KR20070078196A - Anti-cancer agent loaded hydrophobic bile acid conjugated hydrophilic chitosan oligosaccharide nanoparticles and preparation method thereof - Google Patents

Abstract

Provided is an anticancer agent-encapsulated and a hydrophobic bile acid-conjugated hydrophilic chitosan oligosaccharide nanoparticle which shows high encapsulation rate of anti-cancer agent, high cytotoxicity against cancer cells and continuous anti-cancer effect by releasing the anti-cancer agent for a long period of time in vivo so as to be usefully used for chemotherapy. The hydrophilic chitosan oligosaccharide nanoparticle encapsulates an anti-cancer agent and is conjugated with a hydrophobic bile acid which is selected from the group consisting of lithocholic acid, deoxycholic acid, cholic acid, chenodeoxycholic acid, 7-oxo-lithocholic acid and 5beta-cholanic acid. The method comprises the steps of: (a) preparing a bile acid solution by dissolving a hydrophobic bile acid together with an activating agent in a solvent with agitation to activate the bile acid; (b) dissolving a hydrophilic chitosan oligosaccharide in a solvent to prepare a chitosan oligosaccharide solution; (c) reacting the chitosan oligosaccharide solution with the activated bile acid solution to prepare a bile acid-conjugated chitosan oligosaccharide solution; (d) dialyzing and filtering the bile acid-conjugated chitosan oligosaccharide solution to prepare an auto-coagulative type bile acid-conjugated chitosan oligosaccharide nanoparticle solution; and (e) adding an anti-cancer solution to the auto-coagulative type of bile acid-conjugated chitosan oligosaccharide nanoparticle solution so as to encapsulate the anti-cancer agent into the nanoparticle.

Description

Anti-cancer Agent Loaded Hydrophobic Bile Acid Conjugated Hydrophilic Chitosan Oligosaccharide Nanoparticles and Preparation Method Thereof}

1 is a diagram showing the ¹ H-NMR spectrum of the lytocholic acid before activation according to the present invention.

Figure 2 is a diagram showing the ¹ H-NMR spectrum of lytocholic acid-NHS after activation in accordance with the present invention.

Figure 3 is a diagram showing the ¹ H-NMR spectrum of the chitosan oligosaccharide having an average molecular weight of 9 kDa used in the present invention.

Figure 4 is a diagram showing the ¹ H-NMR spectrum of the bile acid-bound chitosan oligosaccharide nanoparticles according to the present invention.

Figure 5 is a diagram showing the size distribution of the bile acid-bound chitosan oligosaccharide nanoparticles according to the present invention.

6 is a view showing the change of I ₃₃₉ / I ₃₃₅ by fluorescence photometer according to the concentration of bile acid-bound chitosan oligosaccharide nanoparticles according to the present invention.

Figure 7 is a diagram showing the size and distribution of paclitaxel-packed bile acid-bound chitosan oligosaccharide nanoparticles according to an embodiment of the present invention.

8 is a diagram showing the results of HPLC analysis of paclitaxel-packed chitosan oligosaccharide nanoparticles and paclitaxel packed with paclitaxel according to an embodiment of the present invention.

9 is a diagram showing a calibration curve of paclitaxel standard used in the present invention.

Figure 10 is a diagram showing the cytotoxicity results for cancer cells of paclitaxel-encapsulated bile acid-bound chitosan oligosaccharide nanoparticles according to an embodiment of the present invention.

11 is a diagram showing the cancer growth inhibitory effect of paclitaxel-containing bile acid-bound chitosan oligosaccharide nanoparticles according to an embodiment of the present invention.

12 is a photograph of an experimental animal after the administration of paclitaxel-encapsulated bile acid-bound chitosan oligosaccharide nanoparticles in accordance with one embodiment of the present invention.

The present invention relates to a hydrophilic chitosan oligosaccharide nanoparticles having an anticancer agent encapsulated, and a method for preparing the hydrophilic chitosan oligosaccharides.

The development of chemotherapy for cancer began in earnest by the use of methotrexate in choriocarcinoma to achieve a cure effect. Today, about 50 anticancer drugs are used, especially choriocarcinoma, leukemia, Wilms' tumor, Ewing's sarcoma, rhabdomyosarcoma, retinoblastoma, lymphoma, mycelial sarcoma, and testicular cancer. Getting

However, the biggest problem with the use of anticancer drugs is that there is no specificity for cancer cells unlike other drugs. In other words, anticancer drugs act on cells that are rapidly dividing or proliferating, thus causing damage to cells that are normally active in cell division (bone marrow cells, gastrointestinal epithelial cells, hair follicles), but the side effects such as bone marrow suppression, gastrointestinal disorders, and hair loss are almost different. Occurs in all patients. However, the effects of anticancer drugs on normal cells and cancer cells are more quantitative than qualitative differences. Because cancer cells respond more sensitively, they are more destroyed than normal cells, and the regenerating ability of normal cells is faster. . On the other hand, anti-cancer drugs have the effect of inhibiting immunity in addition to anti-cancer effects, so they are also used for the purpose of suppressing rejection after organ transplant surgery.

These serious side effects, as described above, have been attempted to reduce the toxicity of anticancer drugs. Representative methods include using micelles or microspheres as carriers of an anticancer agent, and using an anticancer agent in combination with a polymer.

The first is to use micelles or microspheres as carriers for anticancer drugs. This is to reduce the side effects of chemotherapy by enclosing the anticancer drugs in micelles or self-aggregates to release them slowly. . In micelles or self-aggregates, amphiphilic polymers having both hydrophilicity and hydrophobicity are formed through interaction between hydrophobic blocks in the aqueous solution to stabilize interfacial energy. Polymer micelles formed by amphiphilic polymers are known to exhibit different particle sizes, distributions, rheological properties, and thermodynamic stability depending on the degree of hydrophilicity and hydrophobicity. In addition, it has recently been reported that various hydrophobic drugs may be enclosed in the hydrophobic domain inside micelles to induce selective and effective delivery of drugs. This is an attempt to suppress the side effects caused by the action of a large amount of anticancer agent in a short time when the anticancer agent alone is administered to induce the anticancer agent to continue to be released slowly from the micelle or microspheres.

The second method is to connect an anticancer drug to a polymer to make an anticancer drug-polymer complex. The current use of anticancer drugs has many side effects because they act on normal cells due to the lack of effective selectivity for cancer tissues. Therefore, in order to reduce the side effects of these anticancer drugs, a method of effectively transporting anticancer drugs to cancer tissues has been devised. Among them, a method of using a polymer carrier has been used. The advantages of this method are that the distribution of the anticancer agent-polymer complex depends on the nature of the polymer carrier and the longevity of the anticancer agent in plasma and the regulation of the chemical binding properties between the anticancer agent and the carrier in the plasma, thereby controlling the Secretion can be controlled.

Research into what can be used as a carrier for anticancer drugs among various types of polymer materials has been conducted. Among them, immunoglobulins (immunoglobulins) have been studied a lot in terms of high specificity and various applications for cancer tissue. Immunoglobulins, however, have been very limited as carriers for anticancer agents because of their chemical and physical properties. For example, modification of immunoglobulin for use as an anticancer drug carrier often results in precipitation due to the hydrophobicity of the anticancer agent. In addition, it should be done very restrictively when performing modification procedures to prevent denaturation of immunoglobulins.

Recently, many kinds of polymers are designed and used more freely as a polymer carrier of an anticancer agent, and various organic reactions are also used in introducing an anticancer agent into a polymer carrier. Many polymers have been studied in this respect, for example poly (N-2- (hydroxypropyl) methacrylamide), poly (divinyl ether-co- Maleic anhydride) (poly (divinyl ether-co-maleic anhydride)), poly (styrene-co-maleic anhydride) (poly (styrene-co-maleic anhydride)), dextran, poly (Ethylene glycol), poly (L-glutamic acid), poly (aspartic acid) and poly (L-lysine) (poly (L-lysine)).

In addition, the anticancer agent-polymer complex method can be used to treat cancer cells using the pathophysiological characteristics of cancer tissues different from normal cells. In general, blood vessels in cancerous tissues are produced more than normal tissues in order to receive more nutrients than normal tissues, and are large in size but have many defects due to their poor structure, and discharge through lymphatic vessels is significantly lower than normal tissues. . Therefore, the polymer is more easily penetrated into cancer tissue than other tissues and organs, and is not easily discharged from the cancer tissue. This characteristic phenomenon of cancer tissue is called enhanced permeability and retention effect. One of the trials using such anticancer agent-polymer complex is N- (hygoxypropyl) methacrylamide (HPMA) -anticancer complex which is currently undergoing a second clinical trial (US Pat. 5,037,883 (1991)). Anticancer-polymer complexes that form self-assembly have a considerable size compared to anti-cancer drugs that do not have a polymer. A moderately sized anticancer-polymer complex circulates blood vessels without embolization in capillaries and prevents them from exiting the kidneys. It may also be possible to penetrate target cells through blood vessels. This self-assembly form also provides a function of protecting the anticancer agent from enzymatic reactions because the polymer encapsulates the anticancer agent (US Pat. 5,037,883 (1991)).

In addition, the Republic of Korea Patent No. 507968 discloses a hydrophobic anticancer agent-chitosan complex and a method for producing the self-assembly, which is a technique for forming a complex with hydrophilic chitosan using an anticancer agent as a hydrophobic introduction group. At this time, since only the anticancer agent capable of reacting with an amine group may be used as the anticancer agent, the anticancer agent that is available is quite limited, and the activity of the anticancer agent may decrease due to the reaction of the amine group with the anticancer agent.

On the other hand, chitin (chitin), chitosan (chitosan) is a material that has recently attracted attention as a natural functional material as a polysaccharide polymer material existing in nature. Chitosan is a substance obtained by de-acetylating chitin present in the shells of shellfish such as crabs and shrimps under basic conditions, and is a natural polymer in which glucosamine, a monomer, is bonded to β-1,4. Such chitosan has various physiological activities such as blood cholesterol reduction and fat absorption inhibitory effect, anticancer effect, immunity strengthening effect, anti-sugar effect, wound healing effect, and antibacterial activity. In particular, since it shows the effect of promoting the absorption of the drug by binding to the mucous layer of the oral cavity, nasal cavity, and intestinal cavities, the application of protein drug or non-absorbable drug as an oral and absorption formulation in various formulations has been actively studied. In addition, since it is cationic under aqueous solution, it is possible to form a complex with an anionic genetic material, and research into a gene carrier of chitosan or chitosan derivative using the same is being actively conducted.

In addition, chitosan is known as a natural polymer having a very good biocompatibility, and as a cationic polymer, it is much lower than typical cationic polymers such as poly (L-lysine) and polyethylenimine. Known to exhibit cytotoxicity to a negligible or negligible level. In addition, it is a biodegradable polymer that is decomposed into glucosamine as a monomer by an enzyme in vivo. As well as having one primary amine group (-NH ₂ ) for each polymer unit has the advantage of easy chemical modification. Therefore, active biomedical applications such as drug carriers and extracellular matrix for tissue engineering using these characteristics have been attempted.

Meanwhile, paclitaxel, which is a representative anticancer agent, is a substance extracted from the bark of the yew tree at the National Cancer Institute in the 1960s and is known as an excellent drug for ovarian cancer, breast cancer and lung cancer. However, the paclitaxel has a very low solubility in water of 0.2 [mu] g / ml, so it must be dissolved in a mixed solvent of cremophor EL and ethanol when used as an injection. This type of prescription can cause serious side effects such as vasodilation, dyspnea, kidney and neurotoxicity, depending on the solvent used.

Accordingly, the present inventors synthesize a hydrophilic and hydrophobic amphiphilic chitosan derivative using chitosan having biodegradability, biocompatibility and nontoxicity as a natural polymer and bile acid which is a hydrophobic biomaterial, thereby preparing a carrier for encapsulating a drug. The present invention was completed by confirming that the hydrophobic anticancer agent can be encapsulated in particles without using an organic solvent to enhance the solubility and anticancer activity of the anticancer agent.

The present invention is to provide a hydrophilic chitosan oligosaccharide nanoparticles are combined with a hydrophobic bile acid, an anticancer agent.

In addition, the present invention is to provide a method for producing a hydrophilic chitosan oligosaccharide nanoparticles, the hydrophobic bile acid is bound, the anticancer agent is encapsulated.

The present invention provides hydrophilic chitosan oligosaccharide nanoparticles bound to hydrophobic bile acids, which are encapsulated with an anticancer agent.

The present invention also provides a method for preparing hydrophilic chitosan oligosaccharide nanoparticles having hydrophobic bile acids bound to the anticancer agent.

Hereinafter, the present invention will be described in detail.

The present invention provides chitosan oligosaccharide nanoparticles in which bile acids formed through the reaction of carboxylic acids of hydrophobic bile acids with primary amines of hydrophilic chitosan oligosaccharides, which are encapsulated with an anticancer agent.

The bile acid-bound chitosan oligosaccharide nanoparticles can be prepared and used by known methods, methods used in the present invention, or modified methods thereof. Preferably, the critical micelle concentration (CMC) is 0.0010-0.0700 g / l. And chitosan oligosaccharide nanoparticles having a bile acid having a particle size of 150 to 350 nm may be used.

The bile acid-bound chitosan oligosaccharide nanoparticles will have a hydrophobic nucleus and a hydrophilic shell structure, and can be encapsulated with a hydrophobic anticancer agent by using hydrophobic interaction in the hydrophobic nucleus of the nanoparticle.

The hydrophobic bile acid is lithocholic acid (lithocholic acid), deoxycholic acid (deoxycholic acid), cholic acid (cholic acid), kenodioxycholic acid (chenodeoxycholic acid), 7-oxo-lithocholic acid and A bile acid selected from the group containing 5β-cholanic acid may be preferably used, and more preferably, lytocholic acid, deoxycholic acid may be used. In addition, the hydrophobic bile acid is preferably contained in a molar ratio of 1 to 40% relative to the monomer of the chitosan, more preferably 5 to 20% may be included.

Litocholic acid is a chemical formula C ₂₄ H ₄₀ O _{3 It} is contained in the bile acid as a minor component. It acts as a surfactant in the stomach, aids in digestion and absorption of foods that are insoluble in water, and is biosynthesized from ergosterol or cholesterol.

In addition, the deoxycholic acid is a molecular formula C ₂₄ H ₄₀ O ₄ and is present as one of the bile acids in the bile acids of humans, cattle, sheep, goats, dogs, rabbits and the like. Colorless crystals with a melting point of 176 to 178 ° C. It is insoluble in water but soluble in organic solvents and easily soluble in benzene and alkaline aqueous solution. It is synthesized from cholic acid and forms various organic compounds and molecular compounds such as carboxylic acids and their esters, ethers, alcohols and hydrocarbons, which are called choleic acid. Sodium salt is used as a surfactant.

In addition, the hydrophilic chitosan oligosaccharide is a partial decomposition product of chitosan, chitosan oligosaccharides are well absorbed in our body, because it is soluble in water is easy to add to any food and use as a main ingredient. In addition, it is a physiological functional material that attracts attention as it is found to be a wide range of high functional bioactive substances such as immune enhancement, antibacterial action, cholesterol control, and calcium absorption promotion due to its high water solubility. In the present invention, chitosan oligosaccharides having an average molecular weight of 8 to 10 kDa can be preferably used. By using a low molecular weight chitosan oligosaccharide, it is possible to easily apply to the injection and to control the molecular weight by overcoming the difficult application to the injection when using a high molecular weight chitosan.

The critical micelle formation concentration (CMC) is absorbed to the surface in an aqueous solution to significantly increase the concentration of the surfactant, a substance that significantly lowers the surface tension, so that the surfactant, which was simply dispersed, begins to form an aggregate (micro micelle). The concentration means, and the lower the critical micelle concentration, the better the surface active ability. Therefore, chitosan oligosaccharide nanoparticles in which the self-aggregated bile acid-conjugated bile acids according to the present invention, including both hydrophobic and hydrophilic moieties, have a relatively low critical micelle concentration and have an excellent encapsulation rate and thus are useful for encapsulating anticancer agents. It can be used as a carrier.

The anti-cancer agent of the present invention, the bile acid-coupled chitosan oligosaccharide nanoparticles are preferably 300 to 500 nm in size, the sealing rate may be 35 to 75%.

The anticancer agent may be any hydrophobic anticancer agent that can be encapsulated inside the chitosan oligosaccharide nanoparticles in which the bile acid of the self-aggregated form is bound, and preferably, paclitaxel, cis-platin, mitomycin-C, Dow Hydrophobic anticancer agents selected from the group comprising damyomycin, doxorubicin and 5-fluorouracil, more preferably paclitaxel can be used.

Chitosan oligosaccharide nanoparticles with bile acids combined with anticancer drugs of the present invention have high content of encapsulated drugs, exhibit cytotoxicity against cancer cells, and can be expected to have a sustained anticancer effect because the anticancer drugs are continuously released in vivo. have.

In addition, the present invention

(a) dissolving hydrophobic bile acid in a solvent with an active agent and stirring to prepare a bile acid solution to activate the bile acid;

(b) dissolving the hydrophilic chitosan oligosaccharide in a solvent to prepare a chitosan oligosaccharide solution;

(c) adding the activated bile acid solution to the chitosan oligosaccharide solution and reacting to prepare a chitosan oligosaccharide solution in which bile acid is bound;

(d) dialysis and filtration of the bile acid-bound chitosan oligosaccharide solution to produce bile acid-bound chitosan oligosaccharide nanoparticles; And

(e) adding a anticancer agent to the chitosan oligosaccharide nanoparticle solution to which the bile acid is coupled to provide a method for producing a hydrophobic bile acid-bound hydrophilic chitosan oligosaccharide, wherein the anticancer agent is encapsulated. do.

First, in step (a), a hydrophobic bile acid is dissolved in a solvent together with an active agent and stirred to prepare a bile acid solution to prepare a bile acid-activator, which is an active form of bile acid, and the activation process is performed by a substitution reaction as in Scheme 1 below.

The hydrophobic bile acid is lithocholic acid (lithocholic acid), deoxycholic acid (deoxycholic acid), cholic acid (cholic acid), kenodioxycholic acid (chenodeoxycholic acid), 7-oxo-lithocholic acid and A bile acid selected from the group containing 5β-cholanic acid is preferred, and more preferably lithocholic acid and deoxycholic acid can be used. In addition, the solvent may be any solvent capable of dissolving the hydrophobic bile acid, preferably tetrahydrofuran (THF), dichloromethane or chloroform, and the like, and more preferably tetrahydrofuran. The hydrophobic bile acid is added to 5 to 20 g with respect to 100 ml of the solvent to prepare a bile acid solution.

In addition, the active agent is added to increase the efficiency of the reaction between the carboxylic acid of the bile acid and the primary amine of the chitosan, and preferably can be activated using N-hydroxysuccinimide (NHS). The active agent is preferably 1.9 to 2.3 mole times, more preferably 2.1 mole times with respect to the bile acid, and if it does not fall within the above range, the bile acid is not sufficiently activated and the active agent is not used economically because it is used in excess of the above range.

In addition, it is preferable to perform the said stirring for a sufficient time under nitrogen stream, More preferably, it carries out for 15 to 30 hours. Impurities such as dicyclohexylurea produced in the solution after the stirring are removed by a conventional filtration method including a method using a paper filter, etc., and the solution is concentrated by a conventional concentration method including concentration under reduced pressure. In addition, activated bile acids having high purity can be obtained through a step of precipitation using a solvent such as benzene or hexane, vacuum drying, or the like.

Next, in step (b), the hydrophilic chitosan oligosaccharide is dissolved in a solvent to prepare a chitosan oligosaccharide solution.

The hydrophilic chitosan oligosaccharides are partial decomposition products of chitosan, chitosan oligosaccharides are well absorbed by our bodies, and because they are soluble in water, it is easy to add to any food and use as a main ingredient. In addition, it is a physiological functional material that attracts attention as it is found to be a wide range of high functional bioactive substances such as immune enhancement, antibacterial action, cholesterol control, and calcium absorption promotion due to its high water solubility. In the present invention, chitosan oligosaccharides having an average molecular weight of 8 to 10 kDa may be preferably used. By using a low molecular weight chitosan oligosaccharide, it is easy to apply to the injection and to control the molecular weight by overcoming the difficulty of applying to the injection when using a high molecular weight.

The solvent may be any solvent capable of dissolving the hydrophilic chitosan oligosaccharide, and preferably water, DMSO, or a mixed solvent thereof may be used.

The chitosan solution is preferably prepared by adding 1500 to 2500 mg of the hydrophilic chitosan oligosaccharide with respect to 100 ml of the solvent.

Next, in step (c), the bile acid activated in step (a) is added to the chitosan oligosaccharide solution prepared in step (b) and reacted to prepare a bile acid-bound chitosan oligosaccharide solution, as shown in Scheme 2 below. The reaction proceeds by the substitution of the amine of chitosan by bile acid.

The activated bile acid (bile acid-activator) is preferably added at a molar ratio of 1 to 40% with respect to the hydrophilic chitosan oligosaccharide monomer, and the reaction is carried out at room temperature for a sufficient time, preferably 20 to 30 hours.

Next, in step (d), the bile acid-bound chitosan oligosaccharide solution in which the reaction is completed in step (c) is dialyzed and filtered to produce chitosan oligosaccharide nanoparticles in which bile acid is bound.

In order to recover the bile acid-bound chitosan oligosaccharides from the bile acid-bound chitosan oligosaccharide solution, unreacted bile acids and impurities are prepared using various solvents, preferably diethyl ether, acetone, or a mixed solvent thereof. The chitosan oligosaccharides to which bile acids are combined can be precipitated and recovered by centrifugation.

The recovered bile acid-bound chitosan oligosaccharides are dissolved in a water-soluble solvent such as water and dialyzed, and passed through a filter having a 1.0-1.5 μm pore to remove large particles, thereby obtaining chitosan oligosaccharide nanoparticles having bile acids bound thereto. Can be. In addition, it is possible to obtain chitosan oligosaccharide nanoparticles to which bile acids are combined in a solid form by lyophilization. The produced bile acid-bound chitosan oligosaccharide nanoparticles are very good drug carrier because the drug loading rate is very high, such as 70% or more.

Next, in step (e), the anticancer agent is added to the chitosan oligosaccharide nanoparticle solution in which the bile acid is combined in the step (d), and the anticancer agent is encapsulated inside the nanoparticles. Bound chitosan oligosaccharide nanoparticles are prepared.

The bile acid-bound chitosan oligosaccharide nanoparticles will have a hydrophobic nucleus and a hydrophilic shell structure, and can be encapsulated with a hydrophobic anticancer agent by using hydrophobic interaction in the hydrophobic nucleus of the nanoparticle.

The anticancer agent may be any hydrophobic anticancer agent that may be encapsulated inside the chitosan oligosaccharide nanoparticles to which the bile acid of the self-aggregated form is bound, and preferably, paclitaxel, cis-platin, mitomycin-C, daunooma Anticancer agents selected from the group consisting of daunomycin, doxorubicin and 5-fluorouracil may be used, and more preferably paclitaxel may be used. In addition, the solvent for dissolving the anticancer agent may be any organic solvent capable of dissolving it, preferably DMSO can be used, 0.1 to 0.3 times by weight, preferably 0.2 times by weight based on chitosan oligosaccharide nanoparticles with bile acids Can be added.

The hydrophobic anticancer agent selectively penetrates into the chitosan oligosaccharide nanoparticles to which bile acids are bound. In this case, various methods may be used, and nanoparticles containing an anticancer agent may be prepared through a modified solvent evaporation method using ultrasonic irradiation. In addition, since DMSO, an organic solvent preferably used for dissolving anticancer agents, has a very low volatility and a high boiling point, a conventional dialysis method may be used to remove the organic solvent, and the macroparticles may be removed through filtration and concentration. Can be rougher.

The bile acid-bound chitosan oligosaccharide nanoparticles prepared by the method of the present invention described above have both a hydrophilic group and a hydrophobic group to form a self-aggregated form, and the drug encapsulation rate is very high, and thus may be usefully used as a drug carrier. The bile acid-bound chitosan oligosaccharide nanoparticles encapsulated with the anticancer agent of the present invention exhibit cytotoxicity against cancer cells and have an effective anticancer effect of effectively reducing the size of cancer in cancer experimental animals by continuously releasing drugs in vivo. .

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited thereto.

< Production Example  1 to 6 Of the present invention Bile acids Combined  Preparation of Chitosan Oligosaccharide Nanoparticles

3-dicyclohexyl carbodiimide (DCC), including lithocholic acid (LA) and deoxycholic acid (DA), which are bile acids used to prepare bile acid-bound chitosan oligosaccharide nanoparticles ), N-Hydroxy succinimide (NHS), L-lactic acid, D-manitol, and pyrene are available from Sigma ( USA). In addition, tetrahydrofuran (THF), a reaction solvent, was dried for 48 hours using lithium aluminum hydride and distilled. In addition, the solvents used in the experiments n-hexane, benzene, diethyl ether and the like were used without purification by using a first-class and a special reagent.

<1-1> Preparation of Chitosan Oligosaccharides

Chitosan oligosaccharides to be used to prepare chitosan oligosaccharide nanoparticles bound to bile acids were prepared as follows.

Chitosan oligosaccharides were isolated from lactate chitosan purchased from Kittolife Co. Korea. The lactate chitosan was a mixture of chitosan having various molecular weights, and was separated into chitosan oligosaccharides having a narrow molecular weight distribution of various molecular weights (molecular weights 1.5 kDa to 9 kDa) by ultrafiltration, and among them, the present invention showed an average molecular weight of 9 kDa. Fractions of chitosan oligosaccharides were used.

<1-2> Bile acid  Activation

The preparation of chitosan oligosaccharide nanoparticles in which bile acids are bound is carried out through the reaction of carboxylic acids of bile acids with primary amines of chitosan. In order to increase the efficiency, carboxylic acid in bile acids is converted to N-hydroxy succinimide (NHS). ) Was used for the reaction after activation. The activation reaction of bile acids was carried out as follows.

As bile acid, 5 g of lytocholic acid or dioxycholic acid is combined with 3 g of NHS (2.1 molar excess for bile acid) and 5.3 g of 3-dicyclohexyl carbodimide (DCC) (2 molar excess for bile acid). It was dissolved in 100 ml of dry THF. The solution was allowed to react with sufficient stirring for 24 hours under a stream of nitrogen. The dicyclohexylurea (DCU) produced after the reaction was removed using a paper filter, the reaction solution was concentrated under reduced pressure, and then precipitated in excess n-hexane (at least 10 vol. The bile acids Litocholic Acid-NHS and Dioxycholic Acid-NHS were obtained. In order to improve the purity of the obtained product and to remove impurities, the product was further dried in benzene after vacuum drying and then precipitated in n-hexane. The final product was dried in a vacuum for 48 hours to obtain a powder, the reaction and purity was confirmed by using ¹ H NMR in Experimental Example 1-1.

<1-3> Bile acids Combined  Synthesis of Chitosan Derivatives and Preparation of Nanoparticles

The reaction of the activated bile acid and chitosan prepared in the above <1-1> or <1-2> is performed through the coupling between bile acid-NHS and the primary amine (NH _{2 in} C2-position) of chitosan. Was used.

300 mg of chitosan oligosaccharides having an average molecular weight of 9 kDa were dissolved in 15 ml of distilled water / DMSO mixed solvent (10/90, v / v), and then litocholic acid-NHS or deoxycholic acid-NHS was dissolved in chitosan, respectively. The reaction was carried out at room temperature for 24 hours by adding 5%, 10% or 20% in the molar ratio. The reaction was recovered by centrifugation after precipitation in a mixed volume of diethyl ether and acetone (2: 1, v / v) in excess of 10 volumes. The recovered precipitate was dispersed in pure acetone to dissolve unreacted bile acid and impurities in the reaction, and then the pure reactant was recovered by centrifugation.

Next, the recovered reactants were dissolved in water, and then bile acid-bound chitosan nanoparticles were prepared through dialysis (MWCO 3500). After the preparation of the nanoparticles, the solution was passed through a filter having a pore size of 1.2 μm to remove large particles, followed by lyophilization, and the lytocholic acid-chitosan oligosaccharide, which is a chitosan oligosaccharide nanoparticle of the bile acid of the present invention in solid form ( Preparation Examples 1 to 3) and deoxycholic acid-chitosan oligosaccharides (Preparation Examples 4 to 6) were obtained and summarized in Table 1.

Production Example Used bile acid % Of bile acids used for chitosan monomer One Litocholanic acid 5 2 10 3 20 4 Deoxycholic acid 5 5 10 6 20

< Example  1 to 6> the anticancer agent of the present invention is enclosed, Bile acids Combined  Chitosan oligosaccharides Preparation of Nanoparticles 1

Chitosan oligosaccharide nanoparticles conjugated with bile acids prepared in Preparation Examples 1 to 6 have a hydrophobic nucleus and a hydrophilic shell, and paclitaxel, which is a hydrophobic anticancer agent using hydrophobic interaction to the hydrophobic nucleus of the nanoparticles. (paclitaxel) was enclosed as follows.

20 mg of the chitosan oligosaccharides bound to the lytocholic acid or chitosan oligosaccharides bound to the dioxycholic acid prepared in Preparation Examples 1 to 6 were dissolved in a mixed solvent of distilled water and DMSO (2: 8.5, v / v) and then paclitaxel. 4 mg was dissolved in 0.5 ml of DMSO and added. The mixed solution was slowly added dropwise while irradiating 200 ml of distilled water with ultrasonic waves to prepare chitosan oligosaccharide nanoparticles containing bile acids packed with paclitaxel. DMSO, an organic solvent used for encapsulation after preparation, was removed for 24 hours through dialysis (dialysis membrane MWCO: 3500 Da). After removal of the organic solvent, the solution was concentrated using an ultrafiltration apparatus (approximately 10 ml, ultrafiltration membrane MWCO: 10 kDa), followed by lyophilization of lytocholic acid-chitosan oligosaccharide (Examples 1 to 3) or Pa. Oxycholic acid-chitosan oligosaccharides (Examples 4 to 6) nanoparticles were obtained.

After lyophilization, the nanoparticles were dispersed in distilled water to 1 mg / mL and large coarse particles were removed through a filter (0.8 μm syringe filter). After removing the macroparticles, the solution was used to measure the encapsulation efficiency of paclitaxel and the size and distribution of nanoparticles. Finally, the final product was obtained through lyophilization of the solution. In order to facilitate redispersion during lyophilization, 50-100 mg of D-mannitol was mixed per 2 mg of paclitaxel in the solution and then lyophilized.

< Example  7 to 8> the anticancer agent of the present invention Enclosed , Bile acids Combined  Chitosan oligosaccharides Preparation of Nanoparticles 2

Chitosan oligosaccharides to which bile acids obtained in Preparation Example 2 or Preparation Example 5 were combined with cis-platin as an anticancer agent, cis-platin of the present invention was encapsulated by the method of Examples 1 to 6, and bile acids were bound. Chitosan oligosaccharide nanoparticles were prepared.

< Example  9 to 10> the anticancer agent of the present invention Enclosed , Bile acids Combined  Chitosan oligosaccharides Preparation of Nanoparticles 3

Chitosan oligosaccharides bound to bile acids obtained in Preparation Example 2 or Preparation Example 5 and mitomycin-C of the present invention by using the mitomycin-C as an anticancer agent, the bile acids are bound by the method of Examples 1 to 6 Chitosan oligosaccharide nanoparticles were prepared.

< Example  11 to 12> the anticancer agent of the present invention Enclosed , Bile acids Combined  Chitosan oligosaccharides Preparation of Nanoparticles 4

Chitosan oligosaccharides bound to bile acids obtained in Preparation Example 2 or Preparation Example 5 using daunomycin as an anticancer agent, the daunomycin of the present invention is encapsulated by the method of Examples 1 to 6, and bile acid-bound chitosan Oligosaccharide nanoparticles were prepared.

< Example  13 to 14> the anticancer agent of the present invention Enclosed , Bile acids Combined  Chitosan oligosaccharides Preparation of Nanoparticles 5

By using bixolic acid-bound chitosan oligosaccharide obtained in Preparation Example 2 or Preparation Example 5 and doxorubicin as an anticancer agent, the doxorubicin-encapsulated chitosan oligosaccharide nanoparticles of the present invention are encapsulated in the method of Examples 1 to 6 above. Prepared.

< Example  15 to 16> the anticancer agent of the present invention Enclosed , Bile acids Combined  Chitosan oligosaccharides Preparation of Nanoparticles 6

The bile acid, in which 5-fluorouracil of the present invention was encapsulated by the method of Examples 1 to 6, using 5-fluorouracil as a chitosan oligosaccharide combined with bile acids obtained in Preparation Example 2 or Preparation Example 5 and an anticancer agent. This bound chitosan oligosaccharide nanoparticles were prepared.

Table 2 summarizes chitosan oligosaccharide nanoparticles containing bile acids packed with anticancer agents of the present invention prepared in Examples 1 to 16.

Example Used bile acid % Of bile acids used for chitosan monomer Used anticancer One Litocholanic acid 5 Paclitaxel 2 10 3 20 4 Deoxycholic acid 5 5 10 6 20 7 Litocholanic acid 10 Cis-platin 8 Deoxycholic acid 10 9 Litocholanic acid 10 Mitomycin-C 10 Deoxycholic acid 10 11 Litocholanic acid 10 Daunomycin 12 Deoxycholic acid 10 13 Litocholanic acid 10 Doxorubicin 14 Deoxycholic acid 10 15 Litocholanic acid 10 5-fluorouracil 16 Deoxycholic acid 10

< Experimental Example  1> according to the present invention Bile acids Combined  Chitosan oligosaccharides Analysis of Nanoparticles

<1-1> ^One H-NMR Spectrum Analysis

The structure of the bile acid-bound chitosan oligosaccharide nanoparticles prepared in Preparation Examples 1 to 6 and the degree of substitution of the bile acid were determined using a ¹ H-NMR spectrometer (Bruker, 400 MHz). It was. In this case, a mixed solvent of D ₂ 0 / DMSO (1: 3, v / v) was used as the NMR solvent.

The analysis results are shown in the following <1-4>.

<1-2> Measurement of Nanoparticle Size and Distribution

Particle size and distribution of chitosan nanoparticles to which bile acids prepared in Preparation Examples 1 to 6 were measured using a light scattering method. The nanoparticles prepared for measurement were dispersed in distilled water at a concentration of 1 mg / ml, and the scattering (scattering) angle was fixed at 90 ° using OTSUKA ELS-8000 and measured at 632.8 nm with a He-Ne laser.

The analysis results are shown in the following <1-4>.

<1-3> Critical micelle formation concentration ( CMC ) Measure

Chitosan nanoparticles to which bile acids prepared in Preparation Examples 1 to 6 are combined as amphiphiles having hydrophilicity and hydrophobicity in one molecule, thereby forming nanoparticles by self-aggregation in aqueous solution. This self-agglomeration tendency was investigated through the observation of optical behavior of pyrene with concentration using pyrene as a fluorescent dye. For the experiment, pyrene was prepared in acetone 6 × 10 ⁻⁵ M and added to distilled water so as to give a final concentration of 1.2 × 10 ⁻⁶ M. The solution was removed acetone under reduced pressure conditions at 40 ℃ for 2 hours. The pyrene solution and a solution of various concentrations of nanoparticles (1.0 × 10 ⁻⁵ to 1 mg / ml) were mixed at a ratio of 1: 1 so that the final concentration of pyrene was 6.0 × 10 ⁻⁷ M. After leaving the mixed solution at 60 ° C. for about 3 hours, fluorescence characteristics were observed using a fluorescence intensometer. Fluorescence analysis was measured at an absorption wavelength of 390 nm. The critical micelle concentration (CMC) was measured using the measured fluorescence spectra and shift of the emission wavelength. In the case of the dioxycholic acid-chitosan oligosaccharide nanoparticles and the litocholic acid-chitosan oligosaccharide nanoparticles according to the present invention, the strongest characteristic peak was shown at 335 nm at a concentration below CMC. With the increase, it moved to 339 nm at high concentration. Using the intensity ratio of these characteristic peaks, the CMC values of the deoxycholic acid-chitosan oligosaccharide nanoparticles and the lytocholic acid-chitosan oligosaccharide nanoparticles combined with the lytocholic acid were determined.

The analysis results are shown in the following <1-4>.

<1-4> analysis results

<1-4-1> Bile acid  Activation

Representative ¹ H NMR spectra of bile acids (lithocolic acid) and activated bile acids (lithocolic acid-NHS) before activation of Preparation Examples 1 to 3 are shown in FIGS. 1 and 2.

As shown in Fig. 1, the characteristic peaks of bile acids (lithocolic acid) include methyl 19 and 20 (C H ₂ , two next to carboxylic acid) in bile acids (litokolic acid) appearing at 2.0 to 2.3 ppm. The peak by group was confirmed.

In addition, as shown in FIG. 2, it was confirmed that the carboxylic acid in the bile acid was converted to NHS-ester by confirming that the peak around 2.0 to 2.3 ppm was completely shifted to around 2.65 to 2.85 ppm after the activation process.

In addition, the excess NHS was used in the activation process, and it was confirmed that some of the NHS used was not completely removed through the purification process by observing the NHS characteristic methyl peak around 2.85-2.95 ppm.

The purity of the final product could also be calculated using the integral value of the 4.6 ppm 3'-OH -C H peak or 0.64 ppm 18'-C H ₃ peak. The purity of the final activated bile acid-NHS (lithocolic acid-NHS) was 85-90% by weight.

<1-4-2> according to the present invention Bile acids Combined  Analysis of Chitosan Oligosaccharide Nanoparticles

Chemical structure and bile acid substitution degree of the product obtained after the reaction of the activated bile acid and chitosan oligosaccharide in Preparation Examples 1 to 6 were measured using ¹ H NMR spectrum. For the measurement of substitution degree, -CH at the C2 position, which is a characteristic peak of chitosan, was used (FIGS. 3 and 4), and an 18′-CH ₃ peak (FIG. 4) was used as a characteristic peak of bile acid. The bile acid substitution degree was calculated | required using the integral value of each characteristic peak.

In addition, the size and distribution of the bile acid-bound chitosan oligosaccharide nanoparticles prepared in Preparation Examples 1 to 6 were measured using dynamic light scattering.

The results are shown in Table 1, FIG. 5 and FIG.

Characteristics of Chitosan Oligosaccharide Nanoparticles with Litocolic Acid and Chitosan Oligosaccharide Nanoparticles with Deoxycholic Acid Production Example Used bile acid % Of bile acids used for chitosan monomer DS (%) ^a d (nm) ^b CMC (g / L) ^c One Litocholic acid 5 4.5 213.9 0.0385 2 10 8.2 295.7 0.0108 3 20 15.9 292.6 0.0018 4 Deoxycholic acid 5 4.4 174.1 0.0629 5 10 8.1 279.6 0.0130 6 20 16.1 203.4 0.0063 ^a : Calculation of degree of substitution by ¹ H-NMR ^b : Measurement of nanoparticle size using DLS ^c : Measurement of critical micelle concentration by fluorescence photometer

As shown in Table 1, the degree of substitution of bile acids showed results close to the dose of the initial reactants. When the bile acid was reacted 5% in a molar ratio with respect to the chitosan monomer, the use of lithocolic acid as bile acid (Preparation Example 1) showed a substitution degree of 4.5%, and the use of deoxycholic acid as bile acid (Preparation Example 4 ) Shows a substitution degree of 4.4%. In addition, when bile acid was reacted at a molar ratio of 20% with respect to the chitosan monomer, when litocolic acid was used as the bile acid (Preparation Example 3), the substitution degree was 15.9%, and when dioxycholic acid was used as the bile acid (Preparation Example 6) shows a substitution degree of 16.1%. Therefore, it was confirmed that the reaction between the activated bile acid and chitosan oligosaccharide proceeded successfully.

In addition, as shown in Table 1 and Figure 5, the nanoparticles prepared according to the production method of the present invention showed a very narrow particle size distribution, using dioxycholic acid as bile acid and reacted 5% in a molar ratio compared to the chitosan monomer. In case (Preparation 4), a size of about 174 nm is shown. The chitosan oligosaccharide nanoparticles combined with other bile acids showed a particle size of approximately 200-300 nm.

In addition, as shown in Table 1 and Figure 6, I ₃₃₉ / I ₃₃₅ value of the intensity ratio (intensity ratio) of the characteristic peak showed a constant value at low concentrations, but showed a sharp increase with the increase in concentration. In general, the point where a sharp increase in the intensity ratio is set to the value of CMC, it was possible to measure the CMC of the chitosan oligosaccharide nanoparticles with bile acids according to the present invention using the results of FIG. The chitosan oligosaccharide nanoparticles bound to litocolic acid showed a CMC value of 0.0018 to 0.0385 g / L, and the chitosan oligosaccharide nanoparticles bound to deoxycholic acid showed 0.0063 to 0.0629 g / L. In addition, it was confirmed that the CMC value decreases due to the increase in hydrophobicity as the degree of substitution of bile acids increases. In addition, the characteristics of the bile acid itself also confirmed that it affects the CMC value. In this case, the chitosan oligosaccharide nanoparticles having the more hydrophobic litocolic acid bound showed lower CMC values at the same substitution condition than the chitosan oligosaccharide nanoparticles having the dioxycholic acid bound.

< Experimental Example  2> encapsulated anticancer agent of the present invention, Bile acids Combined  Analysis of Chitosan Oligosaccharide Nanoparticles

The particle size and distribution of the paclitaxel-encapsulated nanoparticles prepared in Examples 1 to 6 were measured, and the encapsulation efficiency and drug loading of paclitaxel were measured by HPLC analysis.

<2-1> Particle size and distribution measurement

Particle size and size distribution of the paclitaxel-encapsulated chitosan oligosaccharide nanoparticles were measured by dynamic light scattering method using OTSUKA ELS-8000 after dispersing the prepared nanoparticles in distilled water at a concentration of 1 mg / ml. The scattering angle was fixed at 90 ° and measured at 632.8 nm with a He-Ne laser.

The analysis results are shown in the following <2-3>.

<2-2> Liquid Chromatography High Performance Liquid Chromatograph

Encapsulation rate and drug loading of the nanoparticles were analyzed using HPCL (Shimatzu, Japan). For analysis, the solvent in the sample used a mixed solvent of 40% acetonitrile / 60% H ₂ O, and prepared through appropriate dilution. The analysis was carried out at 25 ° C. at a flow rate of 1 ml / min, and the detection of the drug was measured at 227 nm wavelength using a UV detector. At this time, the column used was a C18 column (4.6 × 250 mm) was used as the particle size of 5 ㎛. The mobile phase was analyzed by changing the composition as follows: 34% acetonitrile / 66% H ₂ O (5 minutes) → 34 to 58% acetonitrile (linear gradient, additional 16 minutes) → 58 to 70 % Acetonitrile (linear concentration, additional 2 min) → 70-34% acetonitrile (linear concentration, additional 4 min) → 34% acetonitrile (additional 5 min). For quantification, a standard curve was prepared by using the concentration of paclitaxel and the area change of the characteristic peak, and then the concentration of paclitaxel in the sample was calculated. In addition, the drug encapsulation rate and the drug loading amount were calculated using the following equations (1) and (2).

The analysis results are shown in the following <2-3>.

<2-3> analysis result

The analysis results of chitosan oligosaccharide nanoparticles in which bile acids packed with paclitaxel of the present invention by the methods of Experimental Examples <2-1> and <2-2> are shown in Table 4 and FIGS. 7 to 9.

Example Weight (mg) of chitosan oligosaccharide nanoparticles to which bile acids of the present invention are bound Paclitaxel (mg) Nanoparticle size (nm) Inclusion Rate (%) Drug loading amount (%) One 20 4 355 68.5 13.4 2 20 4 407 67.0 13.7 3 20 4 436 54.5 11.7 4 20 4 385 38.5 7.5 5 20 4 368 72.3 13.8 6 20 4 445 53.8 12.9

As shown in Table 4 and Figure 7, the paclitaxel-encapsulated nanoparticles showed a distribution in unimodal form. In addition, as the paclitaxel was encapsulated, the size of the nanoparticles was increased to confirm that the nanoparticles having a size of 350 to 450 nm were formed.

In addition, as shown in FIG. 8, paclitaxel and paclitaxel encapsulated in the nanoparticles of Example 2 were confirmed to exhibit the same retention time and peak shape under the same HPLC analysis conditions. In addition, as shown in FIG. 9, in the case of the calibration curve obtained using the paclitaxel standard product, it was confirmed that a calibration curve of orthogonal form was formed in the range of 0.1 μg / ml to 50 μg / ml. The inclusion rate and drug loading of the paclitaxel-encapsulated nanoparticles measured using the calibration curve are shown in Table 4 above.

Among the nanoparticles of Examples 1 to 3, wherein the bile acid was used as the bile acid, in the case of Example 3 having a high degree of bile acid substitution, the nanoparticles were relatively large, and thus, the encapsulation rate was relatively low due to the formation of macroparticles and the removal using a filter. And drug loading. On the other hand, Examples 1 and 2 showed similar encapsulation rates (68.5% and 67%, respectively) and loading amounts (13.4% and 13.7%, respectively).

Also, among the nanoparticles of Examples 4 to 6 in which dioxycholic acid was used as the bile acid, the nanoparticles of Example 4 having a low degree of bile acid substitution had a high loading rate (38.5%) and drug loading (7.5%) for the same reasons as in Example 1. Indicated. On the other hand, Example 6, which has a high degree of bile acid substitution, showed a relatively low loading rate and drug loading amount for the same reason as in Example 3. As a result, the nanoparticles of Examples 4 to 6 using dioxycholic acid as bile acid showed an optimized encapsulation rate (72.3%) and drug loading (13.8%).

Therefore, the chitosan oligosaccharide nanoparticles to which bile acids are bound according to the present invention have excellent drug encapsulation rate and drug supporting ability, and thus, it was confirmed that the bile acid-conjugated chitosan oligosaccharide nanoparticles can be used as a carrier of an anticancer agent such as paclitaxel.

< Experimental Example  3> of the present invention Paclitaxel Enclosed Bile acids Combined  Cytotoxicity Test of Cancer Cells by Chitosan Oligosaccharide Nanoparticles

The anticancer activity of the paclitaxel-embedded bile acid-bound chitosan oligosaccharide nanoparticles was investigated by measuring cytotoxicity using cancer cells.

B16F10 melanoma cells (melanoma cells) were used for the experiment, and the cells were used after culturing with DMEM containing 10% FBS. The experiment used a 96 well plate. About 5000 cells were dispensed for drug treatment and placed in each well with 90 μl of culture, and further cultured for about a day to allow cells to stably attach. Here, the chitosan oligosaccharide nanoparticles containing paclitaxel or the paclitaxel-encapsulated bile acids prepared in Example 2 or Example 5 have various concentrations of the drug solution so that the final concentration is 10, 1, 0.1, 0.01 or 0.001 μg / ml. After administration of 10 μl was further cultured to the experimental group. As a control group, a group treated with PBS was used. After approximately 48 hours of additional culture, 10 μl of MTT solution (5 mg / ml in PBS) was added to each well, followed by incubation for about 2 hours to induce oxidation of MTT by viable cells. After the final culture, the cell culture was removed, and then, 100 μl of DMSO was used to dissolve the dark purple MTT oxide formed by the viable cells, and the absorbance was measured at 570 nm. The percentage of viable cells was calculated using Equation 3 below. At this time, OD _{570 ( no sample)} was used as the measured value of the absorbance of the well itself.

Cell survival rate (%) = [OD _{570 ( Experimental )} -OD _{570 ( Ignore )} ] / [OD _{570 (Control)} -OD _{570 ( Ignore )} ] × 100

The results are shown in FIG.

As shown in FIG. 10, B16F10 melanoma cells, which are cancer cells, showed cellular activity inversely proportional to the concentration of paclitaxel administered. In addition, both paclitaxel and paclitaxel-embedded nanoparticles under the same drug concentration showed similar cytotoxicity. Therefore, it was found that the anticancer agent encapsulated in the chitosan oligosaccharide nanoparticles having the bile acid including the anticancer agent of the present invention effectively exhibited the medicinal effect of the drug itself.

< Experimental Example  4> of the present invention Paclitaxel Enclosed Bile acids Combined  Anti-cancer activity measurement of chitosan oligosaccharide nanoparticles (cancer cancer suppression experiment)

In order to measure the anticancer activity of paclitaxel-encapsulated bile acid-bound chitosan oligosaccharide nanoparticles, the degree of inhibition of cancer proliferation was measured using experimental animals as follows.

Experiments were carried out using cancer-induced C57BL6 mice through B16F10 melanoma cell transplantation. Induction of cancer was induced by implanting about 1 × 10 ⁶ cancer cells in passaged cultures in each mouse. The paclitaxel-encapsulated chitosan oligosaccharide nanoparticles or paclitaxel of the present invention prepared in Examples 2 or 5 are about one week after cancer cell transplantation, and the size of the grown cancer is about 50 to 100 mm 3. Administration was at an amount of 10 mg / kg through the tail vein. At this time, paclitaxel was dissolved in 12 mg / kg in a solution in which cremophor EL: ethanol was mixed 1: 1 and then diluted 1/6, and paclitaxel of the present invention prepared in Examples 2 or 5 above. As the encapsulated bile acid-bound chitosan oligosaccharide nanoparticles, a diluent of 2 mg / kg concentration was used. In addition, the drug was administered at a dose of 125 μl at a time, four times at two-day intervals (day 0, 2, 4, 6), and the saline group was used as a control group. Observation of cancer growth behavior after drug administration was performed by measuring the size (volume) of the cancer, and the size of the cancer was calculated using Equation 4 below.

Arm size (volume, thickness) = [arm short diameter] ² × [arm long diameter] × 1/2

The results are shown in FIGS. 11 and 12.

As shown in Figure 11, in the case of the control group it can be seen that a rapid increase in cancer size over time. In particular, the size of the cancer, which was 50 ~ 70 실험 at the beginning of the experiment, showed a cancer size of about 7700 후 after 16 days through rapid proliferation. Paclitaxel was dissolved in a mixed solvent of Cremophor EL: ethanol 1: 1 mixed with the same administration method, and showed a slight cancer suppression efficiency in animals administered with dilution. After 16 days, the average cancer cell size was 4160. Was. In the group to which the paclitaxel-containing bile acid-binding chitosan oligosaccharide nanoparticles of the present invention prepared in Examples 2 or 5 showing remarkable cancer suppression efficacy were administered, the average cancer sizes after 16 days were 2500 and 2600 mm 3, respectively. About one third of the control group), the growth of cancer was stopped during 0-6 days of continuous administration of the anticancer agent, and the proliferation of the cancer was observed after the end of the administration of the anticancer agent. . In addition, the timing of rapid cancer proliferation was also different. In the case of the control group, a sudden increase in the size of the cancer was observed after about 8 days, but in the group to which the paclitaxel-containing bile acid-binding chitosan oligosaccharide nanoparticles of the present invention prepared in Example 2 or 5 were administered, the administration was slow after the end of the anticancer drug administration. One cancer showed an increase in size.

In addition, as shown in Figure 12, the picture showing the characteristics of the cancer of the experimental animals after the end of the treatment with the cancer drug, the control group was able to confirm a very large size of the cancer and skin necrosis due to excessive proliferation of the cancer. On the other hand, in the group administered with the paclitaxel-supported nanoparticles of the present invention it can be confirmed that the cancer of a relatively small size, no skin necrosis phenomenon. Therefore, it was confirmed through animal experiments that the paclitaxel-supported nanoparticles showed a very excellent cancer growth inhibitory effect.

Hydrophilic chitosan oligosaccharide nanoparticles with hydrophobic bile acids bound to the anticancer agent of the present invention have a high drug encapsulation rate using hydrophilic chitosan oligosaccharide nanoparticles with bile acids, which form a self-aggregated form in water, and are highly resistant to cancer cells. In addition to high toxicity, the drug can be continuously released in vivo for a long time, thus exhibiting a continuous anticancer effect, and thus can be usefully used for chemotherapy for cancer.

Claims (28)

Hydrophilic chitosan oligosaccharide nanoparticles bound to hydrophobic bile acids encapsulated with an anticancer agent. According to claim 1, wherein the hydrophobic bile acid is lithocholic acid (lithocholic acid), deoxycholic acid (deoxycholic acid), cholic acid (cholic acid), chenodeoxycholic acid (chenodeoxycholic acid), 7-oxo- lysoholic acid (7- oxo-lithocholic acid) and 5β-cholanic acid (5β-cholanic acid), characterized in that the nanoparticles selected from the group consisting of. The nanoparticle of claim 2, wherein the hydrophobic bile acid is lithocholic acid or deoxycholic acid. According to claim 1, wherein the hydrophobic bile acid nanoparticles, characterized in that contained in 1 to 40% by molar ratio with respect to the hydrophilic chitosan monomer. According to claim 1, wherein the hydrophilic chitosan oligosaccharides nanoparticles, characterized in that the average molecular weight of 8 ~ 10 kDa. [Claim 2] The nanoparticles according to claim 1, wherein the critical micelle formation concentration (CMC) of the hydrophilic chitosan oligosaccharide nanoparticles to which the hydrophobic bile acid is bound is 0.0010 to 0.0700 g / l. The nanoparticle according to any one of claims 1 to 6, wherein the hydrophilic chitosan oligosaccharide nanoparticles having hydrophobic bile acids bound to the anticancer agent are 300 to 500 nm in size. The nanoparticles according to any one of claims 1 to 6, wherein the encapsulation rate of the hydrophilic chitosan oligosaccharide nanoparticles to which hydrophobic bile acids are bound is 35 to 75%. The nanoparticle of claim 1, wherein the anticancer agent is a hydrophobic anticancer agent. 10. The method of claim 9, wherein the anticancer agent is from the group comprising paclitaxel, cis-platin, mitomycin-C, daunomycin, doxorubicin, and 5-fluorouracil. Nanoparticles, characterized in that selected. The nanoparticle of claim 10, wherein the anticancer agent is paclitaxel. (a) dissolving hydrophobic bile acid in a solvent with an active agent and stirring to prepare a bile acid solution to activate the bile acid; (b) dissolving the hydrophilic chitosan oligosaccharide in a solvent to prepare a chitosan oligosaccharide solution; (c) adding the activated bile acid solution to the chitosan oligosaccharide solution and reacting to prepare a chitosan oligosaccharide solution in which bile acid is bound; (d) dialysis and filtration of the bile acid-bound chitosan oligosaccharide solution to prepare chitosan oligosaccharide nanoparticles in which bile acid is bound to self-aggregated form; And (e) adding an anticancer solution to the bile acid-bound chitosan oligosaccharide nanoparticle solution to encapsulate an anticancer agent inside the bile acid-bound chitosan oligosaccharide nanoparticles. Manufacturing method. The method according to claim 12, wherein the hydrophobic bile acid of step (a) is lithocholic acid (lithocholic acid), deoxycholic acid (deoxycholic acid), cholic acid, chenodeoxycholic acid (chenodeoxycholic acid), 7-oxo- Method for producing nanoparticles, characterized in that selected from the group comprising lysocholic acid (7-oxo-lithocholic acid) and 5β-cholanic acid (5β-cholanic acid). The method of claim 13, wherein the hydrophobic bile acid is lithocholic acid or deoxycholic acid. The method of claim 12, wherein the solvent of step (a) is tetrahydrofuran, dichloromethane or chloroform. 13. The method of claim 12, wherein the hydrophobic bile acid of step (a) is added in an amount of 5 to 20 g based on 100 ml of the solvent. The method of claim 12, wherein the active agent of step (a) is N-hydroxysuccinimide (NHS). 13. The method of claim 12, wherein the active agent of step (a) is added to the hydrophobic bile acid by 1.9 to 2.3 times by weight. The method of claim 12, wherein the hydrophilic chitosan oligosaccharide of step (b) is a method for producing nanoparticles, characterized in that the molecular weight of 8 ~ 10 kDa. The method of claim 12, wherein the solvent of step (b) is water, DMSO, or a mixed solvent thereof. The method according to claim 12, wherein the hydrophilic chitosan oligosaccharide in the step (b) is a method for producing nanoparticles, characterized in that the addition of 1500 ~ 2500mg with respect to 100ml of the solvent. The method according to claim 12, wherein the bile acid activated in step (c) is a method for producing nanoparticles, characterized in that the addition of 1 to 40% to the hydrophilic chitosan oligosaccharide monomer. The method of claim 12, wherein the filtration in step (d) is a method for producing nanoparticles, characterized in that using a filter of 1.0 ~ 1.5 ㎛ pore. The method of claim 12, wherein the anticancer agent is a hydrophobic anticancer agent. 25. The method of claim 24, wherein the anticancer agent of step (e) is paclitaxel, cis-platin, mitomycin-C, daunomycin, doxorubicin, and 5-fluorouracil Method for producing a nanoparticles, characterized in that selected from the group containing. The method of claim 25, wherein the anticancer agent is paclitaxel. 13. The method of claim 12, wherein the anticancer agent of step (e) is added to the bile acid-bound chitosan oligosaccharide nanoparticles by 0.1 to 0.3 times by weight. The method of claim 12, wherein the step of encapsulating the anticancer agent of step (e) is characterized in that the use of ultrasonic irradiation.

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