WO2019007400A1

WO2019007400A1 - Biodegradable film, preparation method therefor and use thereof

Info

Publication number: WO2019007400A1
Application number: PCT/CN2018/094677
Authority: WO
Inventors: 孙雨龙
Original assignee: 孙雨龙
Priority date: 2017-07-07
Filing date: 2018-07-05
Publication date: 2019-01-10
Also published as: CN109206641A

Abstract

Disclosed are a biodegradable film, a preparation method and the use thereof. The preparation method for the biodegradable film comprises placing a mobile phase made of a film-forming raw material in a dry, flat-bottomed container, solidifying the mobile phase and forming a film, such that the film has a weight of 0.1-0.15 mg/square centimetre; the film-forming raw material comprises a biocompatible macromolecular material containing a hydroxyl, an amino and/or a carboxyl group. The biodegradable film can be directly attached effectively to tissues, and has the advantages of an excellent mechanical property, easy bending and a good drug-loading effect, and is capable of effectively preventing tissue leakage and adhesions in the abdomen, heart and blood vessels, spine, tendons, gynecological pelvis, etc., in clinical applications, and can be used as a drug carrier for medical treatments.

Description

Biodegradable film and preparation method and application thereof

Technical field

The invention belongs to the technical field of medical biomaterials, and relates to a biodegradable film which can be closely attached to a tissue, which is used for preventing tissue leakage, tissue adhesion after surgery and releasing a drug locally in a tissue as a carrier.

Background technique

During tissue injury and healing after surgery, interstitial adhesions often occur in the patient's wounds, especially in the abdomen, spine, and flexor tendons. According to statistics, after abdominal surgery, the incidence of abdominal adhesions is between 67% and 93%. After gynecologic pelvic surgery, the incidence of adhesions is as high as 97%. Abdominal surgical adhesions can lead to serious clinical complications such as intestinal obstruction, abdominal and pelvic pain, infertility, etc., increasing the difficulty of reoperation and the potential for further complications. The causes of adhesions are complex and can be summarized as follows: wound bleeding, wound surface exposure, ischemia, foreign body retention, bacterial infection, etc. Therefore, timely implementation of anti-adhesion treatment on tissue wound surface is tissue wound treatment and smooth healing An important part.

The use of physical barriers is currently the most effective method for clinical prevention of adhesions. For example, polylactic acid membranes have been approved for clinical use by multinational drug regulatory agencies to prevent adhesions after tendon, abdominal pelvic, and spinal surgery. A clinical study on the use of poly-DL-lactic acid absorbable medical film in the prevention of adhesions in abdominal surgery found that the clinical signs of postoperative bowel recovery time and exhaust time were better. The experimental group had better postoperative results (Ma Fuping, Shi Ming : Clinical observation of poly-DL_lactic acid film for abdominal surgery to prevent adhesions, "Chinese Medical Engineering", 2012, No. 20, p68-69). Another study also used poly-DL-lactic acid absorbable medical film The curative effect of prevention of postoperative adhesions showed that the abdominal adhesions in the observation group were milder than those in the control group (Luo Yiming, Li Zhihong: "Therapeutic effect of polylactic acid anti-adhesion membrane to prevent postoperative adhesion", "Chinese Practical Medicine" 2012, No. 7, p95‐96.) In addition, clinical studies have also demonstrated that poly-DL-lactic acid absorbable medical membranes have anti-adhesion effects in spinal surgery (Zhong Xihong, Wu Wei: “Poly-DL-lactic acid anti-adhesion membrane for spinal surgery] 51 cases of surgery", "China Medical Frontier" 2012, No. 7, p55).

Although there are many studies showing that the polylactic acid film has a good anti-adhesion effect in clinical practice, the film is hydrophobic and cannot adhere to the surface of the tissue. It is necessary to suture the film with the surrounding tissue with a surgical line for internal fixation. Inconvenient to use.

In order to overcome the insufficiency of the anti-adhesion barrier product and tissue adhesion of the polylactic acid film, the present invention relates to a biomaterial film closely attached to the tissue, which is biodegradable and can effectively prevent tissue contents leakage and tissue adhesion. And can be used as a drug carrier to release the drug locally in the tissue.

In addition to the above mentioned polylactic acid absorbable medical film, as a hemostatic and anti-adhesion material, there are reports and uses, as well as hemostatic sponge, hemostatic powder, semi-fluid/gel material, etc., the preparation method is to make the film-forming material After sufficient cross-linking reaction in the aqueous solution, the desired material product is prepared by a corresponding process, for example, the cross-linked product is spray-dried into a powder, and the cross-linked product liquid is injection-molded and freeze-dried, and then compressed into a hemostatic film, etc. See Chinese patent application CN 201410216904.8.

Although the barrier film is relatively easy to use from the perspective of clinical anti-adhesion treatment, good adhesion of the barrier membrane to the wound tissue (avoiding misalignment during operation), good barrier properties (mechanical properties) and ease of use must be considered. condition. In order to solve the good adhesion of the separator to the wound tissue, the applicant of the present invention disclosed a bio-coupling material in the patent CN 200610095787.X, which is an incomplete coupling of the collagen hydrolyzate with the carbodiimide. The product of the reaction, having fluidity and adhesion, can control the speed and progress of the coupling reaction by adjusting the concentration of the raw material and the reaction temperature, and the incomplete coupling product is applied to the wound to continue the coupling while achieving adhesion to the tissue. Attached to achieve the purpose of anti-adhesion and exudation. Compared with the solid membrane, this incompletely coupled flow dynamic material is more suitable for application in the surgical site during clinical operation, but needs to be prepared temporarily before use, regardless of the reaction timing or clinical use, and has high requirements on the operator. Operational technology can affect the properties and isolation of the separator, such as the adhesion and strength of the membrane, while adjusting the raw materials and concentration, there will be conflicts between the mechanical strength of the final film formation and the degradation time in the body. On the other hand, this Flow dynamic materials do not adequately meet the drug demand.

On the other hand, the hemostatic anti-adhesion material currently used for tissue wounds, whether it is a powder, a sponge or a film, is indispensable in the preparation process, such as a crosslinking agent, a foaming agent, a plasticizer, etc., in order to reduce these The potential risks introduced by the use of additives require the removal of excess additives after the reaction is completed. In order to separate these small molecules from the already formed polymer membrane, dialysis treatment is usually required, which increases the preparation time and the cost of the product.

Summary of the invention

In view of the current status of clinical anti-adhesion materials, the present invention provides a biodegradable film which can be directly attached to tissues, and has the advantages of excellent mechanical properties, easy bending, and good drug-loading effect.

The invention also provides a preparation method of the above biodegradable film, wherein the raw material film forming operation is simple, the additive used only needs to be washed by water, and no dialysis treatment is required, and the operation is simple, and the film property is stabilized.

The invention provides a biodegradable film, which comprises preparing a mobile phase made of a film-forming raw material in a dry flat-bottomed container, solidifying and forming a film, so that the quality of the film is 0.1- 15 mg/cm 2 ; the film forming material comprises a biocompatible macromolecular material comprising a hydroxyl group, an amino group and/or a carboxyl group.

In the biodegradable film material of the present invention, the film forming raw material selects a biocompatible macromolecular material containing a hydroxyl group, an amino group and/or a carboxyl group, and in the specific implementation, the film forming raw material may be selected from the group consisting of gelatin, collagen, and fibrin. , silk fibroin, albumin (albumin), globulin (Globulin), hyaluronic acid, chitin, chitosan, cellulose, polylysine, polyarginine, polyglutamic acid, poly Aspartic acid, alginic acid, starch, deacetylated hyaluronic acid, carboxymethyl chitosan, carboxymethyl starch, carboxymethyl cellulose, polygalacturonic acid, dextran, agar or carrageenan and derivatives thereof . Those skilled in the art can select based on their own basic knowledge. For example, the carboxyl group-containing macromolecule may have polyglutamic acid, polyaspartic acid, hyaluronic acid, alginic acid, carboxymethyl starch, carboxymethyl group. Cellulose, polygalacturonic acid or derivatives thereof, etc.; amino group-containing macromolecules may have polylysine, polyarginine, chitosan and derivatives thereof, etc.; Biocompatible macromolecules with amino groups can be gelatin, collagen, fibrin, silk fibroin, albumin, globulin, deacetylated hyaluronic acid, carboxymethyl chitosan and its derivatives. and many more. When the biomaterial film is produced, the film forming materials may be used singly or in combination, but at least one biomacromolecule may be partially or completely dissolved in water and an aqueous solution. These film-forming materials are generally from 1 to 100% by dry weight of the film.

The biodegradable film of the present invention, the forming raw material (also referred to as a film forming system) may further include a necessary coupling agent and/or a coupling initiator in addition to the film forming raw material, so the preparation method thereof may include forming a film forming raw material. The prepared mobile phase is placed in a dry flat-bottomed container, and a coupling agent and/or a coupling initiator is added after solidification or solidification to cause a coupling reaction, washed by water and dried, and then peeled off to obtain the Film products. Applicants' research has found that under the action of a coupling agent or/and a coupling initiator, biodegradable macromolecules in the feedstock are more conducive to covalent bond coupling, thereby improving the mechanical properties of the biofilm material. The material becomes a scaffold with a certain mechanical strength, which isolates the potentially adherent tissue, and the covalent coupling also increases the stability of the natural biomacromolecules in the scaffold, ensuring that the material scaffold can act as a tissue barrier for a considerable period of time.

In a specific embodiment of the present invention, the coupling agent may include genipin, a proanthocyanidin or an aldehyde compound (more preferably a dialdehyde compound), and the like; the coupling initiator mainly includes a carbodiimide or the like. . The use of a coupling agent enables the coupling of chemical groups that are distant from each other on biodegradable macromolecules, which not only participate in the coupling reaction, but also become part of the coupling product. For example, genipin and proanthocyanidins are biodegradable, low-cytotoxic natural molecules that form a cross-linking bridge with a heterocyclic structure of an amino group in the molecule of the film-forming material. The coupling agent may be used singly or in combination, for example, in a film-forming system, the concentration of genipin is 0.01% to 10%, the concentration of proanthocyanidin is 0.01% to 10%, and glutaraldehyde (or other two) The concentration of the aldehyde compound) may be from 0.001% to 5%.

The chemical coupling initiator used in the present invention may be a carbodiimide coupling initiator, which promotes chemical coupling of a carboxyl group and an amino group which are closely spaced apart on a biodegradable macromolecule under mild conditions in terms of mechanism of action. But it does not become part of the final coupling product. The chemical structure of the carbodiimide contains an R1-N=C=N-R2 group which promotes the formation of a chemical bond between the carboxyl group and the amino group, wherein R1 and R2 are an alkyl group, a cycloalkyl group, an aralkyl group or a substituent thereof. , including but not limited to 1-alkyl-3-(3-dimethylaminopropyl)carbodiimide (wherein the alkyl group contains 1 to 10 carbon atoms), N,N'-diisopropylcarbamate Imine, dicyclohexylcarbodiimide, 1-ethyl-3-(3-(trimethylammonium)propyl)carbodiimide, iodized [1-cyclohexyl-3-(3-trimethylamine) Propyl)carbodiimide], cyclohexyl-β-(N-methylmorpholinyl)ethylcarbodiimide p-toluenesulfonate, 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide methyl iodide, 1,6-hexylene bis(ethylcarbodiimide), p-phenylene bis(ethylcarbodiimide), and the like. The more carbodiimide used may be 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or its hydrochloride, and the concentration of carbodiimide in the film-forming system is generally It is 0.01-10%. The coupling initiator can be used alone or in combination with a coupling agent.

In order to further enhance the effectiveness of a coupling initiator such as carbodiimide to promote coupling of a carboxyl group to an amino group to form a bioconjugating material having a greater degree of coupling, the present invention may also use a coupling initiation synergist such as N-hydroxy amber. Imide (NHS, N-hydroxysuccinimide) or N-hydroxysulfosuccinimide (Sulfo-NHS, N-hydroxysulfosuccinimide), etc., other adjuvants are 1-hydroxybenzotriazole (HOBt), 3-hydroxyl -1,2,3-benzotriazine-4(3H)-one (HOOBt) and/or 1-hydroxy-7-azobenzotriazole (HOAt), these coupling initiation increases in the film-forming system The concentration of the agent can be 0-10%.

In addition to using a coupling agent or/and a coupling initiator to promote covalent bond formation to enhance the mechanical properties of the film, prolong the biodegradation time of the film, and attach properties of the film to the tissue, the present invention can also adjust the film by adding metal ions. Performance realization, these ions include divalent and trivalent metal ions, such as calcium ions, zinc ions, iron ions, etc., usually added as soluble salts.

Applicant's research found that when the film-forming system contains a coupling agent and/or a coupling initiator, the type of film-forming material is appropriately selected, which is more favorable for obtaining biofilm materials with improved adhesion properties and mechanical properties. . For example, the film-forming material may be selected from the group consisting of a biocompatible macromolecular material containing both a carboxyl group and an amino group, a mixture of a macromolecular material containing both a carboxyl group and an amino group, and a macromolecular material containing only a carboxyl group or an amino group (eg, gelatin and a mixture of sodium alginate), or a mixture of a macromolecular material containing only a carboxyl group and a macromolecular material containing only an amino group (for example, a mixture of chitosan and sodium carboxymethylcellulose); and further comprising cellulose, carboxymethyl Cellulose, starch or carboxymethyl starch.

The biodegradable film of the present invention may be a film material directly formed by a fluid dynamic film forming material (for example, gelatin, collagen, hyaluronic acid, etc.), which may be prepared by placing a mobile phase of a film forming raw material. In a dry flat-bottomed container, it is solidified into a film, and after drying, the film is peeled off and taken out; or a film material produced by a film forming system including a film forming raw material and a coupling agent and/or a coupling initiator may be used. The preparation method may be that the mobile phase of the film-forming raw material is first placed in a dry flat-bottomed container, and after being in a solidified state or during the solidification process, a coupling agent and/or a coupling initiator solution are added to perform the coupling. The solution is poured off, and the excess coupling agent and/or coupling initiator is removed by washing with water, and after drying, the film is peeled off and taken out.

In order to promote the formation of the membrane, adjust the mechanical properties and degradation rate of the membrane, biodegradable adjuvants such as starch, cellulose, chitin, dextran, carrageenan, agarose and other natural macromolecules and their derivatives may be added to the film-forming system. Also includes biodegradable synthetic polymers such as polyglycolic acid (PGA), polylactic acid, polyglycolic acid and lactic acid copolymer (PLGA), polyethylene glycol (PEG), polyethylene glycol and polylactic acid copolymerization The auxiliary materials account for 1-90% of the dry weight of the film. The excipients are used in the form of an aqueous solution, a suspension or a dry powder, and the suspension and dry powder materials include nanoparticulate materials.

The preparation method of the present invention does not have strict requirements for the operation of the film forming process, for example, it can be left to stand at room temperature or ambient temperature, and the film can be dried by drying or air drying at ambient temperature or room temperature. The film raw material mobile phase or the film forming system mobile phase may be formulated using water, a dilute acid solution, a salt solution or the like, for example, using deionized water, dilute hydrochloric acid solution, sodium phosphate solution, EMS buffered physiological saline or the like.

The coupling agent and/or the coupling initiator are added to initiate the coupling reaction, and generally it is allowed to stand. In the preparation of the film, the coupling reaction time may be from 0.1 to 24 hours, and after the coupling, it is washed at least once with distilled water to remove excess coupling agent and/or coupling initiator.

The present invention provides a biodegradable film material prepared by any of the above methods. Applicants' research has found that even if only the above-mentioned film-forming material is used to control a film formed by direct solidification of a suitable unit mass, such as a gelatin film, It can effectively adhere to the wound tissue, exert anti-adhesion and hemostatic effects, and has the advantages of excellent mechanical properties and easy bending. When a combination is required, the corresponding drug can be conveniently placed on the surface or the whole of the film, and the film material simultaneously assumes the role of the drug carrier, which facilitates the release of the drug on the treatment surface. For example, antibiotics (such as mitomycin C, hydroxycamptothecin, etc.), anti-inflammatory drugs (such as aceclofenac, diclofenac sodium, dexamethasone, etc.), antioxidants (such as vitamin C, vitamin E, blackening) can be added. Hormone, methylene blue, etc. to prevent bacterial infection and inflammation, add calcium ions, blood coagulation factors, vitamin K and other materials to improve the hemostatic function, add phospholipids, slip protein (lubricin) and other anti-adhesive adhesions, but also can be added to biological factors control Wound healing process.

The biodegradable film material prepared by any of the above methods provided by the present invention may also use a coupling agent and/or a coupling initiator in the film forming process, but a coupling agent and/or a coupling initiator are specified. For delayed introduction, that is, the coupling agent and/or the coupling initiator are added to the film forming system when the mobile phase of the film forming raw material has been in a solidified state, and the obtained film material is clinically adhered to the wound tissue. The effect, or the controlled release effect of the drug and the safety of use, will be more ideal. These clinical manifestations should be confirmed by the microscopic mechanism of film formation and coupling reactions.

It can be understood that the delayed introduction of the coupling agent and/or the coupling initiator during film formation reduces the intramolecular coupling and facilitates the bonding of the film to the tissue in the application (the coupling agent also promotes the film and the contacted tissue). Covalent bonding); conventionally, the coupling agent and/or the coupling initiator are first compounded with the film-forming raw material to form a film together, and the coupling agent and/or the coupling initiator are delayedly introduced. Under solidification conditions, firstly, the range of motion between macromolecules is limited to a limited space, which limits the distance between molecules, enhances interaction, reduces intramolecular coupling during coupling, and increases intermolecular coupling. That is, the delayed introduction of a chemical coupling agent or/and a coupling initiator will lead to coupling between macromolecules, resulting in a material having a unique molecular structure and properties; and forming a chemically active site in the already solidified material. Point, these sites can not only form chemical bonds with the tissue attached to the biomaterial film, but also provide active sites for binding with small molecules, growth factors and other drugs to achieve controlled release of the drug, and To prevent the drug (such as growth factor) from decreasing and losing the activity of the drug due to self-coupling; on the other hand, the coupling agent and/or the coupling initiator are delayedly introduced to act on the surface of the film-forming system which has been in a solid state, redundant The reagent can be efficiently removed by pouring and washing without using an inefficient dialysis method, which not only improves the preparation efficiency, reduces the operation difficulty and cost, but also improves the biosafety of the material.

In a specific embodiment of the present invention, the film is required to have a mass of 0.1-15 mg/cm 2 , preferably 1 from the effective adhesion effect of the film to the wound tissue, the bending resistance during use of the film, and the mechanical properties of the film. -10 mg/cm 2 , for a film with a certain composition, the mass per unit area of the film corresponds to a certain film thickness, and can be made into a triangle, a square, a rectangle, a rectangle, a circle, an ellipse, etc. according to the specific needs of the clinical application. Shapes, as well as films of different sizes, do not require secondary processing for direct use.

The present invention also provides the use of the above biodegradable film in the preparation of a carrier for preventing tissue leakage, an anti-adhesion film after surgery, and a topical drug carrier. The experimental results show that the biomaterial film of the invention can adhere closely to the surface of the tissue, and the non-covalent bond chemistry such as hydrogen bond and/or ionic bond formed by the hydrophilic material in the material and the surface of the tissue can also be passed through the organism. The covalent bond formed between the material film and its attached tissue is reinforced to prevent leakage of gas, body fluids, and tissue contents, and also prevents movement of the material during use. The biodegradable membrane of the present invention can effectively prevent tissue leakage and adhesion at the abdomen, cardiovascular, spine, ankle, gynecological pelvis, etc. in clinical application, and can be used as a drug carrier in medical treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the comparison of the adhesion properties of gelatin films to tissues of different thicknesses (mass per unit area).

In the figure, a 1 x ² cm gelatin film (mass per unit area of ¹ , 5, 10, 15, 20, and 30 mg/cm ² ) was pressed in the small intestine of the dog for 3 seconds, and then the adhesion of the film to the tissue was observed.

Figure 2 is a graph showing the tensile force displacement of a film sample obtained by two different film forming methods (the coupling agent is added at the time of film formation or added after film formation).

In the figure, the film 1 represents a tensile force displacement curve of a film sample obtained by chemical coupling occurring at the same time as the film forming raw material is solidified into a film, and the film 2 represents a tensile force displacement of the film sample obtained by chemically coupling the film forming material to a film before chemical coupling. curve.

Detailed ways

The implementation of the present invention and the implementation of the technical effects thereof are further illustrated by the following specific examples and experimental examples, but are not intended to limit the scope of the present invention.

Parts and percentages referred to in the present invention are by weight unless otherwise indicated.

I. Biomaterial Preparation Example

The following examples are intended to illustrate the preparation of the film of the present invention, but the material composition, amount of raw materials and application are not limited to these examples.

All raw materials are from commercial products.

Example 1

100 mg of gelatin dissolved in 10 ml of 50 ° C deionized water, poured into a square horizontal trough made of polytetrafluoroethylene with a bottom surface size of 5 cm x 8 cm, solidified at room temperature, naturally dried, washed with distilled water as needed, dried After careful removal of the gelatin film from the Teflon cell, the film has a mass per unit area of about 2.5 mg/cm ² .

Example 2

Dissolve 100 mg of gelatin and 10 mg of sodium hyaluronate in 10 ml of deionized water at 50 ° C, pour into a square horizontal trough made of polytetrafluoroethylene 5 cm x 8 cm, solidify at room temperature, dry naturally, if necessary After washing with distilled water, the film was carefully peeled off from the Teflon cell after drying, and the mass per unit area of the film was about 2.75 mg/cm ² .

Example 3

Dissolve 100 mg of gelatin and 10 mg of sodium carboxymethylcellulose in 10 ml of deionized water at 50 ° C, pour into 5 cm x 8 cm square troughs made of Teflon, place at room temperature, dry naturally, as needed After washing with distilled water, the film was carefully peeled off from the Teflon cell after drying, and the mass per unit area of the film was about 2.75 mg/cm ² .

Example 4

2 g of chitosan (deacetylation degree 80%, MW200,000) was placed in 100 ml of dilute hydrochloric acid solution, stirred for 24 hours, the acidity value was maintained at pH 3.0, and the insoluble matter was removed by filtration to prepare a 2% chitosan solution. . 100 mg of gelatin is dissolved in 8 ml of 50 ° C deionized water, then 2 ml of the above 2% chitosan solution is added, mixed, poured into a square horizontal trough made of polytetrafluoroethylene 5 cm x 8 cm, and solidified at room temperature. Dry naturally, carefully peel the gel film from the Teflon cell, and the film unit area is about 3.5 mg/cm ² .

Example 5

5 ml of 2% silk protein solution and 5 ml of 2% chitosan solution were poured into a square horizontal trough made of polytetrafluoroethylene 5 cm x 8 cm, mixed, placed at room temperature, naturally dried until solidified, and then sodium phosphate was used. The aqueous solution was washed once, washed twice with distilled water, and air-dried, and the gel film was carefully peeled off from the polytetrafluoroethylene tank, and the mass per unit area of the membrane was about 5 mg/cm ² .

Example 6

100 mg of gelatin dissolved in 10 ml of deionized water at 50 ° C, poured into a square horizontal trough made of polytetrafluoroethylene 5 cm x 8 cm, allowed to dry to complete solidification, and then added 2 ml of 1% EDC to allow the solution to be evenly spread. The film was allowed to stand at room temperature for 3 hours, and the excess solution was poured out, then washed three times with distilled water, air-dried, and the gel film was carefully peeled off from the Teflon bath, and the mass per unit area of the film was about 3 mg/cm ² .

Example 7

100 mg of gelatin and 10 mg of sodium alginate were dissolved in 10 ml of deionized water at 50 ° C, poured into a square horizontal trough made of polytetrafluoroethylene 5 cm x 8 cm, completely solidified at room temperature, and 1 ml of 1% EDC was added. Allow the solution to spread evenly on the membrane, leave it at room temperature for 3 hours, pour out the excess solution, wash it three times with distilled water, dry the gel film at room temperature, and carefully peel it off from the Teflon tank. The mass per unit area of the membrane is about 3 mg/ Cm ² .

Example 8

Dissolve 100 mg of gelatin in 10 ml of deionized water at 50 ° C, add 50 mg of hydroxypropyl methylcellulose powder, mix well, pour into 5 cm x 8 cm square troughs made of Teflon, place at room temperature After solidification, add 2 ml of 1% EDC, let the solution spread evenly on the membrane, leave it at room temperature for 3 hours, pour out the excess solution, wash it three times with distilled water, dry the gel film at room temperature, and carefully carefully from the Teflon tank. Peeling, the mass per unit area of the film was about 4.25 mg/cm ² .

Example 9

5 ml of 2% chitosan solution was mixed with 5 ml of 1% sodium carboxymethylcellulose aqueous solution containing 50 mg of hydroxyethyl starch, and poured into a square horizontal trough made of polytetrafluoroethylene 5 cm x 8 cm. After standing at room temperature and completely solidified, 1 ml of 1% EDC was added to allow the solution to spread evenly on the film. After standing at room temperature for 3 hours, the excess solution was poured out, washed three times with distilled water, and air-dried, and the gel film was carefully peeled off from the Teflon cell, and the mass per unit area of the film was about 5.25 mg/cm ² .

Example 10

100 mg of gelatin was dissolved in 10 ml of deionized water at 50 ° C, poured into a square horizontal trough made of polytetrafluoroethylene 5 cm × 8 cm, placed at room temperature until completely solidified, and 2 ml of 1% EDC/1% NHS solution was added. Allow the solution to spread evenly on the membrane, leave it at room temperature for 3 hours, pour out the excess solution, wash it three times with distilled water, leave it to dry at room temperature, and carefully peel off the gel film from the Teflon tank. The mass per unit area of the membrane is about 3.5 mg/ Cm ² .

Example 11

200 mg of gelatin dissolved in 10 ml of deionized water at 50 ° C, poured into a square horizontal trough made of polytetrafluoroethylene 5 cm × 8 cm, set to solidify at room temperature, add 2 ml of 0.1% glutaraldehyde, let the solution spread evenly The film was allowed to stand at room temperature overnight, and the solution was poured out, washed three times with distilled water, and then the gel was air-dried and carefully peeled off from the Teflon cell, and the mass per unit area of the film was about 5 mg/cm ² .

Example 12

100 mg of chitosan (deacetylation degree 80%, MW200,000) was added to 10 ml of dilute hydrochloric acid aqueous solution, stirred for 24 hours, the acidity value was maintained at pH 3.0, and poured into 5 cm × 8 cm made of polytetrafluoroethylene. In a square horizontal trough, set to solidify at room temperature, wash three times with aqueous sodium phosphate solution, add 2 ml of 0.2% glutaraldehyde, let the solution spread evenly on the membrane, leave it at room temperature overnight, pour out the excess solution, wash it three times with distilled water, then The gel was air-dried and carefully peeled off from the Teflon cell, and the mass per unit area of the film was about 2.6 mg/cm ² .

Example 13

100 mg of gelatin dissolved in 5 ml of deionized water at 50 ° C, added 50 mg of hydroxyethyl starch, mixed, and mixed with 5 ml of 2% chitosan solution, poured into 5 cm × 8 cm made of polytetrafluoroethylene In a square horizontal tank, set to solidify at room temperature, add 2 ml of 0.1% glutaraldehyde, let the solution spread evenly on the membrane, leave it at room temperature overnight, pour off the excess solution, wash it three times with distilled water, then dry the gel, then The Teflon cell was carefully peeled off and the membrane unit area was about 6.3 mg/cm ² .

Example 14

100 mg of gelatin dissolved in 10 ml of deionized water at 50 ° C, poured into a square horizontal trough made of polytetrafluoroethylene 5 cm × 8 cm, completely solidified at room temperature, and added 2 ml of 2% EDC / 1% genipin solution The solution was evenly spread on the membrane, left at room temperature overnight, and the excess solution was poured out, washed three times with distilled water, and the gel was air-dried at room temperature, and then carefully peeled off from the Teflon cell. The mass per unit area of the membrane was about 3.5 mg/cm. ² .

Example 15

100 mg of gelatin dissolved in 10 ml of deionized water at 50 ° C, poured into a square horizontal trough made of polytetrafluoroethylene 5 cm × 8 cm, completely solidified at room temperature, then added 2 ml of 0.5% EDC / 0.1% glutaraldehyde The solution was allowed to spread evenly on the membrane, left at room temperature overnight, and the excess solution was poured out, washed three times with distilled water, then the gel was air-dried, and carefully peeled off from the Teflon cell. The mass per unit area of the membrane was about 2.8 mg/cm. ² .

The amount of the raw material was adjusted according to the method of any of the above examples, and the mass of the final film formation was attempted to be less than 0.9 mg/cm ² , but it was difficult to peel off to obtain a complete gel film.

II. Example of relationship between film thickness and tissue adhesion

According to the method of Example 1, the gelatin usage amount was adjusted to prepare gelatin films having a basis weight of 1, 5, 10, 15, 20, 30 mg/cm ² which have different thicknesses.

Take a 15cm dog small intestine, put a plastic rod inside, and round the small intestine, then take a different thickness of gelatin film, take a size of 1cm x 2cm, and press it on the dog's small intestine for 3 seconds to observe the attachment.

Results: Gelatin films A, B, and C with thicknesses of 1, 5, and 10 mg/cm ² were able to adhere tightly to the surface of the small intestine after pressing for 3 seconds, and gelatin film D having a thickness of 15 mg/cm ² was only partially attached to the small intestine. On the surface, the pressing time is increased to 15 seconds to adhere the film of the thickness to the surface of the small intestine, and the gelatin films E and F having a thickness of 20 and 30 mg/cm ² are difficult to adhere closely by pressing even if the pressing time is increased. On the surface of the small intestine (refer to Figure 1).

III. Preparation method of biomaterial film and its mechanical properties

Take 300 mg of gelatin and 30 mg of carboxymethylcellulose, mix and dissolve in 15 ml of water at 50 °C.

Take two square horizontal grooves made of polytetrafluoroethylene with a bottom area of 5cm×8cm. Pour 6 ml of the gelatin and carboxymethyl cellulose mixture prepared above into one horizontal tank, and immediately add 4 ml of 0.9%. EDC / 0.45% NHS solution, quickly mix the solution in the tank, dry at room temperature to obtain membrane 1; in another horizontal tank, also pour 6 ml of the gelatin and carboxymethyl cellulose mixture prepared above and 4 ml of water, mixed Evenly, spread in a horizontal tank, air at room temperature until the material is completely solidified, add 4 ml of 0.9% EDC/0.45% NHS solution, and let the solution spread evenly on the membrane. After reacting for 2 hours, pour out the excess solution and wash it three times with distilled water. The gel was then air-dried and peeled off from the Teflon bath to obtain Film 2.

The membranes obtained by the two preparation methods were cut into dumbbell shapes of the same size, the narrowest point was 3.3 mm, and the tensile force was placed on a mechanical stretching machine, and the relationship between the tensile force and the displacement was plotted in Fig. 2. The two preparation methods were used to make a film. The mechanical properties. It was found that the chemical coupling occurred in the film 1 at the time of film formation, and the breaking strength of the formed film was much smaller than that of the film 2 which was chemically coupled after film formation, and the rigidity (slope during the rise of the curve) also showed the same tendency.

In the same manner, the film of the foregoing examples was tested for mechanical properties, and the control group was still the film having the same composition but chemical coupling occurred at the time of film formation, and the same results were obtained.

IV. Tissue adhesion and hemostasis effect examples

Ten New Zealand rabbits were randomly assigned to the control group and the biomaterial group, 5 rats in each group, anesthetized with sodium pentobarbital, laparotomy along the ventral midline under sterile conditions, exposed to the liver, causing 1 cm x 1 cm area on the liver surface. The size of the tissue was damaged, the control group was treated with a disinfecting sponge to stop bleeding, and the biomaterial group applied the gelatin film (3 cm x 3 cm) prepared in the above biological material preparation example 6 to the wound, recorded the hemostasis time, measured the amount of bleeding, and after completing the recording and measurement, The wound was sutured and the animals were given free access to water to observe the survival of the animals.

The hemostasis time of the control group was 526±78 seconds, the hemostasis time of the biomaterial group was 205±56 seconds; the bleeding volume of the control group was 1.83±0.86 ml, and the amount of bleeding of the biomaterial group was 1.49±0.62 ml.

Two weeks after the operation, the animals survived intact, and the wounds recovered well after laparotomy.

This experiment proves that the biomaterial film can adhere to the surface of the tissue, has a hemostatic effect, and has good biocompatibility and can be degraded in the body.

The biomaterial group can also adopt the film prepared in Example 4, and the same operation treatment has a reduced hemostatic time and bleeding amount.

V. Example of preventing adhesion effect

Taking the dog as an experimental model of the animal, the experimental animal dog weighed 20 kg.

The flexor tendons of the second and fifth fingers of the right fore paw of the dog were completely cut off at the forefoot, and the suture was surgically sutured. After the second flexor tendon as a control group was sutured, no treatment was performed; and after the fifth flexor tendon of the test group was sutured, the gelatin film (3 cm x 1 cm) prepared in Example 10 was prepared by the above biological material. After the tendon is sutured and treated, the tendon sheath and skin are sutured layer by layer.

The paws were dissected three weeks after surgery, and the degree of tendon adhesion was observed. The fifth finger of the test group was found. The gelatin film could effectively prevent the flexor tendon from adhering to the surrounding tissue, and the second finger of the control group had tissue adhesion.

Claims

A method for preparing a biodegradable film, comprising: placing a mobile phase made of a film-forming raw material in a dry flat-bottomed container, solidifying and forming a film, so that the mass of the film is 0.1-15 mg/cm 2 ; Membrane materials include biocompatible macromolecular materials containing hydroxyl, amino and/or carboxyl groups.
The production method according to claim 1, further comprising using a coupling agent and/or a coupling initiator, the preparation method comprising: placing a mobile phase made of a film-forming raw material in a dry flat-bottomed container, After the solidification or solidification, a coupling agent and/or a coupling initiator are added to cause a coupling reaction, which is washed with water and dried, and then peeled off to obtain the film product.
The production method according to claim 2, wherein the coupling agent comprises genipin, a proanthocyanidin or an aldehyde compound; and the coupling initiator comprises a carbodiimide.
The preparation method according to any one of claims 1 to 3, wherein the film-forming material is selected from the group consisting of gelatin, collagen, fibrin, silk fibroin, albumin, globulin, hyaluronic acid, chitin, and shell poly Sugar, cellulose, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, alginic acid, starch, deacetylated hyaluronic acid, carboxymethyl chitosan, carboxymethyl Base starch, carboxymethyl cellulose, polygalacturonic acid, dextran, agar or carrageenan and derivatives thereof.
The production method according to claim 3, wherein the film-forming material is selected from the group consisting of a biocompatible macromolecular material containing both a carboxyl group and an amino group, a macromolecular material containing both a carboxyl group and an amino group, and a carboxyl group or an amino group only. a mixture of macromolecular materials, or a mixture of a macromolecular material containing only a carboxyl group and a macromolecular material containing only an amino group; or

The film-forming material is selected from the group consisting of a biocompatible macromolecular material containing a carboxyl group and an amino group, a mixture of a macromolecular material containing a carboxyl group and an amino group, and a macromolecular material containing only a carboxyl group or an amino group, or a carboxyl group only. A mixture of macromolecular materials and macromolecular materials containing only amino groups, and further comprising cellulose, carboxymethyl cellulose, starch or carboxymethyl starch.
The production method according to claim 1 or 2, wherein the drying comprises natural drying or air flow drying at ambient temperature or room temperature.
The preparation method according to claim 2, wherein after the mobile phase prepared by the film-forming raw material is completely solidified in the container, a coupling agent and/or a coupling initiator is further added, and the mixture is allowed to stand for 0.1 to 24 hours. The excess liquid is then removed and washed with distilled water at least once.
A biodegradable film prepared by the method of any one of claims 1-7.
The biodegradable film according to claim 8, wherein the film has a mass of from 1 to 10 mg/cm 2 .
Use of the biodegradable film of claim 8 or 9 in the preparation of a carrier for preventing tissue leakage, a postoperative anti-adhesion film and a topical drug carrier.