阅读说明：本技术 一种预防瘢痕粘连的组合物、术后防粘连材料及应用 (Composition for preventing scar adhesion, postoperative anti-adhesion material and application ) 是由 苑康见 柏桓 赵艳 张春霞 张在庆 闫永丽 于 2021-08-02 设计创作，主要内容包括：本发明涉及一种预防瘢痕粘连的组合物、术后防粘连材料及应用。硬膜外瘢痕粘连是术后腰椎手术失败综合征的重要原因,本发明目的在于提供一种载药型的术后密封材料,在密封伤口的同时实现药物稳定释放。本发明首先提供了一种预防瘢痕粘连的丝裂霉素C-IFNγ-透明质酸钠结合物,所述结合物能够有效抑制瘢痕组织形成,减缓药物降解速度,将其应用于聚乙二醇类密封剂能够获得低溶胀、快速凝结的密封效果,特别适用于椎板切除术等脊椎外科手术,具有良好的临床应用价值。(The invention relates to a composition for preventing scar adhesion, a postoperative anti-adhesion material and application. The invention aims to provide a drug-loaded postoperative sealing material which realizes stable release of a drug while sealing a wound, and provides an epidural scar adhesion which is an important reason for postoperative lumbar spine surgery failure syndrome. The invention firstly provides a mitomycin C-IFN gamma-sodium hyaluronate conjugate for preventing scar adhesion, which can effectively inhibit scar tissue formation and slow down drug degradation speed, can obtain low-swelling and rapid-coagulation sealing effects when applied to a polyethylene glycol sealant, is particularly suitable for vertebral column surgical operations such as laminectomy and the like, and has good clinical application value.)

1. A composition for preventing scar adhesions, which is characterized by comprising mitomycin C, interferon and polysaccharide; the polysaccharide is selected from one of dextran, chitin, chitosan, hyaluronic acid, cellulose, collagen, gelatin, fucan, and mucopolysaccharide.

2. The composition for preventing scar adhesions of claim 1 where the polysaccharide is sodium hyaluronate; the mitomycin C, the interferon and the sodium hyaluronate are combined through covalent bonds, and the mitomycin C-IFN gamma-sodium hyaluronate is a combination of mitomycin C-IFN gamma-sodium hyaluronate.

3. The composition for preventing scar adhesions of claim 1 where the interferon IFN γ is IFN- β.

4. The composition for preventing scar adhesions of claim 2 where the combination of mitomycin C-IFN γ -sodium hyaluronate is prepared as follows: adding sodium hyaluronate into PBS buffer solution with pH6.5, uniformly mixing, adding EDC, and stirring at low speed for 25-35 min to activate the sodium hyaluronate; adding mitomycin C and IFN gamma into the activated sodium hyaluronate buffer solution, and stirring for 6-10 hours at room temperature to obtain a mitomycin C-IFN gamma-sodium hyaluronate conjugate; after the reaction is finished, purifying the product by dialysis or elution.

5. Use of the composition according to any one of claims 1 to 4 for the preparation of a postoperative adhesion prevention material.

6. The use of the composition of claim 5in the preparation of a postoperative adhesion prevention material, wherein the postoperative adhesion prevention material is one of a film preparation, a liquid preparation, and a gel preparation; the gel formulation is a cross-linked product of a polyethylene glycol activated ester.

7. The use of the composition of claim 5 for the preparation of a postoperative adhesion prevention material, wherein the hydrogel formulation comprises the raw materials of polyethylene glycol activated ester, mitomycin C-IFN γ -sodium hyaluronate conjugate, a cross-linking agent and a buffer solution;

the polyethylene glycol activated ester is selected from one of four-arm polyethylene glycol succinimide succinate, four-arm polyethylene glycol succinimide glutarate, four-arm polyethylene glycol succinimide sebacate, six-arm polyethylene glycol succinimide succinate, six-arm polyethylene glycol succinimide glutarate, six-arm polyethylene glycol succinimide sebacate, eight-arm polyethylene glycol succinimide succinate, eight-arm polyethylene glycol succinimide glutarate and eight-arm polyethylene glycol succinimide sebacate, and the molecular weight of the polyethylene glycol activated ester is 3000-20000 daltons;

the cross-linking agent is one or a combination of trilysine and polyethyleneimine;

the buffer solution comprises an acidic buffer solution and an alkaline buffer solution, wherein the pH value of the acidic buffer solution is 3-5, and the acidic buffer solution is one of a phthalic acid buffer solution, a phosphoric acid buffer solution, a citric acid buffer solution and an acetic acid buffer solution;

the pH value of the alkaline buffer solution is 9-11, and the alkaline buffer solution is one of a phosphate buffer solution, a barbital sodium buffer solution, a Tris buffer solution, a boric acid buffer solution, a glycine buffer solution and a carbonic acid buffer solution.

8. The use of the composition according to claim 7 for the preparation of a postoperative adhesion prevention material, wherein the trilysine and the polyethyleneimine are respectively placed in different dissolution systems: the polylysine is placed in a buffer solution, and the polyethyleneimine is dissolved in a polysaccharide solution.

9. A sealant kit for surgical use, comprising the composition for preventing scar adhesion according to any one of claims 1 to 4.

10. The sealant kit for surgical use according to claim 9, further comprising polyethylene glycol activated ester, a cross-linking agent, a buffer solution, a syringe, and a mixing device.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a composition for preventing scar adhesion, a postoperative anti-adhesion material containing the composition, and application of the postoperative anti-adhesion material in spinal surgery.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Trauma to muscle or ligaments requires repair by scar tissue formation. After laminectomy or fenestration, the dura mater, the muscle and the lamina stump contact each other, and in the process of wound repair, dense scar tissue is formed in the vertebral lamina defect area, which is called a vertebral lamina resection membrane. Scar tissue is adhered to the dura mater or nerve root, which can cause the dura mater or nerve root to be drawn and stuck, resulting in postoperative symptom recurrence. The scholars believe that scar tissue can cause pain, and inflammatory reaction of dura mater and nerve root can also participate in adhesion, which can cause nerve tissue nutrient disturbance and conduction dysfunction and is closely related to postoperative pain symptoms. Therefore, epidural scar adhesion after laminectomy is an important factor in the recurrence of postoperative pain.

After the epidural is formed, the difficulty of secondary operation is increased, and the treatment effect is poor. Therefore, prevention and reduction of post-operative epidural scar adhesions associated with the transforaminal spine has been a focus of research in spinal surgery. The main ways of preventing postoperative adhesion in the field comprise implanting anti-adhesion materials, preventing medicines and the like. The implanted anti-adhesion material has good biocompatibility and proper degradation time, and the anti-adhesion materials mainly adopted at present comprise hard materials such as tricalcium phosphate artificial vertebral plate, polyamide composite artificial vertebral plate and the like, membranous soft materials such as polylactic acid film and the like, fluid materials such as sodium hyaluronate, chitosan and the like, and biological materials such as autologous fat and the like. Among the anti-adhesion materials, the anti-adhesion membrane has moderate flexibility and histocompatibility, and has the defects that the membrane material is prepared in advance, the thickness cannot be changed, the contact degree between certain complex wound surface parts and tissues is low, and the anti-adhesion effect is reduced. The anti-adhesion liquid of the chitosans has short degradation time, has undesirable anti-adhesion effect on wound surfaces with long chronic inflammation, has poor anti-pressure capability and large influence on the body position, and is not suitable for preventing and treating the adhesion of parts such as nerves, peripheral tissues, tendons and the like. In the case of a prophylactic method, a drug such as mitomycin C is usually administered directly to the surgical site, and this administration method has a short time for the drug to remain on the wound surface, and thus it is difficult to obtain an ideal therapeutic effect.

The hydrogel is used as a biocompatible material and is mainly used for postoperative adhesion prevention, hemostat, defect tissue filling, tissue fluid leakage prevention, drug slow release and the like. The hydrogel sealing adhesive is present in the form of a liquid precursor without a permanent shape prior to gel formation, and acts directly on the site of action by simple injection or coating. It is determined that the hydrogel sealant has good adaptability to the surgical wound surface, the delivery mode is simple, fast and easy to operate, and the unique amorphous liquid precursor property also endows the hydrogel sealant with the capability of better contacting the action site and firmer adhesion.

Disclosure of Invention

Against the background of the above studies, the inventors thought that the gel could be well attached to the surface of the site of action, and itself had the effect of preventing scar adhesions. And the degradation time of the gel can be regulated and controlled, and the gel is used as a drug carrier to hopefully realize the slow release of the drug until the wound surface is healed. Therefore, the invention provides a drug-loaded gel which can be firmly adhered to wound tissues to realize the stable release effect of drugs and can be used as an postoperative anti-adhesion sealing material.

Based on the technical purpose, the invention provides the following technical scheme:

in a first aspect of the invention, a composition for preventing scar adhesion is provided, which comprises mitomycin C, interferon and polysaccharide; the polysaccharide is selected from dextran, chitin, chitosan, hyaluronic acid, cellulose, collagen, gelatin, fucan or mucopolysaccharide.

It is well known in the art that Interferon (IFN) is a broad-spectrum antiviral agent, does not directly kill or inhibit viruses, but rather primarily inhibits viral replication by allowing cells to produce antiviral proteins through the action of cell surface receptors, and also enhances the activity of natural killer cells (NK cells), macrophages, and T lymphocytes, thereby playing a role in immunomodulation and enhancing antiviral ability. Interferons are a group of active proteins (mainly glycoproteins) with multiple functions, a cytokine produced by monocytes and lymphocytes. They have broad spectrum antiviral, cell growth affecting, differentiation, immunity regulating and other bioactivity on homogeneous cell, and are the main antiviral infection and antitumor biological products.

The research of the invention shows that the mitomycin C and the interferon are compounded to inhibit the formation of scar tissues. Wherein the interferon is preferably type I interferon, and in the scheme with better effect, IFN-beta is adopted. The research of the composition shows that the composition has better effect of inhibiting scar formation by compounding hyaluronic acid, mitomycin C and interferon. The major drawbacks of the prior art using mitomycin C or hyaluronic acid as a postoperative anti-adhesion material are that the degradation rate is too fast and the retention time of the drug on the wound surface is too short. The present invention is conceived to provide a combination form of the above-mentioned drugs, in which the above-mentioned active ingredients are bound by covalent bonds, the degradation time of the active ingredients is effectively slowed down, and the ability to inhibit scar tissue formation is also significantly better than that of the unbound mixed components.

In a second aspect of the invention, the composition of the first aspect is provided for use in preparing a postoperative anti-adhesion material.

Based on the good affinity and drug slow-release effect of the mitomycin C-IFN gamma-sodium hyaluronate conjugate, the mitomycin C-IFN gamma-sodium hyaluronate conjugate has better application significance to operation parts with more dense nerve distribution such as epidural or meningeal. The above conjugate, however, still has insufficient degradation time to support wound healing, and the present invention envisions the application thereof to a polymeric post-operative sealing material to further increase the sustained release effect. In the research process of the composite material, the inventor finds that the swelling effect and the gelling speed can be effectively reduced by adopting the composite cross-linking agent, and further, the inventor adopts different solvent systems to dissolve the cross-linking agent, and through verification, the composite effect of the trilysine and the polyethyleneimine is good, and the polyethyleneimine can be dissolved in a polysaccharide solution to realize a better mixing effect.

The hydrogel preparation has high coagulation speed and low swelling rate, is applied to parts with dense nerve distribution, such as the spine or the brain, and can effectively reduce the compression effect on peripheral tissues and nerves. Has good prevention and treatment effect on common adhesion and scar complications after dural surgery, particularly laminectomy.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a photograph of an in vitro degradation experiment of the present invention;

FIG. 2 is a photograph of an experiment of subcutaneous implantation of the hydrogel described in example 1.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, in order to solve the technical problems as described above, the present invention provides a composition for preventing scar adhesion and the use of the composition in the preparation of a postoperative anti-adhesion material.

In a first aspect of the invention, a composition for preventing scar adhesion is provided, which comprises mitomycin C, interferon and polysaccharide; the polysaccharide is selected from dextran, chitin, chitosan, hyaluronic acid, cellulose, collagen, gelatin, fucan or mucopolysaccharide.

In a preferred embodiment of the composition of the first aspect, the polysaccharide is selected from chitosan, cellulose, dextran, chitosan, or sodium hyaluronate.

In a preferred embodiment, the polysaccharide is chitosan, dextran or sodium hyaluronate.

Wherein, the sodium hyaluronate has good effect of inhibiting scar tissues and ideal biocompatibility. In a further preferred embodiment, the polysaccharide is sodium hyaluronate.

Preferably, the interferon IFN γ is a type i interferon, and further, is IFN- β.

In one embodiment of the above preferred embodiment, the composition is a conjugate of mitomycin C-IFN γ -sodium hyaluronate, mitomycin C, IFN γ and sodium hyaluronate are covalently bound together, and the covalent binding is formed by crosslinking with a crosslinking agent, wherein the crosslinking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) or N-hydroxysuccinimide (NHS).

In a specific embodiment, the mitomycin C-IFN γ -sodium hyaluronate conjugate is prepared as follows: dissolving sodium hyaluronate in PBS buffer solution with pH6.5, adding EDC, and stirring at low speed for 25-35 min to activate sodium hyaluronate; adding mitomycin C and IFN gamma into the activated sodium hyaluronate buffer solution, and stirring for 6-10 hours at room temperature to obtain a mitomycin C-IFN gamma-sodium hyaluronate conjugate; after the reaction is completed, the product can be purified by dialysis or elution.

In a second aspect of the invention, the composition of the first aspect is provided for use in preparing a postoperative anti-adhesion material.

Preferably, the postoperative anti-adhesion material is one of a film preparation, a liquid preparation and a gel preparation.

Further, the postoperative anti-adhesion material is hydrogel which is a cross-linking product of polyethylene glycol activated ester.

In the above embodiment, the raw materials of the hydrogel preparation include polyethylene glycol activated ester, mitomycin C-IFN γ -sodium hyaluronate conjugate, a cross-linking agent, and a buffer solution.

The polyethylene glycol activated ester is selected from quadri-armed polyethylene glycol succinimide succinate (4-arm-PEG-SS), quadri-armed polyethylene glycol succinimide glutarate (4-arm-PEG-SG), quadri-armed polyethylene glycol succinimide sebacate (4-arm-PEG-SSeb), hexa-armed polyethylene glycol succinimide succinate (6-arm-PEG-SS), hexa-armed polyethylene glycol succinimide glutarate (6-arm-PEG-SG), hexa-armed polyethylene glycol succinimide sebacate (6-arm-PEG-SSeb), octa-armed polyethylene glycol succinimide succinate (8-rm-PEG-SS), octa-armed polyethylene glycol succinimide glutarate (8-arm-PEG-SG), or octa-armed polyethylene glycol succinimide sebacate (8-arm-PEG-SSeb) ). The molecular weight is 3000-20000 daltons.

The cross-linking agent is selected from one or the combination of polylysine or polyethyleneimine.

In a more effective embodiment, the cross-linking agent is a combination of polylysine and polyethyleneimine.

Further, the polylysine is a trilysine, a tetrapolylysine or a pentapolylysine.

Preferably, the buffer solution comprises an acidic buffer solution and an alkaline buffer solution, wherein the pH value of the acidic buffer solution is 3-5, and the acidic buffer solution is further one or more of a phthalic acid buffer solution, a phosphoric acid buffer solution, a citric acid buffer solution or an acetic acid buffer solution; the pH value of the alkaline buffer solution is 9-11, and further the alkaline buffer solution is one or more of phosphate buffer solution, barbital sodium buffer solution, Tris buffer solution, boric acid buffer solution, glycine buffer solution and carbonic acid buffer solution.

In an embodiment with a better effect in the above preferred technical solution, the polylysine is trilysine, and the trilysine and polyethyleneimine are respectively placed in different dissolution systems: the polylysine is placed in a buffer solution, while the polyethyleneimine is dissolved in a polysaccharide solution.

In other specific embodiments, the hydrogel formulation further comprises an antioxidant, a coloring agent, or other drugs.

In a fourth aspect of the present invention, there is provided a sealant kit for surgical use, comprising a composition for preventing scar adhesions as described in the first aspect.

Preferably, the kit further comprises polyethylene glycol activated ester, a cross-linking agent or a buffer solution.

Preferably, the kit further comprises a syringe and a mixing device.

In the clinical treatment process, the gel preparation prepared quickly after operation has important significance for reducing the infection probability of the wound surface. Those skilled in the art have an incentive to adopt hydrogel mixing and spraying devices, such as injectors and spray heads, to achieve the rapid mixing and smearing effects of the raw materials.

In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.

Example 1

In this embodiment, a mitomycin C-IFN γ -sodium hyaluronate conjugate is provided, wherein the molecular weight of sodium hyaluronate is 5kDa, and the preparation method of the mitomycin C-IFN γ -sodium hyaluronate conjugate is as follows:

dissolving sodium hyaluronate in PBS buffer solution (pH6.5), adding appropriate amount of EDC, and stirring at room temperature for 30min at low speed to activate carboxyl group of sodium hyaluronate. Dissolving mitomycin C and IFN gamma in PBS buffer solution, slowly adding into activated sodium hyaluronate under stirring, stirring at room temperature for 8h after dropwise addition, purifying the conjugate by PBS dialysis, and freeze-drying the purified product for later use.

Example 2

In this embodiment, a mitomycin C-IFN γ -sodium hyaluronate conjugate is provided, wherein the molecular weight of sodium hyaluronate is 10kDa, and the preparation method of the mitomycin C-IFN γ -sodium hyaluronate conjugate is as follows:

dissolving sodium hyaluronate in PBS buffer solution (pH6.0), adding appropriate amount of EDC, and stirring at room temperature for 35min at low speed to activate carboxyl group of sodium hyaluronate. Dissolving mitomycin C and IFN gamma in a PBS buffer solution, slowly adding the solution into activated sodium hyaluronate under the stirring condition, stirring the solution for 10 hours at room temperature after the dropwise addition is finished, eluting the solution by adopting column chromatography to obtain a mitomycin C-IFN gamma-sodium hyaluronate conjugate, and freeze-drying the purified product for later use.

Example 3

In this embodiment, a mitomycin C-IFN γ -sodium hyaluronate conjugate is provided, wherein the molecular weight of sodium hyaluronate is 10kDa, and the preparation method of the mitomycin C-IFN γ -sodium hyaluronate conjugate is as follows:

sodium hyaluronate was dissolved in PBS buffer (pH6.0), and after adding an appropriate amount of EDC, the mixture was stirred at room temperature at a low speed for 25in to activate the carboxyl group of sodium hyaluronate. Dissolving mitomycin C and IFN gamma in a PBS buffer solution, slowly adding the solution into activated sodium hyaluronate under the stirring condition, stirring the solution for 10 hours at room temperature after the dropwise addition is finished, eluting the solution by adopting column chromatography to obtain a mitomycin C-IFN gamma-sodium hyaluronate conjugate, and freeze-drying the purified product for later use.

Example 4

In this embodiment, a postoperative adhesion prevention material is provided, and the adhesion prevention material is a polyethylene glycol hydrogel preparation, and the hydrogel preparation includes the following components in parts by weight:

(a) 0.4g of four-arm polyethylene glycol succinimide succinate and 0.25g of mitomycin C-IFN gamma-sodium hyaluronate conjugate;

(b) trilysine (10g/L) -borate buffer (65mM)2.5mL, pH 9.8;

(c) 8 percent of polyethyleneimine (molecular weight 2000) and 7 percent of sodium hyaluronate, and 0.1mL of aqueous solution;

(d) phosphate buffer (1.5mM)2.4mL, pH 4.0.

The preparation method of the hydrogel comprises the following steps: mixing the above (a) and (d) to obtain solution A, mixing the above (B) and (c) to obtain solution B, and mixing the solution A and B to obtain gel preparation.

Example 5

In this embodiment, a postoperative adhesion prevention material is provided, and the adhesion prevention material is a polyethylene glycol hydrogel preparation, and the hydrogel preparation includes the following components in parts by weight:

(a) 0.4g of hexa-arm polyethylene glycol succinimide succinate and 0.25g of mitomycin C-IFN gamma-sodium hyaluronate conjugate;

(b) trilysine (10g/L) -borate buffer (65mM)2.5mL, pH 9.8;

(c) 8 percent of polyethyleneimine (molecular weight 2000) and 7 percent of sodium hyaluronate, and 0.1mL of aqueous solution;

(d) phosphate buffer (1.5mM)2.4mL, pH 4.0.

The preparation method of the hydrogel comprises the following steps: mixing the above (a) and (d) to obtain solution A, mixing the above (B) and (c) to obtain solution B, and mixing the solution A and B to obtain gel preparation.

Example 6

In the embodiment, a drug-loaded surgical sealant is provided, wherein the hydrogel in the surgical sealant comprises the following components in parts by weight:

(a) 0.45g of hexa-arm polyethylene glycol succinimide sebacate and 0.2g of mitomycin C-IFN gamma-sodium hyaluronate conjugate;

(b) trilysine (15g/L) -borate buffer (65mM)2.5mL, pH 9.8;

(c) 10 percent of polyethyleneimine (molecular weight is 1800) and 5 percent of chitosan and 0.1mL of aqueous solution;

(d) phosphate buffer (1.5mM)2.4mL, pH 4.0.

The preparation method of the hydrogel comprises the following steps: mixing the above (a) and (d) to obtain solution A, mixing the above (B) and (c) to obtain solution B, and mixing the solution A and B to obtain gel preparation.

Comparative example 1

In this comparative example, a mixed solvent of mitomycin C, IFN γ and sodium hyaluronate was prepared by adding mitomycin C, IFN γ and sodium hyaluronate, which were used in the same amounts in example 1, to a PBS solution and stirring for 30min to mix them uniformly.

Comparative example 2

In this comparative example, a mixed solvent of mitomycin C, IFN γ and chitosan was provided, and mitomycin C, IFN γ and chitosan in the same amounts as in example 1 were added to the PBS solution and stirred for 30min to mix well.

Comparative example 3

In this comparative example, a conjugate of mitomycin C, IFN γ and chitosan was provided, and a mitomycin C-IFN γ -chitosan conjugate was prepared by replacing sodium hyaluronate of the same dose as in example 1 with chitosan of the same molecular weight.

Comparative example 4

In this embodiment, a postoperative adhesion prevention material is provided, and the adhesion prevention material is a polyethylene glycol hydrogel preparation, and the hydrogel preparation includes the following components in parts by weight:

(a) 0.4g of four-arm polyethylene glycol succinimide succinate and 0.25g of mitomycin C-IFN gamma-sodium hyaluronate conjugate;

(b) trilysine (10g/L) -borate buffer (65mM)2.5mL, pH 9.8;

(c) 8 percent of polyethyleneimine (molecular weight 2000) to 0.1mL of aqueous solution;

(d) phosphate buffer (1.5mM)2.4mL, pH 4.0.

The preparation method of the hydrogel comprises the following steps: mixing the above (a) and (d) to obtain solution A, mixing the above (B) and (c) to obtain solution B, and mixing the solution A and B to obtain gel preparation.

Comparative example 5

In this embodiment, a postoperative adhesion prevention material is provided, and the adhesion prevention material is a polyethylene glycol hydrogel preparation, and the hydrogel preparation includes the following components in parts by weight:

(a) 0.4g of four-arm polyethylene glycol succinimide succinate and 0.25g of mitomycin C-IFN gamma-sodium hyaluronate conjugate;

(b) trilysine (10g/L) -borate buffer (65mM)2.5mL, pH 9.8;

(c) phosphate buffer (1.5mM)2.4mL, pH 4.0.

The preparation method of the hydrogel comprises the following steps: mixing the above (a) and (c), injecting into (b), mixing, and injecting into wound surface to be sealed.

The invention inspects the scar inhibition effect of the mitomycin C-IFN gamma-sodium hyaluronate conjugate, and the inspecting method comprises the following steps of measuring the content of collagen fibers in rat epidural scar tissues: selecting healthy mature Wistar rats, performing standard operation according to lumbar laminectomy after anesthesia, placing the rats in a prone position after anesthesia, placing an operation incision in the middle of the dorsal side, removing spinous processes and vertebral plates after separating skin, fascia and muscles, and closing a window after the operation. The compositions described in examples 1-3 and comparative examples 1-3 were placed on the wound bed, respectively. Three postoperative days are drenched antibiotic treatment in succession, and the rat is sacrificed 14 days after the operation, takes epidural adhesion scar tissue, thereby detects the content of collagen fiber through the content of hydroxyproline in the ultraviolet absorbance detection scar tissue after the stoving, and the testing result is as shown in following table 1:

TABLE 1

Group of	Hyp content μ g/mg sample	In vitro degradation time (d)
			Example 1	1.19±0.11	3.4
Example 2	1.27±0.24	4.1
			Example 3	1.08±0.41	4.2
Comparative example 1	1.53±0.14	1.2
			Comparative example 2	1.88±0.31	0.8
Comparative example 3	1.69±0.34	1.5

As can be seen from the results in the table above, the conjugate provided by the invention can effectively prolong the retention time of the drug in the wound and effectively prolong the action time of the drug. As can be seen by measuring the content of hydroxyproline, the conjugate form can effectively inhibit the formation of collagen fibers in scar tissues, but the conjugate prepared by using a mixture form or other polysaccharides can not achieve the effect. In the research process of the invention, tests are also carried out on various polysaccharides such as glucan, gelatin and the like, and the effect of hyaluronic acid combination cannot be realized.

The invention also aims at the detection of the hydrogel in vitro degradation time, swelling rate and gelling speed in the following examples of the hydrogel described in examples 4-6, and the specific detection method is as follows:

in vitro degradation time: spraying the hydrogel into a conical flask, weighing, adding physiological saline according to 0.1g/mL, covering a bottle stopper, sealing by a sealing film, putting into a shaking incubator at 37 +/-1 ℃, and observing the disappearance time of the gel.

Detection of swelling ratio: weighing the prepared hydrogel, transferring the hydrogel into a ground triangular flask, adding a phosphate buffer solution (the formula of the phosphate buffer solution is that 1.36g of monopotassium phosphate is weighed, 79mL of 0.1mol/mL sodium hydroxide solution is added, and water is used for diluting the solution to 200mL, wherein the pH value of the phosphate buffer solution is 7.4) which is preheated to 37 +/-1 ℃, putting the ground triangular flask into an incubator at 37 +/-1 ℃, taking out a sample every few hours, absorbing surface moisture by using filter paper, weighing the sample until the weight is not increased any more, and finishing weighing. The gel swelling ratio was calculated as follows.

Swelling ratio (mass of sample after swelling-sample amount) × 100%/sample amount.

And (3) detection of gelling time: connecting the first syringe (a) with the powder bottle (c), injecting liquid into the powder bottle to dissolve the polyethylene glycol derivative, drawing the liquid back to the first syringe after dissolution is finished, injecting 0.5mL of liquid into the test tube, placing a magneton into the test tube, placing the test tube on a magnetic stirrer to rotate the magneton, then quickly injecting 0.5mL of liquid in the second syringe (b) into the test tube, starting timing, stopping timing when the magneton stops rotating or the speed is obviously changed, and the time is the gelling time. The test results are shown in the following table 2:

TABLE 2

Group of	Gelling time/s	Swelling ratio/%	In vitro degradation time/d
				Example 4	0.67	0.85	7.8
Example 5	0.75	0.97	8.1
				Example 6	0.88	0.78	7.9
Comparative example 4	1.23	2.15	6.4
				Comparative example 5	1.58	3.14	5.4

In order to verify the safety performance of the hydrogel product, the hydrogel prepared in the examples 4 to 6 is implanted under the skin of a New Zealand white rabbit, and the degradation time in a biocompatible organism of the hydrogel product is examined. Experimental rabbits were anesthetized by intraperitoneal injection of ketamine 8mg + sodium pentobarbital (0.2mg/kg), and the hydrogel was injected subcutaneously into the backs of the rabbits at 1mL per site. The change of the rabbit is observed every 2 weeks after the operation, and the skin of the injection point is taken to observe the envelope condition after the rabbit is killed. As shown in figure 2, after the skin of the rabbit is cut at 2 weeks, a thin transparent coating can be observed at the injection point, the coating is soft, and a small amount of capillary vessels are proliferated, which indicates that the hydrogel product provided by the invention has good biocompatibility. The hydrogels described in examples 4-6 had in vivo degradation times of 8-12 weeks.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.