KR101649360B1 - Hydrogel anti-adhesion adjuvant and manufacturing method of the same - Google Patents

Abstract

The present invention provides a hydrogel adhesion inhibitor comprising 0.1 to 10% by weight of a tilamin-modified carboxymethylcellulose derivative, 0.5 to 10% by weight of pullulan and 0.1 to 2% by weight of a horseradish peroxidase, and a preparation method thereof do.
According to the present invention, not only is it possible to improve the usability of the user by improving the adhesiveness to tissues and organs, tissue immunological reaction, ease of manipulation, biocompatibility and biodegradability which are problems of conventional gel for preventing adhesion, The effective adhesion ability can help quick healing of wounds, and the step of removing the adhesion preventive membrane after surgery is omitted, so that the ease of operation of the user can be increased.

Description

Hydrogel adhesion inhibitor and manufacturing method thereof [0002]

The present invention relates to a hydrogel adhesion preventive agent for preventing tissue adhesion in vivo and a method for preparing the same, and more particularly, to a hydrogel adhesion preventive agent containing a derivative of carboxymethyl cellulose, a natural polymer, and pullulan, The present invention relates to a hydrogel adhesion preventive agent and a method for producing the same.

Adhesion is a phenomenon in which the tissue surface of the organ of an organism to be separated is connected to and fused with a fibrous tissue. Usually, adhesion of tissues after surgical operation of a patient is a natural phenomenon occurring in the process of proliferation and regeneration of damaged tissue cells, but abnormally connected adhesions with excessive surrounding tissues may result in organs such as obstruction It can lead to disability, depending on the degree of reattachment surgery is necessary, and in some cases can be a life-threatening factor.

The method of using the adhesion preventive membrane among the adhesion preventive methods currently used in the medical field is to prevent the adhesion by forming a physical barrier at the surgical site and to biodegrade in vivo after the healing of the wound for a certain period is finished, Is absorbed in the body and disappears or is artificially removed to prevent adhesion through the form. Such an anti-adhesion material should be effective in preventing adhesion for this purpose, and the material itself or its degradation products must maintain high safety in vivo without toxicity, irritation, genotoxicity and the like.

Examples of natural polymers used as the adhesion preventive film include bio-derived polymers and non-bio-derived polymers. Specific examples of natural polymers derived from living bodies include carboxymethyl cellulose (CMC), hyaluronic acid (HA), collagen Fibrin, gelatin and alginic acid. Examples of non-living body derived polymers include oxidized regenerated cellulose, polyethylene glycol (PEG), poloxamer, and gore- (Gore-Tex) are known.

The anti-adhesion membrane can be roughly classified into two groups, an intra-peritoneal instillator and an adhesion barrier. Peritoneal drip agents can be divided into a solution type and a gel type, and the adhesion blocking agent is mainly in the form of a film and a membrane.

Intraperitoneal drip agents have the advantage of being widely applicable irrespective of the wound size and shape of the patient, but there is a disadvantage that the anti-adhesion agent applied to the tissue can flow down from the application site due to gravity. Products such as Adept, Dextran 70, Sepracoat, and Spray Gel are commercially available as intraperitoneal instillants.

The intraperitoneal dissolubles are usually prepared in a liquid form. When they are prepared in a liquid form, even if they have a high viscosity, they flow down from the body, are difficult to apply accurately to the wound, flow into other parts before the function of preventing adhesion, And there are many disadvantages that the adhesion preventing function is not properly performed.

In addition, when an anti-adhesion agent is manufactured using non-living body-derived materials, there is a possibility of causing a foreign body reaction in vivo.

In this situation, in order to overcome the disadvantages of the liquid formulation, a gel-type anti-adhesion agent is spotlighted. Such a gel-type anti-adhesion agent can be easily used in the form of injection and spray, thereby increasing the ease of operation. At present, among the most effective gel-type adhesion preventive membranes in Korea, Hanmi Medicare's Guardix is a product composed of hyaluronic acid and carboxymethylcellulose.

However, there is a problem that hyaluronic acid is easily decomposed by hyaluronidase in vivo with a half-life period of 1 to 3 days in vivo. The problem of premature decomposition is that there is a problem in that the duration of staying in the wound tissue is insufficient and the persistence of the adhesion preventing effect can not be ensured because the adhesion preventive agent is removed before the wound of the expected adhesion surface is healed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an object of the present invention is to provide a biodegradable biodegradable and tissue-immunized biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable The present invention also provides a hydrogel adhesion preventive agent and a method for producing the same, which can improve the adhesion of the hydrogel adhesion agent to the surface of the adhesive agent,

In order to achieve the above-mentioned object, the hydrogel adhesion preventive agent according to an embodiment of the present invention includes tyramine modified carboxymethyl cellulose, pullulan and Armoracia rusticana peroxidase.

Preferably, the above-mentioned tyramine-modified carboxymethyl cellulose has a content range of 0.1 to 10% by weight based on the total weight,

Pullulan can have a content range of 0.5 to 10% by weight based on the total weight.

Preferably, the tyramine-modified carboxymethyl cellulose may be represented by the following formula (1).

[Chemical Formula 1]

Preferably, the molecular weight of the tyramine-modified carboxymethylcellulose may be 20,000 to 500,000.

Further, preferably, the molecular weight of the pullulan can be 50,000 to 250,000

Preferably, the hydrogel adhesion inhibitor may have a viscosity of 10,000 to 25,000 cP at room temperature.

In addition, preferably, the hydrogel adhesion preventive agent may have an adhesive strength of 10 to 30 kPa.

In addition, preferably, the hydrogel adhesion preventive agent may have a liquid syringe or spray formulation.

The method for preparing a hydrogel adhesion preventive agent according to an embodiment of the present invention includes the steps of adding carboxymethyl cellulose to a water solvent and stirring to prepare an aqueous solution of carboxymethyl cellulose, and adding to the aqueous carboxymethyl cellulose solution, NHS and stirring the mixture to prepare tyramine-modified carboxymethylcellulose by introducing tyramine into the side chains of carboxymethylcellulose, dialyzing the tyramine-modified carboxymethylcellulose, and lyophilizing to prepare a tyramine-modified carboxymethylcellulose powder Preparing a mixed solution of tyramine-modified carboxymethylcellulose and pullulan, adding the tyramine-modified carboxymethylcellulose powder and pullulan to a water solvent, stirring the mixture, and adding a mixture of horseradish peroxidase and hydrogen peroxide to the mixture, It may include the step of conducting response.

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Preferably, the content of tyramine in the step of preparing the tyramine-modified carboxymethyl cellulose may be 0.01 to 2% by weight.

Further, preferably, in the step of preparing the above-mentioned tyramine-modified carboxymethylcellulose, the substitution degree of tyramine may be 1 to 70% per unit.

Also, the concentration of the horseradish peroxidase may be 0.001 to 1 mg / ml.

Preferably, the tyramine-modified carboxymethyl cellulose may be represented by the following formula (1).

[Chemical Formula 1]

By incorporating carboxymethylcellulose and pullulan, which are naturally occurring natural polymers, into the composition, the present invention can exhibit excellent biocompatibility without causing a tissue immune response in vivo.

Further, by introducing tyramine into the side chain of carboxymethylcellulose and adjusting the degree of introduction, the mechanical strength of the hydrogel can be remarkably increased.

In addition, in particular, the inclusion of pullulan as a natural functional polysaccharide makes it possible to remarkably increase film forming ability, moisturizing effect, tissue adhesiveness and in vivo stability.

Gel formability and gel formation time can be optimized by providing an appropriate blending ratio of horseradish peroxidase (HRP) and hydrogen peroxide in the gelation step.

Therefore, it is possible to improve user convenience by improving adhesiveness, tissue immunological reaction, easy handling, biocompatibility and biodegradability to tissues and organs, which has been a problem of conventional gel for preventing adhesion, And the removal step of the post-operative adhesion preventing film is omitted, thereby increasing the ease of operation of the user.

1 is a view showing a reaction mechanism of a carboxymethyl cellulose according to an embodiment of the present invention in the step of reforming a thymine group.
FIG. 2 is a view showing the enzyme catalytic reaction by the horseradish peroxidase of the present invention. FIG.
3 is a view showing a mechanism of crosslinking reaction by the enzyme catalyst of the present invention.
4 shows the ATR / FT-IR spectrum of the tyramine-modified carboxymethylcellulose of the present invention.
FIG. 5 is a graph showing changes in gel formation time according to the concentration of horseradish peroxidase in the hydrogel adhesion preventive of the present invention. FIG.
6 is a graph showing changes in gel formation time according to the concentration of hydrogen peroxide in the hydrogel adhesion preventive of the present invention.
7 is a diagram showing the results of an in vitro biodegradation test of the hydrogel adhesion preventive agent of the present invention.
FIG. 8 is a photograph of a fracture surface of the hydrogel adhesion preventive agent of the present invention taken by a scanning electron microscope. FIG.
9 is a diagram showing an experimental step for evaluating adhesion of the hydrogel adhesion preventive agent of the present invention.
10 is a view showing the evaluation score of the adhesion score of the hydrogel adhesion preventive agent of the present invention.
11 is a view showing a result of adhesion evaluation of the hydrogel adhesion preventive agent of the present invention.
12 is a diagram showing the results of a tissue immunological evaluation test of the hydrogel adhesion preventive agent of the present invention.
Fig. 13 is a view showing the adhesion evaluation result of the hydrogel adhesion preventive agent of the present invention. Fig.

Hereinafter, the present invention will be described in detail.

The present invention relates to a tylamine-modified carboxymethylcellulose and a pullulan, wherein the tylamine-modified carboxymethylcellulose is obtained by reacting a chain branch of carboxymethylcellulose with N-hydroxysuccinimide (NHS), 1-ethyl-3- Methylaminopropyl) -carbodiimide (EDC) and tyramine, and is a hydrogel adhesion inhibitor in which the tyramine functional groups of carboxymethyl cellulose modified with tyramine are prepared by a cross-linking reaction of horseradish peroxidase.

Herein, the content of tyramine-modified carboxymethylcellulose may be 0.1 to 10% by weight, and the content of pullulan may be 0.5 to 10% by weight.

In addition to carboxymethylcellulose and pullulan, the hydrogel adhesion preventive agent may include additives such as buffers, excipients, dispersants, wetting agents, and viscosity adjusting agents necessary for the production of hydrogels depending on the situation.

The pullulan contained in the present invention is a pale, odorless, amorphous white powder polysaccharide material produced by extracellular production of Aureobasidum pullulans . When referring to the following chemical formula 1, 3 bonded tartrate is formed of linear glucan repeatedly bonded with an alpha-1, 6 bond

[Chemical Formula 1]

A detailed study of the existing pullulan showed that the structure was not formed only from the repetition structure of the maltotriose unit but contained several percent of the sattacharin maltotetraose and the terminal of the molecule was glucose and the structure was different from dextran It turned out.

Pullulan having such a structure is metabolized by various microorganisms and converted into carbonated gas and water which are the final products and discharged. The degradation products do not adversely affect the human body, so that they have excellent biocompatibility characteristics, and even in various toxicity and mutagenicity test results, And it has been approved as a food additive at home and abroad.

In particular, pullulan is known to have an excellent film-forming ability, and when the hydrogel containing pullulan is applied to tissues or organs, not only does it show an effective film-forming ability, but also has excellent adhesion to hydrogel It is possible to impart tissue adhesion. In addition, pullulan has a strong elongation property, so it can easily adapt to fluid movements of organs and tissues to prevent hydrogel breakage.

Here, pullulan has a low viscosity, but its viscosity is less influenced by temperature, salt and pH, so it can be applied in various fields as a stable neutral solution which does not give any strain to the living body. As an example, in the case of titanium or boron ions which are believed to form a chelate with a -OH group in the pullulan molecule at a viscosity of 250-300 cp in the aqueous solution of pullulan (1% aqueous solution at 25 ° C, 30 rpm) in which various metal ions coexist The viscosity is not increased or the gel is not formed in any case.

The molecular weight of the pullulan can be 50,000 to 250,000, preferably 100,000 to 200,000. Further, it may contain 0.5 to 10% by weight, preferably 2 to 7% by weight, based on the total weight. When the pH of the hydrogel is higher than the above range, decomposition of the hydrogel may be promoted in vivo, which may affect the gel stability. If the pH of the hydrogel is lower than the above range, It can be difficult to maintain a role.

The carboxymethylcellulose contained in the present invention is a water-soluble polymer having a relatively small molecular weight, which is produced through chemical modification of plant sugar as a raw material. In general, carboxymethylcellulose has a characteristic that it can be effectively used as an anti-adhesion agent in living body because its absorption rate in vivo is moderate so that it is absorbed early before the wound heals and does not disappear and slowly dissolves and disappears. In addition, the decomposition products of carboxymethyl cellulose do not exhibit adverse effects that adversely affect the human body when they are absorbed into the tissues, and it is not necessary to remove the hydrogel after the operation, so that the manipulation and convenience can be provided.

The carboxymethylcellulose may be a homopolymer of lactic acid or a copolymer derivative of lactic acid and glycolic acid, and may be in the form of a sodium salt, a calcium salt, or the like. In particular, the viscosity of carboxymethyl cellulose may vary depending on the substitution degree of the glycolic acid ether group contained in the molecule as a hydrophilic derivative.

The degree of substitution of carboxymethyl cellulose included in the present invention may be 0.5 to 1.5, preferably 0.8 to 1.2. The preferred viscosity of the carboxymethylcellulose may be 200 to 1000 cP. When the viscosity range is higher than the above range, there arises a problem in ease of operation during operation. On the contrary, when the range is lower than the above range, there is a problem in tissue adhesion and storage stability. In addition, the hydrogel adhesion preventive of the present invention can be used as a spray type, a spray type, or the like, and therefore, it is preferable that the viscosity range is within the above range.

The present invention can also include a step of subjecting the carboxymethyl cellulose to a thiamine reforming to produce a tyramine-modified carboxymethyl cellulose.

Referring to FIG. 1, tyramine-modified carboxymethylcellulose is a mixture of carboxymethylcellulose, tyramine, N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3- dimethylaminopropyl) ) To introduce tyramine into the side chain of carboxymethylcellulose, and has a structure as shown in Formula 2 below.

(2)

Since the tilamine functional group modified to the side chain of the carboxymethyl cellulose is a crosslinking reaction site connecting the polymer and the polymer, the mechanical strength and the gel formation time of the hydrogel may vary depending on the substitution degree of the tilamine introduced into the carboxymethyl cellulose.

In the modification of carboxymethylcellulose, the substitution degree of tyramine may be considered. The degree of substitution of preferable tyramine may suitably be 1 to 70% per unit.

 Controlling these crosslinking points means that the biodegradability and desired physical properties of the hydrogel can be obtained. Moreover, according to recent reports, it is known that controlling the mechanical strength of hydrogels greatly influences the growth rate of cells and the functionalization of tissues.

The tylamine-modified carboxymethylcellulose may have a molecular weight of 20,000 to 500,000, and may be included in an amount of 0.1 to 10% by weight, preferably 0.5 to 5% by weight based on the total weight of the composition. If it is contained at a weight percentage higher than the above range, the adhesive property and the gel-forming ability can be obtained, but the improvement of the adhesiveness is not infinite. In addition, the retention time in the living body is prolonged, It is possible to show an economic incompatibility with the efficacy. On the contrary, when the content is lower than the above range, the tissue adhesion and the gel-forming ability are lowered, so that an effective anti-adhesion effect can not be secured.

In general, in order to utilize the hydrogel as a bio-injectable material, a liquid form preparation that is easy for a user to manipulate in vitro is required, and after the injection, a property of gelation by physical or chemical crosslinking is required. The chemical crosslinking is a form in which the polymer and the polymer are crosslinked by covalent bonding, and the physical crosslinking is a form in which the polymer and the polymer are crosslinked through secondary bonding such as entanglement of the polymer, hydrogen bonding and hydrophobic interaction. Since the physical crosslinking reaction is a reversible reaction, it can be easily decomposed by other external stimuli such as heat. Therefore, the crosslinking reaction between the polymer and the polymer is performed by chemical crosslinking, which is an irreversible reaction that mainly forms a strong covalent bond .

However, when the cross-linking reaction between the polymers is caused by the chemical cross-linking, unfavorable physical properties may be changed due to the concentration or the content ratio of the catalyst or the cross-linking agent contained as a reactant. In addition, when a non-living body-derived crosslinking agent is used, tissue immunological and biomedical restrictions may occur in vivo.

The present invention may include a cross-linking agent derived from a natural substance to overcome the above-mentioned problems of the cross-linking agent. In particular, horseradish peroxidase (HRP), an enzyme extracted from Western horseradish, has excellent biodegradability and biocompatibility, and has a function of effectively controlling the crosslinking reaction.

The present invention includes a process for producing a hydrogel adhesion inhibitor by reacting tyramine-modified carboxymethylcellulose, pullulan, horseradish peroxidase, and hydrogen peroxide. Here, the horseradish peroxidase plays a role as a crosslinking agent.

The reaction consists of two main stages. Referring to FIG. 2, the first step involves the oxidation of the horseradish peroxidase by hydrogen peroxide, and the second step involves the oxidation of the oxidized horseradish peroxidase to the phenol functional group in the tyramine group of the tyramine-modified carboxymethylcellulose ≪ / RTI > to form a radical intermediate.

Referring to FIG. 3, the radical present at the tyramine end of the thiamine-modified carboxymethyl cellulose side chain may be a crosslinking site capable of crosslinking with another polymer. As a result, through the above two steps, a network structure is formed through radial bonding with a tyramine group of another modified carboxymethyl cellulose to produce a hydrogel adhesion preventive agent. At this time, the important factor is that the mechanical strength and gel formation time of the hydrogel can be controlled by the concentration of horseradish peroxidase and hydrogen peroxide. The horseradish peroxidase may comprise 0.1 to 2% by weight based on the total weight, and the concentration may be 0.001 to 1 mg / mL, preferably 0.005 to 0.5 mg / mL. Here, when a value higher than the above-mentioned concentration range is included, the gel formation rate is too fast, so that the user can not show efficient operation easiness in the process of applying the hydrogel to tissues or organs. On the contrary, when a value lower than the above-mentioned concentration range is included, the gel formation rate is too slow, and thus there is a problem that the hydrogel can flow into other tissues or organs in the process of applying the hydrogel to tissues or organs. Therefore, it is important to adjust the optimum concentration of horseradish peroxidase in consideration of gel formation rate and mechanical strength characteristics.

In addition, the adhesion strength of the hydrogel adhesion inhibitor may be 10 to 30 kPa. When the above range is satisfied, it is possible to prevent the hydrogel adhesion preventive agent from being separated from the adhesion surface or being lost. In addition, the preferred viscosity of the hydrogel adhesion inhibitor may be 10,000 to 25,000 cP. When the above range is satisfied, it is easy to use clinically, and when applied to the adhesion surface, it is possible to prevent the phenomenon that gravity flows into other organs or deviates from the adhesion surface in advance.

The hydrogel adhesion preventive agent prepared through the above-mentioned process can be used in the form of a spraying type, a spray type, a gel type, a liquid type and an aerosol. In particular, it may be preferable to use the compound in the form of a syringe, which is an effective drug delivery vehicle which can be easily used in combination with a bio-therapeutic agent such as a pharmaceutically acceptable natural extract, an antibacterial peptide, a physiologically active substance and a drug It is because. In addition, it can be easily applied to laparoscopic surgery using a complex type of adhesion surface or minimally invasive surgery.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, these embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples and experimental examples.

<Examples>

The following materials are prepared for the examples.

Carboxymethylcellulose: manufactured by Sigma Aldrich, molecular weight = 250,000

Pullulan: manufactured by Sigma Aldrich, molecular weight = 100,000

Tiramine: manufactured by Sigma-Aldrich, molecular weight = 173.64

N-hydroxysuccinimide: manufactured by Wako

Preparation of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide:

Western horseradish peroxidase: manufactured by Amresco

Buffer: 1X PBS buffer

Hydrogen peroxide: Daejung Chemical Manufacturing

Tyramine  Modification Carboxymethylcellulose  Produce

Referring to the following Table 1, formulation ratios suitable for each material were specified.

material weight% Carboxymethylcellulose 10 Tyramine 0.034

First, carboxymethylcellulose is added to the third distilled water and stirred at room temperature for 3 hours to dissolve uniformly to prepare a solution. After adding N-hydroxysuccinimide (NHS), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and tilamine to the solution for the introduction of the tyramine group of carboxymethylcellulose And the mixture was stirred for 24 hours to prepare a modified carboxymethylcellulose solution. Dialyzed to remove pesticide residues from the prepared modified carboxymethylcellulose solution and dialyzed to increase purity. The dialysis was carried out through membrane dialysis in 100 mM NaCl solution, 25 v / v% ethanol and distilled water for 2 days, 1 day, 2 days. The dialyzed solution was concentrated by lyophilization to prepare tylamine-modified carboxymethylcellulose.

Hydrogel  Preparation of anti-adhesion agent

Referring to the following Table 2, concentrations and mixing ratios suitable for each material were specified

material weight% Tyramine-modified carboxymethylcellulose 3 Pullulan 3 Western horseradish peroxidase (0.01 mg / ml) 0.4 Hydrogen peroxide 0.4

First, thylamin-modified carboxymethylcellulose and pullulan were added to a 1X PBS buffer, and reacted through stirring for 3 hours to prepare a homogeneous solution, followed by reaction with horseradish peroxidase (HRP) for 30 minutes, Hydrogen peroxide was added thereto and reacted for 60 seconds to finally prepare a hydrogel adhesion preventive.

<Test Results>

ATR / FT - IR Using Carboxymethylcellulose  Confirmation of modification

IR spectra were examined using Attenuated total reflection FT-IR (ATR-FTIR) for the confirmation of the tyramine group modification of the tyramine-modified carboxymethylcellulose of the present invention. As the infrared rays incident through the prism are reflected on the surface of the prism, a part of the infrared wavelength is absorbed on the surface of the material adhered to the prism, in ATR (MIRacle ™, single reflection HATR). The absorbed infrared rays provide information about the chemical composition of the material surface. Therefore, ATR-FTIR is a useful tool for identifying the chemical composition of extreme surfaces of materials. At this time, the prism was MIRACLE Ge crystal (cat. No. 025-2050, MIRacle).

Referring to FIG. 4, the -NH peak of tyramine overlaps the specific -OH peak of carboxymethylcellulose, and the C = C peak and the CN peak of tyramine become C = O of carboxymethyl cellulose Peak and CO peak showed superimposed absorption without showing specific shape.

Measurement of gel formation time according to concentration of Western wasabi peroxidase and hydrogen peroxide

Gel formation time was measured according to the concentrations of horseradish peroxidase and hydrogen peroxide in the hydrogel adhesion inhibitor composition. Referring to FIG. 5, in the case of horseradish peroxidase, the gel formation time rapidly decreased as the concentration of horseradish peroxidase increased, resulting in rapid gel formation. On the other hand, referring to FIG. 6, as the concentration of hydrogen peroxide increases, the gel formation time decreases, but the trend is slower than that of horseradish peroxidase.

In vitro In vitro ) Evaluation of biodegradation

To evaluate the degree of biodegradation of the hydrogel adhesion inhibitor, the hydrogel was applied to an aluminum pan of 1 cm × 0.5 cm size, separated by gelation, placed in a PBS buffer at 37 ° C. in an environment similar to body fluids, 7, 14, 21, 28 days later, and the changed hydrogel weight was measured.

Referring to FIG. 7, it can be seen that weight reduction is clearly observed during the period of 4 to 7 days, which is the early stage of the biodegradation process, and that the biodegradation process is progressing, but the weight change of the hydrogel was not observed between 7 and 28 days. Here, the hydrogel was lyophilized and its fracture surface was observed using a scanning electron microscope.

Referring to FIG. 8, it can be seen that the pores of the hydrogel are maintained at the beginning of the biodegradation process, and the pores gradually collapse as the biodegradation process proceeds. As a result, due to the property of absorbing moisture of the hydrogel, there is no weight loss after a certain time, but in the scanning electron microscopic image, the pores of the hydrogel are collapsed and biodegradation is proceeding normally. Since the wound healing generally stabilizes around 7 to 10 days, the time and degree of biodegradation of the hydrogel adhesion preventive agent according to the present invention are considered to be appropriate.

Evaluation of tissue adhesion grade through animal experiments

An animal experiment was conducted to evaluate the effectiveness of the hydrogel adhesion preventive agent to prevent tissue adhesion. Outbred male Sprague Dawley rats were selected for animal experiments. In the first experiment, five healthy rats (weight: 230-280 g) were used for animal experiments. In the second experiment, 8-week old (weight: 230 ~ 280 g) rats were applied to animal experiments. The rats were maintained for one week so that they could adapt to the laboratory environment before the surgery, and the rats were operated one week later. Referring to FIG. 9, the operation is performed by abdominal wall destruction and a cecum catheter friction model (Renee Kennedy, Darren J. Costain, Vivian C. Meallister and Timothy DG Lee, "Prevention of experimental postoperative peritoneal adhesions by N, O-carboxymethyl chitosan" , Vol. 120, No. 5, 866-870, 1996). After rubbing the peritoneum with a scalpel, the scar was scratched off, and the abdominal wall of the adjacent cecum was rubbed off with sandpaper to induce bleeding, Conditions that could occur were artificially formed. As a test group, rats having an adhesion preventive membrane inserted and rats not having the adhesion preventive membrane inserted as a control group were allowed to regenerate the tissues of the peritoneum and cecum 4 weeks after the surgery, The effects of Vlahos method (Angie Vlahos, Pingyang Yu, Charles E. Lucas, and Anna M. Ledgerwood, "Effect of a composite membrane of chitosan and poloxamer gel on postoperative adhesive interactions", The American Surgeon, Vol. , 15-21, 2001).

10 and Table 3, the adhesion score was evaluated by Knightly's method (Knightly JJ, Agostino D, Cliffton EE. The effect of fibrinolysin and heparin on the formation of peritoneal adhesions. Surgery 1962 ; 52: 250-8.).

Rating Adhesion scale 0 No adhesion occurs One 1 thin adhesion band easily separable between peritoneum and appendix 2 Two or more detachable adhesive bands occur between the peritoneum and the appendix 3 There are two or more adhesion bands that are difficult to separate between the peritoneum and the cecum, 4 Dense adhesions between peritoneum and appendix

Rats after 4 weeks of operation were evaluated for adhesion grade with reference to Table 3 above. Referring to FIG. 11, in the case of the first experiment, the control group showed an average degree of adhesion of 3.6 and the control group showed adhesion grades of 3 and 4, indicating that adhesion was frequently and strongly formed. In the experimental group, the average adhesion grade of 0.2 was shown. As a result, the adhesion of hydrogel was very good. In the second experiment, the control group showed an average adhesion grade of 3.7, and the control group showed adhesion grades of 3 and 4, indicating that adhesion was frequent and strong. In the experimental group, the adhesion grade was 0.9, The hydrogel adhesion inhibitor of the present invention has been shown to exhibit excellent ability to prevent adhesion of damaged tissues in vivo.

Tissue immunological evaluation

The most widely used method for tissue staining is hematoxylin-eosin staining, staining the nucleus with a basic dye, hematoxylin, and then staining with an acid dye, eosin.

The hematoxylin is a dye that binds to phosphate groups contained in many nucleotides, DNA and RNA in tissues. When hematoxin produced by the oxidation process of hematoxylin binds with mordant (Alu), it is converted into positive charge, and it binds with negatively charged phosphate group and is dyed blue. Eosin is an acidic dye that binds to basic cytoplasm or connective tissue and is dyed pink. In the tissue staining, the nucleus is stained with hematoxylin and then contrasted with eosin to prepare the cytoplasm or the cell outer structure. There are various proteins in the nucleus, which are reddish by eosin, but dark blue due to hematoxylin results in the nucleus being purple and the cytoplasm being dyed pink.

For tissue immunological evaluation, first collect the collected tissue in 10% formalin solution and wash it with xylin or other solution. Next, the tissue is put into paraffin, hardened, and cut to a thickness of about 5 탆 using a microtome. If the tissue is floated and dried, the tissue will temporarily stick to it by paraffin, which is then immersed in hematoxylin for 2 minutes and washed in running water. After that, after immersing in eosin for 3 minutes and then washing with water, the nucleus was stained with purple, the cytoplasm was stained with pink, and the result was confirmed with an optical microscope.

12, in the control group, adherence frequently occurred between the cecum and the peritoneum. However, in the experimental group in which the hydrogel according to the embodiment of the present invention was applied to the peritoneum, the wound restoration between the cecum and the hydrogel and between the peritoneum and the hydrogel Wound healing was effective, and no inflammatory reaction occurred.

<Comparative Example>

Comparative Example  One

In Comparative Example 1, a hydrogel adhesion preventive agent in which the content of pullulan was reduced to half in the course of the examples was prepared.

Comparative Example  2

In Comparative Example 2, a hydrogel adhesion preventive agent containing no pullulan was prepared in the procedure of Example.

Adhesive strength evaluation

In order to measure the adhesive strength effect of the hydrogel adhesion inhibitor formed by the inclusion effect of pullulan of the present invention, Comparative Example 1 and Comparative Example 2 in which the content ratio of pullulan was adjusted in the course of the example were prepared , And the adhesive strength was measured.

In order to create an environment as close as possible to the physiological condition, the BioPlus bath of Instron, which can control the temperature accurately at 37 ° C ± 1 ° C, was used as the measuring environment for the adhesive strength. The peeling test was conducted using ASTM F2256 instrument was used. In the experiment, a load cell of 1 to 3 N volume was placed on a flat surface of the upper specimen holder of the instrument and fixed on a moving crosshead, and then contacted with a collagen film attached to the lower specimen holder. For the adhesion of the collagen film and the hydrogel, a constant pressure is applied for 6 seconds to form adhesive bonds. The initial load is 1 N, and the crosshead is moved at a constant speed in the upward direction to separate the two surfaces. . Referring to FIG. 13, the highest adhesive strength was shown in the examples, and the lower the content of pullulan was, the lower the adhesive strength was.

Claims (13)

Tylamine modified carboxymethylcellulose, pullulan and Armoracia rusticana peroxidase;
The thylamine-modified carboxymethylcellulose has a content range of 0.1 to 10% by weight based on the total weight of the hydrogel adhesion preventive agent, the molecular weight is 20,000 to 500,000;
Wherein the pullulan has a molecular weight of 50,000 to 250,000 and has a content range of 0.5 to 10% by weight based on the total weight of the hydrogel adhesion preventive. delete [Claim 2] The hydrogel adhesion preventive agent according to claim 1, wherein the tyramine-modified carboxymethyl cellulose is represented by the following formula (2).
(2)

delete delete The method according to claim 1,
Wherein the viscosity at room temperature is 10,000 to 25,000 cP. The method according to claim 1,
Wherein the adhesive strength is 10 to 30 kPa. The method according to claim 1,
Characterized in that it has a liquid syringe or spray formulation. Adding carboxymethylcellulose to a water solvent and stirring to prepare an aqueous solution of carboxymethylcellulose;
0.01 to 2% by weight of tyramine, EDC and NHS are added to the aqueous solution of carboxymethylcellulose and stirred to introduce tyramine to the side chain of carboxymethyl cellulose in an amount of 1 to 70% per unit of degree of substitution of tyramine, Preparing thylamin-modified carboxymethyl cellulose having a content range of 0.1 to 10% by weight based on the total weight of the adhesion inhibitor and having a molecular weight of 20,000 to 500,000;
Dialyzing the tyramine-modified carboxymethyl cellulose, followed by lyophilization to prepare a tyramine-modified carboxymethyl cellulose powder;
The pullulan having a molecular weight of 50,000 to 250,000 and a content of 0.5 to 10% by weight based on the total weight of the hydrogel adhesion preventive is mixed with the above-mentioned tyramine-modified carboxymethyl cellulose powder, and the mixture is stirred to form a mixture of tyramine-modified carboxymethylcellulose Preparing a mixed liquid of pullulan; And
And adding hydrogen peroxide and a horseradish peroxidase having a concentration of 0.001 to 1 mg / ml to the mixed solution to perform a crosslinking reaction.
delete delete delete 10. The method of claim 9,
Wherein the thiamine-modified carboxymethylcellulose is represented by the following formula (2).
(2)

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