KR102403559B1 - Biodegradable polymer microsphere with hydrophilic surface using plasma treatment technique and method for preparing the same - Google Patents

Abstract

The present invention relates to biodegradable polymer microparticles having a hydrophilic surface, an injection containing the same, and a method for preparing the same.
The biodegradable polymer microparticles prepared according to the method of the present invention have significantly improved hydrophilic surface properties through plasma treatment and active material coating, and thus can be widely used as a material for injectable cell therapeutics.

Description

Biodegradable polymer microsphere with hydrophilic surface using plasma treatment technique and method for preparing the same

The present invention relates to biodegradable polymer microparticles having a hydrophilic surface, an injection containing the same, and a method for preparing the same.

In the development of cell therapeutics including stem cells, it is required to develop a material that is biodegraded to provide a space for cells to grow after cells injected into the human body are located and protected during differentiation.

Patent 10-1112775 has confirmed its effectiveness by presenting a technology of coating a biodegradable polymer suture with a peptide or protein that helps cell adsorption and differentiation through plasma surface treatment and coating for the purpose of solving this problem. However, the technology relates to the manufacture of sutures, the range of the affected area requiring treatment is very limited, and there is a disadvantage in that the procedure is complicated and difficult than injection of a general injection.

Under this background, the present inventors made diligent efforts to develop materials for injectable cell therapeutics, and as a result, by identifying manufacturing conditions that can significantly improve the hydrophilic surface properties of biodegradable polymer microparticles manufactured using plasma treatment technology. The present invention was completed.

One object of the present invention comprises the steps of: (a) preparing a first mixture by mixing a biodegradable polymer and an organic solvent, and preparing a second mixture by mixing a surfactant and water; (b) mixing the first mixture and the second mixture, stirring and washing to separate the biodegradable polymer microparticles; (c) performing plasma surface treatment on the fine particles; And (d) coating the active material; to provide a method for producing a hydrophilic surface-treated biodegradable polymer microparticles comprising a.

Another object of the present invention is to provide a biodegradable polymer microparticles prepared by the above method.

Another object of the present invention is to provide an injection composition comprising the biodegradable polymer microparticles.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object, (a) preparing a first mixture by mixing a biodegradable polymer and an organic solvent, preparing a second mixture by mixing a surfactant and water; (b) mixing the first mixture and the second mixture, stirring and washing to separate the biodegradable polymer microparticles; (c) performing plasma surface treatment on the fine particles; And (d) coating the active material; provides a method for producing a hydrophilic surface-treated biodegradable polymer microparticles comprising a.

The biodegradable polymer of the present invention is poly-L-lactic acid (PLLA), polydioxanone (PDO), polylactic acid (PLA), poly-D-lactic acid ( poly-D-lactic acid (PDLA), poly-ε-caprolactone (PCL), polyglycolic acid (PGA), copolymers thereof, and mixtures thereof may be at least one selected from . These copolymers may be, for example, polylactic acid-glycolic acid copolymer, polydioxanone-caprolactone copolymer, polylactic acid-caprolactone copolymer, and the like. The biodegradable polymer of the present invention may specifically be poly-L-lactic acid (PLLA), wherein the poly-L-lactic acid is a polyester derived from lactic acid and has excellent biocompatibility and toxicity. It has a longer decomposition period compared to other biodegradable polymers, so it is widely used as a filler for molding and a material for drug delivery.

The average molecular weight of the biodegradable polymer in the present invention may be 50,000 to 200,000 Dalton, 70,000 to 150,000 Dalton, or 80,000 to 110,000 Dalton. If the average molecular weight of the biodegradable polymer is less than 80,000 Dalton, the decomposition rate of the biodegradable polymer microparticle increases and may not be suitable for use as a cell carrier material. Since processing is difficult, it may be difficult to manufacture particles having a uniform size and quality.

The organic solvent of the present invention is methylene chloride, HFIP (1,1,1,3,3,3-hexafluoro-2-propanol), acetone, acetic acid, dioxane, It may be one or more selected from the group consisting of ethanol, methanol, isopropyl alcohol (IPA), propanol, tetrahydrofuran (THF), and mixtures thereof. The organic solvent of the present invention may specifically be methylene chloride (dichloromethane), and the methylene chloride is a volatile organic solvent of the formula CH ₂ Cl ₂ , mixed with the biodegradable polymer of the present invention to prepare a first mixture can be used

The "first mixture" of the present invention means a solution in which a biodegradable polymer and an organic solvent are mixed, and the concentration of the biodegradable polymer with respect to the first mixture may be 1 to 10% (w/v). Specifically, the concentration of the biodegradable polymer may be 3 to 7% (w/v), more specifically 5% (w/v), in this case, for example, 5 g of the biodegradable polymer is dissolved in 100 ml of an organic solvent. If the concentration of the biodegradable polymer included in the first mixture is too low, the production efficiency of the biodegradable polymer microparticles may be reduced, and if the concentration of the biodegradable polymer is too high, it may be difficult to obtain microparticles of a uniform size .

The surfactant of the present invention is at least one selected from the group consisting of polyvinyl alcohol, polyoxyethylene sorbitan and its salts, soybean lecithin, monoglyceride, and mixtures thereof. can The surfactant of the present invention may specifically be polyvinyl alcohol, and the polyvinyl alcohol is a water-soluble polymer having a structure of [CH ₂ CH(OH)] _n , has high surface activity and is biodegradable. have. The average molecular weight of the surfactant may be 10,000 to 100,000 Daltons.

"Second mixture" of the present invention means a solution in which a surfactant and water are mixed, and the concentration of the surfactant with respect to the second mixture may be 0.01 to 5% (w/v). Specifically, the concentration of the surfactant may be 0.01 to 3% (w/v), more specifically 0.5% (w/v), in this case, for example, 10 g of the surfactant is dissolved in 2,000 ml of water. When the concentration of the surfactant included in the second mixture is too low, the surface activity is weakened and it may be difficult to manufacture biodegradable polymer microparticles of uniform size. Otherwise, agglomeration between fine particles may occur.

In the present invention, the first mixture and the second mixture are mixed in a volume ratio of 1:15 to 25. When the second mixture is mixed below the above range, it may be difficult to manufacture a biodegradable polymer of a uniform size, and when the second mixture is mixed above the above range, aggregation between the biodegradable polymer microparticles may occur.

In the present invention, mixing and stirring the first mixture and the second mixture may be performed sequentially or substantially simultaneously. For example, the first mixture and the second mixture may be simultaneously or sequentially added to a vessel in which the stirrer rotates, and stirring may be performed at the same time.

The stirring may be performed at 100 to 800 rpm, 100 to 700 rpm, 200 to 700 rpm, 200 to 600 rpm, 300 to 600 rpm, or 300 to 500 rpm. If the stirring of the mixture (eg, stirring speed, rpm) is too slow, the mixing of the first mixture and the second mixture may not be performed smoothly. If the agitation of the mixture is too fast, the uniformity of the particle size of the biodegradable polymer microparticles may be deteriorated. The stirring may be performed for 24 hours or more. As the stirring proceeds for a long time, the organic solvent slowly volatilizes, so that the biodegradable polymer can be gradually precipitated as polymer microparticles under uniform conditions, and thus, the uniformity of the particle size of the biodegradable polymer microparticles can be improved. . Since the stirring is at a low speed of 800 rpm or less, if the stirring time is less than 24 hours, the precipitation of the biodegradable polymer microparticles may not proceed sufficiently.

Following the stirring, the biodegradable polymer microparticles are separated. Separating the microparticles includes precipitating the biodegradable polymer microparticles: washing the microparticles multiple times by replacing the supernatant; And it may include the step of screening the fine particles.

After stirring, the mixture of the first mixture and the second mixture may be left to stand for, for example, 1 hour or more, 2 hours or more, 5 hours or more, or 12 hours or more to precipitate biodegradable polymer microparticles. Then, after removing the supernatant as much as possible, purified water is added, stirred at 100 to 1000 rpm for 1 to 24 hours, and then a washing process of removing purified water can be performed one or more times. By this washing process, it is possible to effectively remove impurities remaining in the biodegradable polymer microparticles.

In the selection of the biodegradable polymer microparticles, the microparticles having an average particle diameter of 20 to 50 μm may be selected in a dry or wet manner using a size sieving machine. If the average particle diameter of the biodegradable polymer microparticles is too large, it may not be suitable for injection.

Next, plasma surface treatment is performed on the separated biodegradable polymer microparticles. The plasma surface treatment is performed by injecting air or helium gas at 1 to 20 cc/min, and applying a voltage of 50 to 110 W so that the plasma-treated air or helium gas is in contact with the biodegradable polymer microparticles. may be carried out. The plasma treatment may be performed for 1 to 60 minutes, specifically for 1 to 50 minutes, and more specifically for 20 to 40 minutes.

In a specific embodiment of the present invention, the surface activation degree of the fine particles of Example 1 surface-treated using helium gas, the fine particles of Example 2 using air, and the fine particles of Comparative Example 1 surface-treated using oxygen gas As a result of comparing the results, it was confirmed that the water contact angle of the microparticles of Examples 1 and 2 subjected to plasma surface treatment using helium gas and air was significantly smaller than that of the microparticles without surface treatment or the microparticles of Comparative Example 1 ( 3), it was found that plasma surface treatment using air and helium gas had a remarkably excellent effect on improving the hydrophilicity of the surface of fine particles.

In another specific embodiment of the present invention, as a result of comparing the degree of surface activation of microparticles subjected to plasma surface treatment by changing plasma treatment conditions such as applied power, gas injection rate, and treatment time, the applied power is 60 W or more It significantly decreased (FIG. 4), the gas injection rate showed the lowest contact angle under the condition of 10 cc/min (FIG. 5), and the best effect was exhibited when the treatment time was 20 minutes or more (FIG. 6).

Next, an active material is coated on the microparticles subjected to plasma surface treatment.

The "active material" of the present invention includes arginine, lysine, fibronectin, fibrinogen, laminin, vitronectin, collagen, gelatin, and these It may be at least one selected from the group consisting of a mixture of The active material may be a cell-affinity material, and includes without limitation as long as it is a hydrophilic material capable of engraftment, adsorption or attachment to cells.

The coating of the active material comprises the steps of mixing an aqueous solution of the active material, and biodegradable polymer microparticles subjected to plasma surface treatment; washing the fine particles; and drying the fine particles. The active material aqueous solution refers to an aqueous solution in which the active material is dissolved in water, and may have a concentration of 0.4 to 1% (w/v), more specifically 0.5% (w/v). If the concentration of the active material aqueous solution is less than 0.4% (w/v), the coating of the active material is not preferably performed, and if it exceeds 1% (w/v), the active material is not uniformly coated on the surface of the biodegradable polymer microparticles. it may not be In a specific embodiment of the present invention, Example 5 coated with an aqueous active material solution at a concentration of 0.5% (w/v), 0.1% (w/v) or 5% (w/v) in an aqueous solution of the active material As a result of comparing the water contact angles of Comparative Examples 7 and 8, it was confirmed that the water contact angle of the microparticles of Example 5 was the most reduced (FIG. 8), the concentration of the active material aqueous solution affecting the hydrophilicity of the coated microparticle surface could see that

After mixing the active material aqueous solution and the biodegradable polymer microparticles subjected to plasma surface treatment, the mixture may be stirred at a speed of 100 to 800 rpm, 100 to 600 rpm, 100 to 400 rpm, or 100 to 300 rpm for 6 to 18 hours. Thereafter, a washing process of separating the biodegradable polymer microparticles, putting them in purified water, and stirring for 0.1 to 3 minutes may be performed one or more times.

After mixing and washing, the fine particles may be dried. Such drying may be carried out, for example, at a temperature of 20 to 50 °C, specifically 25 to 45 °C, 0.01 to 2 atm, specifically 0.05 to 1 atm. The drying time may be, for example, 1 to 48 hours, 5 to 40 hours, or 12 to 30 hours.

In a specific embodiment of the present invention, as a result of comparing the water contact angle of the microparticles of Example 5 coated with the active material and the microparticles of Comparative Example 6 without the coating treatment, it was found that the microparticles coated with the active material had higher hydrophilicity. was confirmed (FIG. 7).

Another aspect of the present invention for achieving the above object provides a hydrophilic surface-treated biodegradable polymer microparticles prepared according to the above method.

The biodegradable polymer microparticles prepared according to the method of the present invention greatly increase the hydrophilic surface properties according to plasma surface treatment and coating of an active material, so that engraftment, adsorption or attachment of cells can be very easy.

The hydrophilic surface property of the present invention can be confirmed by measuring the water contact angle of the fine particles, and it can be inferred that the lower the water contact angle, the better the hydrophilic surface property.

Another aspect of the present invention for achieving the above object provides an injection composition comprising the biodegradable polymer microparticles.

The "injection agent" of the present invention can be used in the form of administration to the skin area of the recipient using an injection device having a fine needle or cannula shape.

The injection composition includes the biodegradable polymer microparticles of the present invention, and may further include a biocompatible carrier. The biocompatible carrier is alginic acid and its salts, hyaluronic acid and its salts, carboxymethyl cellulose and its salts, dextran and its salts, collagen, gelatin. (gelatin), and may include one or more selected from elastin (elastin).

The injection composition may further include a physiologically active substance, a local anesthetic, water for injection, sterile water, or distilled water depending on the use.

The biodegradable polymer microparticles prepared according to the method of the present invention have significantly improved hydrophilic surface properties through plasma treatment and active material coating, and thus can be widely used as a material for injectable cell therapeutics.

1 is a view showing a scanning electron microscope (SEM) image of the biodegradable polymer microparticles prepared according to the method of Preparation Example 1.
2 is a diagram schematically illustrating a plasma processing apparatus.
3 is a view showing the measurement result of the water contact angle according to the plasma surface treatment.
4 is a diagram illustrating a result of measuring a water contact angle according to a gas output.
5 is a view showing the measurement result of the water contact angle according to the gas injection amount.
6 is a view showing the measurement result of the water contact angle according to the treatment time.
7 is a view showing the measurement result of the water contact angle according to the active material coating.
8 is a view showing the measurement result of the water contact angle according to the concentration of the active material aqueous solution.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Preparation Example 1. Preparation of biodegradable polymer microparticles

A first mixture was prepared by dissolving 5 g of poly-L-lactic acid (PLLA) having a weight average molecular weight of 80,000 to 110,000 Dalton as a biodegradable polymer in 100 ml of methylene chloride, and the average as a surfactant A second mixture was prepared by dissolving 10 g of polyvinyl alcohol (PVA) having a molecular weight of 22,000 in 2,000 ml of purified water.

The first mixture and the second mixture were mixed in a volume ratio of 1:20, methylene chloride was evaporated for 24 hours under a stirring condition of 400 rpm, and then maintained for 4 hours with the stirring stopped to precipitate PLLA microparticles, and the upper layer After removing the liquid as much as possible, purified water was added. Thereafter, the mixture was stirred at 400 rpm for 4 hours again, and the process through precipitation and supernatant replacement was repeated 3 times to remove the surfactant contained in the solution. Next, PLLA microparticles having a particle diameter of 20-50 μm were separated and obtained using a sieve, and the microparticles were dried by storing in a drying oven maintained at 37° C. for 24 hours. The PLLA microparticles obtained through the above process are shown in FIG. 1 .

Preparation Example 2. Plasma surface treatment

Example 1

The PLLA fine particles obtained by the method of Preparation Example 1 were subjected to surface treatment using helium gas through the plasma treatment apparatus shown in FIG. 2 , and detailed conditions thereof are shown in Table 1 below.

gas type Print gas injection rate processing time Helium 60 W 10 cc/min 20 min

Example 2

The same surface treatment was performed in Example 1 except that air was used instead of helium as the gas.

Comparative Example 1

The same surface treatment was performed in Example 1 except that oxygen was used instead of helium as the gas.

Comparative Example 2

The same surface treatment was performed except that the output was set to 20 W in Example 1.

Example 3

The same surface treatment was performed except that the output was increased to 100 W in Example 1.

Comparative Example 3

The same surface treatment was performed except that in Example 1, the gas injection rate was increased to 20 cc/min.

Comparative Example 4

The same surface treatment was performed except that in Example 1, the gas injection rate was increased to 40 cc/min.

Comparative Example 5

The same surface treatment was performed except that the plasma treatment time was set to 10 minutes in Example 1.

Example 4

The same surface treatment was performed except that the plasma treatment time was set to 40 minutes in Example 1.

Evaluation Example 1. Effects of Plasma Surface Treatment

Plasma surface treatment was performed for each gas type through Examples 1 and 2 and Comparative Example 1, and the hydrophilic effect was confirmed by measuring the water contact angle to confirm the degree of surface activation through this.

Specifically, in order to prepare a sample for measurement, a carbon tape was attached to the back of a slide glass, and the PLLA microparticles of Examples 1 and 2 and Comparative Example 1 that were plasma-treated and the PLLA microparticles that were not surface-treated were well spread. Samples were prepared to be as flat as possible. Thereafter, the measurement results using a contact angle measuring equipment (Phoenix-MT(M), SEO Co., Ltd.) are shown in FIG. 3 .

As a result, it was confirmed that plasma surface treatment using air and helium gas showed the most excellent effect on improving the hydrophilicity of the surface of the fine particles.

Evaluation Example 2. Plasma surface treatment effect according to output

In Examples 1, 3 and Comparative Example 2, plasma surface treatment was performed for each output, and the water contact angle was measured to confirm the degree of surface activation through this, and the hydrophilic effect was confirmed. The water contact angle was measured using the same method as in Evaluation Example 1, and the results are shown in FIG. 4 .

As a result, it was confirmed that the contact angle decreased to the maximum when the output was 60W or more.

Evaluation Example 3. Plasma surface treatment effect according to gas injection amount

In Example 1, Comparative Examples 3 and 4, plasma surface treatment was performed according to the amount of gas injected, and the water contact angle was measured to confirm the degree of surface activation through this, and the hydrophilic effect was confirmed. The water contact angle was measured using the same method as in Evaluation Example 1, and the results are shown in FIG. 5 .

As a result, it was confirmed that the contact angle was the lowest when the gas injection rate was 10 cc/min.

Evaluation Example 4. Plasma surface treatment effect according to treatment time

Plasma surface treatment was performed according to the treatment time through Examples 1, 4 and Comparative Example 5, and the hydrophilic effect was confirmed by measuring the water contact angle to confirm the degree of surface activation through this. The water contact angle was measured using the same method as in Evaluation Example 1, and the results are shown in FIG. 6 .

As a result, it was confirmed that the contact angle was the lowest when the treatment time was 20 minutes or more.

Preparation Example 2. Active material coating treatment

Example 5

As an active material, arginine was dissolved in water to prepare an aqueous arginine solution as shown in Table 2 below, and 1 g of the PLLA microparticles obtained in Example 1 were added, and the agitation was maintained at 200 rpm and stored at room temperature for 12 hours. Thereafter, the fine particles were separated, put in 1,000 ml of purified water, and stirred at 200 rpm for 1 minute, and the washing process was repeated 3 times in total. Thereafter, the fine particles of Example 5 were obtained which were kept at 37° C. and 0.2 atm and were completely dried for 24 hours.

Arginine Concentration arginine water fine particles 0.5% (w/v) 5g 1,000mL 1 g

Comparative Example 6

Fine particles of Comparative Example 6 were obtained in the same manner as in Example 5, except that arginine was not used, and the arginine coating treatment was not performed.

Comparative Example 7

The same treatment was performed except that in Example 5, an arginine concentration of 0.1% (w/v) solution was prepared and used.

Comparative Example 8

The same treatment was performed except that a 5% (w/v) solution of arginine concentration was prepared and used in Example 5.

Evaluation Example 5. Effects of Active Material Coating Treatment

The water contact angles of the fine particles of Examples 5 and 6 were confirmed in the same manner as in Evaluation Example 1, and are shown in FIG. 7 . In the corresponding figure, 'W/ coating' indicates the particles prepared in Example 5, and 'W/O coating' indicates the contact angle of the particles prepared in Comparative Example 6 with water.

As a result, when the arginine coating was not performed, the contact angle was 82 degrees, whereas it was confirmed that the contact angle was 53 degrees when the hydrophilic material, arginine coating, was performed.

Evaluation Example 5. Coating Effect according to Concentration of Active Material

The water contact angles of the fine particles of Examples 5, 7, and 8 were confirmed in the same manner as in Evaluation Example 1, and are shown in FIG. 8 .

As a result, when the concentration of arginine was 0.1% (w/v) or 5% (w/v), there was no difference in the contact angle, but it was confirmed that the contact angle was reduced when the concentration of 0.5% (w/v) was used.

Therefore, the biodegradable polymer microparticles prepared by the method of the present invention have a hydrophilic surface modified to help the adsorption of cells or living tissues, and are included in the injection to provide a space for cells or living tissues to grow. can be utilized.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (14)

(a) preparing a first mixture by mixing poly-L-lactic acid (PLLA) as a biodegradable polymer, and an organic solvent, and preparing a second mixture by mixing a surfactant and water ;
(b) mixing the first mixture and the second mixture, stirring and washing to separate the biodegradable polymer microparticles;
(c) performing plasma surface treatment on the fine particles; and
(d) coating arginine as an active material; as a method for producing a hydrophilic surface-treated biodegradable polymer microparticles comprising a,
The (c) plasma surface treatment is performed by injecting air or helium gas at 1 to 20 cc/min, and applying a voltage of 50 to 110 W to apply plasma-treated air or helium gas for 20 to 40 minutes. It is carried out so as to be in contact with the biodegradable polymer microparticles,
The (d) coating of the active material is to mix the biodegradable polymer microparticles in an aqueous solution of the active material at a concentration of 0.4 to 1% (w/v), a method for producing biodegradable polymer microparticles.
delete The method of claim 1, wherein (a) the organic solvent is methylene chloride, HFIP (1,1,1,3,3,3-hexafluoro-2-propanol), acetone, acetic acid One selected from the group consisting of ), dioxane, ethanol, methanol, isopropyl alcohol, IPA, propanol, tetrahydrofuran, THF, and mixtures thereof The above method for producing biodegradable polymer microparticles with hydrophilic surface treatment.
The method of claim 1, wherein (a) the average molecular weight of the biodegradable polymer is 80,000 to 110,000 Daltons, the hydrophilic surface-treated biodegradable polymer microparticles.
According to claim 1, wherein (a) the concentration of the biodegradable polymer in the first mixture is 1 to 10% (w / v), the hydrophilic surface treatment of the biodegradable polymer microparticles manufacturing method.
According to claim 1, wherein (a) the surfactant is polyvinyl alcohol, polyoxyethylene sorbitan and its salts, soybean lecithin, monoglyceride, and mixtures thereof A method for producing at least one selected from the group consisting of, hydrophilic surface-treated biodegradable polymer microparticles.
According to claim 1, wherein (a) the concentration of the surfactant in the second mixture is 0.01 to 5% (w / v), the hydrophilic surface treatment of the biodegradable polymer microparticles manufacturing method.
According to claim 1, wherein (b) the average particle diameter of the biodegradable polymer microparticles is 20 to 50㎛, hydrophilic surface-treated biodegradable polymer microparticles manufacturing method.
The method of claim 1, wherein (b) the first mixture and the second mixture are mixed in a volume ratio of 1:15 to 25, hydrophilic surface-treated biodegradable polymer microparticles.
delete delete delete A biodegradable polymer microparticle with a hydrophilic surface treatment prepared according to the method of claim 1.
An injection composition comprising the biodegradable polymer microparticles of claim 13.

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