KR101447130B1 - Methotrexate encapsulated polymeric micelles of chitosan copolymer grafted with methoxy poly(ethylene glycol) and preparation method thereof - Google Patents

Abstract

The present invention relates to a polymeric micelle in which methotrexate is supported on a chitosan copolymer grafted with methoxypolyethylene glycol having improved release properties of methotrexate and stability to normal cells, and a method for producing the same.

According to the present invention, methotrexate, which is a folate antitumor substance, can be easily carried on chitosan copolymer grafted with methoxypolyethylene glycol without a structural change, thereby improving the sustained release of methotrexate, Can be usefully used to increase the efficacy of methotrexate by reducing toxicity.

Soluble chitosan, methotrexate, polymeric micelle, methoxypolyethylene glycol

Description

TECHNICAL FIELD The present invention relates to a polymeric micelle having methotrexate supported on methoxy polyethylene glycol-grafted chitosan copolymer and methotrexate-containing polymeric micelles of chitosan copolymer grafted with methoxy poly (ethylene glycol)

The present invention relates to a polymeric micelle in which methotrexate is supported on a chitosan copolymer in which methoxypolyethylene glycol is grafted as a hydrophilic group to a chain of a water-soluble chitosan having a free amine group.

Chitosan is a β-1,4-linked pyranose unit of glucosamine. The chitosan has a molecular weight of more than 5,000 and has a polysaccharide family of multivalent cations. Biomolecules can be extracted from fisheries including crustaceans such as crab shells and shrimp, and squid. In terms of its molecular structure, it has a structure similar to cellulose, which is a type of polysaccharide. It has excellent biocompatibility, Has been applied to the pharmaceutical industry since it does not occur. Recently, after being certified as a food by the US FDA, chitosan has been applied as an important biological industry and biomedical materials in the 21st century.

In particular, chitosan having a specific molecular weight within a range of 20,000 to 100,000 is known to have a strong physiological activity, so it is expected to be applicable to the fields of health food, food and beverage, cosmetics, healthcare and pharmaceuticals .

However, the chitosan having the above-described characteristics and advantages is a water-insoluble substance which is firmly bonded to a neighboring molecule by strong hydrogen bonds and is conventionally known as a water-insoluble substance. To dissolve chitosan, lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid, And inorganic acids composed of hydrochloric acid, nitric acid, and sulfuric acid, and thus, there is a problem in that application to living bodies is limited. The inventors of the present invention have developed a novel water-soluble chitosan which can solve such a problem and have been registered with a patent application (Korean Patent No. 441270). Specifically, it is as follows.

Korean Patent No. 441270 discloses a method for producing a chitosan oligosaccharide which comprises the steps of: 1) treating a solution of an organic acid or inorganic acid salt of a chitosan oligosaccharide with a base trialkylamine, 2) adding an organic solvent to the solution, 3) The acid-removed chitosan oligosaccharide solution is treated with an inorganic acid and then purified by an activated carbon / ion exchange resin column to obtain a chitosan oligosaccharide of 1,000-100,000 A method for producing pure water-soluble free amine chitosan having a molecular weight of Da ".

On the other hand, methotrexate is a folate antimetabolite, which is used to treat malignant tumors and osteosarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, brain cancer, oral cancer and lung cancer, breast cancer, psoriasis, choriocarcinoma and trophoblastic tumor of children with acute lymphocytosis leukemia Have been used as various therapeutic agents. Nevertheless, it is known that the toxicity of methotrexate to normal cells and the characteristics of drug resistance and nephrotoxicity, myelosuppression, acute and long-term hepatotoxicity, pneumonia and side effects of long-term lung disease. Various studies have been made on the carrier of methotrexate to solve such a problem. Specifically, it is as follows.

Li and Kwon reported that methotrexate binds to PEG-block poly (2-hydroxyethyl L-aspartamide) and that the macromolecule-bound polymeric micelles bind to methotrexate for sustained release (Y. Li, GS Kwon, Pharm. Res. 17 (2000) 607-611). In addition, it was confirmed that the polymeric micelles to which methotrexate was bound were sensitive to pH (Y. Li, G. S. Kwon, Colloids and Surfaces B: Biointerfaces 16 (1999) 217226). Kang et al. Reported that methotrexate binds to poly (2-hydroxyethyl aspartamide) by self-association due to physical chemical bonding (H. Kang, JD Kim, SH Han, IS Chang, J. Control. ).

  Zhang and Zhuo also reported that the incorporation efficiency of methotrexate-bearing polymeric micelles was 73.0% (w / w) using ABA triblock copolymer (Y. Zhang, RXZhuo, Biomaterials 26 (2005) 20892094). It has been reported that the antitumor action depends on the molecular weight of dextran when covalently conjugating a polysaccharide such as dextran with methotrexate (D. Nevozhay, R. Budzynska, U. Kanska, M. Jagiello, MS Omar, J. Boratynski , A. Opolski, Anticancer Res. 26 (2006) 1135-1143). However, these conventional methods chemically bind methotrexate directly to the polymer, thereby affecting intrinsic properties of methotrexate and deteriorating anticancer activity. In addition, the block copolymer used for encapsulating methotrexate is a novel polymer There is a problem that stability such as toxicity and persistence in the human body can not be guaranteed.

Accordingly, the present inventors have succeeded in improving the sustained release of methotrexate by preparing a core-shell type copolymer by grafting MPEG to a low molecular weight water-soluble chitosan and producing methotrexate-loaded polymeric micelles therein, The present inventors have found a method for producing methotrexate-loaded ChitoPEG copolymer polymer micelles that reduce toxicity to normal cells, and completed the present invention.

An object of the present invention is to provide an anticancer agent-carrier complex polymeric micelles carrying methotrexate, which is an anticancer agent. Another object of the present invention is to provide a method for preparing the above-described methotrexate-loaded anticancer drug-delivery complex polymer micelles.

In order to achieve the above object, the present invention provides a chitosan copolymer (ChitoPEG) in which methoxy poly (ethylene glycol) is grafted as a hydrophilic group to a chain of a water-soluble chitosan having a free amine group as a carrier and methotrexate And a method for producing the same.

According to the present invention, the inside of ChitoPEG, in which a hydrophilic group is grafted, has a structural characteristic composed of an ionic complex of a polymer and a drug, and the outside is composed of a hydrophilic methoxypolyethylene glycol (MPEG) The present invention has the effect of securing solubilization and sustained release of hydrophobic drugs and reducing side effects such as toxicity to normal cells of methotrexate, and also has the effect of inhibiting the methotrexate- Micelles are useful as carriers for antitumor agents because they have excellent redispersibility in distilled water after freeze-drying due to the advantages of polymers based on water-soluble chitosan, which is a natural polymer material.

Hereinafter, the present invention will be described in detail.

The present invention provides polymeric micelles in which methotrexate is supported on a chitosan copolymer in which methoxypolyethylene glycol is grafted as a hydrophilic group to a chain of water-soluble chitosan having a free amine group.

The water-soluble chitosan according to the present invention is preferably a water-soluble chitosan having a pure free amine group having a molecular weight of 500 to 100,000 Da, more preferably 1,000 to 50,000 Da.

The form of the methotrexate-loaded ChitoPEG polymeric micelle according to the present invention is in the form of a spherical core-shell in which ChitoPEG surrounds the methotrexate molecules (see FIG. 1). Due to such structural characteristics, methotrexate, which is located inside the core shell, is guaranteed to be sustained release and toxicity to normal cells can be reduced.

Furthermore, in the polymer micelle according to the present invention, the substitution degree of methoxy polyethylene glycol in the ChitoPEG copolymer is preferably 2-12. The degree of substitution can be calculated by the following equation (1). In the present invention, the degree of substitution is an important factor for determining drug loading efficiency in polymer micelles. When the degree of substitution is out of the above range, the drug loading efficiency is lowered. Also, even when methotrexate is loaded, This is a difficult problem.

Substitution degree of MPEG = [(ChitoPEG number average molecular weight - chitosan number average molecular weight) / 2000] x100

The particle size of the polymeric micelle carrying the methotrexate according to the present invention is preferably in the range of 50 to 300 nm. If the size of the micelle particle is less than 50 nm, the methotrexate is difficult to be encapsulated. If the size of the micelle particle is more than 300 nm, the in vivo distribution may not be uniform when administered to a living body, have.

The polymer micelle according to the present invention is thought to be a polymer micelle due to mutual ionic bonding between nitrogen of the free amine group of the water-soluble chitosan and oxygen of the carboxyl group of the methotrexate as shown in the following Chemical Formula 1, But is not limited to theory.

(Where n is 1.7 to 310; 6 , and m is 45.5 to 113.6.)

In addition, the present invention relates to a method for producing a chitosan-containing polymer, which comprises dissolving a water-soluble chitosan having a free amine group in distilled water, adding methoxypolyethylene glycol-N-hydroxysuccinimide dropwise to prepare a copolymer (ChitoPEG) ; And dissolving lyophilized ChitoPEG in distilled water in step 1, and then adding methotrexate dissolved in an organic solvent to the ChitoPEG solution so as to support methotrexate (step 2), thereby preparing a methotrexate-loaded polymeric micelle ≪ / RTI >

Hereinafter, the above manufacturing method will be described step by step.

Step 1 according to the present invention is a step of preparing a copolymer solution by reacting water-soluble chitosan having a yuramine group with methoxypolyethylene glycol. The water-soluble chitosan having a yulia-amine group used as a starting material of the present invention is prepared by treating a solution of an organic acid or inorganic acid salt of a water-insoluble chitosan oligosaccharide with a base and treating the solution with an organic solvent to remove the organic acid or inorganic acid bound to the chitosan oligosaccharide As the water-soluble chitosan obtained by recovering chitosan oligosaccharide removed in the form of base salt and purifying after simple treatment, those disclosed in Korean Patent No. 441,270 of the present inventors can be used.

The water-soluble chitosan according to the present invention preferably uses water-soluble chitosan having a pure free amine group having a molecular weight of 500 to 100,000 Da, more preferably 1,000 to 50,000 Da.

Further, commercially available methoxypolyethylene glycol-N-hydroxysuccinimide can be used for grafting methoxypolyethylene glycol to the water-soluble chitosan according to the present invention.

Step 2 according to the present invention is a step of loading methotrexate, which is an anticancer agent, on the ChitoPEG prepared in step 1 above.

ChitoPEG prepared by freeze-drying in step 1 is dissolved in distilled water, and methotrexate is separately dissolved in an organic solvent. The organic solvent is preferably dimethylsulfoxide, dimethylformamide, dimethylacetamide, 1,4-dioxane, hydrochloric acid or acetic acid, more preferably dimethylsulfoxide. Methotrexate can be carried on ChitoPEG by slowly adding the methotrexate solution to the ChitoPEG aqueous solution. At this time, it is preferable to stir the mixed solution of the two solutions at room temperature for 5 to 30 minutes so that the loading can be smoothly performed. When the support is completed, dialysis is performed while exchanging distilled water several times in order to remove the organic solvent in the mixed solution. When the dialysis process is completed, the methotrexate-loaded polymeric micelles of the present invention can be obtained by lyophilization of the methotrexate-loaded ChitoPEG solution.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

< Example  1> ChitoPEG on Methotrexate Supported  Polymer Micelle  Manufacturing I

Step 1: ChitoPEG  Preparation of Copolymer

100 mg of a low molecular weight water-soluble chitosan having a molecular weight of 1,000 to 50,000 Da was dissolved in 0.2 ml of distilled water to prepare a low molecular weight water-soluble chitosan solution, and 9.8 ml of dimethylsulfoxide (DMSO) was added dropwise. Then, methoxypolyethylene glycol-N-hydroxysuccinimide (MPEG-NHS) dissolved in 2 ml of DMSO was added dropwise to the solution, and the solution was reacted after nitrogen was charged. The reaction solution was dialyzed with distilled water and lyophilized for 24 to 48 hours to obtain a powdered ChitoPEG copolymer (0.16 g, 95.1%, MPEG substitution degree 2.5).

Step 2: Methotrexate Supported  Polymer Micelle  Produce

20 mg of the ChitoPEG copolymer prepared in the step 1 was dissolved in 5 ml of distilled water to prepare a ChitoPEG solution. Then, 0.1 ml of methotrexate dissolved in DMSO was added dropwise to the solution, followed by stirring at 25 ° C for 10 minutes . The reaction solution was dialyzed against distilled water for 12 to 24 hours to prepare polymer micelles carrying methotrexate on ChitoPEG.

< Example  2-1 to 2-3> ChitoPEG on Methotrexate Supported  Polymer Micelle  Manufacturing II

Methotrexate-loaded polymeric micelles were prepared in the same manner as in Example 1, except that the weight ratio of ChitoPEG having a substitution degree of MPEG of 5.15 and methotrexate was 50/5, 50/10, and 50/20, respectively.

< Example  3-4> ChitoPEG on Methotrexate Supported  Polymer Micelle  Manufacturing III

Polymeric micelles carrying methotrexate on ChitoPEG were prepared in the same manner as in Example 1, except that ChitoPEG copolymers having substitution degrees of MPEG of 7.3 and 11.5 were prepared, respectively.

< Experimental Example  1> Methotrexate Supported  Polymer Micelle  Drug content and Of carrying efficiency  Measure

The following experiments were performed to investigate the drug content and the loading efficiency of the methotrexate-loaded ChitoPEG copolymer polymer micelles prepared in Examples 1 to 4.

Methotrexate-loaded ChitoPEG copolymer 5 mg of polymeric micelle was dissolved in 0.5 ml of distilled water, and then 9.5 ml of DMSO was added dropwise. The amount of methotrexate in the solution was calculated using a UV spectrophotometer (303 nm, UV-1601, Shimazu Corp., Japan). The equivalent of ChitoPEG copolymer was used as a blank test and the whole procedure was performed in the dark room three times. The drug content and the loading efficiency were calculated by the following equations (2) and (3), respectively, and the results are summarized in Table 1 below.

ChitoPEG /
Methotrexate
Weight ratio (mg / mg) The degree of substitution of MPEG Drug content
(% (w / w)) Loading efficiency
(% (w / w)) Example 1 50/5 2.5 8.6 94 Example 2-1 50/5 5.15 8.35 91.1 Example 2-2 50/10 5.15 14.0 81.5 Example 2-3 50/20 5.15 24.0 78.8 Example 3 50/5 7.3 8.3 90.1 Example 4 50/5 11.5 6.1 65.2

In Table 1, the degree of substitution of MPEG was measured by the method of Equation (1).

As shown in Table 1, the drug content was 8.6, 8.35, 14.0, 24.0, 8.3 and 6.1% (w / w), and the ChitoPEG copolymer was 94, 91.1, 81.5, 78.8, 90.1, 65.2% / w) &lt; / RTI &gt; in the presence of methotrexate. According to Table 1, when the degree of MPEG substitution is maintained within a predetermined range (2 to 8), the drug content is as much as 8.3% (w / w) at 24.0% (w / w) (W / w) and 78.8% (w / w), respectively. However, when the MPEG substitution degree is 11.5 (Example 4), it is understood that the drug content and the carrying efficiency are lowered.

< Experimental Example  2> Methotrexate Supported  Polymer Micelle ^One H NMR  Measure

The following experiments were performed to obtain ¹ H NMR spectra of the methotrexate-loaded polymeric micelles prepared in Examples 1 to 4 above.

Experimental Example  2-1

First, the hydrophilic substance is ChitoPEG was measured spectrum with 0.5 ~ ¹ H-NMR analyzer at 298 K was dissolved in D ₂ O to 1 mg / ml concentration of (AVANCE 400 (400MH _z), Bruker, Germany) (FIG. 2 (a)).

Next, methotrexate, which is a hydrophobic substance, was dissolved in a mixed solution of D ₂ O and DCl at a concentration of 0.5 to 1 mg / ml, and a ¹ H-NMR spectrum was measured at 298 K (see FIG. 2 (b)).

Next, the polymeric micelle carrying the methotrexate prepared in Example 2-1 was dissolved in D ₂ O at a concentration of 0.5 to 1 mg / ml, and a ¹ H-NMR spectrum was measured at 298 K (see (c ) Reference).

Next, the polymer micelles were dissolved in a mixture of DMSO and D ₂ O and DCl at a concentration of 0.5 to 1 mg / ml, and ¹ H-NMR spectra were measured at 298 K (FIGS. 2 (d) and 2 ) Reference).

As shown in FIG. 2 (a), when the ChitoPEG copolymer was dissolved in D ₂ O, it was confirmed that the characteristic peak of the ChitoPEG copolymer was between 3.0 and 5.0 ppm.

As shown in FIG. 2 (b), when methotrexate was dissolved in D ₂ O / DCl, it was confirmed that the characteristic peak of methotrexate was 1.0 to 2.0, 3.5 to 5.0 and 6.5 to 8.0 ppm.

As shown in FIG. 2 (c), in the spectrum obtained by dissolving methotrexate-loaded ChitoPEG copolymer polymer micelles in D ₂ O, only characteristic peaks of the ChitoPEG copolymer as shown in FIG. 2 (a) there was. From this, it can be seen that, in distilled water, the ChitoPEG copolymer forms a micelle structure in which methotrexate is encapsulated in a core-shell form.

On the other hand, as shown in FIG. 2 (d), when the polymer micelles were dissolved in DMSO as an organic solvent, the characteristic peaks of the ChitoPEG copolymer and the characteristic peaks of methotrexate could be confirmed at the same time. This shows that ionic complexes of ChitoPEG copolymer and methotrexate are partially separated under an organic solvent.

As shown in FIG. 3 (e), when the polymer micelle was dissolved in a mixed solution of D ₂ O and DCl, the characteristic peak of the ChitoPEG copolymer and the characteristic peak of methotrexate could be confirmed at the same time. From this, it can be seen that the efficiency in which methotrexate is carried on the ChitoPEG copolymer is lowered in the acidic atmosphere (D ₂ O / DCl).

On the other hand, by confirming the same methotrexate characteristic peak as unmodified methotrexate (Fig. 3 (b)), it can be seen that no structural change occurs when methotrexate is loaded on the ChitoPEG copolymer.

Experimental Example  2-2

In order to examine the binding pattern between the chitosan main chain and methotrexate, ¹ H-NMR spectrum was measured after dissolving the loaded ChitoPEG copolymer in a D ₂ O / DCl mixed solution with different amounts of methotrexate, .

As shown in FIG. 3, when the characteristic peak (b) of methotrexate is observed in the polymer micelles (c to d), most peaks disappear. 24% (w / w)). From this, it can be seen that the binding between methotrexate and ChitoPEG is weakened in the acidic solvent (D ₂ O / DCl), unlike the case where the methotrexate-carrying polymeric micelle according to the present invention is dissolved in distilled water (FIG. 2 have. Accordingly, it can be seen that the macromolecular micelles according to the present invention encapsulate methotrexate in micelles in the form of an ion complex with chitosan main chain.

< Experimental Example  3> Methotrexate Supported  Polymer Micelle  Particle size, surface voltage and shape measurement

The following experiments were carried out to measure the size and shape of the polymer micelles of the methide lecith-bearing ChitoPEG copolymer prepared in Examples 1 to 4 above.

First, the lyophilized polymeric micelles were dispersed in distilled water at a concentration of 1.0 mg / ml, and then analyzed using an ELS-8000 electrophoretic LS spectrophotometer (NICOMP 380 ZLS zetapotential / particle sizer) equipped with a He-Ne laser Particle size and surface voltage were measured.

The measurement results are shown in Table 2 below.

ChitoPEG /
Methotrexate weight ratio (mg / mg) Particle Size (nm) zeta
electric potential burglar
(intensity) mass
(weight) Number of particles
(number) Example 1 50/5 64.1 ± 16.9 49.4 ± 11.9 43.4 ± 11.5 38.52 Example 2-1 50/5 37.4 ± 7.2 33.6 ± 6.2 31.4 ± 9.0 39.28 Example 2-2 50/10 42.0 ± 9.4 36.0 ± 6.2 31.9 ± 6.7 30.92 Example 2-3 50/20 30.6 ± 6.4 27.3 ± 5.5 24.9 ± 4.5 29.55 Example 3 50/5 35.3 ± 8.0 30.5 ± 6.5 27.5 ± 5.4 27.58 Example 4 50/5 49.0 ± 9.0 44.4 ± 8.0 41.3 ± 10.4 20.34

As shown in Table 2 and FIG. 1 (a), the average particle size of the polymeric micelles carrying methotrexate was smaller than 100 nm, and the particle size distribution showed a narrow distribution.

In addition, the zeta potential was found to have a value in the range of (+) 20 to 40 mV. As the surface of the polymeric micelle shows a (+) value, it can be seen that the surface of the polymer micelle is surrounded by the ChitoPEG copolymer. In addition, the zeta potential was lowered with increasing amounts of methotrexate. As a result, the polymeric micelles of methotrexate-loaded polymeric micelle showed that the main chain of the copolymer formed a complex with methotrexate, and the zeta potential decreased as the complex formation between methotrexate and the copolymer increased.

Next, the polymer micelle was dropped on a copper grid coated with a carbon film, dried at room temperature for 24 hours, and observed for morphology using JEOL JEM-2000 FX II at 120 kV. As shown in FIG. 1, it can be seen that the polymer micelle is a spherical particle of 100 nm or less.

< Experimental Example  4> Methotrexate Supported  Polymer Micelle FT - IR  Measure

IR spectrophotometer (Shimadzu Co., FI-IR 8700) to determine whether the methionine-containing polymeric micelle prepared in Example 1 was encapsulated between the amine group and the hydroxyl group of the polymeric micelle, Is shown in Fig.

Referring to FIG. 4, characteristic peaks near 1200 cm ^{-1 and} near 1400 cm ^-1 appearing in the IR peak (a) of free methotrexate are still observed in (c) when ChitoPEG and methotrexate are physically mixed . However, when the methotrexate was carried on the ChitoPEG copolymer to form micelles, the characteristic peak was not observed in (e). However, as the content of methotrexate was increased, a methotrexate characteristic peak was observed mildly by exceeding the drug encapsulation limit (d).

< Experimental Example  5> Methotrexate Supported  Polymer Micelle  toxicity

In order to examine the toxicity of the polymer micelles prepared in Examples 1 to 4, the following experiment was conducted and the results are shown in FIG.

To test the antitumor effect of methotrexate and polymer micelles, B16F10 melanoma cells and MCF7 breast cancer cells were used. All tumor cells were maintained at 37 ° C in a 5% CO ₂ incubator. Methotrexate was dissolved in 100% DMSO and diluted 100 times in Dulbecco's modified Eagle's medium (DMEM) solution supplemented with 10% serum. Polymer micelles were dissolved in DMEM solution and diluted at the same rate as the concentration of methotrexate. Approximately 5,000 of the cells were dispensed in a 96-well incubator at a rate of 100 μl each, and then cultured in an incubator for 12 hours to stably attach the cells to the incubator. Methotrexate and polymer micelles dissolved in DMEM solution were then added dropwise to 100 mu l of DMEM solution per incubator. After 24-48 hours, methotrexate was added dropwise to the incubator and incubated in an incubator for 4 hours. Thereafter, the supernatant was removed and 100 占 퐇 of DMSO was added dropwise. The toxicity results were determined by absorbance at 560 nm using a microtiter plate reader.

As shown in FIG. 5, the polymer micelles of the present invention showed almost similar toxicity to B16F10 melanoma cells as compared to pure methotrexate. Thus, it can be seen that the inherent toxicity of methotrexate is not affected even if it is carried on a ChitoPEG copolymer.

1 is a graph of particle size distribution of methotrexate-loaded polymer micelles and a transmission electron microscope photograph of polymer micelles,

Figure 2 is a graph showing the effect of the present invention on (a) ChitoPEG copolymer under D ₂ O, (b) methotrexate under D ₂ O / DCl, (c) polymeric micelles carrying the methotrexate of the present invention under D ₂ O, (d) (E) ¹ H-NMR spectrum of the polymeric micelle carrying the methotrexate of the present invention under D ₂ O / DCl, wherein the methotrexate-

Figure 3 shows the results of (a) ChitoPEG copolymer under D ₂ O, (b) free methotrexate under D ₂ O / DCl, (c) 8.4% (w / w) of the inventive methotrexate under D ₂ O / the polymeric _{micelles, (d) D 2 O /} DCl 14.0% ((w / w)) 24.0% ((w / w under the polymeric micelles, and (e) D ₂ O / DCl methotrexate is supported of the invention under a loaded ) &Lt; ¹ &gt; H-NMR spectrum of the macromolecular micelle carrying the methotrexate of the present invention,

4 shows FT-IR spectra of polymeric micelles carrying (a) free methotrexate, (b) ChitoPEG copolymer, (c) a physical mixture of methotrexate and ChitoPEG copolymer, and (d)

Fig. 5 shows the toxicity test results of polymer micelles carrying B16F10 melanoma cells carrying 14.0% (w / w) of methotrexate.

Claims (8)

In a polymer micelle in which methotrexate is carried on a chitosan copolymer (ChitoPEG) in which methoxy poly (ethylene glycol) (MPEG) is grafted as a hydrophilic group to a chain of water-soluble chitosan having a free amine group, The molecular weight of the water-soluble chitosan is 1,000 to 50,000 Da, Wherein the degree of substitution of the methoxy polyethylene glycol of the chitosan copolymer calculated by the following formula (1) is 2.5 to 7.3. &Quot; (1) &quot; Substitution degree of MPEG = [(ChitoPEG number average molecular weight - chitosan number average molecular weight) / 2000] x100 delete delete The methotrexate-loaded polymeric micelle according to claim 1, wherein the particle size of the methotrexate-loaded polymeric micelle is 50 to 300 nm. Soluble chitosan having a free amine group was dissolved in distilled water, and then methoxypolyethylene glycol-N-hydroxysuccinimide was added dropwise thereto to prepare a chitosan copolymer (ChitoPEG (meth) acrylate) grafted with methoxypolyethylene glycol ) And then lyophilization (step 1); And (Step 2) of dissolving the lyophilized ChitoPEG in the distilled water in the step 1, adding the methotrexate dissolved in the organic solvent to the ChitoPEG solution to carry the methotrexate, The molecular weight of the water-soluble chitosan is 1,000 to 50,000 Da, The method of claim 1, wherein the degree of substitution of the methoxy polyethylene glycol of the chitosan copolymer calculated by the following formula (1) is 2.5 to 7.3. &Quot; (1) &quot; Substitution degree of MPEG = [(ChitoPEG number average molecular weight - chitosan number average molecular weight) / 2000] x100 [7] The method of claim 5, wherein the methotrexate-containing solution of step 2) is prepared by stirring the mixture of ChitoPEG and methotrexate at room temperature for 5 to 30 minutes, dialyzed with distilled water and lyophilized. A method for producing micelles. 7. The method of claim 6, wherein the distilled water is exchanged several times to remove the organic solvent. The method of claim 5, wherein the organic solvent is dimethyl sulfoxide. 7. The method of claim 1, wherein the organic solvent is dimethyl sulfoxide.

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