KR101690179B1 - Water-soluble chitosan nanoparticles conjugated with amifostine - Google Patents

Abstract

The present invention relates to a method for preparing radio-responsive chitosan nanoparticles with which amifostine as a radio-protectant is covalently bound and crosslinked by 3,3-diselenodipropionic acid (DSePA). Particularly, the method for preparing radio-responsive chitosan nanoparticles comprises the steps of: carrying out covalent bonding between DSePA and amifostine; carrying out covalent bonding between the resultant covalently bound product with water-soluble chitosan to obtain a chitosan derivative; allowing methoxy poly(ethylene glycol) to be bound with the chitosan derivative; and crosslinking the chitosan derivative with DSePA. The radio-responsive chitosan nanoparticles to which amifostine is bound according to the method can protect the human body from exposure to radiation, and discharges amifostine only when being irradiated with radiation, thereby minimizing side effects to the human body.

Description

Water-soluble chitosan nanoparticles conjugated with amifostine is a cross-linked water-soluble chitosan nanoparticle.

The present invention relates to water-soluble chitosan nanoparticles covalently bonded with amifostine, a method of supporting a radioprotective agent or a drug using the nanoparticles, and a nanoparticle for radiation protection using the nanoparticles .

The human body is exposed to various types of radiation such as artificial radiation, including natural radioactivity (1 to 3 mSv / year), but the research to defend it is still in the early stage, and the human body by radiation exposure through medically used radiation The development of a protective agent to reduce and inhibit harmfulness and side effects is active worldwide, but the development of domestic radioprotection agents has not been developed yet.

In recent years, there has been an increase in interest in radiation protection products due to the increase in the use of industrial radiation, including medical diagnosis and treatment, and nondestructive testing, as well as radiation exposure due to the accident in Fukushima nuclear power plant in Japan. In addition, frequent accidents in domestic nuclear power plants have increased the disbelief of the safety of nuclear power generation by ordinary citizens, resulting in the closure of Kori Nuclear Power Plant and fear of radiation exposure. In particular, the number of domestic radiation workers is more than 100,000 in various fields such as medical, education, nuclear power plant, manufacturing and nondestructive inspection, and the annual average is increasing to 6% (ICRP Publication 115, 2012) It is used not only for diagnosis but also for diagnosis and treatment of cancer. T-99m, P-32, and Sr-89, which are used for radiological diagnosis / treatment (X-ray, CT, PET-CT etc.) , Y-90) and the like, which causes human injury.

Radioprotective agents for protecting the human body from such radioactivity can be roughly classified into radioprotectors, radiomitigators, treatments, and radiosensitizers. Radioprotectors are drugs that prevent normal cell damage by taking it before radiation exposure. Typical drugs include WR-1065 (amifostine) and WR-2721 (phosphorilated amifostine) (Mucositis, esophagitis, proctitis, acute chronic dry mouth, dysphagia, pneumonia, cystitis, dermatitis, etc.) without radical scavenging of the radicals produced by exposure and protection of cancer cells. (81%) and complete response rates (81%) (International Journal of Radiation Oncology · Biology · Physics, 2006). In addition, studies on synthetic agents such as dipyridamole, adenosine monophosphate, tetracychlorodecaoxide, eriuron, and deoxyspergualin have been conducted. Radiomitigators are substances that mitigate the damage of normal cells after radiation exposure, such as Palifermin (Kepivance, marketed by Biovitrum, DNA repair), Captopril (ACE inhibitor, radiation-induced nephropathy mitigation) . Treatments are drugs that improve the damage of normal cells and tissues exposed to radiation, such as pentoxyfilline and vitamin E (antifibrotic treatment). Radiosensitizers are substances that reduce the radiation dose during radiotherapy. They include 5-Nitroimidazole (Nimorazole), which is used for radioactive treatment of small cell carcinoma, (Tirapazamine (SR-4233)), diagnostic reagents containing radioactive nuclides, and chelating drugs (Radiogardase ^ⓡ -Cs, Ca-DTPA, etc.) that help release internal exposure agents.

Development and industrialization of radiopharmaceuticals are progressing actively in developed countries. Although they mainly focus on pure single chemical components such as amifostine, tempol, etc., (Eg, Palifermin, 5,000 / one-time treatment), while its use is not generalized due to preemption of the protective agent market . Amifostine (Ethyol ^ⓡ ), which is one of the most widely used radioprotective agents, has been proven to have a radiation protection effect and has been used in clinical practice. However, it has been reported that hypotension, polymorphic erythema, hypersensitivity, (Toxic epidermal necrolysis), Stevens-Johnson syndrome, and so on. Recently, biomedical efficacy screening of biological materials that can minimize toxicity or side effects has been required. Especially, development of radiation biohazardous materials has been required. There has been growing interest in industrialization through efficient development of radiation protection materials. Researches on the effects of radiation on living organisms and radiation protection materials in KAERI, nuclear medicine hospitals, and some universities have been limited. HemoHIM, a herbal medicine extract, has been introduced as a physiological activity promoting substance such as immunity enhancement / hematopoietic cell proliferation. The extract has been recognized as a bioactive defense health functional food for radiation ("Development of Functional Foods Using Radiation Protection" - Report, KAERI). However, the extract is limited to simple extracts of herbal medicines, and thus has a limited protection efficacy. Especially, it is difficult to perform hydroxy radical treatment which is essential for radiation protection. Therefore, it is urgently required to develop a new protective agent for protection of normal cells prior to exposure to radiation, high protective properties (DRF) and damaged DNA, and cultivation of advanced core functional foods or pharmaceuticals industry through commercialization.

Chitosan is a β-1,4-linked pyranose unit of glucosamine. The chitosan is composed of a polysaccharide group with more than 5,000 glucosamine residues and a molecular weight of more than 1 million and a polyvalent cation Is a biomolecular material that can be extracted from fisheries including crustaceans such as crab shells and squid and squid. Its molecular structure is similar to that of cellulose, which is a type of polysaccharide, and has excellent biocompatibility. It is not applied and is applied to the pharmaceutical industry. In addition, chitosan has been applied as an important biochemical industry and biomedical materials in the 21st century after recently being certified as a food by the US FDA. In particular, chitosan having a specific molecular weight range of 20,000 to 100,000 has a strong physiological activity And it can be expected to be applied to the fields of health food, food and beverage, cosmetics, healthcare, and pharmaceuticals.

Nanodrug, nanoparticle, and nanomedicine technologies can be easily injected or administered into human body by making materials with a diameter of several hundred nanometers (nm) using polymers, nanoparticles, etc., Active research is underway in the world because of its ability to selectively deliver drugs to specific cells and organs. In particular, it is possible to reduce accumulation of toxic or adverse drug substances in the liver or spleen such as anticancer drugs and radioprotective agents, to maintain the drug concentration in the blood for a long time, and to maintain physiochemical properties such as irradiation, magnetic field, pH, It can be manufactured to respond to stimulation, and it is attracting attention as a next-generation anticancer agent or a protective agent. Reflecting these recent research and development trends, it is expected that a new radioprotective agent with a level of physiologically active function that is stabilized by using a new nanopharmaceutical technology can be put to practical use, a technique capable of protecting normal cells, There is a need to develop nanoparticle technology for controlled release of radioprotective agents.

On the other hand, as prior art for chitosan nanoparticles bound with amphostin, International Patent Application No. WO2012 / 163290A1 discloses an anti-free radical pharmaceutical polymer having an aminothiol moiety, ANKITA A. NAGVEKAR has described the development and characterization of an efficient nanopar- tivate delivery system for aminostatin, a radioprotective drug (ANKITA A. NAGVEKAR, Creighton University, 2008), Sarala Pamujula et al. (Sarala Pamujula et al., JPP 2008, 60: 283-289) discloses the preparation of PGLA and chitosan-fused microcapsules. However, it has been reported that the nanoparticles are selectively collapsed by the irradiation of the present invention, There is no known hitherto known water-soluble chitosan nanoparticles covalently bonded to postin and DSePA.

The inventors of the present invention have developed a water-soluble chitosan nanoparticle covalently bound with aminosthtin and DSePA, and found that the nanoparticles can be separated from the nanoparticles Aminostatin is released, thereby completing the present invention.

It is an object of the present invention to provide a method for preparing water-soluble chitosan nanoparticles covalently bonded with amifostine, wherein release of the radioprotective agent is controlled in accordance with exposure to radiation, and a nano drug for radiation protection using the same .

In order to achieve the above object, the present invention provides a chitosan derivative comprising a chitosan derivative covalently bonded to a water-soluble chitosan and a conjugate of 3,3'-diselenodipropionic acid (DSePA) and amifostine and methoxypolyethylene glycol and amphostin crosslinked chitosan nanoparticles comprising a chitosan copolymer grafted with methoxy poly (ethylene glycol) (MPEG).

In addition,

1) synthesizing a covalent bond between 3,3'-diselenodipropionic acid (DSePA) and amifostine;

2) covalently linking the covalently bound DSePA of step 1) with water-soluble chitosan to synthesize a chitosan derivative;

3) synthesizing chitosan-methoxypolyethylene glycol graft copolymer in which amphostin is covalently bonded by covalently bonding the chitosan derivative of step 2) to methoxy poly (ethylene glycol) step; And

4) cross-linking the graft compatibilizer of step 3) with DSePA. The present invention also provides a method for producing aminosthin-crosslinked chitosan nanoparticles.

In addition, the present invention provides a nano drug for radioprotectants, wherein said amphostin comprises crosslinked chitosan nanoparticles.

The water-soluble chitosan nanoparticles covalently bonded to the amifostine and 3,3'-diselenodipropionic acid (DSePA) of the present invention can be selectively incorporated into the nanoparticles Can be used to protect the human body from exposure to radiation by collapsing and controlling the release of the drug.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a step of covalently bonding 3,3'-diselenodipropionic acid (DSePA) and amifostine.
Fig. 2 is a schematic diagram showing a step of covalently bonding a DSePA-aminostatin compound to water-soluble chitosan.
3 is a diagram schematically showing the step of graft copolymerization of methoxy poly (ethylene glycol) to a chitosan derivative.
Fig. 4 is a schematic diagram showing a step of cross-linking a chitosan derivative graft-copolymerized with methoxypolyethylene glycol with DSePA. Fig.
Figure 5 shows the particle size change of chitosan nanoparticles cross-linked with DSePA prepared according to one embodiment of the present invention and amipostin covalently linked by DSePA:
(A): a photograph of the morphology of the nanoparticles before irradiation with a transmission electron microscope;
(B): a photograph of the morphology of nanoparticles observed by transmission electron microscope after irradiation (10 Gy);
(C): Changes in size of nanoparticles according to irradiation dose.
6 is a graph showing the effect of irradiation on the release rate of aminostatin from the chitosan nanoparticles of the present invention.
7 shows the cell viability of chitosan nanoparticles of the present invention after irradiation of hepatocytes with irradiation:
(A): Changes in survival rate of hepatocytes after irradiation;
(B): Changes in apoptosis and necrosis of hepatocytes after irradiation.
8 is a graph showing the survival rate of rats after intravenous injection of chitosan nanoparticles of the present invention into a rat (BALb / C) and irradiated with them;
(A): Survival rate of rats;
(B): Changes in normal hepatocytes and abnormal hepatocytes in liver tissue photographs and liver tissues;
(C): Changes in protein expression of Bax and Bcl-2 in liver tissue.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing a chitosan derivative having methoxypolyethylene glycol (methoxypolyethylene glycol) by adding a chitosan derivative covalently bonded to a water-soluble chitosan with a combination of 3,3'-diselenodipropionic acid (DSePA) and amifostine, ; ≪ / RTI > MPEG) grafted chitosan copolymers.

The DsePA and amipostin are preferably covalently bonded and cross-liked to the chitosan copolymer.

In addition,

1) synthesizing a covalent bond between 3,3'-diselenodipropionic acid (DSePA) and amifostine;

2) covalently linking the covalently bound DSePA of step 1) with water-soluble chitosan to synthesize a chitosan derivative;

3) synthesizing chitosan-methoxypolyethylene glycol graft copolymer in which amphostin is covalently bonded by covalently bonding the chitosan derivative of step 2) to methoxy poly (ethylene glycol) step; And

4) cross-linking the graft compatibilizer of step 3) with DSePA. The present invention also provides a method for producing aminosthin-crosslinked chitosan nanoparticles.

In the above method, the covalent conjugate of DSePA and amopostin in step 1) is preferably used by dissolving DSePA in a solvent in an amount of 1 to 15% by weight, and is preferably dissolved in DMSO (dimethyl sulfoxide) in an amount of 10% by weight It is preferable to add 1 to 5% by weight of aminostatin, more preferably 3% by weight.

The chitosan derivative in step 2) is preferably dissolved in a solvent in an amount of 10 to 25% by weight, more preferably in an amount of 20% by weight in DMSO, more preferably in a solution in which water-soluble chitosan is dissolved in water To 15% by weight and added to the compound solution, more preferably 10% by weight to the compound solution.

The covalent bond in step 3) is preferably dissolved in a solvent such that the amount of methoxypolyethylene glycol is 3 to 7% by weight, more preferably 5% by weight in DMSO. Of the chitosan derivative is dissolved and dissolved in DMSO or water to 3 to 7% by weight, and it is more preferable to dissolve the chitosan derivative in 5% by weight of DMSO.

The chitosan nanoparticles of step 4) may be prepared by dissolving DSePA in a solvent in an amount of 1 to 5% by weight, preferably 3% by weight, in DMSO, It is preferable that the covalently bonded chitosan-methoxy polyethylene glycol graft copolymer is dissolved in DMSO or water in an amount of 1 to 5 wt%, more preferably 3 wt% in DMSO.

The solvent may be selected from the group consisting of water (H ₂ O), ethanol, dimethyl acetamide (DMAc), N, N-dimethylformamide (DMF), N-methyl-2-pyrrolidinone NMP), dimethyl sulfoxide (DMSO), acetone, tetra-hydrofuran (THF), ethylene carbonate (EC), diethyl carbonate (DEC) (DMC), ethyl methyl carbonate (EMC), propylene carbonate (PC), acetic acid, formic acid, chloroform, dichloromethane ) And DMP, but the present invention is not limited thereto.

In a specific embodiment of the present invention, the present inventors have found that a method for producing a chitosan derivative, comprising covalently bonding DSePA and amphostin, covalently bonding the covalent bond and water-soluble chitosan to synthesize a chitosan derivative, , And a step of cross-linking the chitosan derivative with DSePA was used to prepare amphostin-crosslinked chitosan nanoparticles (see FIGS. 1 to 4).

The present invention also provides a nano drug for radioprotectants, wherein said amphostin comprises crosslinked chitosan nanoparticles.

It is preferable that the nano drug dissociates the cross-linking between DSePA and chitosan by radiation exposure to release amopostin.

The radiation may be, but is not limited to, electromagnetic waves or particles having the ability to directly or indirectly ionize, including, but not limited to, alpha rays, beta rays, gamma rays, protons, neutrons, heavy atomic nuclei, X rays, heat rays, If it is a line, it can be all.

Such exposures may include, but are not limited to, those involving experiments, accidents, industrial sites, and medical activities, including chemotherapy, and may be all situations that can be directly or indirectly affected by radiation.

The radiation protection solvent may be selected from the group consisting of Amifostine, Ebselen, phosphorilated amifostine, dipyridamole, adenosine monophosphate, tetracychlorodecaoxide, ), Vitamin C (vitamin C), cortisol (Captopril), pentoxyfilline, vitamin E, dexyspergualin, dexyspergualin, , Vitamin A (vitamin A), 5-nitroimidazole, tirapazamine, Prussian blue, pentetate calcium trisodium, scleroglucan ( scleroglucan, beta-glucan, diselenium, selenocystamine, selenocystine, diselenodipropionic acid, curcumin, epiglass, Catechin epigallate (Ep igallocatechin gallate, Caffeic acid phenethyl ester, and derivatives thereof. However, the present invention is not limited thereto.

In a specific example of the present invention, it was confirmed that when the chitosan nanoparticles prepared by the above method were irradiated with radiation, the morphology of the nanoparticles was destroyed. Further, as a result of measuring the size of the nanoparticles according to the irradiation dose, When irradiated with radiation, it was confirmed that the crosslinking was completely broken and the nanoparticles were completely destroyed by the radiation dose of 20 Gy (see FIG. 5). Further, as a result of confirming the change in the rate of release of amipostin from the chitosan nanoparticles by irradiation, it was confirmed that the release rate of the drug was accelerated by breaking of the internal bonds of the nanoparticles from about 2 hours after irradiation 6).

In addition, the cytotoxicity of the chitosan nanoparticles was confirmed by using hepatocyte. As a result, it was confirmed that the viability of the cells against the irradiation was higher, and the apoptosis and necrosis of the cells were also significantly decreased (See FIG. 7).

As a result of confirming the radiation protection effect using a rat animal model, the chitosan nanoparticles showed a survival rate of 60% or more when injected. After immunohistochemical staining with the liver tissue of the rat, , The group administered with the chitosan nanoparticles was found to be in good condition, and the liver parenchymal cells were reduced to 9% or less after irradiation. Furthermore, as a result of western blot analysis, it was confirmed that the expression ratio of BAX / Bcl-2, a protein showing apoptosis, was the lowest when chitosan nanoparticles were injected (see Fig. 8 (c)).

Therefore, it is confirmed that the amphostin of the present invention has an effect of releasing a drug by specifically reacting with cross-linked chitosan nanoparticles to radiation, and confirming that the radiation protection effect is excellent, thereby protecting the human body from radiation exposure However, it can be usefully used as a radioprotective nano drug which can release only amipostin when radiation is irradiated to minimize adverse effects on the human body.

The aminosthin-crosslinked chitosan nanoparticles of the present invention may further contain at least one active ingredient exhibiting the same or similar functions, and may further include a pharmaceutically acceptable additive, Acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, sugar, gum arabic, pregelatinized starch, corn starch, powdered cellulose, Propyl cellulose, starch glycolate, starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc.

That is, the nano drug of the present invention can be administered in various formulations of oral or parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, May be formulated using excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are commonly used simple diluents . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The nano drug of the present invention can be administered orally or non-orally in accordance with the desired method, and can be administered orally or parenterally in the case of non-oral administration by intraperitoneal or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, It is desirable to select the method.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

< Example  1> Aminostatin  Preparation of covalently bound chitosan nanoparticles

<1-1> Di-selenodipropionic  mountain( DSePA )and Aminostatin  Covariance

The covalent bonding of aminostatin with 3,3'-diselenodipropionic acid (DSePA) was carried out by the following method and represented by the formula in FIG. Specifically, DSePA was dissolved in DMSO (dimethyl sulfoxide) in an amount of 3% by weight, dimethyaminopropyl ethyl carbodiimide (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide) Hydroxysuccinimide was added to the reaction mixture to activate the carboxyl groups at both ends of the DSePA with hydroxysuccinimide and then 2% by weight of amphostin was added thereto. The mixture was allowed to react for 24 hours to form a diselenodipropionic acid -Aminopostin covalent bond (Figure 1). As a result of confirming the binding of the conjugate using proton nuclear magnetic resonance spectroscopy, the ethylene specific peak of amphistin appeared at 2.6 to 3.0 ppm, and the peak at 1.6 ppm and 2.2 ppm The characteristic peak of DSePA was confirmed, confirming that DSePA and amphostin were combined.

<1-2> DSePA - Aminostatin  Synthesis of Chitosan Derivatives Covalently Coupled

Synthesis of chitosan derivatives covalently bonded to DSePA-aminosthtin conjugate was carried out by the following method and represented by the formula in Fig. Specifically, the DSePA-aminostatin compound was dissolved in 20% by weight of DMSO, the water-soluble chitosan was dissolved in 10% by weight of water, added to the compound solution, and stirred with a magnetic stirrer for 24 hours at room temperature. Thereafter, the reaction product was placed in a dialysis membrane and dialyzed against water for 48 hours or longer to remove the organic solvent, followed by lyophilization to obtain amphostin-covalently bonded chitosan derivatives (FIG. 2). In order to confirm the binding of the derivative, the ethylene characteristic peak of amphostin was confirmed at 2.5 to 3.0 ppm and the characteristic peak of DSePA at 1.6 ppm and 2.2 ppm was confirmed by nuclear magnetic resonance spectroscopy. The characteristic peaks of chitosan were confirmed at 1.5 ppm and 3 ~ 5 ppm, and it was confirmed that the chitosan was bound.

<1-3> Methoxy Polyethylene glycol Graft Copolymerized  Production of chitosan

The chitosan obtained by graft-copolymerizing methoxy polyethelene glycol was prepared by the following method, and the formula was shown in Fig. Specifically, methoxypolyethylene glycol (methoxypolyethylene glycol) was dissolved in DMSO in an amount of 5% by weight, dimethyaminopropyl ethyl carbodiimide and hardoxysuccinimide were added thereto, and the mixture was stirred at room temperature for 3 hours at room temperature. After stirring for a period of time, the chitosan derivative of Example <1-2> was dissolved in DMSO to a concentration of 5% by weight, and the mixture was stirred at room temperature for 24 hours or more with a magnetic stirrer. Then, the reaction product was put into a dialysis membrane, dialyzed with water to remove the organic solvent, and lyophilized to obtain a graft copolymerization chitosan derivative with methoxypolyethylene glycol (FIG. 3). In order to confirm the binding of the derivative, the ethylene peak of methoxypolyethylene glycol was observed at 3.7 ppm using a nuclear magnetic resonance spectrometer, and it was confirmed that the bond was formed.

<1-4> By DSePA Cross-linked  Preparation of chitosan nanoparticles

Crosslinked chitosan nanoparticles with DSePA were prepared by the following method and are shown in FIG. 4 as chemical formulas. Specifically, DSePA was dissolved in DMSO at 1% by weight, dimethyaminopropyl ethyl carbodiimide and hardoxysuccinimide were added twice as much as the solution, and the mixture was stirred at room temperature for 3 hours with a magnetic stirrer. Then, the amorphostatin covalently bonded chitosan-methoxy polyethylene glycol graft copolymer prepared in Example <1-3> was dissolved in DMSO at 3% by weight and added to the solution. Then, the mixture was stirred at room temperature in a magnetic stirrer For 24 hours or more. Then, the reactant was put into a dialysis membrane and dialyzed with water to remove the organic solvent. Lyophilization was performed to synthesize spherical chitosan nanoparticles crosslinked with DSePA and bound with amphostin. The chitosan nanoparticles were freeze-dried or stored in the refrigerator for use in the present invention Respectively.

< Experimental Example  1> Confirmation of changes of chitosan nanoparticles by irradiation

The nanoparticles prepared in Example <1-4> were dispersed in distilled water at 1 wt% to prepare a crosslinked chitosan nanoparticle solution by DSePA. As a result of microscopic examination, as shown in FIG. 5 (a) Likewise, spherical chitosan nanoparticles were confirmed and the nanoparticles were found to have a particle size of about 100 nm. However, as shown in FIG. 5 (B), X-ray radiation (CLINAC ix 4229, Varian Co. Inc. USA), it was confirmed that the nanoparticles were destroyed when irradiated with 20 Gy of radiation to the nanoparticles.

As shown in FIG. 5 (c), the binding of the complexes in the chitosan crosslinked by DSePA was cut off until the irradiation dose of 7 Gy, and the particle size of the chitosan nanoparticles was measured. It was confirmed that the particle size was increased by forming loose bonds and when the irradiation of 10 Gy or more was irradiated, the cross-linking was completely broken and the diameter of the particles was reduced to 80 nm or less. According to the irradiation dose of 20 Gy, (Fig. 5).

< Experimental Example  2> by irradiation Aminostatin  Identification of change in release rate

The chitosan nanoparticles prepared in Example <1-4> were dispersed in a buffer solution to a concentration of 5% by weight in order to confirm the rate of release of amphopustine from the chitosan nanoparticles by irradiation with radiation, As shown in FIG. 6, the release rate of aminopostin at 37 ° C was measured by a UV spectrophotometer. As shown in FIG. 6, from about 2 hours after irradiation, the inner bond of nanoparticles was destroyed, (Fig. 6).

< Experimental Example  3> Cytotoxicity of chitosan nanoparticles

The nanoparticles prepared in Example <1-4> were treated with a Chang liver cell line (ATCC) to confirm cytotoxicity. Specifically, rat hepatocytes were used, and 3 × 10 ⁴ cells were cultured in a 96-well plate. Then, the cells were treated with the nanoparticles at a concentration of 500 mg / ml and irradiated with radiation . Thereafter, after further incubation for 24 hours, the survival rate of the cells was evaluated using MTT assay, and apoptosis analysis was performed to evaluate the degree of cell suicide or necrosis. As a result, as shown in Fig. 7 (a), when cells treated with aminopostin and aminostatin-bound nanoparticles were found to have higher viability of cells for irradiation, Fig. 7 , Apoptosis and necrosis of the cells were also markedly decreased (Fig. 7).

 < Experimental Example  4> Effect of chitosan nanoparticles on animal model

The chitosan nanoparticles prepared in Example <1-4> were intravenously injected into the tail of BALb / C rats and irradiated with radiation (10 Gy), and the survival rate of the mice was confirmed. Rats were fed 20 g, 5-week-old BALb / C mice (Orient Bio, Korea) and the rats were free to feed water and feed during breeding. Ten rats were used per treatment group. Breeding conditions, experimental conditions, and sacrifice were strictly observed in the animal ethics committee. As a result, as shown in Fig. 8 (A), the rats that had been irradiated with radiation and had no treatment died after 12 days, while the group receiving amifostine (Amifostine) showed a survival rate of 40% (ChitoSe + Ami) showed a survival rate of 60% or more. The group (Empty NP) injected with empty aminopthin-coated nanoparticles (Empty NP) showed 30 % Survival rate (Fig. 8 (A)).

As shown in FIG. 8 (b), immunohistochemical staining experiments were performed on the liver tissues of the rats. As a result, it was possible to detect damage or necrosis of the liver tissues in the control group after irradiation, When the particles were injected, it was confirmed that the state of the tissue was good. After irradiation, abnormal hepatic parenchymal cells in the liver of the control rats were increased up to 25%, but decreased to 14% when the aminostatin was injected. In the case of injecting aminostatin-loaded chitosan nanoparticles 9% or less (Fig. 8 (B)).

As a result of western blot analysis, the expression ratio of BAX / Bcl-2, which is a protein showing apoptosis, as shown in Fig. 8 (c) was lowest when amipostin-bound chitosan nanoparticles were injected (FIG. 8 (c)). As a result, it was confirmed that amitophene-bound chitosan nanoparticles react with radiation specifically to release the drug, and the radiation protection effect is excellent.

Claims (10)

(Methoxypolyethylene glycol) (methoxypolyethylene glycol) was added to chitosan derivatives in which amide-bonded complexes of 3,3'-diselenodipropionic acid (DSePA) and amifostine were combined with water-soluble chitosan- ; MPEG) chitosan copolymer grafted with an amide bond.
delete delete 1) synthesizing an amide bonded conjugate using 3,3'-diselenodipropionic acid (DSePA) and amifostine as a solvent in DMSO;
2) synthesizing a chitosan derivative by amide bonding amiopostin-amide bonded DSePA of step 1) to water-soluble chitosan using DMSO and water as a solvent;
3) Chitosan derivatives of step 2) above were amide bonded to methoxy poly (ethylene glycol) using DMSO as a solvent to prepare amidephosphine amide-bonded chitosan-methoxypolyethylene glycol graft copolymer ( graft copolymer; And
4) crosslinking the graft compatibilizer of step 3) with an amide bond with DSePA using DMSO as a solvent.
[6] The method according to claim 4, wherein the covalent conjugate of DSePA and amopostin in step 1) is prepared by dissolving DSePA in DMSO in an amount of 1 to 15% by weight and adding 1 to 5% by weight of amphostin Wherein the chitosan nanoparticles have an average particle size of about 10 nm.
The method according to claim 4, wherein the chitosan derivative of step 2) is prepared by dissolving a DSePA-aminostatin compound in DMSO in an amount of 10 to 25% by weight, dissolving the water-soluble chitosan in 5 to 15% &Lt; / RTI &gt;
[Claim 4] The method according to claim 4, wherein the covalent bond of step 3) is dissolved in DMSO such that 3 to 7% by weight of methoxypolyethylene glycol is used, and the chitosan derivative of step 2) is dissolved in DMSO to 3 to 7% Wherein the chitosan nanoparticles are added to the chitosan nanoparticles.
[5] The method of claim 4, wherein the chitosan nanoparticles in step 4) are prepared by dissolving DSePA in a solvent in an amount of 1 to 5% by weight and using the amidopolysethrin-amide bonded chitosan-methoxypolyethylene glycol graft copolymer Is dissolved in DMSO in an amount of 1 to 5% by weight.
A nano drug for radioprotectants comprising the chitosan nanoparticles of claim 1.
[Claim 11] The nano drug according to claim 9, wherein the amide bond of the chitosan is cleaved by exposing the nano drug to radiation to release amipostin.

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