CN106362222A - Temperature-sensitive injectable postoperative anti-adhesion material and preparation method thereof - Google Patents

Abstract

The invention relates to a temperature-sensitive injectable postoperative anti-adhesion material and a preparation method thereof. Gel prepared by dissolving modified xyloglucan into water is adopted as the anti-adhesion material. The temperature-sensitive injectable postoperative anti-adhesion material has the reversible temperature-sensitive behaviors of being sol at low temperature and being gel at high temperature. The invention further provides the preparation method of the injectable temperature-sensitive physical gel with the low critical gel concentration and the high gel strength. According to the preparation method, the natural polysaccharide-xyloglucan is taken as the raw material, a green enzymolysis reaction of a water system is adopted, and usage of organic reagents and generation of chemical reactions are completely avoided; the preparation method is simple, the process is easy to operate, the prepared postoperative anti-adhesion material achieves the good biocompatibility, biodegradability and in-situ injectability, the convenience of postoperative operation is improved when the material is used for preventing postoperative adhesion, and the material is an ideal postoperative anti-adhesion material. The preparation method is easy, the process is easy to operate, the raw material xyloglucan is high in reserve volume and wide in source, and commercialization is easy to achieve.

Description

A temperature-sensitive injectable postoperative anti-adhesion material and its preparation method

technical field

The invention relates to a temperature-sensitive injectable postoperative anti-adhesion material and a preparation method thereof, belonging to the fields of polymer materials and biomedical materials.

Background technique

Postoperative adhesions are abnormal fibrous bands formed by the connection of connective tissue and adjacent tissues or organs after the injury of normal tissue in a separated state. Postoperative adhesion is a common problem in the field of surgery, and it is also one of the medical problems that have not been solved for a long time in clinical practice. The incidence of abdominal adhesions after general abdominal surgery is 76%, while the incidence of adhesions after open gynecological pelvic surgery is as high as 97%. Adhesions after surgery may cause serious complications and increase the difficulty of secondary surgery, for example, adhesions after abdominal surgery may cause chronic pain, intestinal obstruction and other complications; adhesions caused by pelvic surgery can lead to ectopic pregnancy, Infertility and other symptoms; secondary abdominal adhesions after liver resection in the treatment of liver cancer will make it difficult to implement second-stage resection and reoperation for recurrent liver cancer; in addition, in tendon repair surgery, tendon adhesions may also affect the patient's movement function is affected.

Due to the prevalence of postoperative adhesions and the severity of the consequences of adhesions, the prevention and treatment of adhesions has become an important clinical problem that needs to be solved urgently. At present, many methods to prevent postoperative adhesions have emerged, such as postoperative local drug therapy, application of postoperative wound physical isolation materials, and the synergistic treatment of the two. Among them, due to the rapid clearance of the drug in the abdominal space, its anti-adhesion effect is not ideal. Solid film materials are currently the most researched type of anti-adhesion products in physical isolation materials. Compared with non-degradable polytetrafluoroethylene films, degradable solid films have received more attention because they avoid secondary operations. At present, the chitosan-based anti-adhesion film of Yantai Wanli Medical Products Co., Ltd.; the post-operative anti-adhesion film of Beijing Bailikang Biochemical Co., Ltd.; Polylactic acid viscose absorbable medical film of Pianfan Medical Technology Co., Ltd.; polylactic acid DIKFILM (Dikang) absorbable medical film of Chengdu Dikang Zhongke Biomedical Materials Co., Ltd., etc. Although these film materials are biodegradable, they are difficult to apply in minimally invasive surgery due to their solid form. More importantly, due to the complex shape and structure of human organs, surgical wounds often present irregular geometric surfaces, so it is difficult for solid-state isolation materials to achieve complete coverage of wounds in clinical use, thus affecting their effectiveness in clinical treatment. . In addition, because the solid film is easy to adhere to the surface of the object with liquid, it is easy to stick to the operator's gloves, surgical instruments and other wet non-wound parts during the operation, so it is not convenient to use in practice. Although liquid anti-adhesion materials improve the convenience of surgical operations and the coverage of wounds, their easy flowability makes it difficult to maintain a physical barrier on the wound for a long time, so the anti-adhesion effect is not ideal.

Gel materials can form a hard-to-flow solid in situ after injection into the wound, and are not affected by peritoneal exudate and body position changes, and can maintain sufficient retention time in the acute phase of wound repair, so as to play a good anti-adhesion effect. Compared with solid-state and solution-based anti-adhesion materials, gel-based materials have greater advantages and potentials in terms of ease of operation and effectiveness in preventing postoperative adhesions. However, most of the current preparations of injectable gels are still based on click chemical reactions such as Schiff base reaction and Michael addition. The occurrence of chemical reactions during in vivo use and the residue of excess precursors with functional groups are in terms of biological safety. is still a problem that cannot be ignored. The temperature-responsive physical gel can realize the transition from solution to gel only by changing the temperature, without the participation of any chemical reaction, thus completely avoiding the use of chemical initiators, cross-linking agents and chemical reactions. The biological safety of the system is fully guaranteed.

Chinese patents with application numbers 200810300757.7, 200910304210.9, and 201010138739.0 disclose polyether-polyester block polymers composed of polyethylene glycol (PEG) and polycaprolactone (PCL) as thermosensitive hydrogels for postoperative adhesions. prevention. However, the use of PCL as a hydrophobic block causes the problem of premature gelation of such materials due to crystallization during storage and use. In order to improve this problem, Chinese patent 201110020196.7 proposes to use degradable polyester and PEG to prepare a block polymer with temperature-sensitive properties and use it as an anti-adhesion material after surgery. However, a large number of studies have proved that the degradation products of aliphatic polyesters such as PLA and PLGA will produce an acid accumulation effect, so the above-mentioned polyether-polyester block polymers have the hidden danger of triggering the body's immune response to a certain extent. Therefore, the development of anti-adhesion materials with simple preparation methods, convenient use, good biological safety and anti-adhesion effects is still an urgent problem in the field of postoperative anti-adhesion.

Contents of the invention

The purpose of the present invention is to provide a temperature-sensitive injectable postoperative anti-adhesion material and its preparation method to solve the problems of poor operation convenience, poor postoperative adhesion prevention effect and in vivo use of existing anti-adhesion materials. Biosafety hazards and other issues. The invention adopts natural polysaccharide xyloglucan with good biocompatibility and biodegradability as raw material, adopts green enzymolysis reaction of water system to prepare injectable modified xyloglucan with controllable sol-gel transition temperature The sugar thermosensitive gel is used as postoperative anti-adhesion material, and the anti-adhesion material prepared by the invention has good biocompatibility and biodegradability, high gel strength and excellent postoperative anti-adhesion effect.

The present invention is achieved through the following technical solutions:

A temperature-sensitive injectable postoperative anti-adhesion material is a gel made of modified xyloglucan dissolved in water.

Preferably, the sol-gel transition temperature of the postoperative anti-adhesion material is 5-37°C.

Specifically, the solvent of modified xyloglucan in the postoperative anti-adhesion material is pure water, physiological saline, buffer solution, cell culture medium, simulated body fluid, animal or human body fluid, or a mixture of more than one solution.

The present invention also discloses a preparation method of the above-mentioned temperature-sensitive injectable postoperative anti-adhesion material, which is characterized in that it includes the following steps:

(1) dissolving tamarind gum in deionized water at 15-70°C, stirring for 4-48 hours to obtain a solution with a mass fraction of 0.5-50%;

(2) Centrifuge the solution obtained in step (1) at a speed of 1000 to 20000rpm for 1 to 30 minutes, then drop the supernatant obtained by centrifugation into absolute ethanol, wherein the volume of the supernatant is equal to that of absolute ethanol The volume ratio is 1:2-10, and the precipitated product xyloglucan is obtained;

(3) vacuum-drying or freeze-drying the xyloglucan obtained in step (2) at a temperature of 15-80° C. to obtain dried xyloglucan;

(4) Dissolve the xyloglucan obtained in step (3) in a buffer solution with a pH in the range of 3 to 7.4, and then add β-galactosidase to obtain a xyloglucan concentration of 0.2 to 10% (g /mL), the concentration of β-galactosidase is 0.01～0.5% (g/mL) solution;

(5) The solution system obtained in step (4) was mechanically stirred and reacted at 50°C and 10-1000rpm for 12-96 hours, then heated to 100-150°C and kept for 10-30 minutes to terminate the enzymatic hydrolysis reaction, and then the reaction product Freeze-drying can obtain modified xyloglucan with side chain galactose removal rate of 35-95%;

(6) Dissolving the modified xyloglucan obtained in step (5) in water at a temperature lower than the sol-gel transition temperature to prepare a modified xyloglucan solution with a mass fraction of 0.1 to 50%, namely Thermosensitive injectable post-operative anti-adhesion materials are available.

Specifically, the molecular weight of the xyloglucan obtained in the step (2) is 50,000-5 million.

Specifically, the β-galactosidase in the step (4) is derived from one of Aspergillus oryzae, Aspergillus niger, Saccharomyces fragilis, Lactic acid yeast or Candida tropicalis.

The temperature-sensitive injectable postoperative anti-adhesion material of the present invention is a modified xyloglucan hydrogel with reversible "low-temperature sol, high-temperature gel" temperature-sensitive behavior. The preparation method uses natural polysaccharide xyloglucan as raw material, adopts the green enzymatic hydrolysis reaction of water system, completely avoids the use of organic reagents and the occurrence of chemical reactions, the preparation method is simple, the process is easy to operate, and the prepared postoperative anti-adhesion The material has good biocompatibility, biodegradability and in-situ injectability, and improves the convenience of postoperative operation when it is used to prevent postoperative adhesion, and is an ideal postoperative anti-adhesion material.

Compared with the prior art, the present invention has the advantages that: (1) the postoperative anti-adhesion material prepared by the present invention has a lower critical gel concentration, and can effectively reduce foreign matter in the body when used as an anti-adhesion material in the body. Rejection. (2) The postoperative anti-adhesion material prepared by the present invention has a faster gel time and high gel strength (Figure 2) at physiological temperature, and can last for a long time after being injected into the wound to form a hydrogel in situ. On the wound surface and maintain good integrity (Figure 6), thus exerting excellent postoperative anti-adhesion effect (Figure 7). (3) The postoperative anti-adhesion material prepared by the present invention uses the natural polysaccharide xyloglucan as a raw material, and the preparation method adopts the enzymatic hydrolysis reaction of the water system, which completely avoids the use of organic reagents and the occurrence of chemical reactions. The biocompatibility of the prepared postoperative anti-adhesion material is guaranteed in terms of raw materials and preparation methods ( FIG. 3 ). (4) The postoperative anti-adhesion material prepared by the present invention has good biodegradability, and its degradation products are small molecular monosaccharides such as glucose, xylose and galactose, and low molecular weight oligosaccharides, etc., and have good biophasic properties. capacitive and bioabsorbable. (5) The preparation method of the postoperative anti-adhesion material provided by the present invention is simple, the process is easy to operate, the raw material xyloglucan has abundant reserves, wide sources, easy commercialization, and has good clinical application in the field of postoperative anti-adhesion prospect.

Description of drawings

Fig. 1 is the apparent state of the modified xyloglucan solution prepared in Example 1 of the present invention at different temperatures.

Fig. 2 is the rheological result of postoperative anti-adhesion material prepared in Example 1 of the present invention. A: Storage modulus (G') and loss modulus (G") variation curves with temperature; B: Storage modulus (G') and loss modulus (G") variation curves with time at 37°C .

Fig. 3 is the effect of the postoperative anti-adhesion material extract prepared in Example 1 of the present invention on the cell activity of L929 fibroblasts.

Fig. 4 is the variation curve of storage modulus (G') and loss modulus (G") with temperature of postoperative anti-adhesion material prepared in Example 2 of the present invention.

Fig. 5 is the variation curve of storage modulus (G') and loss modulus (G") with temperature of postoperative anti-adhesion material prepared in Example 3 of the present invention.

Fig. 6 is a photo of the postoperative anti-adhesion material prepared in Example 1 of the present invention when used in the SD rat abdominal wall-cecum injury adhesion model. A: normal abdominal wall and cecum; B: rat abdominal wall-cecum injury adhesion model C, D: use of thermosensitive injectable postoperative anti-adhesion materials.

Fig. 7 is a photograph of the postoperative anti-adhesion effect of the anti-adhesion material prepared in Example 1 of the present invention in the SD rat abdominal wall-cecum injury adhesion model 14 days after the operation. A: Adhesions occurred between the damaged cecum and abdominal wall in the control group using normal saline; B: No adhesions occurred in animals using thermosensitive injectable postoperative anti-adhesion materials.

detailed description

The technical solution of the present invention will be further described below in conjunction with the examples and accompanying drawings, but it is not limited to the following embodiments. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be done. included in the protection scope of the present invention.

Example 1

(1) dissolving tamarind gum in deionized water at 25°C, stirring for 12 hours to obtain a solution with a mass fraction of 1%;

(2) Centrifuge the solution obtained in step (1) at a speed of 9000rpm for 15 minutes, and then drop the supernatant obtained by centrifugation into absolute ethanol whose volume is 3 times its volume to obtain the precipitated product xylogluglucan sugar;

(3) freeze-drying the xyloglucan obtained in step (2) to obtain dried xyloglucan;

(4) dissolving the xyloglucan obtained in step (3) in the sodium acetate buffer solution of pH 4.5, then adding the β-galactosidase derived from Aspergillus oryzae to obtain a xyloglucan concentration of 2% ( g/mL), the concentration of β-galactosidase is a solution of 0.1% (g/mL);

(5) The solution system obtained in step (4) was mechanically stirred and reacted at 50°C and 500rpm for 24 hours, then heated to 100°C and kept for 30 minutes to terminate the enzymolysis reaction, and then the reaction product was freeze-dried to obtain the modified wood Dextran;

(6) Dissolve the modified xyloglucan obtained in step (5) in water at 20°C to prepare a solution with a modified xyloglucan concentration of 4% (g/mL) to obtain a thermosensitive Anti-adhesion material after injection.

For the temperature-sensitive injectable postoperative anti-adhesion material obtained in this example, the results of vial inversion method show that the temperature-sensitive injectable postoperative anti-adhesion material obtained in this example has a reversible "low-temperature sol, high-temperature gel" transition behavior; temperature scanning The rheological results show that the temperature-sensitive injectable postoperative anti-adhesion material obtained in this example has temperature responsiveness, and can spontaneously change from sol to gel as the temperature increases, and the sol-gel transition temperature is 23°C; the rheological results of time scanning show that the thermosensitive injectable postoperative anti-adhesion material obtained in this example can be gelled in 20 seconds at 37°C, and the strength of the gel after complete gelation is as high as 780Pa . The temperature-sensitive injectable postoperative anti-adhesion material obtained in this example was biologically evaluated with reference to the experimental method described in GB/T 16886. The results showed that different concentrations of extracts of the material had no effect on the cell viability of L929 fibroblasts.

Example 2

(1) dissolving tamarind gum in deionized water at 15°C, stirring for 48 hours to obtain a solution with a mass fraction of 2%;

(2) Centrifuge the solution obtained in step (1) at a speed of 20,000 rpm for 5 minutes, and then drop the supernatant obtained by centrifugation into absolute ethanol whose volume is 10 times its volume to obtain the precipitated product xylogluglucan sugar;

(3) Drying the xyloglucan obtained in step (2) under constant temperature and vacuum at a temperature of 25° C. to obtain dried xyloglucan;

(4) dissolving the xyloglucan obtained in step (3) in the sodium acetate buffer solution of pH 3.5, then adding the β-galactosidase derived from Aspergillus oryzae to obtain a xyloglucan concentration of 3% ( g/mL), the concentration of β-galactosidase is a solution of 0.15% (g/mL);

(5) The solution system obtained in step (4) was mechanically stirred and reacted at 50°C and 100rpm for 48 hours, then heated to 120°C and kept for 15 minutes to terminate the enzymolysis reaction, and then the reaction product was freeze-dried to obtain the modified wood Dextran;

(6) Dissolve the modified xyloglucan obtained in step (5) in water at 25°C to prepare a solution with a modified xyloglucan concentration of 2% (g/mL) to obtain a thermosensitive Anti-adhesion material after injection.

For the temperature-sensitive injectable postoperative anti-adhesion material obtained in this example, the rheological temperature scanning results show that the temperature-sensitive injectable postoperative anti-adhesion material obtained in this example has a temperature response of "low-temperature sol, high-temperature gel". The transition from sol to gel can occur spontaneously with increasing temperature, and the sol-gel transition temperature is 33 °C.

Example 3

(1) dissolving tamarind gum in deionized water at 70°C, stirring for 4 hours to obtain a solution with a mass fraction of 1.5%;

(2) Centrifuge the solution obtained in step (1) at a speed of 1000rpm for 30 minutes, and then drop the supernatant obtained by centrifugation into absolute ethanol twice its volume to obtain the precipitated product xylogluglucan sugar;

(3) Drying the xyloglucan obtained in step (2) under constant temperature and vacuum at a temperature of 80° C. to obtain dried xyloglucan;

(4) dissolving the xyloglucan obtained in step (3) in the sodium acetate buffer solution of pH 5.5, then adding the β-galactosidase derived from Aspergillus niger to obtain a xyloglucan concentration of 1% ( g/mL), the concentration of β-galactosidase is a solution of 0.05% (g/mL);

(5) The solution system obtained in step (4) was mechanically stirred and reacted at 50°C and 1000rpm for 12 hours, then heated to 150°C and kept for 10 minutes to terminate the enzymolysis reaction, and then freeze-dried the reaction product to obtain the modified wood Dextran;

(6) Dissolve the modified xyloglucan obtained in step (5) in water at 25°C to prepare a solution with a modified xyloglucan concentration of 3% (g/mL) to obtain a thermosensitive Anti-adhesion material after injection.

For the temperature-sensitive injectable postoperative anti-adhesion material obtained in this example, the rheological temperature scanning results show that the temperature-sensitive injectable postoperative anti-adhesion material obtained in this example has a temperature response of "low-temperature sol, high-temperature gel". The transition from sol to gel can occur spontaneously with increasing temperature, and the sol-gel transition temperature is 28 °C.

Example 4

In this example, the temperature-sensitive injectable postoperative anti-adhesion material prepared by the present invention is applied in the abdominal wall-cecum injury adhesion model of SD rats to investigate its postoperative anti-adhesion effect.

The specific method is to anesthetize and disinfect the experimental animal SD rats, open the abdomen at the position of the alba linea, and use ophthalmic scissors to peel off the superficial muscle with a size of ¹ ×2cm2 on the abdominal wall to form a bleeding surface; The surface was rubbed with a surgical brush until the serosa layer of the cecum was destroyed and there were obvious spotting; then the mesentery of the cecum was sutured and fixed at the upper right corner of the abdominal wall wound with a 3-0 suture to ensure that the wound surfaces of the abdominal wall and cecum were mutually connected. sufficient contact between them. Before closing the abdomen, inject 1 mL of the thermosensitive injectable postoperative anti-adhesion material prepared in Example 1 to the wound surface of the abdominal wall and cecum.

The abdomen was opened 14 days after the operation, and the animals using the temperature-sensitive injectable anti-adhesion material prepared in Example 1 did not have any form of adhesion, and the damaged cecum and abdominal wall had completely healed; while in the control group using normal saline Extensive and dense adhesions developed between the damaged cecum and the abdominal wall.

At the same time, the temperature-sensitive injectable postoperative anti-adhesion materials prepared according to the present invention such as Examples 2, 3 and others not described in detail have also been shown in similar experiments using rats, mice, New Zealand white rabbits, etc. as experimental animals. A similar anti-adhesion effect is produced.

Claims (7)

1. A thermosensitive injectable postoperative anti-adhesion material, characterized in that the postoperative anti-adhesion material is a gel made of modified xyloglucan dissolved in water. 2. The material according to claim 1, characterized in that the sol-gel transition temperature of the postoperative anti-adhesion material is 5-37°C. 3. The material according to claim 1, characterized in that, the solvent of modified xyloglucan in the postoperative anti-adhesion material is pure water, physiological saline, buffer solution, cell culture medium, simulated body fluid, animal or A mixed solution of one or more than one of the body fluids of the human body. 4. A method for preparing the temperature-sensitive injectable postoperative anti-adhesion material according to any one of claims 1-3, characterized in that it comprises the following steps: (1) dissolving tamarind gum in deionized water at 15-70°C, stirring for 4-48 hours to obtain a solution with a mass fraction of 0.5-50%; (2) Centrifuge the solution obtained in step (1) at a speed of 1000 to 20000rpm for 1 to 30 minutes, then drop the supernatant obtained by centrifugation into absolute ethanol, wherein the volume of the supernatant is equal to that of absolute ethanol The volume ratio is 1:2-10, and the precipitated product xyloglucan is obtained; (3) vacuum-drying or freeze-drying the xyloglucan obtained in step (2) at a temperature of 15-80° C. to obtain dried xyloglucan; (4) Dissolve the xyloglucan obtained in step (3) in a buffer solution with a pH in the range of 3 to 7.4, and then add β-galactosidase to obtain a xyloglucan concentration of 0.2 to 10% (g /mL), the concentration of β-galactosidase is 0.01～0.5% (g/mL) solution; (5) The solution system obtained in step (4) was mechanically stirred and reacted at 50°C and 10-1000rpm for 12-96 hours, then heated to 100-150°C and kept for 10-30 minutes to terminate the enzymatic hydrolysis reaction, and then the reaction product Freeze-drying can obtain modified xyloglucan with side chain galactose removal rate of 35-95%; (6) Dissolving the modified xyloglucan obtained in step (5) in water at a temperature lower than the sol-gel transition temperature to prepare a modified xyloglucan solution with a mass fraction of 0.1 to 50%, namely Thermosensitive injectable post-operative anti-adhesion materials are available. 5 . The method for preparing a temperature-sensitive injectable postoperative anti-adhesion material according to claim 4 , wherein the xyloglucan obtained in step (2) has a molecular weight of 50,000-5 million. 6. the preparation method of thermosensitive injectable postoperative anti-adhesion material according to claim 4, is characterized in that, the β-galactosidase of step (4) is derived from Aspergillus oryzae, Aspergillus niger, Saccharomyces fragilis, lactic acid One of yeast or Candida tropicalis. 7. The temperature-sensitive injectable postoperative anti-adhesion material according to any one of claims 1-3, characterized in that, the postoperative anti-adhesion material can be used to prevent abdominal adhesions and uterine horn adhesions after surgery. and tendon adhesions.