CN116271265A

CN116271265A - Temperature-sensitive hydrogel for preventing postoperative tissue adhesion and preparation method thereof

Info

Publication number: CN116271265A
Application number: CN202111569575.1A
Authority: CN
Inventors: 张凌; 朗菩新
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2023-06-23

Abstract

The invention relates to a temperature-sensitive hydrogel for preventing postoperative tissue adhesion and a preparation method thereof. The hydrogel consists of a temperature-sensitive hydrogel material and a medicament for treating tissue adhesion. When in preparation, the two components are mixed according to a certain sequence and proportion to obtain the temperature-sensitive hydrogel for preventing postoperative tissue adhesion. The hydrogel material prepared by the invention can be sprayed for administration, so that the adhesion of abdominal tissues after operation can be prevented and treated. The hydrogel has good cell compatibility and an effect of preventing abdominal tissue adhesion.

Description

Temperature-sensitive hydrogel for preventing postoperative tissue adhesion and preparation method thereof

Technical Field

The invention belongs to the technical field of high molecular medical biological materials, and particularly relates to a temperature-sensitive composite hydrogel capable of preventing postoperative tissue adhesion and a preparation method thereof.

Background

Surgery is one of the more frequently used ways to treat diseases. But surgery can also cause damage to vascular tissue at the surgical site. In the postoperative recovery process, vascular endothelial cells, fibroblasts and myofibroblasts at the operation site grow fibrous tapes while tissue collagen repair is performed under the promotion of cell growth factors in blood. The fibrous tape will bind together the abdominal organs and tissues with which the surgical site is in contact, forming abdominal tissue adhesions. The incidence of postoperative abdominal tissue adhesions was counted to be over 90%. Abdominal tissue adhesions can cause serious postoperative complications such as abdominal pain, pelvic pain, infertility, and ileus.

A variety of medical products have been developed to prevent post-operative tissue adhesions. Mainly comprises drug treatment, physical barrier and a mode of combining the drug and the physical barrier. The common medicines include anti-inflammatory medicines such as ibuprofen and aspirin and anticoagulants such as heparin and dimethyl sulfoxide. Common physical barriers include hydrogels such as chitosan and polylactic acid, and film materials such as polycaprolactone and polyethylene glycol. However, materials have been developed to prevent post-operative tissue adhesions, which have relatively limited therapeutic effects and have some side effects. There is a lack of products with a wide range of acceptance.

From the mechanism of postoperative tissue adhesion, the barrier material for preventing adhesion needs to meet the following requirements: 1) A material for preventing adhesion is stably fixed at a surgical site for a period of time; 2) The adhesion preventing material has good biodegradability. Thus, controlling the degradation behavior and biological properties of the anti-adhesion barrier is an effective strategy for preventing post-operative tissue adhesions.

Disclosure of Invention

Aiming at the problems, the invention aims to provide a nontoxic and degradable temperature-sensitive hydrogel and a preparation method thereof. Meanwhile, the hydrogel has good cell compatibility, simple preparation process and low cost.

In order to solve the technical problems, the invention adopts the following technical scheme.

The hydrogel with the effect of preventing postoperative tissue adhesion is characterized by comprising heparin sodium, poloxamer, carboxymethyl chitosan and calcium chloride. The preparation method comprises the following steps:

1) Dissolving heparin sodium in pure water to prepare heparin sodium aqueous solution;

2) Adding carboxymethyl chitosan into the solution prepared in the step 1) to prepare a solution;

3) Adding poloxamer into the solution prepared in the step 2);

4) Adding calcium chloride into the solution prepared in the step 3) to prepare the hydrogel.

The concentration of the heparin sodium solution in the step 1) is 30000-200000 IU/L;

the concentration of the carboxymethyl chitosan in the step 2) is 2.5% -3.5%;

the dissolution temperature of the carboxymethyl chitosan in the step 2) is 2-8 ℃;

the dissolution time of the carboxymethyl chitosan in the step 2) is 15-48 hours;

the deacetylation degree of the carboxymethyl chitosan in the step 2) is more than or equal to 90%, and the substitution degree is more than or equal to 80%;

the concentration of poloxamer in the step 3) is 22% -25%;

the average molecular weight of poloxamer in the step 3) is 8000-18000;

the dissolution temperature of the poloxamer in the step 3) is 2-8 ℃;

the dissolution time of the poloxamer in the step 3) is 70-90 hours;

the addition amount of the calcium chloride in the step 4) is 0.09-0.12%.

Compared with other methods, the temperature-sensitive hydrogel for preventing postoperative tissue adhesion and the preparation method thereof have the advantages that:

1) Based on the gel forming mechanism of the hydrogel, the thermal-induced curing mechanism is selected, and the ionic crosslinking mechanism is introduced, so that the prepared hydrogel has excellent temperature-sensitive property and good mechanical strength, can meet the requirement of maintaining at a wound for a certain time, and effectively isolates abdominal tissues;

2) In the use process, not only isolating tissues or organs from contacting with the operation part, but also absorbing redundant tissue exudates at the wound part;

3) The preparation method of the hydrogel is simple, the preparation process is stable and controllable, and the mass production is easy.

Drawings

FIG. 1 is a graph showing the sol-gel phase transition of a temperature-sensitive hydrogel for preventing postoperative tissue adhesion under temperature change.

Fig. 2 is an in vitro hydrolysis performance graph of a temperature-sensitive hydrogel material for preventing post-operative tissue adhesion.

Fig. 3 is a graph of the evaluation results of the abdominal adhesion experiment on the temperature-sensitive hydrogel rat for preventing postoperative tissue adhesion.

Fig. 4 is a graph showing the change of the body weight of mice in 14 days after the abdominal adhesion experiment of the temperature-sensitive hydrogel rat for preventing postoperative tissue adhesion.

Fig. 5 is a graph of experimental cytotoxicity of abdominal adhesion of a temperature-sensitive hydrogel rat for preventing postoperative tissue adhesion.

Detailed Description

The preparation method of the hydrogel for preventing postoperative tissue adhesion comprises the following steps:

3) Adding poloxamer into the solution prepared in the step 2);

The present invention will be described in further detail below by reference to the following detailed description. The scope of the invention is not therefore limited to the examples described below. The reagents and instruments used in the examples and the experimental conditions can be further adjusted according to the actual situation, the implementation conditions are not noted as conventional experimental conditions, and the reagents and instruments of manufacturers are not noted as conventional products which can be purchased through the market.

Main reagents, instruments and laboratory animals in examples

Poloxamer (Mn-14600, shanghai Ala Biochemical technologies Co., ltd.); carboxymethyl chitosan (CAS: 83512-85-0, chengdu Melin Biotechnology Co., ltd.); heparin sodium; calcium chloride; SD rats (Chengdu laboratory animal Co., ltd.).

Example 1

1) Dissolving 0.067g of heparin sodium into 79.96g of pure water to obtain heparin sodium solution;

2) 8.219g of carboxymethyl chitosan is added into the solution prepared in the step (1), and the mixture is placed in a refrigerator at the temperature of 4 ℃ to be swelled for 60 hours;

3) 65.76g of poloxamer is added into (2) and placed in a refrigerator at 4 ℃ to be swelled for 96 hours;

4) 0.20g of calcium chloride was added to 9.80g of pure water to prepare a calcium chloride solution having a concentration of 2%;

5) And (3) mixing the solutions in the step (3) and the step (4) to prepare the hydrogel.

Example 2

1) Dissolving 0.067g of heparin sodium into 89.96g of pure water to obtain heparin sodium solution;

3) 65.76g of poloxamer is added into the step (2), and the mixture is placed in a refrigerator at the temperature of 4 ℃ to be swelled for 96 hours, thus obtaining the hydrogel.

Example 3

1) Dissolving 0.016g of heparin sodium into 28g of pure water to prepare heparin sodium solution;

2) Adding 1.217g of carboxymethyl chitosan into the solution prepared in the step (1), placing the solution in a refrigerator at the temperature of 4 ℃ and swelling the solution for 48 hours;

3) Adding 8.825g of poloxamer into the step (2), and placing the mixture into a refrigerator at the temperature of 4 ℃ to swell for 80 hours;

4) 0.2g of calcium chloride was added to 9.8g of pure water to prepare a calcium chloride solution having a concentration of 2%;

5) Mixing 2ml of the solutions of the step (3) and the step (4) to prepare the hydrogel.

Example 4

2) 0.2g of calcium chloride was added to 9.8g of pure water to prepare a calcium chloride solution having a concentration of 2%;

3) Mixing the solution in the step (1) and 2ml of the solution in the step (2);

4) Adding 1.217g of carboxymethyl chitosan into the solution prepared in the step 3), placing the solution in a refrigerator at the temperature of 4 ℃ and swelling the solution for 48 hours;

5) 8.825g of poloxamer was added to (2) and put in a refrigerator at 4℃to swell for 80 hours, to prepare a hydrogel.

Example 5

1) Dissolving 0.019g of heparin sodium into 27.65g of pure water to prepare a heparin sodium solution;

2) Adding 0.825g of carboxymethyl chitosan into the solution prepared in the step 1), placing the solution in a refrigerator with the temperature of 4 ℃ and swelling the solution for 48 hours;

3) Adding 9.626g of poloxamer into the step (2), and placing the mixture into a refrigerator at the temperature of 4 ℃ to swell for 80 hours;

5) Mixing the solution in the step (3) and 2ml of the solution in the step (4) to prepare the hydrogel.

Example 6

1) Dissolving 0.027g of heparin sodium into 29.2g of pure water to obtain heparin sodium solution;

2) Adding 0.844g of carboxymethyl chitosan into the solution prepared in the step (1), placing the solution in a refrigerator at the temperature of 4 ℃ and swelling for 60 hours;

3) 8.345g of poloxamer is added into the step (2), and the mixture is placed in a refrigerator at the temperature of 4 ℃ to be swelled for 96 hours;

mixing the solution in the step (3) and 2ml of the solution in the step (4) to prepare the hydrogel.

Example 7

In vitro degradation experiments

Two 50ml conical flasks were filled with 15g of the hydrogels prepared in example 1 and example 2, respectively, and placed in an environment of 37℃for 20min, the hydrogels were subjected to sol-gel conversion, 20ml of PBS solution (pH 7.4) was added, the temperature (37.+ -. 0.5) DEG C, the shaking speed was 100r/min, and 2,4,7,24,36,48,72,96,120,144,168,192,216,240,264,288,312,36,360h were sequentially poured out of the culture medium, the hydrogel weight was weighed, and the same volume of PBS with equal temperature and equal volume was supplemented. According to degradation formula W _t =1-(M _t /M ₀ ) (%) and in vitro degradation curves were plotted. The results are shown in FIG. 2.

As can be seen from FIG. 2, the in vitro degradation effect of the hydrogel prepared in example 1 is significantly better than that of the hydrogel prepared in example 2. Cacl ₂ The addition of (3) can effectively enhance the strength of the hydrogel. The hydrogel prepared in example 1 can meet the requirement of the product in the in vitro degradation time of the hydrogel.

Example 8

Cytotoxicity test

NIH/3T3 cells were cultured at 3X 10 ⁵ Is inoculated in 96-well plates at 37 ℃ and 5% CO ₂ After 24 hours of environmental culture, the original medium was removed, and the culture medium containing 0mg/ml, 0.1mg/ml, 1mg/ml, 10mg/ml and 100mg/ml of the hydrogel of example 1 was added, respectively, to culture for 24 hours. The cytotoxicity of the hydrogels was characterized using cck-8 reagent. The results are shown in FIG. 5.

1) Grouping of laboratory animals

Rat abdominal tissue adhesion model: the 12 female rats were randomly divided into three groups: control group (n=2), model group (n=5) and experimental group (n=5).

2) Rat abdominal tissue adhesion

SD rats of 6-8 weeks were preoperatively cultured for three days, the control group did not undergo any treatment, the model group and the experimental group, were preoperatively anesthetized with 0.7ml of 10% paraformaldehyde solution, and the anesthetized rats were subcutaneously injected with 0.5ml of 17mg/ml ampicillin for preventing postoperative infection. Fixing the mouse on an operation plate, removing abdominal hair, sterilizing skin surface with iodophor, making 3cm incision along the white line of abdomen, taking out cecum, wiping the surface of cecum with sterile gauze until 3cm appears ² The experimental group coated the appropriate amount of the hydrogel prepared in example 1 on the wound surface (the model group coated with the same amount of physiological saline) and then put the cecum back into the abdominal cavity, and suture the abdominal cavity tissue and the skin tissue with absorbable sutures of 2-0 and 3-0. Mice were weighed at two-day post-operative intervals for evaluation of hydrogel biocompatibility. On the 14 th day after the operation, all mice were euthanized and abdominal tissue adhesion was evaluated by open abdomen.

3) Results of animal experiments

The evaluation criteria for abdominal tissue adhesion were as follows:

1) The tissue was not adhered and was marked as 0;

2) The tissue adhesion area is 0-100mm ² Is marked as 1;

3) The tissue adhesion area is 100-300mm ² Marked as 2;

4) Tissue adhesion at 300-500mm ² And is designated 3.

From fig. 3, it can be seen that the abdominal tissue adhesion degree of mice in the experimental group is significantly better than that of mice in the model group, and 60% of mice in the experimental group are not adhered. 40% of the mice in the model group developed severe adhesions. The temperature-sensitive hydrogel can effectively prevent postoperative tissue adhesion.

As can be seen from fig. 4, the weight of mice after surgery was significantly lower than that of normal mice, and the weight of experimental mice was superior to that of model mice in the middle and late stages. As is clear from FIG. 5, the cell activities were higher than 90% in the concentration range of 0.1-100mg/ml, thus demonstrating that the cytotoxicity of the hydrogels was low. Therefore, the hydrogel has higher biocompatibility.

The above examples are provided for illustrating the technical construction and features of the present invention, and are intended to enable those skilled in the art to understand the contents of the present invention and to implement the same, not to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.

Claims

1. A temperature-sensitive hydrogel for preventing postoperative tissue adhesion, characterized in that: the hydrogel comprises heparin sodium, carboxymethyl chitosan, poloxamer and calcium chloride.

2. The hydrogel for preventing postoperative tissue adhesion according to claim 1, wherein the concentration of heparin sodium is 30000 UI/L to 200000 UI/L.

3. The hydrogel for preventing post-operative tissue adhesions of claim 1, wherein carboxymethyl chitosan is added at a concentration of 2.5% -3.5%.

4. The hydrogel for preventing postoperative tissue adhesion according to claim 1, wherein poloxamer is added at a concentration of 22% -25%.

5. The hydrogel for preventing postoperative tissue adhesion according to claim 1, wherein the calcium chloride is added at a concentration of 0.09% -0.12%.

6. The temperature-sensitive hydrogel for preventing post-operative tissue adhesions of any one of claims 1-5, prepared by the method of: 1) Adding heparin sodium into water, and stirring to fully dissolve the heparin sodium; 2) Adding carboxymethyl chitosan into the solution 1), and fully dissolving; 3) Adding poloxamer into the solution 2), and fully dissolving; 4) Adding the calcium chloride solution into the solution obtained in the step 3), and stirring to fully mix the calcium chloride solution to obtain the hydrogel.

7. A method for preparing a hydrogel for preventing postoperative tissue adhesion, comprising the steps of:

1) Adding heparin sodium into pure water, and stirring to fully dissolve the heparin sodium;

2) Adding carboxymethyl chitosan into the solution 1), and fully dissolving;

3) Adding poloxamer into the solution 2), and fully dissolving;

4) Adding the calcium chloride solution into the solution obtained in the step 3), and stirring to fully mix the calcium chloride solution to obtain the hydrogel.

8. The method for preparing the hydrogel for preventing postoperative tissue adhesion according to claim 7, wherein: the dissolution temperature in the step 2) and the step 3) is 2-8 ℃.

9. The method for preparing the hydrogel for preventing postoperative tissue adhesion according to claim 7, wherein: the dissolution time of the carboxymethyl chitosan added in the step 2) is 15-48h.

10. The method for preparing the hydrogel for preventing postoperative tissue adhesion according to claim 7, wherein: the dissolution time of the poloxamer added in the step 3) is 70-100h.