysis of treatment performance of photosensitizer-fucoidan conjugate on coronary artery smooth muscle cells

A stent is used to widen the area of blood vessels narrowed by atherosclerosis, and vascular restenosis is a problem due to the proliferation of smooth muscle cells in this area, so anticancer drugs Development of a mounted degradable stand is ongoing. Therefore, it was evaluated through cell experiments whether the photosensitizer-fucoidan conjugate has a therapeutic effect on smooth muscle cells. Figure 22A is treated with various concentrations of photosensitive agent-fucoidan conjugate (Ce6-Fucoidan) and free photosensitive agent (free Ce6) in coronary artery smooth muscle cells (human primary coronary artery smooth muscle cells, HCASMC) and measures cell viability. One result. It was confirmed that the cell viability decreased as the concentration of the photosensitizer-fucoidan conjugate increased, which appears to be a therapeutic effect on the fucoidan in the conjugate.

22B is a cell survival rate when the photosensitive agent-fucoidan conjugate and photodynamic therapy are simultaneously performed on coronary artery smooth muscle cells (HCASMC). Light was irradiated using a laser having a wavelength of 670 nm, and the power density of light was 10 J/cm ² . The HCASMC cells were treated with Ce6 as a control group and the photosensitizer-fucoidan conjugate according to the present invention for 6 hours according to the concentration, and then exchanged with a new medium and subjected to photodynamic treatment with 10 J/cm ² with a 670 nm laser. After treating the treated cells in a CO ₂ incubator for an additional 24 hours, the viability of the cells was analyzed by the CCK-8 assay analysis method. As shown in the figure, it was found that the photosensitizer-fucoidan conjugate is superior to HCASMC cells compared to the conventional Ce6, and the concentration (IC ₅₀ ) of the substance required to kill about half of the cells is It was analyzed to be 1.05 μM.

Claims (5)

A fluorescent dye is covalently linked to fucoidan. The conjugate according to claim 1, wherein the carboxyl group of the fucoidan and the amine group of the fluorescent dye are covalently bonded using a coupling agent. The method of claim 1, wherein the fluorescent dye is cyanine (cyanine), rhodamine (rhodamine), coumarin (cumarine), EvoBlue (EvoBlue), oxazine (oxazine), bodipi (BODIPY), carbopyronine, naphthalene, A fluorescent dye-fucoidan conjugate that is a fluorescent dye selected from the group consisting of biphenyl, anthracene, phenanthrene, pyrene, or carbazole. The method according to claim 1, wherein the fluorescent dye is fluorescein (Fluorescein), CR110: Carboxyrodamine 110: Rhodamine Green, TAMRA: Carboxytetramethylrodamine: TMR, Carboxyrodamine 6G: CR6G, BODIPY FL: 4, 4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 493/503: 4,4-difluoro-1,3,5 ,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, BODIPY R6G: 4,4-difluoro-5-(4-phenyl-1,3-butadier Neil)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 558/568: 4,4-difluoro-5-(2-thienyl)-4-bora-3a ,4a-diaza-s-indacene-3-propionic acid, BODIPY 564/570: 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3 -Propionic acid, BODIPY 576/589: 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 581/591: 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, EvoBlue10, EvoBlue30, MR121, ATTO 655, ATTO 680, ATTO 700, ATTO MB2, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Flamma 496, Flamma 507, Flamma 530, Flamma 552, Flamma 560, Flamma 575, Flamma 581, Flamma 648, Flam ma 675, Flamma 749, Flamma 774, Flamma 775, Rhodamine Red-X, Texas Red-X, 5(6)-TAMRA-X, 5TAMRA, Cy5 ^TM , Cy5.5 ^TM , Cy7 ^TM or Licor NIR ^TM ,IRDye38 ^TM Fluorescent dye-fucoidan conjugate that is a fluorescent dye selected from the group consisting of, IRDye78 ^TM , IRDye80 ^TM , LaJolla Blue ^TM , Licor NIR ^TM , Indocyanine green (ICG) and ZW800-1C. A composition for diagnostic fluorescence imaging comprising the conjugate of any one of claims 1 to 4.

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