KR20080020222A - Multilayer film comprising hyaluronic acid derivatives - Google Patents

Abstract

A multi-layer film comprising hyaluronic acid derivatives is provided to obtain a multi-layer film that is safe inside a human body and contains hyaluronic acid derivatives whose decomposition speed is controlled. A multi-layer film comprising hyaluronic acid derivatives includes a tissue adhesion layer and a porous support layer. The tissue adhesion layer contains hyaluronic acid derivative that is obtained by reacting polymer selected among polylactic acid, polyglycolic acid and lactic acid-glycolic acid polymer with the hyaluric acid. The porous support layer contains hyaluronic acid in the form of hydrogel and is disposed on the tissue adhesion layer. The polymer is coupled to the carboxyl group of the hyaluronic acid by interposing a linker therebetween.

Description

Multilayer film comprising hyaluronic acid derivatives

1 is a graph showing the result of quantifying the hydrazide group introduction rate in hyaluronic acid by nuclear magnetic resonance method.

Figure 2 is a graph showing the result of analysis with HPLC after dialysis of the hyaluronic acid derivative.

Figure 3 is a graph showing the results of analyzing the degradation products of hyaluronic acid and hyaluronic acid derivatives treated with hyalurodinase by GPC.

4A and 4B are graphs showing the results of analysis of carbazole assay and GPC for degradation products of hyaluronic acid derivatives in the form of hydrogel treated with hyaluronidase.

The present invention relates to a multilayer membrane including a hyaluronic acid derivative, and more particularly, to a multilayer membrane including a hyaluronic acid derivative having a controlled rate of decomposition in the body.

In order to treat alveolar bones damaged by periodontal disease, recent attempts have been made to introduce new membranes into damaged periodontal tissues to promote healing, induce restoration of complete periodontal tissues, and improve bone graft results to induce the production of new alveolar bones. .

As part of such an attempt, a collagen shielding membrane, which is a natural polymer, has been applied as an induction tissue regeneration procedure or a dressing material for skin or mucosal tissue. However, collagen may cause inflammation and allergic reactions if the antigenicity is not completely removed as a protein, and the degradation rate in vivo is not constant. In addition, when collagen is derived from an animal, there are many risks of causing diseases such as mad cow disease.

The technical problem to be achieved by the present invention is to provide a multilayer membrane including a hyaluronic acid derivative that is safe in vivo and the rate of degradation is controlled.

According to an aspect of the present invention, there is provided a multilayer film including a hyaluronic acid derivative obtained by reacting hyaluronic acid with a polymer selected from at least one selected from polylactic acid, polyglycolic acid, and lactic acid-glycolic acid copolymer. And a porous support layer disposed on the tissue adhesion layer and the hyaluronic acid derivative in the form of a hydrogel.

In this case, the polymer of the tissue adhesive layer may be bonded to the carboxyl group of the hyaluronic acid through a linker.

The linker may comprise at least two amino groups, one of the amino groups may be bonded to the carboxyl group of the hyaluronic acid, and the other of the amino groups may be bonded to the carboxyl group of the polymer.

Such a tissue adhesive layer may include at least one unit represented by the following Chemical Formula 1.

Formula 1

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, and R ₄ is a polylactic acid, polyglycolic acid and lactic acid-glycolic acid aerial At least one selected from coalescing polymers. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

In addition, the hyaluronic acid derivative in the form of a hydrogel of the porous support layer may be obtained by reacting hyaluronic acid with suverate.

The suverate may be bonded to the carboxyl group of the hyaluronic acid via a linker, and the suverate may preferably be bis (sulfosuccinimidyl) suverrate.

The linker comprises at least two amino groups, one of the amino groups may be bonded to the carboxyl group of the hyaluronic acid, and the other of the amino groups may be bonded to the carbonyl group of the suverate.

The porous support layer may include at least one unit represented by the following formula (2).

Formula 2

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, and y is an integer from 0 to 10. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

In addition, the tissue adhesive layer may be formed, for example, by solvent extraction, and the porous support layer may be formed by freeze drying on the tissue adhesive layer.

The multilayer membrane including the tissue adhesive layer and the porous support layer as described above may be applied to, for example, a periodontal tissue regeneration shielding membrane.

According to another aspect of the present invention, there is provided a multilayer membrane including a hyaluronic acid derivative obtained by reacting hyaluronic acid with a polymer selected from at least one selected from polylactic acid, polyglycolic acid, and lactic acid-glycolic acid copolymer. And a porous support layer on the tissue adhesion layer and including a hyaluronic acid derivative obtained by reacting hyaluronic acid with methacrylic anhydride.

The polymer of the tissue adhesive layer may be bonded to the carboxyl group of the hyaluronic acid through a linker.

In addition, the linker may include at least two amino groups, one of the amino groups may be bonded to the carboxyl group of the hyaluronic acid, and the other of the amino groups may be bonded to the carboxyl group of the polymer.

Such a tissue adhesive layer may include at least one unit represented by the following Chemical Formula 1.

Formula 1

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, and R ₄ is a polylactic acid, polyglycolic acid and lactic acid-glycolic acid aerial At least one selected from coalescing polymers. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

In addition, the anhydrous methacryl of the porous support layer may be bonded to the carboxyl group of the hyaluronic acid via a linker.

The linker may comprise at least two amino groups, one of which may be bonded to the carboxyl group of the hyaluronic acid, and the other of which may be bonded to the carbonyl group of the anhydrous methacryl.

The porous support layer may include at least one unit represented by the following formula (3).

Formula 3

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, and y is an integer from 0 to 10. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

The linker may be bonded to the carboxyl group of the hyaluronic acid in a solution containing 0 to 60% by volume of at least one organic solvent, for example, ethanol may be used as the organic solvent.

In addition, the hyaluronic acid derivative included in the porous support layer may be in the form of a hydrogel crosslinked by a dithiol-based crosslinking agent. In this case, the dithiol-based crosslinking agent may be a peptide including an array of arginine, glycine and aspartic acid, for example, each of which contains cysteine between them.

The porous support layer may include at least one unit represented by the following formula (4).

Formula 4

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, C is cysteine, R is arginine, G is glycine, D is aspart Acid, and (-S-) represents sulfur derived from a mercapto group contained in cysteine. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

In addition, the tissue adhesive layer may be formed, for example, by solvent extraction, and the porous support layer may be formed by freeze drying on the tissue adhesive layer.

The multilayer membrane including the tissue adhesive layer and the porous support layer as described above may be applied to, for example, a periodontal tissue regeneration shielding membrane.

Specific details of other embodiments are included in the detailed description and the drawings. Accordingly, the advantages and features of the present invention, and methods for achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms, and only the embodiments make the disclosure of the present invention complete, and the scope of the invention to those skilled in the art. It is provided for the purpose of full disclosure, and the invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout.

Hereinafter, a multilayer film according to an embodiment of the present invention will be described in detail.

The multilayer film according to an embodiment of the present invention includes a tissue adhesive layer and a porous support layer positioned on the tissue adhesive layer.

First, the tissue adhesion layer in the multilayer film will be described.

The tissue adhesive layer has a function of inducing tissue regeneration in the side facing the damaged tissue. This tissue adhesive layer comprises a hyaluronic acid derivative obtained by reacting hyaluronic acid with a polymer selected from at least one of polylactic acid (PLA), polyglycolic acid (PGA) and lactic acid-glycolic acid copolymer (PLGA). .

Hyaluronic acid is a type of glucosamide glycan consisting of a disaccharide unit in which D-glucuronic acid and N-acetylglucosamine are linked by β (1 → 3) glycoside bonds, as shown in the following formula (5). Hyaluronic acid has no differences in chemical and physical structure, humans have a metabolic system, and is the safest biomaterial in terms of immunity and toxicity. Hyaluronic acid can be produced in large quantities, for example by fermenting microorganisms, in which case there is no risk of viral contamination, allergic reactions, and can maintain a certain level of quality.

Formula 5

The term "hyaluronic acid" in the present invention is a concept that includes not only hyaluronic acid itself but also salts thereof. That is, "hyaluronic acid" in the present invention includes hyaluronic acid, a hyaluronic acid salt or a mixture of hyaluronic acid and hyaluronic acid. Hyaluronic acid salts include both inorganic salts such as sodium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronic acid and the like, and organic salts such as tetrabutylammonium hyaluronic acid and the like. In some cases, at least two combinations thereof may be used. Although the molecular weight of hyaluronic acid in this invention is not specifically limited, For example, it may be 100,000-10,000,000.

Hyaluronic acid and polylactic acid (PLA), polyglycolic acid (PGA), or glycolic acid copolymer (PLGA) included in the tissue adhesive layer are chemically covalently bonded. Therefore, hyaluronic acid combined with polylactic acid (PLA), polyglycolic acid (PGA), and lactic acid-glycolic acid (PLGA) in the tissue adhesive layer may be separated from each other by environmental changes such as salt concentration and pH change even after administration in vivo. It is not separated.

The molecular weight of the polymer selected from polylactic acid (PLA), polyglycolic acid (PGA) and lactic acid-glycolic acid copolymer (PLGA) is not particularly limited, but in terms of reaction efficiency with hyaluronic acid, it is usually 100,000 viscosity average molecular weight The thing of Dalton or less, for example, the thing of 1,000-50,000 dalton can be used.

Such hyaluronic acid may be bound via a linker when polylactic acid (PLA), polyglycolic acid (PGA), or lactic acid-glycolic acid copolymer (PLGA) is bonded. Linkers are introduced into hyaluronic acid to regulate the rate of degradation of the hyaluronic acid derivative in vivo.

Hyaluronic acid derivatives are decomposed by hyalurodinase, a hyaluronic acid degrading enzyme present in the body, by recognizing hyaluronic acid by a carboxyl group of hyaluronic acid. Therefore, the rate of decomposition of the hyaluronic acid derivative by hyalurodinase can be controlled by binding the linker to the carboxyl group of hyaluronic acid to control the number of exposure of the carboxyl group. For example, the higher the proportion of the linker introduced into hyaluronic acid, the lower the degradation rate of the hyaluronic acid derivative in vivo. Therefore, by controlling the introduction rate of the linker introduced into the hyaluronic acid, it is possible to control the decomposition rate of the hyaluronic acid derivative in the body as desired.

Such linkers may comprise at least two amino groups. The linker may be, for example, a dihydrazide compound, a diamine compound, a hydrazine compound comprising a plurality of amino groups. The linker is for example H ₂ N- (CH ₂ ) x-NH ₂ (where x is an integer from 0 to 10), H ₂ NNHCO- (CH ₂ ) -CH _2- (O-CH ₂ -CH ₂ ) is a diamine compound represented by x-NH ₂ (wherein x is an integer of 0 to 10), and H ₂ NNHCO- (CH ₂ ) x-CONHNH ₂ (wherein x is an integer of 1 to 10). represented dihydrazide compound, or NH (R ₁₎ -NH (R ₂₎ may be a hydrazine compound represented by (wherein, R ₁ and R ₂ are each a hydrogen atom or a C _{1 -6} alkyl group).

The position at which polylactic acid (PLA), polyglycolic acid (PGA) or lactic acid-glycolic acid copolymer (PLGA), or linker is introduced into hyaluronic acid may be, for example, the carboxyl group of hyaluronic acid. When introduced into a carboxyl group, the polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), by forming an amide group, hydrazide group, diacylhydrazide group, carboxylic acid ester group Or a linker moiety can be introduced into the hyaluronic acid. Hyaluronic acid derivatives formed by introducing polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), or linkers into hyaluronic acid to form carboxylic acid esters with hyaluronic acid The desorption of the polymer may occur in a relatively short time due to hydrolysis in vivo. Therefore, polylactic acid (PLA), polyglycolic acid (PGA), or lactic acid-glycolic acid copolymer (PLGA) is introduced into hyaluronic acid via a linker containing an amide group, a hydrazide group, or the like having a slow hydrolysis rate. It is preferable.

As a method of chemically graft bonding polylactic acid (PLA), polyglycolic acid (PGA), or lactic acid-glycolic acid copolymer (PLGA) to hyaluronic acid, first, the carboxyl group of hyaluronic acid is activated. For example N, N'-carbonyldiimidazole (CDI), N, N'-dicyclohexylcarbonylimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-di Hydroxynoline (EEDQ), 4- (4,6-dimethoxy-1,3,5-triazine) -4-methylmorpholium (DMT-MM), 2-benzotriazole-1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy-tris Pyrrolidino-phosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) or 1-ethyl-3- (3-dimethylamino Condensation agents, such as propyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS), are used alone or in combination as appropriate to active esterify the carboxyl group of hyaluronic acid.

A linker containing a functional group reactive with active esters such as a hydrazide group and an amino group is introduced therein, and then ion-exchanged with a tetrabutylammonium (TBA) salt, and then in 1-ethyl- in dimethyl sulfoxide (DMSO). The method of reacting with the polymer which activated the carboxyl group by 3- (3-dimethylaminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS) can be used. In this case, the linker may be introduced into the carboxyl group of hyaluronic acid in a solution containing distilled water or at least one organic solvent. The organic solvent may be, for example, ethanol, and the proportion of the organic solvent in the total solution may be about 60% by volume or less of the solution, and preferably about 50% by volume.

In addition, another method of chemically graft-bonding polylactic acid (PLA), polyglycolic acid (PGA), or lactic acid-glycolic acid copolymer (PLGA) to hyaluronic acid, first, the terminal carboxyl group of the polymer is 1-ethyl-3- ( Activated with 3-dimethylaminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS) and reacted with a linker so that it has a diamine or dihydrazide end, and reacts with a carboxyl group of hyaluronic acid. A method of bonding with a condensing agent such as -ethyl-3- (3-methylaminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide can be used. In this case, the linker may be introduced into the carboxyl group of hyaluronic acid in a solution containing distilled water or at least one organic solvent. The organic solvent may be, for example, ethanol, and the proportion of the organic solvent in the total solution may be about 60% by volume or less of the solution, and preferably about 50% by volume.

The tissue adhesive layer of the multilayer film according to the embodiment of the present invention as described above may include a repeating structure of the unit represented by the following formula (1) in the molecule.

Formula 1

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, and R ₄ is a polylactic acid, polyglycolic acid and lactic acid-glycolic acid aerial At least one of the polymers is selected. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

Such tissue adhesive layer can be processed into a film using, for example, solvent extraction.

The porous support layer located on the tissue adhesion layer allows the bone cells to integrate toward the porous support layer in contact with the bone tissue. This porous support layer comprises hyaluronic acid in the form of a hydrogel. In more detail, the hyaluronic acid derivative in the form of a hydrogel obtained by reacting hyaluronic acid with suverrate is included. The suverate reacted with hyaluronic acid may be, for example, Bis [sulfosuccinimmidyl] suberate.

Hyaluronic acid is chemically covalently bonded to suverate. Therefore, hyaluronic acid derivatives in which hyaluronic acid and suverate are combined are not separated from each other by environmental changes such as salt concentration and pH change even after administration in vivo.

Such hyaluronic acid may be bound via a linker when combined with subvertate. The linker may be introduced into the hyaluronic acid in combination with the carbonyl group of the suverate, and serves to control the degradation rate of the porous support layer in vivo. That is, the rate of decomposition of the porous support layer administered in vivo may vary depending on the proportion of the linker introduced into the hyaluronic acid. Since the linker is substantially the same as the linker included in the tissue adhesive layer, duplicate description thereof will be omitted.

The position at which the suverate or linker is introduced into the hyaluronic acid can be, for example, a carboxyl group of hyaluronic acid. When introduced to a carboxyl group, the subvertate or linker moiety can be introduced to the hyaluronic acid by forming an amide group, hydrazide group, diacylhydrazide group, carboxylic acid ester group. In the hyaluronic acid derivative formed by the introduction of a submerate or a linker to the hyaluronic acid by forming a carboxylic acid ester with the hyaluronic acid, the desorption of the suverate may occur in a relatively short time due to hydrolysis in the administration solution and in vivo. Therefore, it is preferable to introduce submerate into hyaluronic acid via a linker containing an amide group, a hydrazide group, or the like having a slow hydrolysis rate.

As a method of chemically graft-bonding submerate to hyaluronic acid, the carboxyl group of hyaluronic acid is activated first. For example N, N'-carbonyldiimidazole (CDI), N, N'-dicyclohexylcarbonylimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2- Dihydroxynoline (EEDQ), 4- (4,6-dimethoxy-1,3,5-triazine) -4-methylmorpholium (DMT-MM), 2-benzotriazole-1,1,3 , 3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy- Tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) or 1-ethyl-3- (3-dimethyl A condensing agent such as aminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS), alone or in appropriate combination, is used for active esterification of the carboxyl group of hyaluronic acid.

After introducing a linker containing a functional group reactive with active esters such as a hydrazide group and an amino group, a submerate may be reacted with the terminal of the linker to react the carbonyl group of the suverate group with the terminal of the linker. have. In this case, the linker may be introduced into the carboxyl group of hyaluronic acid on a solution containing distilled water or at least one organic solvent, the organic solvent may be, for example, ethanol, and the ratio of the organic solvent in the total solution is about 60% by volume or less. And preferably about 50% by volume.

As described above, the porous support layer of the multilayer membrane according to the exemplary embodiment of the present invention may include a repeating structure of a unit represented by the following Chemical Formula 2 in a molecule.

Formula 2

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, and y is an integer from 0 to 10. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

Such a porous support layer may be, for example, poured a precursor solution of a hyaluronic acid derivative on a tissue adhesive layer and then crosslinked, and then placed in a deep freezer and rapidly cooled at about −70 ° C., followed by a freeze dryer. It can be completed by drying in).

The multilayer membrane including the tissue adhesive layer and the porous support layer as described above may be used, for example, for the purpose of tissue regeneration induction shielding membrane. In other words, the inner surface of the shielding membrane, that is, the porous support layer, may be inserted or overlaid on the damaged periosteal bone or implanted jaw area and then fixed to the root with surgical biodegradable sutures or firmly attached to bone tissue using a biocompatible adhesive. Bone tissue that requires induction regeneration is located on the side, and the connective tissue or epithelial tissue is directly contacted and covered on the outer surface, that is, the tissue adhesive layer.

Subsequently, a multilayer film according to another embodiment of the present invention will be described.

The multilayer film according to another embodiment of the present invention includes a tissue adhesive layer and a porous support layer positioned on the tissue adhesive layer.

Since the tissue adhesive layer of the multilayer film according to another embodiment of the present invention is substantially the same as the tissue adhesive layer included in the multilayer film according to an embodiment of the present invention, redundant description thereof will be omitted. Therefore, here, a multilayer film according to another embodiment of the present invention will be described based on the porous support layer.

The porous support layer positioned on the tissue adhesive layer includes a hyaluronic acid derivative obtained by reacting hyaluronic acid with methacrylic anhydride.

Hyaluronic acid is chemically covalently bonded to anhydrous methacryl. Therefore, hyaluronic acid derivatives in which hyaluronic acid and anhydrous methacryl are combined are not separated from each other by environmental changes such as salt concentration and pH change even after administration in vivo.

Such hyaluronic acid may be bound via a linker when combined with anhydrous methacryl. The linker can be introduced into hyaluronic acid in combination with the carbonyl group of anhydrous methacryl and controls the rate of degradation in vivo of the porous support layer. Since the function and type of the linker, the position of introduction of the linker, the method of introducing the linker, and the like are substantially the same as those of the multilayer film according to the exemplary embodiment of the present invention, redundant descriptions are omitted here.

As described above, the porous support layer of the multilayer membrane according to another embodiment of the present invention may include a repeating structure of a unit such as Chemical Formula 3 in a molecule.

Formula 3

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, and y is an integer from 0 to 10. In this case, preferably, R ₁ and R ₂ may each be a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

Such a porous support layer may be completed, for example, by pouring a precursor solution of a hyaluronic acid derivative on a tissue adhesive layer and then performing a crosslinking reaction, placing the same in a freezer refrigerator, rapidly cooling at about -70 ° C, and then drying in a freeze dryer. have.

The multilayer membrane including the tissue adhesive layer and the porous support layer as described above may be used, for example, for the purpose of tissue regeneration induction shielding membrane.

Subsequently, a multilayer film according to another embodiment of the present invention will be described.

The multilayer film according to another embodiment of the present invention includes a tissue adhesive layer and a porous support layer positioned on the tissue adhesive layer.

Since the tissue adhesive layer of the multilayer film according to another embodiment of the present invention is substantially the same as the tissue adhesive layer included in the multilayer film according to an embodiment of the present invention, redundant description thereof will be omitted. In addition, the porous support layer of the multilayer film according to another embodiment of the present invention is another embodiment of the present invention in that the hyaluronic acid derivative included in the porous support layer of the multilayer film according to another embodiment of the present invention is cross-linked to have a hydrogel form There is a difference from the multilayer film according to the example. Therefore, here, a multilayer film according to another embodiment of the present invention will be described based on the porous support layer.

The hyaluronic acid derivative included in the porous support layer of the multilayer membrane according to another embodiment of the present invention has a hydrogel form in which the hyaluronic acid derivative according to an embodiment of the present invention is crosslinked by a dithiol-based crosslinking agent.

The dithiol-based crosslinking agent may be, for example, a peptide having a cysteine containing a mercapto group (-SH) at both ends and having an arrangement of arginine, glycine and aspartic acid therebetween. Both ends of the crosslinking agent are each combined with an α, β-unsaturated carbonyl group derived from anhydrous methacryl of the hyaluronic acid derivative to form a hyaluronic acid derivative in hydrogel form.

Hyaluronic acid derivatives may be improved in cell adhesion through the introduction of peptides comprising arginine, glycine and aspartic acid sequences. Cell adhesion is caused by integrin, a receptor on the cell surface in vivo, and an adhesion protein present in the ECM. Peptide arrays that directly attach cells to the adhesion protein This is an arrangement of arginine, glycine and aspartic acid. Therefore, cell adhesion can be effectively induced by introducing a dithiol-based crosslinking agent comprising an arrangement of arginine, glycine and aspartic acid into the hyaluronic acid derivative.

The porous support layer of the multilayer membrane according to another embodiment of the present invention formed as described above may include a repeating structure of a unit as shown in Formula 4 in the molecule.

Formula 4

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, C is cysteine, R is arginine, G is glycine, D is aspart Acid, and (-S-) represents sulfur derived from a mercapto group contained in cysteine. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

Such a porous support layer may be completed, for example, by pouring a precursor solution of a hyaluronic acid derivative on a tissue adhesive layer and then performing a crosslinking reaction, placing the same in a freezer refrigerator, rapidly cooling at about -70 ° C, and then drying in a freeze dryer. have.

The multilayer membrane including the tissue adhesive layer and the porous support layer as described above may be used, for example, for the purpose of tissue regeneration induction shielding membrane.

Hereinafter, the present invention will be described in more detail through experimental examples. However, it is to be understood that the experimental examples are for illustrating the present invention and the present invention is not limited by the following experimental examples.

[ Experimental Example  1] Preparation of hyaluronic acid derivative

[ Experimental Example  1-1] Hydrazide  Preparation of Introduced Hyaluronic Acid

Hyaluronic acid solutions having concentrations of hyaluronic acid (Denkikagaku Kogyo Co. (Tokyo, Japan)) in distilled water were prepared at 2 mg / ml and 5 mg / ml, respectively. In this case, when the hyaluronic acid solution includes only distilled water, ethanol was prepared in a ratio of 25% by volume and 50% by volume with respect to distilled water, respectively.

Here, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (Sigma-Aldrich (St. Louis, MO, USA) was used to activate the carboxyl group of hyaluronic acid, followed by adipic dihydra. Zide (ADH) (Sigma-Aldrich (St. Louis, Mo., USA)) was added and reacted with 1N hydrochloric acid for about 2 hours at room temperature while maintaining the pH at about 4.8, where the amount of EDC and ADH added was hyaluronic. It was 4 times and 40 times in molar ratio with respect to the carboxyl group of lonic acid, respectively.

The reaction product was dialyzed with 100 mM sodium chloride solution, 25% ethanol solution, distilled water, and lyophilized to obtain hyaluronic acid (HA-ADH) to which a hydrazide group was introduced.

The result of quantifying the hydrazide group introduction rate in each obtained hyaluronic acid by ¹ H-NMR (DPX300, Bruker, Germany) is shown in FIG. 1 (N-acetyl group (α) of hyaluronic acid is about 2.1 ppm, a) The methylene groups (β, γ) of the diffic acid moiety are about 1.7 ppm, 1.8 ppm, 2.4 ppm, 2.5 ppm).

As shown in Figure 1, it was found that the introduction rate of the hydrazide group increases as the ratio of ethanol in the hyaluronic acid solution increases.

[ Experimental Example  1-2] Hydrazide  Quantification of Introduced Hyaluronic Acid

Hyaluronic acid (HA-ADH) to which the hydrazide group was introduced, obtained by setting the hyaluronic acid concentration to 5 mg / ml and the ethanol ratio to 50 vol% in Experimental Example 1-1, was analyzed by HPLC. Shown.

At this time, the HPLC conditions were that the eluate was 34 mM phosphate buffer (pH 6.6) / methanol = 80:20 (v / v), and the Waters 1525 binary HPLC pump, the Waters 2487 dual λ adjuvant detector, the Waters 717 plus autosampler ( Milford, MA, USA)), and the columns were ultrahydrogel 250 and 1000 columns (7.8 mm id x 30 cm), flow rate was 0.5 mL / min, sample injection volume was 10 μl, sample concentration was 5 mg / ml. Detection wavelength was 210 nm.

[ Experimental Example  1-3] Hydrazide  Enzyme Resistance Evaluation of Introduced Hyaluronic Acid

Hyaluronic acid, hyaluronic acid in which hydrazide groups were introduced in distilled water (HA-ADH), and hyaluronic acid in which hydrazide groups were introduced on a solution containing about 50% by volume of ethanol (stealth HA-ADH) were respectively about 4.57 mg / ml. Each was dissolved in 1.8 ml of 0.2 M sodium phosphate buffer (pH 6.2). To each of these solutions, 200 μl of a bovine testicular hyaluronidase solution at a concentration of 1500 U / ml was added and incubated at about 37 ° C. for 48 hours.

Hyaluronic acid (HA-ADH) in which hyaluronic acid or a hydrazide group is introduced is decomposed by N-acetalhexosaminide bonds by hyalurodinase, as shown in Scheme 1 below.

Scheme 1

After treating hyaluronic acid (HA-ADH) into which hyaluronic acid and a hydrazide group were introduced with hyalurodinase, a double bond in the decomposition product was quantified by ultraviolet spectroscopy, and the result of analyzing the decomposition rate is shown in FIG. 3. GPC analysis was used to analyze degradation products.

Figure 3 (a) is a result of GPC analysis showing before and after treatment of hyalurodinase with respect to hyaluronic acid, (b) is the hyaluronic acid (HA-ADH) of the hyaluronic acid (HA-ADH) in which hydrazide groups are introduced in distilled water GPC analysis results before and after treatment, (c) GPC analysis results before and after the treatment of hyalurodinase for hyaluronic acid (stealth HA-ADH) in which hydrazide groups were introduced on a solution containing about 50% by volume of ethanol. to be.

In GPC, the peak of a high molecular weight material comes out first, and the peak of a low molecular weight material comes out later. The decomposition products decomposed by the enzyme move backward because the molecular weight decreases. The greater the degree of change in the position of the peak, the greater the decomposition. The hyaluronic acid concentration in all of (a), (b) and (c) was about 5 mg / ml, and the hydrazide group introduction rates in (b) and (c) were 70.47% and 82.82%, respectively.

As shown in FIG. 3, the higher the introduction ratio of the hydrazide group to the hyaluronic acid, the lower the degree of degradation by the enzyme of the hyaluronic acid.

[ Experimental Example  1-4] Hydrazide  In introduced hyaluronic acid Subversate  Enzyme Resistance Evaluation of Introduced Hyaluronic Acid Derivatives

12 mg of each of hyaluronic acid having a hydrazide group introduction rate of 69.39 mol%, 79.61 mol%, and 84.98 mol%, respectively, was dissolved in 270 µl of PBS (0.01 M, pH 7.4, 25 ° C.). After complete dissolution, 180 μg of each solution was placed in a syringe with the front cut off.

Bis (sulfosuccinimidyl) severate was dissolved in 20 μl of PBS, added to the hyaluronic acid solution in the syringe and mixed uniformly. The amount of added bis (sulfosuccinimidyl) subrate was 20 mol% of ADH introduced into hyaluronic acid.

This precursor solution was incubated at 37 ° C. for one hour to complete the crosslinking reaction.

Each completed hydrogel form hyaluronic acid (about 200 μl) was placed in a 4 ml vial. 0.9 ml of 0.2 M sodium phosphate buffer (pH 6.2) was added to each vial. To each vial was added 0.9 ml of 0.2 M sodium phosphate buffer (pH 6.2) containing 180 U of hyaluronate lyase. It was incubated at 37 ° C. for 96 hours.

At the designated sampling time, 200 μl of supernatant was removed from each vial and 200 μl of 0.2 M sodium phosphate buffer (pH 6.2) was added again. The dilution of the supernatant occurring at each sampling time was candidate. Each 200 μl of the extracted supernatant was immersed in a water bath containing boiling water for about 3 minutes to stop the enzyme activity.

Hydrolysis of hyaluronic acid derivatives in the form of hydrogels by hyaluronidase is analyzed by measuring the amount of glucuronic acid that has been decomposed into the supernatant through carbazole assay and GPC, respectively, and the results are shown in FIGS. 4b In the carbazole assay, the degree of hyaluronic acid degradation can be quantitatively analyzed by measuring the absorbance of the sample in which the color reaction has occurred using an ultraviolet spectrophotometer.

4A and 4B, hyaluronic acid derivative (◆) having a hydrazide group introduction ratio of 69.39 mol%, hyaluronic acid derivative (■) of 79.61 mol%, and hyaluronic acid derivative (▲) having 84.98 mol% The degree of degradation by hyaluronidase was found to progress slowly as the introduction rate of hydrazide groups increased. That is, the higher the introduction ratio of the hydrazide group, it was confirmed that the degradation by the degradation enzyme is suppressed.

[ Experimental Example  1-5] Hydrazide  Preparation of hyaluronic acid derivatives in which methacrylic anhydride is introduced into hyaluronic acid introduced

Hyaluronic acid (HA-ADH) to which the hydrazide group was introduced was dissolved in 2.25 mL of distilled water, and then 0.25 mL of phosphate buffer (200 mM, pH 8.0) was added to adjust the pH to 8. A 20 molar excess of anhydrous methacryl (MW = 154.17) relative to the hydrazide group was added to the hyaluronic acid (HA-ADH) solution into which the hydrazide group was introduced. After stirring for 4 hours at room temperature, the mixture was precipitated using ethanol to obtain a hyaluronic acid derivative (HA-MA). After ethanol washing three times, dried at room temperature. The introduction rate of methacrylic anhydride into hyaluronic acid can be obtained through ¹ H NMR analysis.

[ Experimental Example  1-6] Hyaluronic acid derivatives with anhydrous methacryl introduced Hydrogel  Manufactured in form

11 mg of the hyaluronic acid derivative (HA-MA) obtained in Experimental Example 1-5 was dissolved in PBS (240 µl, 10 mM, pH 7.4) containing 16 µl / ml of TEA for 2 hours. Adding TEA to PBS changes the pH to 9.5. Hyaluronic acid derivative (HA-MA) solution by dissolving a peptide containing cysteine-arginine-glycine-aspartic acid-arginine-cysteine in an amount of 1: 2 molar ratio of anhydrous methacryl to thiol as a crosslinking agent in DMSO After addition to the solution, the mixture was stirred and vortexed immediately to generate a vortex. 250 μl of this precursor solution was placed in a syringe with the front cut and incubated at 37 ° C. until the crosslinking reaction was completed.

[ Experimental Example  2] comprising a porous support layer and a tissue adhesive layer Multilayer  Produce

[ Experimental Example  2-1] Fabrication of Tissue Adhesion Layer

In order to introduce lactic acid-glycolic acid copolymer into hyaluronic acid, lactic acid-glycolic acid copolymer-NHS (PLGA-NHS) was synthesized by first coupling NHS to lactic acid-glycolic acid copolymer using DCC (Scheme 2). .

Next, hyaluronic acid (HA-ADH) having a hydrazide group introduction rate of 80 mol% or more was dissolved in DMSO, and then reacted with the lactic acid-glucolic acid copolymer-NHS (PLGA-NHS) solution at 40 ° C for 24 hours. (Scheme 3).

The hyaluronic acid derivative (HA-PLGA-NHS) obtained through the reaction has different behavior in solution depending on the degree of introduction of lactic acid-glycolic acid copolymer (PLGA) bound to hyaluronic acid. Hyaluronic acid derivatives (HA-PLGA-NHS), which have a low degree of substitution of lactic acid-glycolic acid copolymer (PLGA) (below 1.5 mole percent), behave in a more compact coil form than hyaluronic acid. PLGA) 's strong hydrophobic interaction. In contrast, the high degree of substitution of the lactic acid-glycolic acid copolymer (PLGA) (7.8 mole percent) hyaluronic acid derivative (HA-PLGA-NHS) has a sharp drop in affinity for water. In other words, by having a strong ionic bond, the solubility in water decreases, and it can be seen that it disperses as a gel in the solution, and as a result, it dissolves well in DMSO.

Dissolve the hyaluronic acid derivative (HA-PLGA-NHS), which has a high degree of substitution of lactic acid-glycolic acid copolymer (PLGA), in a volatile organic solvent, transfer the appropriate amount of lactic acid-glycolic acid to a small chalet, and then slowly evaporate the solvent. A tissue adhesive layer in the form of a film was obtained.

Scheme 2

Scheme 3

[ Experimental Example  2-2] Preparation of Porous Support Layer on Tissue Adhesion Layer

Hyaluronic acid (HA-ADH) introduced with hydrazide groups was dissolved in PBS (0.01M, pH 7.4, 25 ° C). Subsequently, a small amount of bis (sulfosuccinimidyl) severate in a molar ratio of bis (sulfosuccinimidyl) suvrate to 40 mg / ml of ADH was 20%. )).

After spraying a bis (sulfosuccinimidyl) severate solution on the tissue adhesive layer prepared in Experimental Example 2-1 in Petri dish, the bis (sulfosuccinimidyl) severate solution is uniformly distributed using spin coating or the like. . A hyaluronic acid (HA-ADH) solution with hydrazide groups is poured onto a tissue adhesive layer coated with a bis (sulfosuccinimidyl) suverate solution, and the bis (sulfosuccinimidyl) severrate solution naturally diffuses into the solution layer. Crosslinking reaction occurs. Incubate at 37 ° C. for one hour to complete the crosslinking reaction.

Petri dishes made with a hydrogel-type hyaluronic acid derivative on a tissue adhesive layer are rapidly cooled at about -70 ° C. in a freezer refrigerator, and then put in a freeze dryer and vacuumed while maintaining a low temperature to obtain moisture from the hydrogel-type hyaluronic acid derivative. This exited the porous support layer on the tissue adhesive layer.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above embodiments and can be variously modified and implemented by those skilled in the art without departing from the technical scope of the present invention.

As described above, the multilayered film including the hyaluronic acid derivative formed by using a hyaluronic acid derived from a microorganism and introducing a linker containing a predetermined amino group is safe in vivo, has excellent biocompatibility, and also controls the decomposition rate. Possible, for example, can be applied as a membrane for regenerating periodontal tissue.

Claims (34)

A tissue adhesive layer comprising a hyaluronic acid derivative obtained by reacting hyaluronic acid with a polymer selected from at least one of a polylactic acid, a polyglycolic acid, and a lactic acid-glycolic acid copolymer; And Located on the tissue adhesive layer, a multi-layer membrane comprising a porous support layer comprising a hyaluronic acid derivative in the form of a hydrogel. The method of claim 1, The polymer is a multilayer film bonded to the carboxyl group of the hyaluronic acid via a linker. The method of claim 2, The linker comprises at least two amino groups, one of the amino groups bonded to a carboxyl group of the hyaluronic acid, and the other of the amino groups bonded to a carboxyl group of the polymer. The method of claim 3, wherein The tissue adhesive layer is a multilayer film including at least one unit represented by the following formula (1). Formula 1

(Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH ₂ ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, R ₄ is polylactic acid, polyglycolic acid and lactic acid-glycolic acid At least one polymer in the copolymer) The method of claim 4, wherein R ₁ and R ₂ are each a hydrogen atom, and R ₃ is -NHCO- (CH ₂ ) y-CONH-. The method of claim 1, The hydrogel-type hyaluronic acid derivative is a multilayer film obtained by reacting hyaluronic acid with suverate. The method of claim 6, The submerate is a multilayer film bonded to the carboxyl group of the hyaluronic acid via a linker. The method of claim 6, Said subvertate is a bis (sulfosuccinimidyl) subvertate. The method of claim 7, wherein The linker comprises at least two amino groups, one of the amino group is bonded to the carboxyl group of the hyaluronic acid, the other one of the amino group is bonded to the carbonyl group of the suverate. The method of claim 9, The porous support layer is a multilayer membrane comprising at least one unit represented by the following formula (2). Formula 2

(Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH ₂ ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10) The method of claim 10, R ₁ and R ₂ are each a hydrogen atom, and R ₃ is -NHCO- (CH ₂ ) y-CONH-. The method of claim 7, wherein The linker is a multilayer film bonded to the carboxyl group of the hyaluronic acid in a solution containing 0 to 60% by volume of at least one organic solvent. The method of claim 12, Wherein said organic solvent is ethanol. The method of claim 1, The tissue adhesive layer is a multilayer film formed by a solvent extraction method. The method of claim 1, The porous support layer is a multilayer film formed by freeze drying on the tissue adhesive layer. The method of claim 1, The multilayer film is a multilayer film that is a shielding film for periodontal tissue regeneration. A tissue adhesive layer comprising a hyaluronic acid derivative obtained by reacting hyaluronic acid with a polymer selected from at least one of polylactic acid, polyglycolic acid, and lactic acid-glycolic acid copolymer; And Located on the tissue adhesion layer, a multilayer membrane comprising a porous support layer comprising a hyaluronic acid derivative obtained by reacting hyaluronic acid with anhydrous methacryl. The method of claim 17, The polymer is a multilayer film bonded to the carboxyl group of the hyaluronic acid via a linker. The method of claim 18, The linker comprises at least two amino groups, one of the amino groups bonded to a carboxyl group of the hyaluronic acid, and the other of the amino groups bonded to a carboxyl group of the polymer. The method of claim 19, The tissue adhesive layer is a multilayer film including at least one unit represented by the following formula (1). Formula 1

(Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH ₂ ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, R ₄ is polylactic acid, polyglycolic acid and lactic acid-glycolic acid At least one polymer in the copolymer) The method of claim 20, R ₁ and R ₂ are each a hydrogen atom, and R ₃ is -NHCO- (CH ₂ ) y-CONH-. The method of claim 17, An anhydrous methacryl is a multilayer film which couple | bonded with the carboxyl group of the hyaluronic acid via a linker. The method of claim 22, The linker comprises at least two amino groups, one of the amino group is bonded to the carboxyl group of the hyaluronic acid, the other of the amino group is bonded to the carbonyl group of the anhydrous methacryl. The method of claim 23, The porous support layer is a multilayer membrane comprising at least one unit represented by the following formula (3). Formula 3

(Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH ₂ ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10) The method of claim 24, R ₁ and R ₂ are each a hydrogen atom, and R ₃ is -NHCO- (CH ₂ ) y-CONH-. The method of claim 22, The linker is a multilayer film bonded to the carboxyl group of the hyaluronic acid in a solution containing 0 to 60% by volume of at least one organic solvent. The method of claim 26, Wherein said organic solvent is ethanol. The method of claim 17, The porous support layer is a multi-layer film comprising a hyaluronic acid derivative in the form of a hydrogel crosslinked by a dithiol-based crosslinking agent. The method of claim 28, The dithiol-based crosslinking agent is a multi-layered film comprising a cysteine at each end, and a peptide comprising an arginine, glycine and aspartic acid therebetween. The method of claim 29, The hyaluronic acid derivative is a multilayer film comprising at least one unit represented by the following formula (4). Formula 4

(Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C _{1 -6} alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH ₂ ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, y is an integer from 0 to 10, C is cysteine, R is arginine, G is glycine, D is Aspartic acid, (-S-) represents sulfur derived from the mercapto group contained in cysteine) The method of claim 30, R ₁ and R ₂ are each a hydrogen atom, and R ₃ is -NHCO- (CH ₂ ) y-CONH-. The method of claim 17, The tissue adhesive layer is a multilayer film formed by a solvent extraction method. The method of claim 17, The porous support layer is a multilayer film formed by freeze drying on the tissue adhesive layer. The method of claim 17, The multilayer film is a multilayer film that is a shielding film for periodontal tissue regeneration.

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