KR20130127232A - Glycol chitosan and fullerenes conjugate as photosensitizer for photodynamic therapy, method for preparing the same, and photodynamic therapy using the same - Google Patents

Abstract

The present invention provides a fullerene-glycol chitosan conjugate in which fullerene and a water-soluble glycol chitosan are conjugated as a photosensitive agent for photodynamic therapy that generates reactive oxygen species by light irradiation. The conjugate of the present invention can improve the poor solubility of fullerene and provide the size of the particle stability and easy removal in vivo as well as selectively act only on the diseased area by local light irradiation to maximize the therapeutic effect.

Description

Glucol Chitosan and Fullerenes Conjugate As Photosensitizer for Photodynamic Therapy, Method for Preparing the Same, and Photodynamic Therapy Using the Same}

The present invention relates to a conjugate of fullerene and glycol chitosan, a preparation method thereof, and a photodynamic therapy method using the same as a photosensitive agent for photodynamic therapy.

Current cancer therapies such as radiotherapy and chemotherapy serve to attack rapidly proliferating cells. This attack not only destroys cancer cells, but also destroys some normal cells. As a result, not only do they have serious side effects that can be life-threatening, but they can actually lower natural anti-tumor defenses. Radioactivity and chemotherapy damage the rapidly dividing cells of the immune system and suppress anti-tumor and anti-infection responses. In addition to causing these side effects, current therapies do not specifically attack cancer cells only, and generally do not achieve the desired effect intensity. As a result, chemotherapy or radiation therapy, or a combination thereof, cannot completely cure cancer. Therefore, the main treatment of current cancer is surgical removal of cancer cells. This method is often used in combination with radiotherapy and chemotherapy, and treatment requires the surgical cutting of the patient's body and the use of highly toxic therapies to destroy all cancer cells.

Photodynamic therapy (PDT) has been developed in an effort to minimize the detriment of such cancer treatments and to improve overall efficacy. PDT is a method of destroying cancer cells by using reactive oxygen species (ROS) generated when light is irradiated to a photosensitive material to destroy cancer cells. PDT involves first administering a photosentizing agent to the human body to localize to cancer cells, then moving the photosensitizer to the cancer cell site and then irradiating light of a specific and appropriate wavelength to the cancer cells containing the photosensitive material. It consists of Thus, PDTs can site-specifically apply activating light of appropriate wavelengths, and therefore can use a combination of photosensitive materials and site-specific light irradiation to produce therapeutic responses in certain tissues such as cancer.

Conventional aromatic molecules or dye molecules have been used as photosensitive materials in the last decades the medical community it has the capability to generate _{ROS, TiO 2, ZnO, Au} , CNT (carbon nanotube; CNT), a porous silicon (PSi) Nanomaterials have been reported as novel PDT photosensitive materials capable of producing ROS.

However, conventionally used photosensitive materials have various problems. For example, conventionally used materials have a relatively large size, which makes it difficult to excrete and remove them from the body after treatment, and may also cause nonspecific cytotoxic effects with tissues because they remain in vivo for a long time. .

Korean Patent Laid-Open Publication No. 2008-0047070 discloses a technique in which fullerene nanoparticles in which fullerenes and silica are covalently linked to each other are used as biocontrast contrast agents or drug carriers because they are harmless to the living body and exhibit strong fluorescence. However, since it is non-biodegradable, it is not only difficult to excrete and remove from the body after treatment, but may also cause non-specific cytotoxic action with tissues because such substances remain in vivo for a long time.

Korean Patent Publication No. 2008-0047070

It is an object of the present invention to provide a photosensitive agent for photodynamic therapy that can improve the poor solubility of fullerene and can be effectively removed from the living body.

Another object of the present invention is to provide a method for preparing a photosensitive agent for photodynamic therapy according to the present invention.

Still another object of the present invention is to provide a photodynamic therapy method using a photosensitive agent for photodynamic therapy according to the present invention.

In order to achieve the above object, the present invention provides a fullerene-glycol chitosan conjugate in which fullerene and water-soluble glycol chitosan are conjugated as a photosensitive agent suitable for photodynamic therapy by generating reactive oxygen species by light irradiation.

In one embodiment, the water soluble glycol chitosan may be one containing an amine residue.

In one embodiment, the fullerene-glycol chitosan conjugate may be a covalent bond of a double bond of fullerene and an amine group of a water-soluble glycol chitosan.

In one embodiment, the degree of substitution, which is the number of fullerene molecules relative to the repeating unit of the water-soluble glycol chitosan, may be 0.001 to 0.2.

In one embodiment, the fullerene-glycol chitosan conjugate may generate monooxygen or free radicals when irradiated with light in a wavelength range of 580 to 700 nm.

In one embodiment, the flaren is C ₆₀ flaren, the water-soluble glycol chitosan may have a feature that accepts water-soluble and insoluble materials, but is not limited thereto.

In one embodiment, the fullerene-glycol chitosan conjugate may have a size of 5 to 500 (nm).

In one embodiment, the fullerene-glycol chitosan conjugate can be selectively accumulated in cancer tissue.

The present invention also provides a nanocarrier comprising a fullerene-glycol chitosan conjugate in which fullerene and water-soluble glycol chitosan are conjugated.

In one embodiment, the nanocarrier may include high molecular weight proteins, peptides, nucleic acid molecules, sugars, lipids, compounds, or inorganic substances, or a high molecular weight compound or inorganic substance may be bound to a surface thereof.

In one embodiment, the compound or inorganic may be an anticancer agent.

The present invention also provides a photodynamic therapy method for animals other than humans using the fullerene-glycol chitosan conjugate.

In one embodiment, the method comprises administering the above-described photodynamic agent for photodynamic therapy to the subject, and irradiating light with a wavelength of 580 to 700 nm percutaneously.

In one embodiment, the light may be irradiated for 1 to 60 minutes at 1 to 200 mW / cm ² light intensity.

In one embodiment, the photodynamic therapy method of the present invention can be preferably used for the treatment of solid cancer.

The present invention also provides a method for preparing a fullerene-glycol chitosan conjugate as a photosensitizer for photodynamic therapy comprising the following steps.

In one embodiment, the method for preparing a fullerene-glycol chitosan conjugate comprises: 1) reacting a glycol chitosan having a fullerene and an amine residue with a triethylamine catalyst in a mixed solvent of anhydrous benzene and dimethylsulfoxide, and 2) After removal, the result is dialysis and vacuum drying.

Existing photosensitizers are difficult to remove from living organisms because they have properties that do not degrade in the body, thereby causing nonspecific cytotoxicity, whereas the fullerene-glycol chitosan conjugate, a photosensitive agent for photodynamic therapy according to the present invention, is biodegradable. It can be easily removed from the body as a sex. In addition, by adding water solubility to fullerene to improve the poor solubility, it exhibits the ability to kill cells through the generation of singlet oxygen with high optical properties, and increase the particle stability in the blood, thereby increasing the drug efficiency and maximizing the therapeutic effect. In addition, it is excellent in biocompatibility and high selectivity for the disease site can increase the local therapeutic effect.

Therefore, the photosensitizer according to the present invention penetrates well in the target cells and accumulates, thereby inducing cell death or necrosis by a photodynamic effect in the target cells. Photodynamic therapy is particularly effective in treating cancer, minimizing damage to surrounding normal cells, and having the advantage of being able to remove tumors without resorting to surgical procedures.

1 is a schematic diagram (a) of a process for preparing a fullerene-glycol chitosan conjugate (GC- g- C ₆₀ ) and a peak of ¹ H-NMR of GC- g- C ₆₀ prepared according to Example 1 of the present invention. .
2 is a graph showing the characteristics of the GC- g -C ₆₀ prepared in Example 1; (a) particle size distribution of GC- g- C ₆₀ (GC-F2, GC-F3, GC-F4, and GC-F5), (b) FE-SEM image of GC-F1 and GC-F5, ( c) glass C ₆₀ Or an optical image of a PBS solution (150 mM, pH 7.4, 1 mg / ml) in which GC-F5 is dispersed.
3 is a UV / visible spectral graph of GC-F5 or glass C60.
Figure 4 is a graph showing the monooxygen generation of GC- g -C ₆₀ prepared in Example 1.
5 is an in vitro anticancer effect of GC- g -C ₆₀ ; (a) CCK on KB cells treated with free C60 (0.1-5 μg / ml), GC-F1 (equivalent C60 0.1-5 μg / ml), and GC-F5 (equivalent C60 0.1-5 μg / ml) - is a graph phototoxicity measured using an 8 assay. (b) Fluorescence images of KB tumor cells treated with free C60 (1 μg / ml), GC-F1 (equivalent C60 1 μg / ml), and GC-F5 (equivalent C60 1 μg / ml). Annexin V-RITC staining (red) indicates apoptosis. (c) KB cells treated with free C60 (0.1-5 μg / ml), GC-F1 (equivalent C60 0.1-5 μg / ml), and GC-F5 (equivalent C60 0.1-5 μg / ml) without light irradiation It is a graph showing the survival rate (%) of the.
Figure 6 is obtained from nude mouse KB tumor cells administered with GC-F1 (equivalent C60 10 mg / kg body), GC-F5 (equivalent C60 10 mg / kg body), PBS alone (ion strength = 0.15, pH 7.4) In vivo non-invasive fluorescence image.

Unless defined otherwise, all technical terms used in the present invention have the following definitions and are consistent with the meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.

In the present specification, the term 'photodynamic therapy' refers to a photosensitive agent (photosensitizer) that reacts to light on the skin, and then emits light of a specific wavelength to selectively accumulate light only on diseased cells, thereby producing a therapeutic effect. Refers to the indicated therapy. Photodynamic therapy is used for the diagnosis and treatment of cancer, autologous bone marrow transplantation, antibiotics, AIDS treatment, skin transplant surgery or arthritis, and the like, but is not limited thereto.

The term 'photosensitizer' refers to the conversion of oxygen molecules (O ₂ ) to reactive oxygen species such as singlet oxygen ( ¹ O ₂ ), the creation of new radicals, or Means a substance that produces a chemical species.

The term "fullerene" refers to a molecule in which carbon atoms are alternately arranged in pentagons and hexagons. In the present invention, fullerene may be used alone or as a mixture of any carbon number such as C60, C70, C74, C76, C78, C80, C82, C88, C90, C96. In addition, nano-tube flaren, various high-dimensional flaren, etc., which are pure carbon materials, can be used, and metal-containing flaren is also possible.

The term "cancer cell" refers to a cell that abnormally grows, divides, or proliferates, and is interchangeable with "tumor cell." The term "cancer" refers to a complex disease resulting from uncontrolled division or proliferation of transformed cells and disordered growth, and in the present invention refers to solid cancer for photodynamic therapy. Solid cancer refers to cancer composed of all masses except blood cancer. Solid tumors include brain tumor, low-grade astrocytoma, high-grade astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, CNS lymphoma, Oligodendroglioma, Craniopharyngioma, Ependymoma, Brain stem tumor, Head & Neck Tumor, Larygeal cancer, Oropharyngeal cancer, Nasal / Nasal cavity / PNS tumor, Nasopharyngeal tumor, Salivary gland tumor, Hypopharyngeal cancer, Thyroid cancer, Oral cavity tumor, Chest Tumor , Small cell lung cancer, non-small cell lung cancer (NSCLC), thymic cancer (Thymoma), mediastinal tumor, esophageal cancer, breast cancer, male breast cancer ), Abdominal tumor (Abdomen-pelvis Tumor), Gastric cancer (Stomach cancer), Hepatoma, Gall bladder cancer, Biliary tract tumor, Pancreatic cancer, Small intestinal tumor, Large intestinal tumor, Anal cancer, Bladder cancer, Renal cell carcinoma, Prostatic cancer, Cervical cancer, Endometrial cancer, Ovarian cancer, Uterine sarcoma, Skin Cancer, and the like, but are not limited thereto.

The term 'about' refers to 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, by reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length. By amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by 4, 3, 2 or 1%.

Throughout this specification, the words "comprises" and "comprising", unless otherwise indicated in the context, include a given step or component, or group of steps or components, but any other step or component, or It is to be understood that it does not exclude a step or group of components.

Hereinafter, the present invention will be described in detail.

For photodynamic therapy Photosensitizer

One aspect of the present invention relates to a photosensitive agent for photodynamic therapy that generates reactive oxygen species by light irradiation.

Photosensitive agents for photodynamic therapy should preferably have the following conditions. First, the quantum yield to produce reactive oxygen species (ROS) must be high, second, the wavelength of absorbing light must be long, third, low toxicity without irradiation, and fourth, selectively bound to a specific target, It should not be combined with specific organizations.

The photosensitizer in the present invention that meets these requirements is a fullerene-glycol chitosan conjugate in which fullerene and water-soluble glycol chitosan are conjugated.

Fullerene absorbs near-infrared light and produces reactive oxygen species (ROS), which are suitable for photodynamic therapy. Since fullerenes are much smaller than conventional materials, they are suitable as photosensitizers. It has poorly soluble properties and limitations of absorption / distribution / metabolism / removal in vivo. That is, fullerene is insoluble in water, which makes it difficult to administer in vivo. Thus, in the present invention, as a biodegradable polymer, glycol chitosan, which promotes biocompatibility and particle stability, was conjugated to fullerene.

Glycol chitosan is one of the water-soluble chitosan derivatives, and exhibits water solubility at neutral pH upon introduction of a hydrophilic ethylene glycol group. Previous studies have reported that glycol chitosan exhibits noncytotoxicity, biocompatibility and stimulates the growth of chondrocytes at low concentrations (Carreno-Gomez. B, Duncan. R, Int. J. Pharm. 1997 , 148, 231; DK Knight, SN Shapka, BG Amsden, J. Biomed.Mater.Res. Part A. 2007, 83, 787). Chitosan is a basic polysaccharide prepared by N-deacetylation by treating chitin with a high concentration of alkali, and is known to be superior to other synthetic polymers in terms of cell adsorption capacity, biocompatibility, biodegradability and moldability. The glycol chitosan used in the present invention preferably has a molecular weight of 250,000 to 400,000, but is not limited thereto.

Since the glycol chitosan contains a lot of positrons in the polymer chain, the cancer tissue accumulation efficiency is very high, and the amine group of the glycol chitosan is covalently bonded with the double bond of the photosensitive agent fullerene to facilitate chemical modification.

The conjugate of the present invention enhances drug efficiency and increases therapeutic effect through solubilization of fullerene and enhancement of particle stability in blood. In addition, it exhibits high optical properties, biocompatibility, and bioremoval rate, and the targeting of cancer cells by glycol chitosan improves the possibility of selective personalized treatment.

Although the type of the fullerene monomer used in the present invention is not particularly limited, it is preferable to use C ₆₀ flaren from the advantages of low toxicity and ease of supply and handling. In the present invention, a plurality of fullerene-glycol chitosan conjugates may be bonded or aggregated to form aggregates.

The fullerene-glycol chitosan conjugates of the present invention may be from 5 to 500 nm in size, preferably up to 200 nm, since they can be effectively removed in vivo when within this diameter range and are suitable for clinical and diagnostic use. Generally, in order to inject particles into blood vessels, the size of the injected particles should be 200 nm or less in consideration of the size of blood vessels.

In the conjugate of the present invention, a plurality of fullerene molecules are conjugated to a polymer of water-soluble glycol chitosan, and the substitution degree, which is the number of fullerene molecules relative to the repeating unit of the water-soluble glycol chitosan, may be 0.001 to 0.2. When an excess of fullerene is bound to the repeating unit of the water-soluble glycol chitosan, the hydrophobicity of the fullerene may cause aggregation to increase the size of the conjugate. Therefore, there is a possibility to be manufactured in a size out of the blood vessel size.

The fullerene-glycol chitosan conjugate according to the present invention covalently bonded the double bond portion of the fullerene with the amine residue of glycol chitosan to improve the poor solubility and enhance the biocompatibility and in vivo removal rate, thereby increasing the therapeutic effect.

If desired, the target-oriented material may be further bound to the fullerene-glycol chitosan conjugate of the present invention. It is possible to target specific binding molecules directly to the fullerene or glycol chitosan moiety. That is, it means binding a tumor specific ligand to the conjugate in order to increase the selectivity or target of a specific cell group such as tumor cells to the membrane receptor. This targeting allows the uptake of the conjugates intracellularly through receptor-mediated endocytosis without cell destruction.

In the present invention, the target-oriented substance may include, for example, folic acid, antibodies, cobalamin, vitamin A, vitamin C, vitamin B12, and the like. Among these, especially folic acid can be used preferably. The corresponding target substance, folic acid receptor, is a useful target for tumor specific drug delivery. That is, folate receptors are expressed on tumor cells in ovarian cancer, colon, mammary gland, brain, colon, lung, kidney-cellular cancer, metastasis of epithelial tumor to brain, bone marrow blood cells in leukemia and neuroendocrine cancer. On the other hand, the expression of folic acid receptors in normal tissues is severely limited so that access to the folic acid receptors in normal breakfast rarely occurs. That is, cells of normal tissue express only a very small amount of folic acid receptors with a few exceptions, while in malignantly transformed cells the amount of receptors for folic acid increases on their surface. Due to the quantitative increase in folate receptors it is possible to effectively bind a significant amount of folic acid. In addition, folic acid shows high affinity with the folate receptor on the cell surface. The binding of folic acid and macromolecules can improve delivery to folate receptor-expressing cancer cells in vitro in almost all tested conditions.

On the other hand, the fullerene-glycol chitosan conjugate of the present invention exhibits target orientation, biocompatibility, and biodegradability, and includes a high molecular weight protein, a peptide, a nucleic acid molecule, a sugar, a lipid, a compound, or an inorganic compound, or a high molecular weight compound on its surface. Or inorganic matter may be combined and used as their nanocarriers. The compound or inorganic may be various anticancer agents such as, for example, doxorubicin, paclitaxel, docetaxel, camptothecin and cisplatin.

In addition, the fullerene-glycol chitosan conjugate of the present invention may be formulated by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. It may be prepared in a dosage form or incorporated into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The pharmaceutically acceptable carrier is conventionally used in the preparation, 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, and the like. Other suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

Photodynamics  Treatment method

Another aspect of the present invention relates to a photodynamic therapy method using the photosensitive agent for photodynamic therapy described above. The method of treatment of the present invention comprises the following steps.

1) administering the aforementioned fullerene-glycol chitosan conjugate to the subject; And

2) transdermally irradiating light with a wavelength of 580 to 700 nm;

The fullerene-glycol chitosan conjugate of the present invention in step 1) is preferably administered parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, intratumoral injection or intralesional injection.

Suitable dosages of the photodynamic therapy compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient. Typically, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the invention, the daily dose is 0.0001-1000 mg / kg.

In the step 2), the light wavelength is preferably in the range of 580 to 700 nm. If the wavelength is less than 580nm, the laser wavelength is short, so it does not penetrate deep into the tissue. Therefore, only the photodynamic treatment near the skin is possible. Not desirable

The light irradiation can activate the fullerene-glycol chitosan conjugate of the present invention, and should be such that the target cell can be destroyed or weakened, and preferably irradiated for 1 to 60 minutes at a light intensity of 1 to 200 mW / cm ^2. Can be. When irradiating an animal to be treated under the above conditions, reactive oxygen species are generated in the cancer cells injected with the fullerene multimer present in the body of the animal, and the cancer cells are killed or necrotic.

The photodynamic therapy method of the present invention can be preferably used for the treatment of solid cancer through photodynamic removal of cells from malignant cells, but is not limited to these diseases.

Method for preparing fullerene-glycol chitosan conjugate

Another aspect of the present invention relates to a method for preparing the fullerene-glycol chitosan conjugate described above. In one embodiment, the process of the present invention comprises the following steps.

1) reacting a glycol chitosan having a fullerene and an amine group residue in a mixed solvent of anhydrous benzene and dimethyl sulfoxide using a triethylamine catalyst; 2) dialysis and vacuum drying the result after removing the solvent;

The reaction temperature is about -20 to 100 ° C, preferably 0 to 50 ° C, more preferably room temperature to 37 ° C, and the reaction time is about 1 to 72 hours, preferably 10 to 50 hours.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

material

Fullerene (C ₆₀ ) is manufactured by NanoLab Inc. (Waltham, MA, USA), purchased from glycol chitosan (GC, Mw = 500 kDa), dimethyl sulfoxide (DMSO), triethylamine (TEA), anhydrous benzene, sodium borate (Na2B4O7), 9,10- Dimethylanthracene (DMA), NN'-dicyclohexylcarbodimid (DCC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Welgene, Inc. (Seoul, South Korea). Chlorine e6 (Ce6) was purchased from Frontier Scientific, Inc. (Logan, UT, USA). Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies Inc (Kumamoto, Japan). Annexin V-RITC fluorescence microscope kit was purchased from BD Pharmingen ™ (USA).

Example 1 Synthesis of Glycol Chitosan and Fullerene Conjugate (GC-g-C60)

C60 (0.35-71 mg) and GC (100 mg) were reacted for 2 days at room temperature in a co-solvent of anhydrous benzene (10 ml) / DMSO (10 ml) containing TEA (1 ml) (FIG. 1a). After the reaction, benzene was removed using a rotary evaporator and the resulting solution was placed in a pre-expanded dialysis membrane tube (Spectra / Por®MWCO 15K) and dialyzed on borate buffer (pH 7.4) to remove unreacted compound. The solution removed from the dialysis membrane tube was lyophilized for 2 days under reduced pressure. The obtained GC-g-C60 conjugate was subjected to 1H-NMR (DMSO- d6 with TMS) with a peak of δ 7.26 ppm [-C H in C60 part of GC- g -C60] and δ 3.50 ppm [-C H 2 in GC] Evaluated as the peak of (see FIG. 1B).

GC-g-C ₆₀ Evaluation method of characteristics

The particle size distribution of GC-gC ₆₀ (0.1 mg / ml) was measured at a fixed scattering angle of 90 ° at a wavelength of 633 nm with a Zetasizer 3000 instrument (Malvern Instruments, Westborough, MA, USA) equipped with a He-Ne laser beam. The morphology of GC-gC ₆₀ (10 mg / ml) was confirmed by FE-SEM (Hitachi s-4800, Tokyo, Japan). UV / visible spectra of GC-gC ₆₀ conjugates (0.05 mg / ml) and free C ₆₀ (0.05 mg / ml) in benzene / DMSO (50/50 vol.%) Cosolvent were monitored at 300800 nm.

The generation of monooxygen at GC-gC ₆₀ (0.1 mg / ml) was confirmed using 9,10-dimethylanthracene (DMA) (Park et al., 2011, 2012; Oh et al., 2012). DMA (20 mmol) was mixed in GC- g- C60 (0.1 mg / ml) and PBS (150 mM, pH 7.4). The solution was irradiated with a 670 nm laser source for 10 minutes at 100 mW / cm ² light intensity. DMA fluorescence intensity (measured at λex 360 nm and λem 380-550 nm using a Shimadzu RF-5301PC spectrofluorometer) reached the plateau after 1 hour, and the fluorescence intensity (Fs) of each sample was subtracted from the total DMA fluorescence intensity. DMA fluorescence intensities (Ff-Fs) were plotted (Ff, meaning no monooxygen, without GC- g -C60 or C60) (Park et al., 2011, 2012; Oh et al., 2012)

In vitro Phototoxicity ( Phototoxicity ) Assessment Methods

To assess phototoxicity for light irradiation of C ₆₀ multimers, human nasopharyngeal epidermal carcinoma KB cells were cultured in a humidified standard incubator in RPMI-1640 medium with 2 mM L-glutamine, 1% penicillin-streptomycin, and 10% FBS. Was maintained at 37 ° C. in a 5% CO ₂ atmosphere. Or tested on RAW264.7 cells. Cells grown in a single layer prior to testing were obtained via trypsinization with 0.25% (w / v) trypsin / 0.03% (w / v) EDTA solution. in vitro KB cells suspended in RPMI-1640 medium were planted in well plates and incubated for 24 hours prior to cell testing (Park et al., 2011, 2012; Oh et al., 2012).

Luminescent GC- g- C60 phototoxicity was tested in KB tumor cells. GC- g- C60 or free C60 dispersed in RPMI 1640 medium was injected into cells transplanted into 96-well plates. The cells were incubated for 4 hours in each sample and then washed three times with PBS (pH 7.4). The cells were irradiated for 10 minutes using a 670 nm laser source at an intensity of 100 mW / cm ² , and further incubated for an additional 6 hours. Cell viability was measured using Cell Counting Kit-8 (CCK-8 assay). Cell viability tests of non-luminescent KB cells treated with GC- g- C60 were performed to assess GC- g- C60 intact toxicity. Cells were incubated with GC- g -C60 for 24 hours and then evaluated via CCK-8 analysis.

Tumor cell apoptosis was also visualized using the Annexin V-RITC fluorescence microscope kit (Park et al., 2011, 2012; Oh et al., 2012). Cells were incubated with each sample (equivalent C60 5 mg / ml) for 4 hours and then washed three times with PBS (pH 7.4). The tumor cells were irradiated with a 670 nm laser source at a intensity of 100 mW / cm ² for 10 minutes, washed twice with PBS and stained with 1 ml of Annexin V-RITC (10 wt.%) For 15 minutes at room temperature. . After staining, tumor cells were washed twice with PBS and fixed using 3.7% formaldehyde in PBS. Cover slip was mounted on microscope slides with a drop of anti-fade mounting medium (5% N-propyl gallate, 47.5% glycerol and 47.5% Tris-HCl, pH 8.4) to reduce fluorescence photobleaching. . Cell death was visualized using fluorescence microscopy (E-SCOPE 1500F at λex 570 nm and λem 595 nm) (Park et al., 2011, 2012; Oh et al., 2012).

Animal model

In vivo The study was performed on 46 week old female BALB / c nude mice (Institute of Medical Science, Tokyo, Japan). Mice were treated according to the guidelines of the approved protocol of the Institutional Animal Care and Use Committee (ICAC).

In vivo Neon Imaging

Prior to animal experiments, fluorescent dyes (Ce6) (1 mg) in DMSO (1 ml) preactivated with DCC (2 mg) and NHS (2 mg) were treated with GC- g- C60 (10 ml) in DMSO (10 ml) at room temperature. 100 mg) and stirred for 4 hours. The solution was filtered to remove dicyclohexylurea (DCU), dialyzed with a dialysis membrane bag (Spectra / Por®MWCO 1K) and lyophilized.

In vivo KB tumor cells were induced by subcutaneous injection of 1 × 10 ⁴ cells suspended in PBS pH 7.4 (ion strength: 0.15) medium in female nude mice for animal experiments. When tumor volume reached about 30 mm ³ , tumor-grafted nude with Ce6-tagged GC- g- C60 conjugate (equivalent C60 10 mg / kg body) or PBS solution alone (ionic strength = 0.15, pH 7.4) Mice were injected intravenously through the tail canal. Using a 12-bit CCD camera (Image Station 4000 MM; Kodak, Rochester, NY, USA) equipped with a special C-mount lens and long wavelength emission filter (600700 nm; Omega Optical, Brattleboro, VT, USA) Live fluorescence images were captured (Park et al., 2011, 164 2012; Oh et al., 2012; Lee et al., 2011a, 2011b).

Statistical analysis

All results were analyzed for diversity at the significance level of p <0.01 or p <0.05 by Student's t-test, and 14 statistical software distributed by MINITAB was used (Minitab, State College, PA, USA).

Experimental Example 1 Characteristic Evaluation Results of GC-g-C60

GC-g-C60 was prepared through a chemical reaction between the free amine group of GC and the C═C double bond of C ₆₀ . The degree of substitution (defined as the number of C60 molecules relative to the repeat units of DS and GC) was 5x10 ^-4 to 0.16 (see Table 1), δ 7.26 ppm [-C H in C60 part of GC- g- C60] to δ 1H-NMR (DMSO- d6 at 3.50 ppm [-C H 2 in GC] with TMS) peak.

The conjugation of C60 is intended to give the polysaccharide conjugate abundant lipophilic for photodynamic activity and self-tissue structure. GC-g-C60 self-organizes in PBS (150 mM, pH 7.4) by increasing C60 molecules conjugated to GC from 5 × 10 ⁻⁴ to 0.16 molecules relative to one GC repeat unit.

As shown in Figure 2a, the average particle diameter of the GC-g-C60 nanoparticles (using GC-F2, GC-F3, GC-F4, GC-F5 in Table 1) was about 10 ~ 23nm. These GC-g-C60 nanoparticles were stable for over a month under optimized conditions without any precipitation. Beyond this period, the particle size and transparency of GC-g-C60 nanoparticles did not change (data not shown).

Morphology images obtained with FE-SEM were found to be nearly spherical GC-g-C60 nanoparticles (GC-F5) at pH 7.4 (FIG. 2B). GC-F2, GC-F3, GC-F4, and GC-F5 nanoparticles showed no difference in morphology (data not shown). However, in the case of GC- g- C60 (GC-F1 in Table 1) having 5x10 ^-4 C60 molecules compared to the GC repeat unit, it was completely dissolved in water, showing no image on the FE-SEM image (FIG. 2B).

In addition, FIG. 2C shows the excellent colloidal stability of the GC-F5 nanoparticles, since the hydrodynamic GC cloud surrounds the C60 molecules, while the free C60 molecules aggregate quickly within 5 minutes and due to the low density PBS Suspended in solution.

Experimental Example 2 Photosensitive GC-g-C60

The UV / visible spectrum of the GC-g-C60 conjugate shows a clear absorption band at 300-800 nm due to the presence of GC and C ₆₀ (see FIG. 3). GC-gC ₆₀ conjugates exhibit strong optical absorption at 328 nm, which is different from the strong optical absorption at 408 nm of free C ₆₀ molecules. Interestingly, the improved light absorption of GC-g-C60 in the near-infrared (NIR) region, 670 nm, is believed to provide increased excited energy to the oxygen molecule, which may lead to active oxygen species (ROS) such as monooxygen. Generate. (Anton et al., 1996; Bosi et al., 2003; Wei et al., 2010; Zhu et al., 2008; Hahn et al., 2007; Nakamura and Isobe, 2003).

4 shows that monooxygen was generated from GC-g-C60 conjugates during light irradiation. For testing, 9,10-dimethylanthracene (DMA) was used as a chemical collector of extremely fast monooxygen (Park et al., 2011, 2012; Oh et al., 2012). Fluorescent DMA selectively reacts with monooxygen to form endoperoxide (Gomes et al., 2005), thereby reducing the phosphor of the DMA. In this experiment, we applied a 670 nm laser source at a light intensity of 100 mW / cm ² u for 10 minutes for GC- g -C60 conjugates (equivalent C60 0.1 mg / ml) or free C60 molecules (0.1 mg / ml). It was investigated using. The change in DMA phosphor intensity in the GC- g- C60 conjugate or free C ₆₀ molecule was monitored to confirm the occurrence of monooxygen. The change in DMA phosphor intensity (Ff-Fs) means that substantially more singlet oxygen is generated (Park et al., 2011, 2012; Oh et al., 2012). GC- g- C60 conjugates (GC-F1, GC-F5) stabilized in PBS (150 mM, pH 7.4) generated more monooxygens than C ₆₀ molecules aggregated in PBS.

Experimental Example 3 Antitumor Activity of GC-g- C60

5 is a GC-g-C60 in the In vitro anti-cancer treatment. The test was performed with human cervical carcinoma KB tumor cells for photodynamic cell removal of GC-g-C60 after light irradiation. GC-g-C60 conjugate or free C60 is dispersed in in RPMI-1640 medium and injected into cells transplanted into 96-well plates. Cells are incubated for 4 hours each in each sample and then washed three times with PBS pH 7.4. Cells were irradiated for 10 minutes using a 670 nm laser source at a light intensity of 100 mW / cm 2 u and further incubated for 6 hours. After all the processes, GC-F5 or GC-F1 led to relatively high levels of KB cell death (see FIG. 5A), which caused photodynamic damage to cells due to the generation of immense monooxygen from GC-F5 or GC-F1. This reflects a significant improvement. Annexin V-RITC staining (Park et al., 2011, 2012; Lee et al., 2011b) shows large apoptotic cell populations induced by GC-F1 or GC-F5, indicating strong red fluorescence in KB tumor cells (See FIG. 5B). However, free C60 produces less phototoxicity against KB tumor cells. It should be noted that neither the GC- g -C60 conjugate nor the free C60 show any cytotoxicity before light irradiation (see Figure 5c). In order to further evaluate the potential of GC- g -C60 as a tumor sensitizer, KB tumors extracted from BALB / c nu / nu female mice in vivo Efficacy was evaluated.

Fluorescent dye (Chlorin e6: Ce6) is bound to GC- g -C60 in vivo Fluorescence images were obtained (Park et al., 2011, 2012; Oh et al., 2012; Lee et al., 2011a, 2011b). Live fluorescent images of nude mice were captured using a 12-bit CCD camera (Image Station 4000 MM; Kodak) equipped with a special lens and a long wavelength emission filter (600 700 nm) (Park et al., 2011, 164 2012; Oh et al., 2012; Lee et al., 2011a, 2011b).

Ce6 the GC- g -C60 conjugate (GC-F1-F5 or GC) or of PBS alone via the tail pipe of a nude mouse by intravenous injection support 4 hours after KB tumors induced in nude mice In labeling vivo Fluorescence images were obtained. High-resonance fluorescence images of 240 volume (˜30 mm 3) small tumor sites were obtained from nude mice administered GC-F5 (FIG. 6B), which were treated with a water soluble GC-F1 (see FIG. 6A) administration group or PBS solution. Compared to rapid clearance in one baseline condition (FIG. 6C). The high accumulation rate of GC-F5 at this tumor site can be explained by GC-F5 extravasation in tumor vessels due to enhanced permeability and retention (EPR) (Maeda and Matsumura, 2011). These data indicate that the GC- g- C60 conjugate of the present invention is in It can be used for the selective delivery of anticancer drugs to tumor cells in vivo .

In summation of the above results, GC- g- C ₆₀ of the present invention The conjugate was confirmed to increase the solubility of C ₆₀ , exhibit the ability to remove photodynamic tumor cells, and can effectively deliver C ₆₀ into the body. GC- g -C ₆₀ This effect of the conjugate is expected to significantly increase the utility of C ₆₀ in photodynamic chemotherapy.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (15)

A photosensitive agent for photodynamic therapy that generates reactive oxygen species by light irradiation, comprising a fullerene-glycol chitosan conjugate conjugated with fullerene and a water-soluble glycol chitosan. The method of claim 1,
The water-soluble glycol chitosan is characterized in that it comprises an amine residue, photosensitive agent for photodynamic therapy. 3. The method of claim 2,
The fullerene-glycol chitosan conjugate is a photosensitive agent for photodynamic therapy, characterized in that a double bond of fullerene and an amine group of water-soluble glycol chitosan are covalently bonded. The method of claim 1,
The degree of substitution, which is the number of fullerene molecules relative to the repeating unit of the water-soluble glycol chitosan, is 0.001 to 0.2, a photosensitive agent for photodynamic therapy. The method of claim 1,
The fullerene-glycol chitosan conjugate is characterized in that to generate a single oxygen or free radicals when irradiated with light in the wavelength range of 580 to 700 nm, photodynamic therapy for photodynamic therapy. The method of claim 1,
The fullerene is a C ₆₀ fullerene, the water-soluble glycol chitosan is characterized in that the water-soluble and insoluble materials are soluble, photodynamic therapy photosensitive agent. The method of claim 1,
The fullerene-glycol chitosan conjugate is characterized in that the size of 5 to 500 (nm), photodynamic therapy photosensitive agent. The method of claim 1,
The fullerene-glycol chitosan conjugate is characterized in that the accumulation selectively in cancer tissue, photodynamic therapy for photodynamic therapy. A nanocarrier comprising a fullerene-glycol chitosan conjugate to which fullerene and water-soluble glycol chitosan are conjugated. 10. The method of claim 9,
The nanocarrier comprises a high molecular weight protein, peptide, nucleic acid molecules, sugars, lipids, compounds or inorganic substances, or characterized in that the high molecular weight compound or inorganic material is bonded to the surface. 10. The method of claim 9,
The compound or the inorganic substance, characterized in that the anticancer agent, nano-carrier. Administering to the subject a photosensitive agent for photodynamic therapy according to any one of claims 1 to 8; And
Transdermally irradiating light with a wavelength of 580 to 700 nm;
Containing, photodynamic therapy method for animals except humans. The method of claim 12,
The light is irradiated for 1 to 60 minutes at 1 to 200 mW / cm ² light intensity. The method of claim 12,
The photodynamic therapy method is characterized in that used for the treatment of solid cancer. 1) reacting a glycol chitosan having a fullerene and an amine residue in a mixed solvent of anhydrous benzene and dimethyl sulfoxide using a triethylamine catalyst; And
2) dialysis and vacuum drying the result after removing the solvent;
A method for producing a photosensitive agent for treating photodynamics according to claim 3, comprising:

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