KR102053913B1 - Manufacturing method of cross-linking medium-viscous-solution using polysaccharides - Google Patents

Abstract

According to one embodiment of the present invention, a method for manufacturing a cross-linked medium viscosity solution comprises: a step of manufacturing an aqueous solution by dissolving high molecular polysaccharides in an alkaline solution; a step of spraying nitrogen or inert gas to the aqueous solution; and a step of manufacturing a cross-linked medium viscosity solution by adding a cross-linking agent to the nitrogen or inert gas-sprayed aqueous solution. According to the present invention, the cross-linked medium viscosity solution has low swelling properties, fluidity, and high biocompatibility when being applied to the body.

Description

Manufacturing method of crosslinking medium solution using polysaccharides {Manufacturing method of cross-linking medium-viscous-solution using polysaccharides}

The present invention relates to a method for preparing a crosslinking medium solution using polysaccharide, and more particularly, to a method for preparing a crosslinking medium solution having low swelling property by forming intramolecular or intermolecular crosslinking in a polymer polysaccharide or derivatives thereof. will be.

Natural polymer polysaccharides have higher biocompatibility than synthetic polymers, and thus, attempts to use them for various applications have been continued for a long time. At present, attempts have been made to denature natural polymer polysaccharides in various fields, particularly in the field of medical materials. Polymer polysaccharides widely studied for this purpose include animal-derived polysaccharides such as carboxymethyl cellulose, cellulose sulfate, agarose, and alginic acid. (hyaluronic acid) and heparin (heparin, poly-α-glucosamine sulfate), such as microorganism-derived polysaccharides such as dextran (dextran). All of them have a common biocompatibility and good rheological properties such as gel formation ability.

Hyaluronic acid is a linear polymer in which β-N-acetyl-D-glucosamine and β-D-glucuronic acid are alternately bonded, and is a polysaccharide having a molecular weight of 50,000 to 10,000,000 Da or more. Hyaluronic acid is the basic material of bio connective tissue, mainly the skin of mammals, synovial fluid, vitreous humor of the eyes, umbilical cord, serum, and chicken's comb. It is known to exist in the capsular membrane of streptococci, etc.). Common methods for obtaining hyaluronic acid include extracting from chicken crest, umbilical cord, etc., culturing Streptococcus lances group A and C, genetically recombined Bacillus subtilis, etc. Etc.

Natural hyaluronic acid is a polydisperse acid in proportion to molecular weight, has no species specificity and does not have tissue or organ specificity, and thus shows excellent biocompatibility when implanted or injected into a living body regardless of its origin. It is used for such.

Dextran is composed of glucose, and is mainly a straight chain of α-D- (1-6) bonds, and has a short branched structure by a small amount of α-D- (1-6) bonds. Dextran is obtained by incubating Leuconostoc mesenteroides microorganisms and then extracting it from there. The molecular weight is usually 1,000 to 2,000,000 Da, of which 40,000 Da and 60,000 Da and 70,000 Da are well purified dextran, which are currently used for medical purposes, more specifically as plasma volume expanders for more than 50 years. have. In addition, medical dextran having molecular weights of 40,000 Da and 70,000 Da is known to prevent pulmonary embolism occurring after surgery, trauma, or burn.

In order to enhance the advantages and applicability of these natural polymers, a method of forming a crosslink or attaching a functional group to the polymer polysaccharide has been attempted. Among them, a bifunctional crosslinking agent such as divinyl sulfone, bisepoxide or formaldehyde It is a highly swellable crosslinked hyaluronic acid gel obtained using

However, most of these results have the disadvantage of poor fluidity in the form of a highly swellable gel or a solid product. These disadvantages are disadvantageous to use for the purpose of spreading to a wide area such as anti-adhesion and the like.

[Patent Document 1] US Registered Patent US6,635,267B1

The present invention has been made to solve the above problems, and an object thereof is to provide a method for producing a crosslinking midpoint solution using a polysaccharide.

It is an object of the present invention to provide a method for producing a crosslinking midpoint solution in which nitrogen or an inert gas is injected into an aqueous solution in which a polymer polysaccharide is dissolved, and vacuum treatment is performed.

The objects of the present invention are not limited to the above-mentioned objects, and other objects that are not mentioned will be clearly understood from the following description.

In order to achieve the above objects, a method for preparing a crosslinking medium solution according to an embodiment of the present invention comprises the steps of dissolving a polymer polysaccharide in an alkaline solution to prepare an aqueous solution; Injecting nitrogen or an inert gas into the aqueous solution; And adding a crosslinking agent to the aqueous solution sprayed with nitrogen or an inert gas to prepare a crosslinking midpoint solution. It may include.

In an embodiment, the method of preparing the crosslinking medium solution may further include performing a vacuum treatment on the aqueous solution before preparing the crosslinking medium solution.

In an embodiment, the method for preparing a crosslinking medium solution may include adding a diluent to the crosslinking medium solution after the step of preparing the crosslinking medium solution, and then removing the diluent after a predetermined time. It may include.

In an embodiment, the preparing of the aqueous solution may include dissolving the polymer polysaccharide in a weak base alkali solution and dissolving the polymer polysaccharide, and then adding a strong base alkali solution to prepare the aqueous solution. The pH of the solution may be greater than the pH of the weak base alkaline solution.

In an embodiment, the step of injecting nitrogen or inert gas may include injecting the nitrogen or inert gas into the aqueous solution using a nozzle or a sparger.

In an embodiment, the step of injecting the nitrogen or inert gas may include the step of injecting the nitrogen or inert gas into the aqueous solution while stirring the aqueous solution.

In an embodiment, the inert gas may include at least one of neon and argon.

Specific details for achieving the above objects will be apparent with reference to the embodiments to be described later in detail with the accompanying drawings.

However, the present invention is not limited to the embodiments disclosed below, but may be configured in a variety of different forms, to make the disclosure of the present invention complete and those skilled in the art to which the present invention belongs ( In the following, the description is provided to fully inform the scope of the invention to the ordinary technician.

According to an embodiment of the present invention, by injecting nitrogen or an inert gas into the aqueous solution in which the polymer polysaccharide is dissolved, and performing a vacuum treatment, the swelling property is not high, the fluidity property, when applied to the body is high biocompatibility, Compared to natural polymers, crosslinked midpoint solutions with improved body persistence can be prepared.

The effects of the present invention are not limited to the above-described effects, and the tentative effects expected by the technical features of the present invention will be clearly understood from the following description.

1 is a flowchart illustrating a method of preparing a crosslinking midpoint solution according to an embodiment of the present invention.

The present invention may be modified in various ways and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail.

Various features of the invention disclosed in the claims may be better understood in view of the drawings and detailed description. The apparatus, methods, recipes, and various embodiments disclosed herein are provided for purposes of illustration. The disclosed structural and functional features are intended to enable those skilled in the art to specifically practice the various embodiments, and are not intended to limit the scope of the invention. The terms and phrases disclosed are intended to explain various features of the disclosed invention in an easy-to-understand manner, and are not intended to limit the scope of the invention.

In the following description of the present invention, if it is determined that the detailed description of the related known technology may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

Hereinafter, a method of preparing a cross-linking medium-viscous-solution using a polymer polysaccharide according to an embodiment of the present invention will be described.

1 is a flowchart illustrating a method of preparing a crosslinking midpoint solution according to an embodiment of the present invention.

Step S101 is a step of preparing an aqueous solution by dissolving the polymer polysaccharide in an alkaline solution. In one embodiment, an aqueous solution having a concentration of 1 to 12% by weight (w / v) may be prepared by dissolving a polymer polysaccharide having a molecular weight of 1,000 to 10,000,000 Da in an alkaline solution of pH 9.0 to 14.0. In one embodiment, the aqueous solution may be stirred well using a stirrer to facilitate dissolution of the polymer polysaccharide.

In this case, when the pH of the alkaline solution is high, the crosslinking reaction occurs quickly, but at the same time, since the decomposition of the polymer polysaccharide is also promoted, the polymer polysaccharide is first dissolved in the weak base alkali solution, and the polymer polysaccharide is completely dissolved, and then the strong base alkali solution is added. To prepare an aqueous solution. At this time, the pH of the strong base alkaline solution may be greater than the pH of the weak base alkali solution. For example, an aqueous solution having a high pH of 12 to 14 may be prepared by first dissolving the polymer polysaccharide in a weak base alkaline solution of pH 9.0 and completely dissolving the polymer polysaccharide, and then adding a strong base alkali solution. In one embodiment, the strong base alkaline solution may comprise at least one of sodium hydroxide and potassium hydroxide.

For example, the polymer polysaccharide is dextran having a molecular weight of 1,000 to 2,000,000 Da, hyaluronic acid having a molecular weight of 50,000 to 10,000,000 Da, or an alkali salt thereof, alginic acid having a molecular weight of 1,000 to 200,000 Da, or an alkali thereof. At least one of the salts.

Step S103 is a step of injecting nitrogen or an inert gas into the aqueous solution. In one embodiment, while continuously stirring the aqueous solution in which the polymer polysaccharide is completely dissolved, the density of the aqueous solution may be lowered by spraying nitrogen gas or inert gas into the aqueous solution. Small nozzles or spargers can be used for the application, and the use of a porous injector is advantageous because of the fast gas distribution. For example, the inert gas can include at least one of neon and argon.

Step S105 is a step of performing a vacuum treatment in an aqueous solution. In this way, when the vacuum is applied to the aqueous solution, the density of the aqueous solution can be further lowered. In some embodiments, step S103 and step S105 may be performed in the reverse order, or may be performed simultaneously. In other words, although FIG. 1 illustrates a case where step S105 is performed after step S103 is performed, this is merely an example for description, and steps S103 and S105 may be performed regardless of the order or at the same time.

Step S107 is a step of preparing a crosslinking midpoint solution by adding a crosslinking agent (or crosslinking monomer) to an aqueous solution having a low density. Through this, the polymer polysaccharide and the crosslinking agent included in the aqueous solution may form a crosslink. In one embodiment, crosslinking is temperature dependent so that the temperature of the aqueous solution can be adjusted at 5 to 60 ° C. to control the rate of crosslinking. For example, the crosslinking agent may include at least one of divinyl sulfone, epichlorohydrin, and epibromohydrin.

Step S109 is a step of removing the diluent after a certain time after adding the diluent to the crosslinking medium solution. That is, the crosslinking agent remaining in the crosslinking midpoint solution in which the formation of the crosslinking is completed may be diluted and removed in the diluent. For example, the diluent may include at least one of purified water, saline, and phosphate buffer solution. In one embodiment, ethanol may be mixed with distilled water. At this time, the concentration of ethanol used may be 1 to 40% by volume (v / v). In one embodiment, when a phosphate buffer solution is used as the diluent, the pH of the crosslinking concentration solution may be adjusted to 5.5 to 8.5.

As such, the polymer solution having a high swelling property but not high swelling property can be prepared while forming intramolecular or intermolecular crosslinks in the natural polymer polysaccharide or derivatives thereof through the process of FIG. 1.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the present invention is not limited only to these embodiments.

Example 1

In one embodiment, sodium hyaluronate may be used as the polymer polysaccharide. In this case, 6 g of sodium hyaluronate having an intrinsic viscosity value of 1.8 m ³ / kg and a molecular weight of about 1,300,000 Da is dissolved in 200 ml of sodium hydroxide solution having a concentration of 0.05 mol / l at a room temperature of 3% by weight (w / v). Aqueous sodium hyaluronate solution can be prepared. The pH can be adjusted to 13 by adding a concentrated sodium hydroxide solution to an aqueous sodium hyaluronate solution.

The porous injection tube can be put in an aqueous sodium hyaluronate solution, and compressed nitrogen having a supply pressure of 1 kgf / cm 2 can be injected into the aqueous solution.

To the aqueous sodium hyaluronate solution, 0.5 g of divinyl sulfone is added as a crosslinking agent and stirred. In one embodiment, the crosslinking reaction may occur by stirring for 10 minutes. Thereafter, the stirring can be stopped and the crosslinking reaction can be continued for 30 minutes. Through this reaction, a crosslinking midpoint solution can be prepared.

Put 800 ml of purified water as a diluent in the crosslinking medium solution and leave for 30 minutes to remove purified water. Repeat this process twice. Here, 800 ml of 10% by volume ethanol aqueous solution (v / v) was added and left for 30 minutes to remove the ethanol aqueous solution. Repeat this process twice. Put phosphate buffer solution 800ml and leave for 30 minutes, then remove the phosphate buffer solution. Repeat this process twice.

Phosphoric acid buffer solution was added to the crosslinking medium solution, the final volume was adjusted to 400 ml, stirred for 12 hours, and then pH and viscosity were measured. Viscosity measurement was performed using a Brookfield viscometer spindle No. 51 and measured at 1.5 rpm. In addition, the cytotoxicity was confirmed as follows. 2 ml of the crosslinking concentration solution was taken and placed in the well of a 6-well dish. 1x10 ⁴ cells were inoculated into each well to start culture, and 2, 4, 6, and 8 days after the start of the culture, cell density was observed under a microscope to confirm cytotoxicity. Viscosity and pH cytotoxicity can be confirmed as shown in Table 1 below.

Example 2

In Example 1, after nitrogen was injected into the porous spray pipe, a vacuum of 600 mTorr was applied to the dissolution tank. And the same process as Example 1 was implemented.

Example 3

In one embodiment, dextran may be used as the polymer polysaccharide. In this case, 6 g of dextran having a molecular weight of about 70,000 Da is dissolved in 200 ml of sodium hydroxide solution having a concentration of 0.05 mol / l at room temperature to prepare an aqueous solution of dextran having a concentration of 3% by weight (w / v). And the same process as Example 1 was implemented.

Example 4

In one embodiment, dextran may be used as the polymer polysaccharide. In this case, 6 g of dextran having a molecular weight of about 70,000 Da is dissolved in 200 ml of sodium hydroxide solution having a concentration of 0.05 mol / l at room temperature to prepare an aqueous solution having a concentration of 3% by weight (w / v). And the same process as Example 2 was performed.

Example 5

In one embodiment, sodium salts of alginic acid may be used as the polymer polysaccharide. In this case, 6 g of sodium salt of alginic acid having a molecular weight of about 100,000 Da is dissolved in 200 ml of sodium hydroxide solution having a concentration of 0.05 mol / l at room temperature to prepare an aqueous solution having a concentration of 3% by weight (w / v). And the same process as Example 1 was implemented.

Example 6

In one embodiment, sodium salts of alginic acid may be used as the polymer polysaccharide. In this case, 6 g of sodium salt of alginic acid having a molecular weight of about 100,000 Da is dissolved in 200 ml of sodium hydroxide solution having a concentration of 0.05 mol / l at room temperature to prepare an aqueous solution having a concentration of 3% by weight (w / v). And the same process as Example 2 was performed.

Comparative Example 1

In Examples 1 and 2, the nitrogen gas injection step and the vacuum step were omitted. And the same process as in Example 1 was carried out, the cytotoxicity test was not performed.

Comparative Example 2

In Examples 3 and 4, the nitrogen gas injection step and the vacuum step were omitted. And the same process as in Example 1 was carried out, the cytotoxicity test was not performed.

Comparative Example 3

In Examples 5 and 6, the nitrogen gas injection step and the vacuum step were omitted. And the same process as in Example 1 was carried out, the cytotoxicity test was not performed.

Polymer polysaccharide concentration (% by weight) Viscosity (cP) pH Cytotoxicity
○: Yes
X: none Example 1 1.5 1,810 6.8 X Example 2 1.5 1,680 6.8 X Example 3 1.5 1,580 6.9 X Example 4 1.5 1,510 6.9 X Example 5 1.5 1,270 6.8 X Example 6 1.5 1,220 6.8 X Comparative Example 1 1.5 38,330 6.9 - Comparative Example 2 1.5 31,520 6.9 - Comparative Example 3 1.5 30,440 6.9 -

Referring to <Table 1>, it can be seen that Examples 1 to 6, which performed at least one of a nitrogen gas injection step and a vacuum step, have a lower viscosity (cP) than Comparative Examples 1 to 3. That is, the crosslinked mid-range solution having high fluidity and high swellability can be prepared through the nitrogen gas injection step and the vacuum step.

In addition, it can be seen that the viscosity of Examples 2, 4, and 6, which performed both the nitrogen gas injection step and the vacuum step, is lower than those of Examples 1, 3, and 5, which performed only the nitrogen gas injection step.

The above description is merely illustrative of the technical idea of the present invention, and those skilled in the art may make various changes and modifications without departing from the essential characteristics of the present invention.

Therefore, the embodiments disclosed herein are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the present invention is not limited by these embodiments.

The protection scope of the present invention should be interpreted by the claims, and all technical ideas within the scope equivalent thereto should be understood as being included in the scope of the present invention.

Claims (7)

Dissolving the polymer polysaccharide in an alkaline solution to prepare an aqueous solution;
Injecting nitrogen or an inert gas into the aqueous solution; And
Preparing a crosslinking medium solution by adding a crosslinking agent to the aqueous solution sprayed with nitrogen or an inert gas;
Including,
Process for the preparation of crosslinking viscosity solution.
The method of claim 1,
Prior to preparing the crosslinking midpoint solution,
Performing vacuum treatment on the aqueous solution;
Further comprising,
Process for the preparation of crosslinking viscosity solution.
The method of claim 1,
After preparing the crosslinking medium solution,
Adding a diluent to the crosslinking medium solution and removing the diluent after a predetermined time;
Further comprising,
Process for the preparation of crosslinking viscosity solution.
The method of claim 1,
Preparing the aqueous solution,
Dissolving the polymer polysaccharide in a weak base alkali solution and dissolving the polymer polysaccharide, and then adding a strong base alkali solution to prepare the aqueous solution;
Including,
PH of the strong base alkali solution is greater than the pH of the weak base alkali solution,
Process for the preparation of crosslinking viscosity solution.
The method of claim 1,
Injecting the nitrogen or inert gas,
Injecting the nitrogen or the inert gas into the aqueous solution using a nozzle or a sparger;
Including,
Process for the preparation of crosslinking viscosity solution.
The method of claim 1,
Injecting the nitrogen or inert gas,
Injecting said nitrogen or inert gas into said aqueous solution while stirring said aqueous solution,
Including,
Process for the preparation of crosslinking viscosity solution.
The method of claim 1,
The inert gas, at least one of neon and argon,
Process for the preparation of crosslinking viscosity solution.

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