KR101107313B1 - Nanoparticles formed from alkanoylated sulfate groups-containing ploysaccharides and a process for the preparation thereof - Google Patents

Abstract

The present invention provides a nanoparticle formed by sulfuric acid group-containing polysaccharides C ₂ -C ₄ alkanoylated and amphiphilized sulfuric acid group-containing polysaccharides self-aggregate while encapsulating a drug, a preparation method thereof, and an injection including the nanoparticles. .

The nanoparticles according to the present invention are not toxic and biocompatible, and can significantly be used as drug carriers having high long-term formulation stability since the aggregation phenomenon between nanoparticles is greatly reduced. In addition, when used as a drug carrier of an anticancer agent, it is possible to accumulate anticancer agents into cancer tissue by inherent passive targeting of nanoparticles, and the anticancer agent may well be contained in cancer cells due to the cell affinity of sulfate-containing polysaccharides constituting the nanoparticles. There is an advantage to be able to work effectively inflow. In addition, since it is easy to introduce a variety of functional polymer materials to the nanoparticles, it is easy to develop a more advanced multi-functional drug carrier.

Description

Nanoparticles formed from alkanoylated sulfate groups-containing ploysaccharides and a process for the preparation according to the present invention.

The present invention relates to a new nanoparticles and a method for producing the same, and more particularly to a nanoparticle formed by self-aggregation of amphiphilized sulfuric acid group-containing polysaccharides by the introduction of a hydrophobic group and a method for producing the same.

The incidence of cancer continues to increase, and accordingly, the development of cancer treatment methods and drugs is continuously made. Currently used cancer treatments include surgery to remove only cancer cells by surgery, radiation therapy, and chemotherapy using anticancer drugs showing cytotoxicity. Chemotherapy with anticancer drugs is a widely used method for cancer treatment, but since it shows cytotoxicity not only to cancer cells but also to normal cells, there is a risk of side effects. Therefore, in the field of chemotherapy using anticancer drugs, researches are continuously conducted to reduce the side effects of anticancer drugs while maximizing cancer treatment effects.

Nanoparticles are one of drug carriers for selective target site delivery of drugs, including anticancer agents, and refers to heterogeneous dispersed particles on a colloid having a large surface area having a particle size of several nm to several hundred nm in diameter. Nanoparticles encapsulated with anticancer agents can be targeted to cancer tissues for cancer treatment without adverse effects on normal tissues without any cancer cell target residues. This targeting effect of the nanoparticles is obtained by the specific pathophysiological characteristics of cancer cells called enhanced permeability and retention (EPR) effects. The malignant neoplasm in the body grows faster than normal tissue, and requires a lot of nutrients and oxygen supply, which causes the rapid production of new blood vessels around cancer cells. In other words, because blood vessels are generated rapidly around cancer cells, these neovascularized blood vessels have large outflow holes sufficiently for the nanoparticles to escape from the blood vessels, so that the nanoparticles accumulate in the cancer tissue.

A method is known in which nanoparticles are formed by micellization of amphiphilized polysaccharides by imparting hydrophobicity to biocompatible polysaccharide materials, and using such nanoparticles as drug carriers (CT Lee et al. 2007, Biomolecular Engineering. 24, 131-139). Biocompatible polysaccharides have been reported to have hydrophilicity and cell affinity for polysaccharide materials with many hydroxyl groups, because they are hydrophilic and contain bioactive moieties (Rodrigues NS, Santos-Magalhaes NS, Coelho. LCBB, Couvreur.P., Ponchel.G., Gref.R. 2003. J. Contr.Rel. 92, 103-112). Such functional groups of biocompatible polysaccharides are known to be characterized by the ability to recognize specific mucosal adhesions and receptors (Torchilin. V. P., 2000, Eur. J. Pharm. Sci. 2, s81-s91). Therefore, nanoparticles formed by amphiphilizing biocompatible polysaccharides can be used as excellent drug carriers due to the biocompatibility of biocompatible polysaccharides in addition to the pharmaceutical properties as nanoparticles themselves.

Thus, although efforts have been made to develop nanoparticles prepared by amphiphilizing various biocompatible polysaccharides, nanoparticles cannot be easily generated by simply hydrophobizing virtually any biocompatible polysaccharide. Therefore, in order to prepare the desired nanoparticles by amphiphilizing the desired biocompatible polymers, intensive research for the selection of suitable biocompatible polymers and the selection of hydrophobization moieties is required.

Accordingly, the present inventors have studied intensively for the development of nanoparticles using biocompatible polysaccharides having excellent properties as drug carriers, and thus, nanoparticles formed by amphiphilizing sulfate-containing polysaccharides by introduction of hydrophobic moieties and then self-aggregating. It was developed to complete the present invention.

It is therefore an object of the present invention to provide nanoparticles formed from amphiphilized sulfuric acid group containing polysaccharides.

Another object of the present invention is to provide a method for preparing nanoparticles formed of the amphiphilized sulfate group-containing polysaccharide.

It is another object of the present invention to provide an injection comprising nanoparticles formed from the amphiphilized sulfate group-containing polysaccharide.

In order to achieve the above object, the present invention provides a nanoparticle formed by self-aggregation of sulfuric acid group-containing polysaccharides C ₂ -C ₄ alkanoylated and amphiphilized sulfuric acid group-containing polysaccharides encapsulating the drug.

The nanoparticles

Dissolving sulfuric acid group-containing polysaccharide and C ₂ -C ₄ alkanoic anhydride in a solvent and reacting to obtain a C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharide product;

Mixing the product with water to obtain C ₂ -C ₄ alkanoylated sulfuric acid group-containing polysaccharides to precipitate;

The obtained C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharide and the drug are dissolved in a solvent, and then put in a dialysis membrane, and then dialyzed in water so that water is introduced into the dialysis membrane, and the polysaccharide encapsulates the drug and forms a nanoparticle. Making; And

It may be prepared by a method comprising the step of recovering the nanoparticles by lyophilizing the formed nanoparticles.

The present invention also provides an injection comprising the nanoparticles formed by self-aggregation of the sulfuric acid group-containing polysaccharide is C ₂ -C ₄ alkanoylated and the amphiphilized sulfuric acid group-containing polysaccharide encapsulates the drug.

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

In the present invention, the C ₂ -C ₄ alkanoyl group is esterified by the nucleophilic substitution reaction of the hydroxyl group of the biocompatible hydrophilic polymer containing a sulfate group, and is suitable as a drug carrier by self-aggregation. It has been found that nanoparticles can be formed.

Accordingly, in one aspect, the present invention provides nanoparticles in which the sulfate-containing polysaccharide is C ₂ -C ₄ alkanoylated and amphiphilized sulfate-containing polysaccharide is self-aggregated while encapsulating the drug.

As the sulfate-containing polysaccharide, chondroitin sulfate, dermatan sulfate, heparan sulfate, curdlan sulfate, etc. may be used, but are not limited thereto, and nanoparticles may be formed by C ₂ -C ₄ alkanoylation. Any sulfate-containing polysaccharide present. Among the sulfate-containing polysaccharides, chondroitin sulfate is particularly preferable.

Chondroitin sulfate is an acidic mucopolysaccharide present in cartilage, skin, cornea, and placenta, and is a type of glycosaminoglycan (GAG), and has excellent biocompatibility and cell compatibility. The form of sulfate chondroitin may be any, and may be, for example, chondroitin-4-sulfate or chondroitin-6-sulfate. Among the chondroitin sulfates, chondroitin sulfates such as the following Chemical Formula 1, in which D-glucuronic acid, N-acetylgalactosamine and sulfate residues are present in an equivalent molar amount, may be used.

Sulfuric acid group-containing polysaccharides, such as sulfate chondroitin, are hydrophilic, including many sulfuric acid residues, and contain hydroxyl groups (-OH), and thus amphiphilicity can be imparted to sulfate-containing polysaccharides through alkanoylation of this portion. Amphiphilic sulfate chondroitin is well soluble in organic solvents and can be formed by nanoparticles through dialysis in water. The degree of alkanoylation of the sulfuric acid group-containing polysaccharide is not particularly limited, but may be reacted with 200-600 moles of C ₂ -C ₄ alkanoic acid per mole of sulfuric acid group-containing polysaccharide, and preferably 250-500 moles of C _2- React with C ₄ alkanoic acid.

C ₂ -C ₄ alkanoylation of the sulfuric acid group-containing polysaccharide can be accomplished by any method known to those skilled in the art, for example, by reaction of sulfuric acid group-containing polysaccharides with C ₂ -C ₄ alkanoic anhydride. have. The C ₂ -C ₄ among alkanoylation is preferably acetyl upset.

The drug encapsulated in the nanoparticles according to the present invention may be any drug that can be enclosed in the nanoparticles, for example, may be a hydrophobic drug. Such hydrophobic drugs can be selected from the group consisting of doxorubicin, taxol, camptothecin, adriamycin, paclitaxel, cis-platin, mitomycin-C, daunomycin and 5-fluorouracil. .

In another aspect of the present invention,

Dissolving sulfuric acid group-containing polysaccharide and C ₂ -C ₄ alkanoic anhydride in a solvent and reacting to obtain a C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharide product;

Mixing the product with water to obtain C ₂ -C ₄ alkanoylated sulfuric acid group-containing polysaccharides to precipitate;

The obtained C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharide and the drug are dissolved in a solvent, and then put in a dialysis membrane, and then dialyzed in water so that water is introduced into the dialysis membrane, and the polysaccharide encapsulates the drug and forms a nanoparticle. Making; And

Freeze-drying the formed nanoparticles to recover the nanoparticles, the sulfuric acid group-containing polysaccharides C ₂ -C ₄ alkanoylated amphiphilized sulfuric acid group-containing polysaccharides are self-aggregated nanoparticles formed by encapsulating the drug It provides a method of manufacturing.

Hereinafter, the manufacturing method will be described in more detail step by step.

1) sulfate-containing polysaccharides and C ₂ -C ₄ Alkaline anhydride is dissolved in a solvent and reacted to obtain an alkanoylated sulfate group-containing polysaccharide product.

The C ₂ -C ₄ alkanoic anhydride is preferably acetic anhydride, and has a structural formula as shown in the following formula (2).

The C ₂ -C ₄ alkanoic acid anhydride is sulfuric acid as Sikkim through a nucleophilic substitution reaction with the chondroitin sulfate -CH ₂ OH moiety changing the -CH ₂ OH moiety in the chondroitin sulfate as -CH ₂ OCOC _1-3 alkyl Hydrophobicity can be imparted to chondroitin. After the reaction of chondroitin sulfate and alkanoic anhydride is terminated, the residues of the alkanoic anhydride are given acidity. Pyridine or the like may be added to the reaction solvent to neutralize the acidity.

As the solvent used in the reaction, any solvent which does not inhibit the alkanoylation of the sulfuric acid group-containing polysaccharide and all the sulfuric acid group-containing polysaccharides and C ₂ -C ₄ alkanoic anhydride can be used, for example, formamide And the like can be used.

Although the ratio of reacting the sulfuric acid group-containing polysaccharide with C ₂ -C ₄ alkanoic acid is not particularly limited, it can be reacted with 200-600 mol of C ₂ -C ₄ alkanoic acid per mole of the sulfuric acid group-containing polysaccharide, preferably 250- React with 500 moles of C ₂ -C ₄ alkanoic acid.

The reaction can be carried out at room temperature, the reaction time can be carried out for about 8-16 hours.

2) adding water to the product, ₂ -C ₄ Obtaining by precipitating an alkanoylated sulfate group containing polysaccharide

In order to separate the alkanoylated sulfate group-containing polysaccharide produced in the step 1), the alkanoylated sulfate group-containing polysaccharide product may be mixed with water to precipitate only the alkanoylated sulfate group-containing polysaccharide given hydrophobicity. have. Since alkanoylated sulfuric acid group-containing polysaccharides have been given hydrophobicity, when the product is added to hydrophilic water, only the alkanoylated sulfuric acid group-containing polysaccharides can be dissolved and precipitated. Thus, the precipitated product can be easily separated from other solvents, unreacted substances and the like, and preferably, the precipitated product can be easily separated by centrifugation.

3) C obtained above ₂ -C ₄ Dissolving the alkanoylated sulfate group-containing polysaccharide and drug in a solvent, and then placing it in a dialysis membrane, and then dialyzing it in water so that water is introduced into the dialysis membrane, and the polysaccharide encapsulates the drug to self-aggregate to form nanoparticles.

The precipitate obtained through the step 2) (C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharides) and the drug is dissolved in a solvent, and then put into the dialysis membrane can be prepared nanoparticles by dialysis in water. Through this dialysis process, the organic solvent moves out of the dialysis membrane and distilled water moves into the membrane by osmotic pressure between the organic solvent in the dialysis membrane and the distilled water outside the membrane. Amphiphilic sulfate chondroitin imparted with hydrophobicity during this process self-aggregates to form nanoparticles. In the process of forming nanoparticles, the hydrophobic portion of the nanoparticles and the hydrophobic portion of the drug bind to each other to form a drug. It can be enclosed in the interior of the. A schematic diagram of nanoparticle formation of such C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharide is shown in FIG. 1.

When using doxorubicin as the drug, it is preferable to remove hydrochloric acid by adding triethylamine before performing the nanoparticle formation process in order to remove hydrophilicity of commercially available doxorubicin hydrochloride.

As the solvent for dissolving the precipitate and the drug, any solvent that does not inhibit the formation of the drug-encapsulated nanoparticles may be used. For example, dimethyl sulfoxide, 1,4-dioxane, or the like may be used. .

The alkanoylated sulfate group-containing polysaccharide and the external water used for dialysis of the mixed solution of the drug dissolved in the solvent in the dialysis membrane may be used as an aqueous solution containing a buffer appropriately selected by those skilled in the art according to the characteristics of the drug used. have. For example, in case of using doxorubicin, it is preferable to use a buffer solution having a pH of 9 or higher in order to maintain stability of the amine group of doxorubicin, and for example, a borate buffer salt buffer solution can be used.

The dialysis membrane used for dialysis may use a dialysis membrane through which a drug which is not encapsulated, and a solvent used to dissolve the precipitate and the drug may be penetrated, and a dialysis membrane in the range of MWCO 500-1000 may be used. When preparing nanoparticles encapsulated with a hydrophobic anticancer agent such as doxorubicin, it may vary depending on the type of hydrophobic anticancer agent, but a dialysis membrane of a range in which only a substance having a molecular weight of 1,000 or less can be removed may be used. By the dialysis process, the amphiphilic alkanoylated sulfate group-containing polysaccharide can form nanoparticles by self-aggregation by entraining the drug by water introduced through the dialysis membrane. With the formation of such nanoparticles, impurities and solvents that can be formed as by-products can be removed, and unsealed drugs and unpolymerized alkanoic anhydrides can be removed.

4) recovering the nanoparticles by lyophilizing the formed nanoparticles.

By lyophilizing the nanoparticles produced and purified by the dialysis process, only pure nanoparticles encapsulated with the drug can be recovered.

The nanoparticles according to the present invention can be used as a carrier of various drugs, including hydrophobic drugs, sulfuric acid group-containing polysaccharide constituting the nanoparticles are non-toxic, biodegradable biodegradable, and a large number of sulfate groups in the polysaccharides It is included, the potential of the nanoparticles has a negative charge can significantly reduce the aggregation phenomenon between the particles. Thus, the nanoparticles according to the present invention can be used as drug carriers that are biocompatible and exhibit long-term formulation stability in which long-term storage does not occur between particles. In addition, the sustained release of the drug, which is a natural property of nanoparticles, can be expected. Moreover, the use of the drug carrier as an anticancer agent can reduce various side effects of the anticancer agent by passive cancer targeting, and also accumulate in the cancer tissue by passive cancer targeting due to the cell affinity of the sulfate-containing polysaccharide constituting the nanoparticles. As the nanoparticles are well introduced into the cancer cells and are broken down in the cells, the encapsulated anticancer agent acts to effectively kill only the cancer cells. In addition, the alkanoylated sulfate group-containing polysaccharide constituting the nanoparticles has a variety of functional groups, it is easy to introduce a functional polymer material by using such a functional group it is easy to develop a more advanced multi-functional drug carrier.

The nanoparticles according to the present invention can be administered to a living body by injection. Thus, in another aspect, the present invention provides an injection comprising the nanoparticles according to the present invention.

Such injections may be prepared according to conventional methods for preparing injections known in the art. Injectables according to the present invention may be in a form dispersed in a sterile medium so that it can be used as it is when administered to a patient, or may be administered in a form in which distilled water for injection is added and dispersed in an appropriate concentration.

As described above, the nanoparticles according to the present invention are not toxic and biocompatible, and the aggregation phenomenon between the nanoparticles is greatly reduced, so that the nanoparticles can be used as drug carriers having high long-term formulation stability. In addition, when used as a drug carrier of an anticancer agent, it is possible to accumulate anticancer agents into cancer tissue by inherent passive targeting of nanoparticles, and the anticancer agent may well be contained in cancer cells due to the cell affinity of sulfate-containing polysaccharides constituting the nanoparticles. There is an advantage to be able to work effectively inflow. In addition, since it is easy to introduce a variety of functional polymer materials to the nanoparticles, it is easy to develop a more advanced multi-functional drug carrier.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Example 1 Preparation of Chondroitin Sulfate Nanoparticles Containing Doxorubicin

1) Hydrophobicity of Chondroitin Sulfate (Acetylation of Chondroitin Sulfate)

Dissolve 1 g of chondroitin sulfate (Carl ROTH, Germany) in 10 ml of formamide (kanto chemical, Japan) at a temperature of 40 ° C, add 3 mL of pyridine (Junsei, Japan), and after 30 minutes, acetic anhydride (Junsei, Japan) ) Was added 7 ml. The reaction was carried out for 12 hours after adding acetic anhydride.

2) Obtaining Chondroitin Sulfate

In order to obtain only chondroitin sulfate acetate prepared in 1), the reaction solution was precipitated in 300 ml of distilled water. The precipitate was separated only by centrifugation (3000 rpm, 15 minutes) to separate only chondroitin sulfate. The precipitate was dissolved in DMSO and dialyzed (cut off 1000) for 3 days, and then lyophilized to obtain solid chondroitin sulfate in solid form.

3) Preparation of Nanoparticles Containing Doxorubicin

0.5 mg of doxorubicin (Sigma-aldrich, USA) was dissolved in 1 ml of DMSO, and 2 μl of triethylamine (Sigma-aldrich, USA) was added and reacted for about 12 hours. Separately, 10 mg of chondroitin sulfate obtained in 2) above was dissolved in 2 ml of DMSO, and then mixed with the doxorubicin solution. The mixed solution was dialyzed using MWCO 1,000 dialysis membrane in aqueous pH 9.5 borate buffer solution. Dialysis was carried out using a pH 9.5 and an aqueous solution of borate buffer solution. Since the pKa value of doxorubicin is 8.4, the amine group (-NH ₂ ) of doxorubicin is hydrophilic and soluble in water at pH below 8.4. In the process of encapsulating doxorubicin in the particles, it is a process to maximize the hydrophobicity and hydrophobic-hydrophobic interaction of the nanoparticles of doxorubicin.

By the dialysis process, the drug-encapsulated nanoparticles were formed, and at the same time, triethylamine used as a catalyst for removing hydrochloric acid of the organic solvent dimethyl sulfoxide, doxorubicin hydrochloric acid, and unsealed doxorubicin were removed.

The aqueous nanoparticle solution thus prepared was dried using a freeze dryer to recover only pure nanoparticles.

Example 2: Preparation of Nanoparticles Without Anticancer Agent

The same procedure as in Example 1 was performed except that the nanoparticles were prepared without containing doxorubicin, thereby obtaining nanoparticles without the drug.

Experimental Example 1: verification of chondroitin sulfate acetate synthesis

FT-IR (Nicolet, Magna IR 550) was used to verify the synthesis of sulfate chondroitin acetate. Chondroitin sulfate prepared in 1) of Example 1 was mixed with a mortar of chondroitin sulfate with KBr in a ratio of KBr = 1: 5 (wt%), and then ground using a mortar to make pellets. The pellets in solid form were placed in the instrument and measured. The results are shown in FIG.

As can be seen in Figure 2, the reduced carbonyl 1800cm ^-1 (C = O) in the vicinity are created, the methyl groups of 1300cm ^-1 vicinity of the hydroxyl group (-OH) 3400cm ^-1 in wave number (-CH ₃₎ It was confirmed that the generated. It was confirmed that chondroitin sulfate was acetylated by confirming the reduction of the hydroxyl group and the formation of the carbonyl group and the methyl group.

Experimental Example 2: Characterization of Nanoparticles

The size of chondroitin sulfate nanoparticles was measured by dispersing chondroitin acetate nanoparticles in deionized water at a concentration of 1 mg / ml and then using zeta potential zetasizerSZ (Malvern, UK). Drug encapsulation efficiency and drug content of the nanoparticles was measured by absorbance of doxorubicin at 490 nm wavelength using a UV-VIS spectrophotometer (UV-2450, Shimadzu). Encapsulation efficiency is an index of the weight of the drug encapsulated in the nanoparticles compared to the weight of the drug, and drug content is an index of the weight of the drug to the weight of the obtained nanoparticles.

The drug loading amount of the nanoparticles was measured by absorbing doxorubicin, a drug encapsulated in the nanoparticles, at a concentration of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.007813, 0.003906, 0.001953, or 0.000977 at a concentration of g / L. The absorbance value of the doxorubicin-encapsulated nanoparticles obtained in Example 1 was introduced into a standard calibration curve to determine the concentration of the drug, and then the amount of the encapsulated drug was calculated based on the calculated concentration of the drug. The same test was performed on the nanoparticles of Example 2 for comparison. The results are shown in Table 1 below.

Particle size (nm) Polydispersity coefficient Encapsulation efficiency
(%) Drug content
(%) Zeta potential
(mV) Example 2 502.23 0.16 W 0.03 - - -41.4 Example 1 234.93 ㅁ 58.30 0.54 91 ㅁ 0.17 4.58 -36.93

Experimental Example 3: Measurement of Critical Aggregate Concentration

Pyrene 98% (Simga aldrich, USA), a fluorescent material, was used to measure the minimum critical concentration value at which nanoparticles can be formed, and the fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) Fluorescence was measured. Pyrene stock solution (6.0 x 10 M) was prepared in acetone for measurement of fluorescence spectroscopy and stored at 5 ° C. until use. Pyrene solution under acetone was added to distilled water to make a pyrene concentration of 12.0 × 10 ⁻⁷ M for the measurement of steady state fluorescence, resulting in 100 ml of solution, followed by a solution at 60 ° C. for 1 hour to remove acetone from the solution. Was distilled off. Acetone-free pyrene solutions were mixed with self-aggregating nanoparticles in concentrations ranging from 0.00025 mg / ml to 1.0 mg / mL. 1 ml of pyrene solution and 1 ml of sample solution were mixed to form a final 2 ml solution. The final concentration of pyrene in each sample was 6.0 x 10 ^-7 M, which is about the same as the solubility in water at 25 ° C. The result is shown in FIG. 3. Figure 3 (A) is a graph measuring the change in fluorescence spectrum according to the concentration of each sample, Figure 3 (B) is a graph showing the shift (fluorescence) of the fluorescence spectrum according to the concentration of the sample through the formula.

Experimental Example 4: Long-term stability measurement of nanoparticles

The nanoparticles prepared in Example 1 were dispersed in deionized water at a concentration of 1 mg / ml, and then 1 ml of the sample was taken over time, and the particle size was analyzed using zetasizerSZ (Malvern, UK).

The results are shown in FIG. As shown in FIG. 4, it can be seen that the particle size of 350 nm is maintained for 22 days. This is because the particles are negatively charged by the sulfuric acid residue contained in chondroitin sulfate, which causes repulsion between particles to prevent unstable aggregation of nanoparticles.

Experimental Example 5: Drug Release Test in Nanoparticles

2 ml of the doxorubicin-encapsulated nanoparticles obtained in Example 1 were placed in a dialysis membrane (MWCO 1,000, Spectra), and the dialysis membrane was measured in a 10 mM physiological saline solution in 10 ml of 37 ° C. and 50 rpm. The experiment was carried out by replacing the physiological saline every specific time, and the doxorubicin was measured by measuring the absorbance of doxorubicin dissolved in the replaced saline at a wavelength of 490 nm using a UV-VIS spectrophotometer (UV-2450, Shimadzu). Quantification The result is shown in FIG. 5.

As can be seen in Figure 5, it can be seen that the drug release proceeded for 60 hours.

Experimental Example 6: Cytotoxicity Test

When the nanoparticles according to the present invention is encapsulated with an anticancer agent (Example 1) and no drug at all (Example 2) 3X per plate on 96 plates to see what toxic differences between HeLa cells, cancer cells Cells were aliquoted at 10 ⁻⁵ cell concentration and treated with 20 μl of nanoparticles after 24 hours. Each well was treated so that the concentration was 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, or 0.125 mg / ml. After 48 hours of nanoparticle treatment, 20 μl of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (Sigma Aldrich) was treated and 4 hours later, the cells were removed by an inhaler. All solutions were removed. After the solution was removed, 150 μl of DMSO (Junsei Co., Ltd.) was treated, and the cell viability was confirmed by measuring absorbance at a wavelength of 595 nm using a microplate reader.

The results are shown in FIG. According to Figure 6, in the case of the nanoparticles prepared in Example 1, the cell viability is reduced to 50% at 1 mg / ml, whereas in Example 2 it was shown that there is no cytotoxicity, the nanoparticles according to the present invention (drugs) Drug carriers other than the components) can be seen that the drug carriers excellent in biocompatibility.

Experimental Example 7: Intracellular influx of anticancer drug-containing chondroitin sulfate nanoparticles

In order to find out whether chondroitin sulfate-containing nanoparticles containing doxorubicin can be introduced into cancer cells, the same method was carried out in the same manner except that 30 mg of chondroitin sulfate (Fluorescein isothiocyanate isomer) was used to fluoresce with 0.15 mg of FITC. Nanoparticles were prepared as shown in 1. Doxorubicin did not process because it has its own fluorescence.

HeLa cells were dispensed at a concentration of 1 × 10 ⁻⁵ cells on 6 plates of cover glass and treated with 100 μl of nanoparticles after 24 hours. After 4 hours of nanoparticle treatment, the cells were washed three times with physiological saline, and the cells were fixed for 10 minutes using 4% paraformaldehyde (Yakuri pure chemicals, Japan), and then rinsed with physiological saline 3 Wash the cells several times and fix the cover glass to which the cells are attached to the slide glass using a mounting solution (Dako, Japan), and then observed using a confocal microscope (Leica TCS NT, Leica, Germany). Photographs of the cells were taken.

The taken picture is shown in FIG. 7. FIG. 7A shows staining of the intracellular nucleus, and FIG. 7B shows the intracellular distribution of chondroitin sulfate acetate nanoparticles by fluorescent labeling (FITC). In addition, Figure 7c confirms the intracellular distribution of doxorubicin, Figure 7d shows an optical image. 7E is a superimposed view of FIGS. 7A-7E. According to these photos, the inflow of chondroitin sulfate nanoparticles into the cells can be confirmed, and the anticancer drug was also confirmed that the inflow into the cells.

1 is a schematic diagram of nanoparticle formation of C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharides according to one embodiment of the present invention.

Figure 2 is a graph showing the results of the FT-IR instrument analysis of chondroitin sulfate sulfate prepared according to an embodiment of the present invention.

3 is a graph showing the results of measuring the critical aggregation concentration of chondroitin sulfate nanoparticles according to an embodiment of the present invention. Figure 3 (A) is a graph measuring the fluorescence spectrum change according to the concentration of each sample, Figure 3 (B) is a graph showing the shift (fluorescence) of the fluorescence spectrum according to the concentration of the sample through the formula.

Figure 4 is a graph showing the results of measuring the particle size over time in the aqueous solution of chondroitin sulfate nanoparticles according to an embodiment of the present invention.

5 is a graph showing the results of measuring the drug release rate in water of chondroitin sulfate nanoparticles according to an embodiment of the present invention over time.

Figure 6 is a result of measuring the cytotoxicity test for cancer cells of nanoparticles (Example 1) and doxorubicin-encapsulated nanoparticles (Example 2) in the nanoparticles according to an embodiment of the present invention. The graph shown.

7 is a confocal microscopy of cancer cells treated with doxorubicin-encapsulated nanoparticles prepared from FITC fluorescently labeled chondroitin acetate to determine whether the nanoparticles enter cancer cells according to an embodiment of the present invention. The picture was taken by observation.

Claims (10)

Nanoparticles formed by the coagulation of sulfate-containing polysaccharides by C ₂ -C ₄ alkanoylation and amphiphilization of sulfate-containing polysaccharides by encapsulation of drugs. The nanoparticle according to claim 1, wherein the sulfate-containing polysaccharide is chondroitin sulfate, dermatan sulfate, heparan sulfate, or curdlan sulfate. The nanoparticle according to claim 1, wherein the sulfate group-containing polysaccharide is acetylated. The nanoparticle of claim 1, wherein the drug is a hydrophobic drug. The group of claim 4, wherein the hydrophobic drug is composed of doxorubicin, taxol, camptothecin, adriamycin, paclitaxel, cisplatin, mitomycin-C, donomycin, porphyrin, chlorine, and 5-fluorouracil. Nanoparticles, characterized in that selected from. Dissolving sulfuric acid group-containing polysaccharide and C ₂ -C ₄ alkanoic anhydride in a solvent and reacting to obtain a C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharide product; Mixing the product with water to obtain C ₂ -C ₄ alkanoylated sulfuric acid group-containing polysaccharides to precipitate; The obtained C ₂ -C ₄ alkanoylated sulfate group-containing polysaccharide and the drug are dissolved in a solvent, and then put in a dialysis membrane, and then dialyzed in water so that water is introduced into the dialysis membrane, and the polysaccharide encapsulates the drug and forms a nanoparticle. Making; And A method for producing a nanoparticle according to any one of claims 1 to 5, comprising recovering the nanoparticles by lyophilizing the formed nanoparticles. The method of claim 6, wherein the C ₂ -C ₄ alkanoic anhydride is acetic anhydride. 7. The process according to claim 6, wherein the solvent used in obtaining the C ₂ -C ₄ alkanoylated sulfuric acid group containing polysaccharide product is formamide. 7. The method of claim 6 wherein the C ₂ -C ₄ alkanoyl to obtain a sulfate group-containing polysaccharide is anecdotal characterized in that by separating the precipitate containing the a C ₂ -C ₄ alkanoylation sulfate polysaccharide centrifugation . An injection comprising the nanoparticles according to any one of claims 1 to 5.

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