KR20180058430A - Pullulan acetate microsphere containing bioactive substance and preparing method thereof - Google Patents

Abstract

The present invention relates to pullulan acetate microspheres containing dexamethasone as bioactive substances and to a manufacturing method thereof, wherein a method for manufacturing the pullulan acetate microspheres can manufacture micro-sized microspheres of a uniform size compared to a conventional method for manufacturing microspheres, and it was confirmed that chitosan-treated pullulan acetate microspheres according to the manufacturing method exhibit high biocompatibility and biodegradability. Accordingly, a three-dimensional cell culture support using the pullulan acetate microspheres of the present invention can effectively induce osteoblast differentiation of stem cells through dexamethasone, which is eluted from the microspheres during stem cell culture.

Description

[0001] The present invention relates to a pullulan acetate microparticle containing a physiologically active substance and a preparation method thereof,

The present invention relates to a pullulan acetate microparticle containing dexamethasone as a physiologically active substance and a method for producing the same.

In order to restore the function of damaged bone, new bone tissue is required to be developed. The primary treatment modality is limited in that it can be used for treatment because of autologous bone grafting, damage to the donor site, and limited supply. Because of this problem, bone grafting has been used by others, but there is a disadvantage that supply can be limited and communicable diseases can be transmitted.

In addition, calcium phosphate such as tricalcium phosphate and hydroxyapatite has also been used as a bone-oil synthetic material, but it has not been widely used clinically because of its low physical properties.

For bone formation, a three-dimensional culture system similar to the body environment is required instead of a two-dimensional cell culture technique using a culture dish.

These three-dimensional structures can maintain the three-dimensional culture form of the cells for a long time, so that the efficiency and reproducibility of the experiment can be enhanced. In addition, in the case of the cells whose physiological characteristics change during the two-dimensional culture, the three- Thereby enabling stable culture.

Pullulan is a neutral and linear extracellular polysaccharide linked by - (1-4) or (1-6) glucose produced by the strain Aureobasidium pullulans.

Since such pullulan is excellent in biocompatibility, it has been extensively studied as a biomaterial through various chemical simulations. Typically, the Sumatomo research team in Japan has developed cholesterol groups in pullulan to prepare self-aggregated nanoparticles and studied their physico-biochemical properties. In addition, Na et al. Reported that pullulan (conjugated with a sulfonamide) The nanoparticles, which are sensitive to the change in pH around the cancer cells, were prepared using the column, and the interaction between the nanoparticles and the cancer cells was changed according to pH, and the change in the release characteristics of the anticancer drug was also reported.

However, since pullulan, which is a kind of polysaccharide, is a water-soluble polymer, it can not be used by itself to produce microparticles. Therefore, it is essential to impart hydrophobicity to pullulan in order to produce microparticles.

Korean Patent Publication No. 2010-0054750 (Published May 25, 2010)

The present invention provides a 3-dimensional scaffold for inducing bone differentiation of stem cells by preparing a pullulan acetate microparticle encapsulating dexamethasone, which is a bone differentiation inducing substance.

The present invention provides a microparticle consisting of pullulan acetate, wherein the microparticle is encapsulated with a physiologically active substance and surface-modified with chitosan.

The present invention relates to a method of preparing a discrete phase by dissolving pullulan acetate and a physiologically active substance in an organic solvent (first step); Dissolving polyvinyl alcohol in distilled water to prepare a continuous phase (second step); Flowing the discontinuous phase and the continuous phase together into a fluidic device to obtain a precipitate that has fallen into a particulate form while the discontinuous phase flows into the continuous phase (Step 3); And lysing the precipitate and treating the surface of the precipitate with a chitosan solution (step 4).

The present invention also provides a three-dimensional cell culture support for inducing bone differentiation containing the pullulan acetate microparticles as an active ingredient.

According to the present invention, it is possible to produce microsized microspheres of uniform size at a lower cost than conventional microsphere production methods. The chitosan-treated pullulan acetate microspheres according to the above production method have high biocompatibility and biodegradability And it was confirmed that it represents sex. Accordingly, the three-dimensional cell culture supporter using the pullulan acetate microparticle of the present invention can effectively induce osteoblast differentiation of stem cells through dexamethasone eluted from the microspheres during stem cell culture.

1 is a chemical structural formula for producing pullulan acetate by adding acetic anhydride to pullulan.
FIG. 2 is a schematic representation of a process for preparing deuloxanthosine-encapsulated pullulan acetate microparticles using a fluidic device.
FIG. 3 is a scanning electron microscope (SEM) photograph showing the surface of a pullulan acetate micropart filled with dexamethasone treated with or without chitosan.
FIG. 4 shows the results of absorbance analysis of dexamethasone-containing pullulan acetate microparticles containing dexamethasone.

The present invention provides a microparticle composed of pullulan acetate, wherein the microparticle has a physiologically active substance encapsulated therein and is surface-modified with chitosan.

The physiologically active substance may be an osteogenic differentiation-inducing substance.

The physiologically active substance may be selected from the group consisting of dexamethasone, bone morphogenetic protein (BMP), transforming growth factor (TGF-β) and ascorbic acid.

The pullulan acetate microparticles may have an average diameter of 50 to 150 micrometers in size.

In the present invention, 'pullulan' refers to a neutral and linear extracellular polysaccharide in which glucose produced by a strain of Aureobasidium pullulans is linked with α (1 → 4) or (1 → 6) , And this pluran is an edible natural polysaccharide having no toxicity and no mutagenicity and has a characteristic of being easily decomposed by fluranease and isofluranease.

In detail, pullulan (molecular weight 100,000 Da; Hayashibara) was added to formamide and the mixture was completely dissolved at room temperature. To the mixture was added pyridine Sigma) and acetic anhydride (Sigma), reacted at room temperature for 48 hours, added with water to precipitate, remove impurities and freeze-dried.

The present invention relates to a method of preparing a discrete phase by dissolving pullulan acetate and a physiologically active substance in an organic solvent (first step); Dissolving polyvinyl alcohol in distilled water to prepare a continuous phase (second step); Flowing the discontinuous phase and the continuous phase together into a fluidic device to obtain a precipitate that has fallen into a particulate form while the discontinuous phase flows into the continuous phase (Step 3); And a step of lyophilizing the precipitate and treating the chitosan solution on the surface of the precipitate (step 4). The present invention also provides a method for producing a pullulan acetate microparticle containing a physiologically active substance.

More specifically, the physiologically active substance may be selected from the group consisting of dexamethasone, bone morphogenetic protein (BMP), transforming growth factor (TGF-β), and ascorbic acid .

The discontinuous phase may be one in which 50 mg / ml to 100 mg / ml of pullulanacetate and 1 mg / ml to 2 mg / ml of dexamethasone are dissolved in an organic solvent and the continuous phase is added to distilled water in a concentration of 1 to 5% The alcohol may be dissolved.

More specifically, the organic solvent may be selected from the group consisting of dichloromethane and chloroform, more preferably dichloromethane, but is not limited thereto.

The chitosan solution may be dissolved in 5 to 20 parts by weight of chitosan relative to 100 parts by weight of the acetic acid solution.

The present invention also relates to a microparticle comprising pullulan acetate, wherein the microparticle is encapsulated with a physiologically active substance and surface-modified with chitosan. The present invention relates to a method for inducing bone differentiation of stem cells containing pluran acetate microparticles as an active ingredient Dimensional cell culture support.

In the present invention, 'dexamethasone' is a synthetic steroid that exhibits glucocorticoid-like physiological activity and is used for drugs having strong anti-inflammatory action or anti-shock action. It inhibits the production of adrenocorticotropic hormone (ACTH) Like the hormone, it is transferred into the nucleus along with the receptor and exerts its physiological activity by promoting or inhibiting the transcription. In addition, it has been reported that it has an effect of promoting differentiation from stem cells to bone cells.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Acetated Pullulan  Produce

Pullulan was acetated by the same procedure as in Fig. 1 to introduce an amphipathic effect.

First, 2 g of pullulan (molecular weight: 100,000 Da; Hayashibara) was added to 20 mL of formamide and stirred vigorously at room temperature to dissolve. 5 mL of pyridine (Sigma) and 10 mL of acetic anhydride And reacted at room temperature for 48 hours.

Then, 200 mL of water was added to precipitate, and the product was recovered by filtration.

The process was repeated three or more times to remove impurities from the resulting product and lyophilized to yield the final product.

< Example  2> dexamethasone Enclosed Pullulan  acetate Microparticle  Produce

A pullulan acetate microparticle encapsulating dexamethasone (DEX), which is a bone differentiation inducing substance, was prepared.

2% polyvinyl alcohol (PVA) solution was used as a continuous phase. In a discontinuous phase, dichloromethane, a volatile solvent, was added to the solution in an amount of 50 mg / ml to 100 mg / ml Dexamethasone 1 mg / ml or 2 mg / ml, and completely dissolved.

The continuous phase / discontinuous phase solution was fixed to a fluid device and the solution was flowed to prepare a pullulan acetate microparticle.

As shown in FIG. 2, the end of the discontinuous phase syringe was connected to the 34G needle, and the needle was connected to a glass tube connected to the rubber tube. The glass tube connected to the rubber tube was placed on the surface of 50 ml of a 2% polyvinyl alcohol solution in a beaker. The unconnected rubber tube was fixed to the continuous phase, and the rubber tube was connected to the discontinuous phase.

The continuous and discontinuous phases were inserted into the syringe and fixed to the fluid device, respectively, and the continuous phase and the discontinuous phase were connected by a rubber tube. The fluid device in which the continuous phase and the discontinuous phase were fixed was operated to cause the discontinuous phase in which the pullulan and the dexamethasone dissolved to flow into the polyvinyl alcohol to be dropped into the microparticle form to prepare the pullulan acetate microparticles encapsulating dexamethasone.

The prepared microspheres were stirred to remove the volatile solvent Dichloromethane, washed with distilled water, and lyophilized to obtain microspheres.

The thus obtained microspheres were placed in a 10 g / L acetic acid solution containing 1 g / L of chitosan dissolved therein and lyophilized to prepare chitosan-coated microspheres.

< Example  3> Microsphere  Observing the surface and checking the diameter

In order to confirm the surface state of the microspheres according to the presence or absence of the chitosan coating, the microspheres prepared in Example 2 were coated with platinum for 45 seconds and the surface and diameter of the microspheres were confirmed using a scanning electron microscope (SEM).

As a result, it was confirmed that the microspheres of Example 2 were about 50 to 150 mu m in size as shown in Fig. On the other hand, it was confirmed that the chitosan-free microparticles had a smooth surface, but the chitosan-coated microparticles had rough surfaces.

< Example  4> Microspheres  Check dexamethasone inclusion rate

To confirm the dexamethasone inclusion rate of pullulan acetate microspheres, a dexamethasone standard curve was prepared.

First, acetonitrile in which dexamethasone (DEX) 1 mg / ml was dissolved was diluted to 0.03125 mg / ml, and diluted by ½ times to 0.000244 mg / ml.

The dexamethasone diluted by the above method was measured for its absorbance at 234 nm using a UV spectrophotometer, and a standard curve was prepared as shown in Fig.

10 mg of the microparticle prepared in Example 2 was dissolved in 1 ml of water and quantified at 234 nm using a UV spectrophotometer.

The dexamethasone inclusion rate was calculated using the quantitative value and the equation.

[Mathematical Expression]

Encapsulation efficiency = {dexamethasone mass in the microsphere} / {mass of dexamethasone in the microsphere production} x 100 (%)

As a result, the mass of dexamethasone in the microfiber was 15.67 mg, and the mass of dexamethasone used was 20 mg, indicating a filling efficiency of 78.35%.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (11)

Wherein the microparticle is a physiologically active substance encapsulated therein and is surface-modified with chitosan. The pullulan acetate microparticle according to claim 1, wherein the physiologically active substance is an osteogenic differentiation agent. [2] The method of claim 1, wherein the physiologically active substance is selected from the group consisting of dexamethasone, Bone Morphogenetic Protein (BMP), Transforming Growth Factor (TGF-?) And ascorbic acid Characterized by pullulan acetate microparticles. The pullulan acetate microparticle of claim 1, wherein the pullulan acetate microparticles have an average diameter of 50 to 150 micrometers. Preparing a discontinuous phase by dissolving pullulan acetate and a physiologically active substance in an organic solvent (first step);
Dissolving polyvinyl alcohol in distilled water to prepare a continuous phase (second step);
Flowing the discontinuous phase and the continuous phase together into a fluidic device to obtain a precipitate that has fallen into a particulate form while the discontinuous phase flows into the continuous phase (Step 3); And
Lyophilizing the precipitate and treating the surface of the precipitate with a chitosan solution (step 4). The method for producing a pullulan acetate microparticle according to claim 1, [6] The method of claim 5, wherein the physiologically active substance is selected from the group consisting of dexamethasone, bone morphogenetic protein (BMP), transforming growth factor (TGF-?) And ascorbic acid &Lt; / RTI &gt; [Claim 6] The method according to claim 5, wherein the discontinuous phase is prepared by dissolving 50 to 100 mg / ml of pullulan acetate and 1 to 2 mg / ml of dexamethasone in an organic solvent. [Claim 7] The method according to claim 7, wherein the organic solvent is selected from the group consisting of dichloromethane and chloroform. [Claim 6] The method according to claim 5, wherein the continuous phase comprises polyvinyl alcohol dissolved in distilled water at a concentration of 1 to 5% by weight. [Claim 6] The method according to claim 5, wherein the chitosan solution is dissolved in 5 to 20 parts by weight of chitosan per 100 parts by weight of the acetic acid solution. A support for three-dimensional cell culture for inducing bone differentiation containing pullulan acetate microparticles according to claim 1 as an active ingredient.

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