CN114028604B - A multi-component wound repair hemostatic dressing based on polymeric amino acid and its application - Google Patents

Abstract

The invention discloses a multicomponent wound repair hemostasis dressing based on polymeric amino acid, which is characterized in that the structure of carboxymethyl chitosan is modified by polyglutamic acid to obtain polyglutamic acid crosslinked carboxymethyl chitosan, excessive activating agent can further activate carboxyl in the polyglutamic acid crosslinked carboxymethyl chitosan during reaction to enable the carboxyl to react with octadecylamine, octadecylamine is introduced on a polyglutamic acid crosslinked carboxymethyl chitosan grid structure, hydrophobic alkyl in the octadecylamine can enable a material to be changed into an amphiphilic structure, the affinity with skin is better, and the amphiphilic structure can obviously increase the load rate of subsequently added hydrophobic compounds such as graphene oxide, berberine and tea polyphenol.

Description

A multi-component wound repair hemostatic dressing based on polymeric amino acid and its application

technical field

The invention relates to the technical field, in particular to a multi-component wound repair hemostatic dressing based on a polymeric amino acid and an application thereof.

Background technique

The skin is the body's first line of defense against various diseases and is directly related to the human body's life and health. In daily life, the skin of the human body often encounters various degrees of trauma. In the process of skin wound healing, the human body is extremely vulnerable to various bacteria. For diabetic patients, patients with partial blood supply deficiency, the elderly and infants Etc., due to poor physical fitness and poor ability to resist diseases, it is easy to cause skin wounds that cannot be healed, which further leads to secondary bacterial infection of the patient's skin, excessive loss of water and protein, and severe wounds may appear. Rot symptoms, threatening patient's life and health. In addition, in surgical operations, wounds are often difficult to heal, secondary bleeding, bacterial infection, etc., which affect postoperative recovery.

At present, there are various types of wound repair products on the market, such as medical gauze, Band-Aid, gel products, hemostatic sponges, etc. Medical gauze, Band-Aid and hemostatic sponge products are all covered on the surface of the wound to play a role in The effect of hemostasis and wound protection, but these products need to be replaced regularly. During the replacement process, the wound that has just healed is damaged again, causing secondary bleeding and prolonging the healing time of the wound. On the one hand, the existing products are not compatible with the water absorption effect and the ventilation effect. On the other hand, they usually have no or poor antibacterial performance, and have poor air permeability. If they are not replaced for a long time, the wound is easily infected with various bacteria. Although loading the antibacterial agent in the polymer material can achieve the purpose of water absorption and antibacterial, the loading rate of the conventional polymer material for the antibacterial agent is not high, the polymer material and the antibacterial agent are separated, and the antibacterial agent cannot be loaded in the polymer material. Therefore, it cannot exert its antibacterial properties well when used.

Therefore, it is of great significance to develop new wound repair materials, which can not only have a good hemostasis effect, but also have excellent antibacterial properties and air permeability, shorten the time of wound healing, and effectively promote wound healing.

SUMMARY OF THE INVENTION

In order to solve the above problems, the purpose of the present invention is to provide a multi-component wound repair hemostatic dressing based on polymeric amino acid and its application.

The present invention is achieved by the following technical solutions in order to achieve the above object:

A multi-component wound repair hemostatic dressing based on polymeric amino acids is prepared according to the following steps:

① In parts by weight, add 15 parts of polyglutamic acid to 300 to 400 parts of solvent, maintain the temperature at 20 to 40°C, stir for 10 to 20 minutes, add 5 to 9 parts of condensing agent and 7 to 10 parts of N- Hydroxysuccinimide, stir for 0.5 to 3 hours, add 15 to 20 parts of carboxymethyl chitosan and 20 to 25 parts of octadecylamine, heat up to 45 to 75 ° C, and stir for 6 to 24 hours. The solution was dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 24-72 hours, and freeze-dried to obtain polyglutamic acid cross-linked modified carboxymethyl chitosan;

The solvent is water and/or N,N-dimethylformamide;

The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide ;

② Take 1-3 parts of chitosan and add it to 900-1000 parts of deionized water, heat up to 20-40°C, ultrasonicate for 10-30 minutes, stir and disperse for 10-30 minutes, add 0.5-2 parts of graphene oxide, stir for 5 For ~15 minutes, add the polyglutamic acid cross-linked modified carboxymethyl chitosan obtained in step (1), heat up to 45 ~ 55 ° C, add 1 ~ 2 parts of acetic acid, stir for 3 ~ 6 hours, use dialysis with a molecular weight cut-off of 3500 The bag is dialyzed for 24-72 hours, and freeze-dried to obtain gel freeze-dried powder;

3. Dissolving the gel freeze-dried powder obtained in step 2. in 900-1000 parts of deionized water, warming up to 20-40° C., adding 0.5-2 parts of berberine hydrochloride and 0.2-1 part of tea polyphenols, ultrasonicating for 30-60 minutes , stirring for 12-48 hours, adding 4-6 parts of collagen, stirring for 4-10 hours, and freeze-drying to obtain a multi-component wound repair hemostatic dressing based on polymeric amino acids.

Preferably, the polyglutamic acid is γ-polyglutamic acid with a molecular weight of 50,000 to 100,000.

Preferably, the solvent is obtained by mixing water and N,N-dimethylformamide in a mass ratio of 1-3:2-3.

Preferably, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

Preferably, the average molecular weight of the chitosan is 150,000 to 250,000, and the degree of deacetylation is greater than 90%.

Preferably, the freeze-drying temperature in step ③ is not lower than -50°C.

The present invention also includes the application of the above-mentioned multi-component wound repair hemostatic dressing based on polymeric amino acid in the preparation of wound dressing or wound repair material.

Compared with the prior art, the present invention has the following advantages:

In the multi-component wound repair hemostatic dressing based on polymeric amino acids of the present invention, the structure of carboxymethyl chitosan is modified with polyglutamic acid to obtain polyglutamic acid cross-linked carboxymethyl chitosan. Excessive activator can further activate the carboxyl groups in polyglutamic acid cross-linked carboxymethyl chitosan, making it react with octadecylamine, on the grid structure of polyglutamic acid cross-linked carboxymethyl chitosan The introduction of octadecylamine, the hydrophobic alkyl group in octadecylamine can make the material into an amphiphilic structure, which has better affinity with the skin, and this amphiphilic structure can significantly increase the subsequent addition of graphene oxide and berberine. and the loading rate of hydrophobic compounds such as tea polyphenols.

The multi-component wound repair hemostatic dressing based on the polymeric amino acid of the present invention can quickly absorb the moisture in the blood at the wound, rapidly increase the concentration of coagulation factors, thrombin and the like in the blood at the wound, and adhere to the wound through gelation On the surface of the wound, it can achieve rapid hemostasis. The structure contains a large number of carboxyl groups, amino groups, amide bonds, etc., which can combine with berberine hydrochloride and tea polyphenols through hydrogen bonds and van der Waals force, and improve the berberine hydrochloride and tea polyphenols. Adsorption efficiency; the cross-linked network of the dressing has good permeability, which can ensure air exchange with the outside world while adhering to the wound surface, preventing the wound from closing; the material has a good moisturizing effect and can provide moisture to the wound. environment to promote wound healing.

When the wound repair dressing of the present invention is used, it only needs to cover the surface of the wound, the dressing can quickly absorb the water in the blood, quickly stop bleeding, and gel at the same time, forming a protective film on the wound, effectively preventing the invasion of bacteria and proliferation. The wound repair dressing of the invention has a super three-dimensional mesh structure, has good air permeability, does not affect the respiration of the wound, and has a certain water retention effect, provides a suitable moist environment for wound healing, and is beneficial to wound healing.

Description of drawings

Fig. 1 is the cytotoxicity test result diagram of the multi-component wound repair hemostatic dressing based on polymeric amino acid obtained in Example 4;

2 is a graph showing the effect of the wound repair dressing and Celox material prepared in Example 4 on hemostasis of rat femoral artery.

Detailed ways

The object of the present invention is to provide a kind of multi-component wound repair hemostatic dressing based on polymeric amino acid and application thereof, which is realized by the following technical solutions:

A multi-component wound repair hemostatic dressing based on polymeric amino acids is prepared according to the following steps:

① In parts by weight, add 15 parts of polyglutamic acid to 300 to 400 parts of solvent, maintain the temperature at 20 to 40°C, stir for 10 to 20 minutes, add 5 to 9 parts of condensing agent and 7 to 10 parts of N- Hydroxysuccinimide, stir for 0.5 to 3 hours, add 15 to 20 parts of carboxymethyl chitosan and 20 to 25 parts of octadecylamine, heat up to 45 to 75 ° C, and stir for 6 to 24 hours. The solution was dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 24-72 hours, and freeze-dried to obtain polyglutamic acid cross-linked modified carboxymethyl chitosan;

The solvent is water and/or N,N-dimethylformamide;

The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide ;

② Take 1-3 parts of chitosan and add it to 900-1000 parts of deionized water, heat up to 20-40°C, ultrasonicate for 10-30 minutes, stir and disperse for 10-30 minutes, add 0.5-2 parts of graphene oxide, stir for 5 For ~15 minutes, add the polyglutamic acid cross-linked modified carboxymethyl chitosan obtained in step (1), heat up to 45 ~ 55 ° C, add 1 ~ 2 parts of acetic acid, stir for 3 ~ 6 hours, use dialysis with a molecular weight cut-off of 3500 The bag is dialyzed for 24-72 hours, and freeze-dried to obtain gel freeze-dried powder;

3. Dissolving the gel freeze-dried powder obtained in step 2. in 900-1000 parts of deionized water, warming up to 20-40° C., adding 0.5-2 parts of berberine hydrochloride and 0.2-1 part of tea polyphenols, ultrasonicating for 30-60 minutes , stirring for 12-48 hours, adding 4-6 parts of collagen, stirring for 4-10 hours, and freeze-drying to obtain a multi-component wound repair hemostatic dressing based on polymeric amino acids.

The present invention uses γ-polyglutamic acid to modify the structure of carboxymethyl chitosan to prepare polyglutamic acid cross-linked carboxymethyl chitosan. Membrane, moisturizing and cohesive, suitable for the preparation of degradable hemostatic materials for surgery. Polyglutamic acid cross-linked carboxymethyl chitosan is a complex cross-linked network structure in structure, which has excellent adsorption and moisturizing effects. The concentration of factors, thrombin, etc. rises rapidly, and adheres to the surface of the wound through gelation to quickly stop bleeding; the structure contains a large number of carboxyl groups, amino groups, amide bonds, etc., which can interact with berberine hydrochloride and tea polyphenols through hydrogen. bond and van der Waals force to improve the adsorption efficiency of berberine hydrochloride and tea polyphenols; the cross-linked network of the dressing has good permeability, which can ensure air exchange with the outside world while adhering to the wound surface. Prevent wound closure; the material has a good moisturizing effect, which can provide a moist environment for the wound and promote wound healing.

The present invention also adds graphene oxide to the material, as a derivative product of graphene, graphene oxide exhibits a certain antibacterial effect, has a good inhibitory effect on bacteria, fungi and plant pathogens, and does not have drug resistance; In addition, graphene oxide has a large conjugated planar structure, and berberine hydrochloride and tea polyphenols can be efficiently stacked on the surface of graphene oxide by π-π stacking, improving the loading of berberine hydrochloride and tea polyphenols At the same time, the combination of this non-covalent bond can ensure the slow release of the two drugs, and prevent the antibacterial performance from being reduced due to the rapid release of the drugs.

The present invention creatively uses chitosan, graphene oxide and polyglutamic acid cross-linked carboxymethyl chitosan to prepare gel freeze-dried powder. Bond, van der Waals force, etc., to form a more complex network space structure; on the other hand, the freeze-dried gel powder can absorb a large amount of water in the blood, and play a role in the rapid hemostasis of the wound surface.

The invention adds berberine hydrochloride and tea polyphenols to the wound repair dressing at the same time. Berberine hydrochloride has excellent antibacterial effect, can effectively inhibit the increase of bacteria in the wound, and at the same time has good antioxidative performance, which can effectively reduce the incidence of patients. , especially the oxidative stress effect in diabetic wounds; tea polyphenols also have excellent antibacterial effects, and synergistically with berberine can significantly inhibit bacterial proliferation and inflammatory response in diabetic wounds, and promote the healing of diabetic wounds. The combined effect of the two drugs can play a synergistic effect and effectively prevent the secondary infection of the wound.

Preferably, the polyglutamic acid is γ-polyglutamic acid with a molecular weight of 50,000 to 100,000.

Preferably, the solvent is obtained by mixing water and N,N-dimethylformamide in a mass ratio of 1-3:2-3.

Preferably, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

Preferably, the average molecular weight of the chitosan is 150,000 to 250,000, and the degree of deacetylation is greater than 90%. Although the polyglutamic acid cross-linked carboxymethyl chitosan prepared by the invention has a high drug loading rate, its gelation effect is general, which is not conducive to the rapid water absorption of the material. Therefore, in a preferred scheme, chitosan with specific molecular weight and degree of deacetylation, graphene oxide and polyglutamic acid cross-linked carboxymethyl chitosan are mixed to prepare gel freeze-dried powder, and chitosan is combined with acetic acid Easy to gel, giving the material excellent water-swelling properties. Then, the three-dimensional network structure of the gel freeze-dried powder and the π-π stacking effect of graphene oxide are used to efficiently load berberine hydrochloride and tea polyphenols. Finally, collagen is added to utilize a large number of carboxyl and amino groups contained in collagen. , covered on the surface of the material by hydrogen bonds and van der Waals forces. In addition, collagen has excellent compatibility with cells, plays a very important role in the growth and adhesion of seed cells, can effectively promote the proliferation of cells in the wound, and has the advantages of non-toxicity and degradability.

Preferably, the freeze-drying temperature in step ③ is not lower than -50°C.

The present invention also includes the application of the above-mentioned multi-component wound repair hemostatic dressing based on polymeric amino acid in the preparation of wound dressing or wound repair material.

The present invention will be further described below with reference to specific examples. The molecular weight range of polyglutamic acid selected in the following examples is 50,000 to 100,000.

Example 1

A multi-component wound repair hemostatic dressing based on polymeric amino acids is prepared according to the following steps:

①Add 1.5kg of polyglutamic acid to 30kg of solvent, maintain the temperature at 20°C, stir for 10 minutes, add 0.5kg of condensing agent and 0.7kg of N-hydroxysuccinimide, stir for 0.5 hours, add 1.5kg of carboxymethyl shell Polysaccharide and 2kg of octadecylamine were heated to 45°C, stirred and reacted for 24 hours, the obtained mixed solution was dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 24 hours, and freeze-dried to obtain a polyglutamic acid cross-linked modified carboxymethyl shell. Glycans;

The solvent is water;

The condensing agent is N,N'-diisopropylcarbodiimide;

② Take 0.1kg of chitosan and add it to 90kg of deionized water, heat up to 20°C, sonicate for 10 minutes, stir and disperse for 10 minutes, add 0.05kg of graphene oxide, stir for 5 minutes, add the polyglutamic acid cross-linking obtained in step ① The modified carboxymethyl chitosan was heated to 45°C, 0.1 kg of acetic acid was added, stirred for 3 hours, dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 24 hours, and freeze-dried to obtain gel freeze-dried powder;

3. dissolving step 2. the obtained gel freeze-dried powder in 90kg deionized water, be warming up to 20 ℃, add 0.05kg berberine hydrochloride and 0.02kg tea polyphenol, ultrasonically 30 minutes, stir 12 hours, add 0.4kg collagen, Stir for 4 hours and freeze-dry to obtain a multi-component wound repair hemostatic dressing based on polymerized amino acids.

Example 2

A multi-component wound repair hemostatic dressing based on polymeric amino acids is prepared according to the following steps:

①Add 1.5kg of polyglutamic acid to 40kg of solvent, maintain the temperature at 40°C, stir for 20 minutes, add 0.9kg of condensing agent and 1.0kg of N-hydroxysuccinimide, stir for 3 hours, add 2kg of carboxymethyl chitosan Sugar and 2.5kg of octadecylamine were heated to 75°C, stirred and reacted for 24 hours, the obtained mixed solution was dialyzed with a dialysis bag with a molecular weight cutoff of 3500 for 72 hours, and freeze-dried to obtain a polyglutamic acid cross-linked modified carboxymethyl shell Glycans;

The solvent is N,N-dimethylformamide;

The condensing agent is dicyclohexylcarbodiimide;

② Take 0.3kg of chitosan with an average molecular weight of 250,000 and a degree of deacetylation greater than 90%, add it to 100kg of deionized water, heat up to 40°C, sonicate for 30 minutes, stir and disperse for 30 minutes, add 0.2kg of graphene oxide, stir for 15 minutes minutes, add the polyglutamic acid cross-linked modified carboxymethyl chitosan obtained in step 1, heat up to 55 ° C, add 0.2 kg of acetic acid, stir for 6 hours, dialyze with a dialysis bag with a molecular weight cut-off of 3500 for 72 hours, freeze-dry, obtain gel freeze-dried powder;

3. dissolving step 2. gained gel freeze-dried powder in 100kg deionized water, be warming up to 40 ℃, add 0.2kg berberine hydrochloride and 0.1kg tea polyphenol, ultrasonically 60 minutes, stir 48 hours, add 0.6kg collagen, Stir for 10 hours and freeze-dry to obtain a multi-component wound repair hemostatic dressing based on polymeric amino acids.

Example 3

A multi-component wound repair hemostatic dressing based on polymeric amino acids is prepared according to the following steps:

①Add 1.5kg of γ-polyglutamic acid to 32kg of solvent, maintain the temperature at 25°C, stir for 12 minutes, add 0.78kg of condensing agent and 0.85kg of N-hydroxysuccinimide, stir for 1 hour, add 1.6kg of carboxymethyl Chitosan and 2.2kg of octadecylamine were heated to 50°C, stirred and reacted for 10 hours, the obtained mixed solution was dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 40 hours, and freeze-dried to obtain polyglutamic acid cross-linked modified carboxymethyl base chitosan; the molecular weight of γ-polyglutamic acid is 50,000 to 100,000;

Described solvent is that water and N,N-dimethylformamide are mixed and obtained according to mass ratio 1:3;

The condensing agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride;

② Take 0.15kg of chitosan with an average molecular weight of 150,000 and a degree of deacetylation greater than 90%, add it to 98kg of deionized water, heat up to 25°C, sonicate for 15 minutes, stir and disperse for 15 minutes, add 0.15kg of graphene oxide, stir for 8 minutes, add the polyglutamic acid cross-linked modified carboxymethyl chitosan obtained in step 1, heat up to 48 ° C, add 0.12 kg of acetic acid, stir for 4 hours, dialyze with a dialysis bag with a molecular weight cut-off of 3500 for 30 hours, freeze-dry, obtain gel freeze-dried powder;

3. dissolving step 2. gained gel freeze-dried powder in 94kg deionized water, be warming up to 25 DEG C, add 0.08kg berberine hydrochloride and 0.08kg tea polyphenol, ultrasonically 40 minutes, stir 15 hours, add 0.45kg collagen, Stir for 6 hours, freeze-dry at -50--55° C. to obtain a multi-component wound repair hemostatic dressing based on polymerized amino acids.

Example 4

A multi-component wound repair hemostatic dressing based on polymeric amino acids is prepared according to the following steps:

①Add 1.5kg of γ-polyglutamic acid to 35kg of solvent, maintain the temperature at 30°C, stir for 15 minutes, add 0.75kg of condensing agent and 0.8kg of N-hydroxysuccinimide, stir for 2 hours, add 1.8kg of carboxymethyl Chitosan and 2.4kg of octadecylamine were heated to 60°C, stirred and reacted for 20 hours, the obtained mixed solution was dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 60 hours, and freeze-dried to obtain polyglutamic acid cross-linked modified carboxymethyl base chitosan; the molecular weight of γ-polyglutamic acid is 50,000 to 100,000;

Described solvent is that water and N,N-dimethylformamide are mixed and obtained according to mass ratio 3:2;

The condensing agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride;

② Take 0.2kg of chitosan with an average molecular weight of 200,000 and a degree of deacetylation greater than 90%, add it to 95kg of deionized water, heat up to 30°C, ultrasonicate for 20 minutes, stir and disperse for 20 minutes, add 0.1kg of graphene oxide, stir for 12 minutes minutes, add the polyglutamic acid cross-linked modified carboxymethyl chitosan obtained in step 1, heat up to 50 ° C, add 0.15 kg of acetic acid, stir for 5 hours, dialyze with a dialysis bag with a molecular weight cut-off of 3500 for 50 hours, freeze-dry, obtain gel freeze-dried powder;

3. dissolving step 2. gained gel freeze-dried powder in 95kg deionized water, be warming up to 30 ℃, add 0.1kg berberine hydrochloride and 0.05kg tea polyphenol, ultrasonically 50 minutes, stir 30 hours, add 0.5kg collagen, Stir for 5 hours, freeze-dry at -50--60° C. to obtain a multi-component wound repair hemostatic dressing based on polymerized amino acids.

Water absorption test

The water absorption rate of the wound repair dressing can directly reflect the ability of the material to absorb water in the blood at the wound, and is closely related to the hemostatic performance of the material. Existing hemostatic material Celox hemostatic powder, accurately weigh 1g with an electronic balance, add it to a beaker, add 50g of water, make the sample fully absorb water, take the sample after 10 minutes, filter the water, and weigh it on the electronic balance again. The weight of the material is calculated by dividing the increased weight of the material by the weight of the material without water absorption × 100%, which is the absorption rate of the material. Each sample experiment is repeated 3 times, and the average value is calculated. The results are shown in Table 1.

Table 1 Water absorption test results

sample name Average water absorption (%) Example 1 924.2 Example 2 931.5 Example 3 945.2 Example 4 956.2 Control substance Celox hemostatic powder 854.6

It can be seen from the results in Table 1 that the multi-component wound repair hemostatic dressings based on polymeric amino acids obtained in Examples 1 to 4 of the present invention have higher water absorption efficiency than the existing hemostatic material Celox hemostatic powder, and the effect is better than that of the existing hemostatic material Celox hemostatic powder. The existing hemostatic material Celox hemostatic powder.

Coagulation Index (BCI) Determination

The methods for testing the coagulation index of the multi-component wound repair hemostatic dressings and Celox hemostatic powder based on polymeric amino acids obtained in Examples 1 to 4 are as follows:

Weigh 300 mg of the sample (multi-component wound repair hemostatic dressing or Celox hemostatic powder based on polymeric amino acids obtained in Examples 1 to 4), put it into a petri dish, incubate at 37°C for 5 minutes, and draw 100 μL of sodium citrate Coagulated rabbit blood was added dropwise to the sample, and 20 μL of 0.2M calcium chloride aqueous solution was quickly added dropwise to the sample. After 5 minutes, 25 mL of deionized water was added to dilute, incubated at 37°C for 5 minutes, centrifuged, and the supernatant was collected. The OD value of the sample solution was measured on a UV spectrophotometer, and the test wavelength was 545 nm. No test sample material was added to the blank group, and other operations were the same as those of the experimental group. The formula for calculating the coagulation index is as follows:

BCI=OD value of experimental group/OD value of blank group×100%

Each sample experiment was repeated three times, and the average value was calculated. The results are shown in Table 2.

Table 2 BCI test result table

As can be seen from the results in Table 2, the polyamino acid-based multi-component wound repair hemostatic dressing obtained in the present invention has a lower coagulation index than Celox hemostatic powder, which proves that the material is easily combined with hemoglobin in blood, so that blood coagulates, and The coagulation index of Examples 3 and 4 is better than that of Examples 1 and 2.

Since the effects of water absorption and BCI coagulation index in Example 4 are the best examples, a comparative test is designed based on Example 4, and other performance tests are carried out.

Comparative Example 1

For the multi-component wound repair hemostatic dressing, the test procedure adopted is the same as that of Example 4, except that no octadecylamine is added.

Comparative Example 2

For the multi-component wound repair hemostatic dressing, the test procedure adopted is the same as that of Example 4, except that graphene oxide is not added.

Comparative Example 3

The multi-component wound repair hemostatic dressing adopts the same test procedure as Example 4, except that berberine hydrochloride is not added.

Comparative Example 4

The multi-component wound repair hemostatic dressing adopts the same test procedure as in Example 4, except that no tea polyphenols are added.

The water absorption rate of the multi-component wound repair hemostatic dressings obtained in Comparative Examples 1 to 4 was tested. The test process was to accurately weigh 1 g of the multi-component wound repair hemostatic dressings obtained in Comparative Examples 1 to 4 with an electronic balance and add it to a beaker. , add 50g of water to make the sample fully absorb water, take the sample after 10 minutes, filter the water, weigh its mass on the electronic balance again, and calculate the water absorption rate of the material. The experiment is repeated 3 times for each sample, and the average value is calculated. As shown in Table 3.

Table 3 The water absorption test results of the multi-component wound repair hemostatic dressings obtained in the comparative example

The coagulation index was tested for the multi-component wound repair hemostatic dressings obtained in Comparative Examples 1 to 4. The test process was the same as the coagulation index test method for the multi-component wound repair hemostatic dressings based on polymeric amino acids obtained in Examples 1 to 4, except that The samples were replaced with the multi-component wound repair hemostatic dressings obtained in Comparative Examples 1 to 4, and the results are shown in Table 4.

Table 4 BCI test result table

sample name BCI(%) Comparative Example 1 30.32 Comparative Example 2 18.65 Comparative Example 3 18.58 Comparative Example 4 18.64 Example 4 18.62

It can be seen from the results in Table 4 that in Comparative Example 1, since no octadecylamine was added, the coagulation index of the material increased a lot compared to Example 4, and the index was on the high side. It can also be seen from the results of Tables 3 and 4. , octadecylamine has a great influence on the water absorption rate and coagulation index of coagulation materials, while other raw materials such as graphene oxide, berberine hydrochloride and tea polyphenols have little effect on the water absorption rate and coagulation index of coagulation materials. This is because if the coagulation material lacks octadecylamine, the molecule does not have an amphiphilic structure, and the network structure formed by the condensation of polyglutamic acid and carboxymethyl chitosan is too dense, which is not conducive to adsorption and coagulation.

Loading efficiency test of materials on berberine hydrochloride

Test process: Weigh 2 mg of the polyamino acid-based multi-component wound repair hemostatic dressing obtained in Example 4 or the multi-component wound repair hemostatic dressing obtained in Comparative Examples 1 to 2 and Comparative Example 4, and add them to 2 ml of anhydrous methanol. , ultrasonically dissolved, filtered with a 0.2-micron microporous membrane, 20 microliters of the filtrate was taken, HPLC was injected, the peak area of berberine hydrochloride was measured, the test wavelength was 349 nm, and the content of berberine in the material was calculated. The mass of berberine is calculated by the loading efficiency (the content of berberine hydrochloride in the material ÷ the mass of berberine hydrochloride added × 100%), the calculation method and the drawing of the standard curve refer to the method reported in the literature (HPLC method for the determination of Shenshuining) The content of berberine hydrochloride in pills, China Modern Distance Education of Traditional Chinese Medicine, 2013, 11(019): 148), the results are shown in Table 5.

Table 5 Loading efficiency results of berberine hydrochloride

sample name Loading efficiency of berberine hydrochloride (%) Example 4 75.56 Comparative Example 1 43.94 Comparative Example 2 59.23 Comparative Example 4 89.40

It can be seen from the results in Table 5 that in Example 4, since the material contains octadecylamine and graphene oxide, the loading efficiency of berberine hydrochloride is as high as 75.56%. The material prepared in Comparative Example 1 does not contain octadecylamine, so the loading efficiency of berberine hydrochloride is reduced to 43.94%. The material prepared in Comparative Example 2 does not contain graphene oxide, so the loading efficiency of berberine hydrochloride is reduced to 43.94%. The efficiency decreased to 59.23%, which indicated that the presence of octadecylamine and graphene oxide could significantly improve the loading efficiency of berberine hydrochloride. Since tea polyphenols are not added to the material prepared in Comparative Example 4, berberine hydrochloride has no competitors and can fully contact the material, so its loading efficiency is increased to 89.40%. Since tea polyphenols are mixtures, the HPLC method is not suitable for testing the loading efficiency, so the loading efficiency of tea polyphenols is no longer measured in the present invention.

Cytotoxicity test

The polyamino acid-based multi-component wound repair hemostatic dressing obtained in Example 4 with the lowest coagulation index was selected for further in vitro cytotoxicity test. The experiment was divided into sample group, Celox control group, positive control group, negative control group and blank group.

Preparation of the extract solution of the sample group: Weigh 500 mg of the polyamino acid-based multi-component wound repair hemostatic dressing obtained in Example 4, add it to a centrifuge tube, add 15 mL of complete DMEM medium, incubate at 37°C for 24 hours, centrifuge, and take the sample. The supernatant was filtered with a microporous membrane, and the filtrate was used as a sample extract with a concentration of 100%, and diluted to obtain a sample extract with a concentration of 75%, 50% and 25%, which was used for cytotoxicity experiments.

Preparation of Celox control group extract: 500mg Celox sample was added to 15mL complete DMEM medium, incubated at 37°C for 24 hours, centrifuged, and the supernatant was collected and filtered with a microporous membrane to obtain the Celox control group extract.

Preparation of extract solution of positive control group: add 500 mg of zinc diethyldithiocarbamate to 15 mL of complete DMEM medium, incubate at 37°C for 24 hours, centrifuge, take the supernatant, filter it with a microporous membrane, A positive control group extract was obtained.

Preparation of extract solution of negative control group: Add 500 mg of high-density polyethylene to 15 mL of complete DMEM medium, incubate at 37°C for 24 hours, centrifuge, take the supernatant, filter it with a microporous membrane, and obtain the extract of negative control group. Extraction.

The L-929 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin dual antibody at 37°C and 5% CO ₂ . When the cells adhered to 80%, the medium was aspirated, washed three times with PBS, digested with 1 mL of trypsin, passaged, adjusted to a cell density of 10 ⁵ cells per mL, and inoculated into a 96-well plate. Continue to cultivate for 24 hours, suck out the original medium, and add 100 μL of the extract of the sample group (including four concentrations of 100%, 75%, 50% and 25%), the extract of the positive control group, and the extract of the negative control group. Then, 100 μL of DMEM medium was added to each well and cultured for 24 hours. Six replicate wells were set for each sample. The cells in the blank group were added with 200 μL of DMEM medium and cultured in an incubator for 24 hours. After culturing for 24 hours, add 20 μL MTT to each well, continue to culture for 4 hours, aspirate the original medium, add 150 μL DMSO to each well, shake for 10 minutes, measure the OD value of each well on a full-wavelength microplate reader, and calculate the test wavelength at 490 nm. The survival rate of L-929 cells is calculated as follows:

Cell viability = OD value of experimental group / OD value of blank group × 100%;

The cytotoxicity test results of the samples are shown in Figure 1.

It can be seen from the results in Fig. 1 that the wound repair dressing prepared in Example 4 of the present invention has low cytotoxicity. When the concentration of the extract is 100%, the cell survival rate is all above 80%. When the concentration of the extract decreases, the cell viability gradually increases, while the commercial product Celox, when the concentration of the extract is 100%, has a cell viability of less than 80%, which is slightly more toxic than this product.

Antibacterial test

The antibacterial activity of the samples was determined by the bacteriostatic zone method. Examples 1 to 4 groups, comparative examples 1 to 4 sample groups, Celox and blank control groups were set. Take the Escherichia coli strain, inoculate it into a solid LB medium with a temperature of 50-60°C, shake it evenly, and cool down to solidify. A circular hole with a diameter of 0.5 cm was dug in the middle of the culture medium, and the sample solutions of Examples 1 to 4 (40 mg/mL), the samples of Comparative Examples 1 to 4 (40 mg/mL), the Celox sample solution (40 mg/mL), and the samples without Bacteria water (blank control group) 200 μL. After culturing for 12 hours, the size of the inhibition zone was observed to judge the antibacterial effect. Diameter of inhibition zone=diameter of total inhibition zone-diameter of sample hole, measured three times and averaged, and the results are shown in Table 6.

Table 6 Antibacterial test results

sample name Inhibition zone diameter (mm) Example 1 13 Example 2 13 Example 3 14 Example 4 15 Comparative Example 1 11 Comparative Example 2 12 Comparative Example 3 11 Comparative Example 4 11 Celox 11 Blank control group 0

From the experimental results in Table 6, it can be seen that the diameter of the inhibition zone of the multi-component wound repair hemostatic dressings based on polymeric amino acids obtained in Examples 1 to 4 is 13 to 15 mm, which is much higher than that of the reference substance Celox. The loading rate of berberine hydrochloride is low, so its antibacterial performance is far less than that of Examples 1 to 2. Comparative Example 3 and Comparative Example 4 do not add berberine hydrochloride and tea polyphenols respectively, although the loading rate of a single substance is It may increase, but berberine hydrochloride and tea polyphenols cannot play a synergistic antibacterial effect, so the antibacterial performance is not high.

Hemostatic effect of femoral artery in rats

The male SD rats that had been fasted for 12 hours were anesthetized, fixed on the operating table, the villi in the groin of the thigh were cut off, the femoral artery was found, and the femoral artery was cut off with a scalpel, and the thigh bleeds rapidly. Then, the multi-component wound repair hemostatic dressing based on polymeric amino acids prepared in Example 4 of the present invention was taken, and pressed to the bleeding place of the femoral artery of the rat for 30s to observe the hemostatic effect. The results are shown in Figure 2.

As can be seen from Figure 2, after the rat femoral artery was cut off, the multi-component wound repair hemostatic dressing based on polymeric amino acids prepared in Example 4 was pressed for 30 seconds, and the wound at the rat femoral artery no longer bleeds. Seconds later, there is still a small amount of blood exuding, which proves that the hemostatic performance of the wound repair dressing prepared in Example 4 is better than that of Celox.

Experiment of wound healing in diabetic rats

Select male SD rats with a body weight of 180-200 g, and after a week of normal feeding, inject 2% streptozotocin (STZ) solution (dissolve STZ in 0.1 mol/L citric acid-sodium citrate buffer solution, pH =4.5), the dose was 30 mg/kg, the fasting blood glucose value was measured 7 days later, and the rats with blood glucose >11.1 mmol/L were selected for wound healing experiments.

Anesthetize the above-mentioned rat fasted for 12 hours, fix it on the operating table, cut off the villi on the back, cut a circular wound with a diameter of 1 cm on the skin, cut it to the skin fascia layer, and apply the obtained Example 4 of the present invention respectively. The multi-component wound repair hemostatic dressing based on polymerized amino acid, the multi-component wound repair hemostatic dressing and Celox material obtained in Comparative Example 3 and Comparative Example 4, three parallel experiments were performed for each group, and a blank control experiment was performed at the same time. , 7 and 14 days to take pictures to observe the wound healing. The results are shown in Table 7.

Table 7 Healing of skin wounds in rats

According to the results in Table 7, after using the multi-component wound repair hemostatic dressing based on polymeric amino acid in Example 4, the healing speed of the diabetic rat skin on the 7th and 14th days was significantly accelerated, and the wound was free of infection, while the blank The group (diabetic rats) healed slowly, and the wounds appeared infection and suppuration. In Comparative Examples 3 and 4, without adding berberine hydrochloride and tea polyphenols, the wound healing speed is obviously slowed down, but it is also faster than the healing speed of Celox wound repair material. This shows that adding berberine hydrochloride and tea polyphenols to the material of the present invention can significantly improve the wound healing effect of diabetic rats.

Claims (6)

1. a multi-component wound repair hemostatic dressing based on polymeric amino acids, is characterized in that: prepare according to the following steps: ① In parts by weight, add 15 parts of polyglutamic acid to 300 to 400 parts of solvent, maintain the temperature at 20 to 40°C, stir for 10 to 20 minutes, add 5 to 9 parts of condensing agent and 7 to 10 parts of N- Hydroxysuccinimide, stir for 0.5 to 3 hours, add 15 to 20 parts of carboxymethyl chitosan and 20 to 25 parts of octadecylamine, heat up to 45 to 75 ° C, and stir for 6 to 24 hours. The solution was dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 24-72 hours, and freeze-dried to obtain polyglutamic acid cross-linked modified carboxymethyl chitosan; The solvent is a mixed solvent of water and N,N-dimethylformamide; The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide ; ② Take 1-3 parts of chitosan and add it to 900-1000 parts of deionized water, heat up to 20-40°C, ultrasonicate for 10-30 minutes, stir and disperse for 10-30 minutes, add 0.5-2 parts of graphene oxide, stir for 5 For ~15 minutes, add the polyglutamic acid cross-linked modified carboxymethyl chitosan obtained in step (1), heat up to 45 ~ 55 ° C, add 1 ~ 2 parts of acetic acid, stir for 3 ~ 6 hours, use dialysis with a molecular weight cut-off of 3500 The bag is dialyzed for 24-72 hours, and freeze-dried to obtain gel freeze-dried powder; 3. Dissolving the gel freeze-dried powder obtained in step 2. in 900-1000 parts of deionized water, warming up to 20-40° C., adding 0.5-2 parts of berberine hydrochloride and 0.2-1 part of tea polyphenols, ultrasonicating for 30-60 minutes , stirring for 12-48 hours, adding 4-6 parts of collagen, stirring for 4-10 hours, and freeze-drying to obtain a multi-component wound repair hemostatic dressing based on polymeric amino acids. 2 . The multi-component wound repair hemostatic dressing based on polymeric amino acids according to claim 1 , wherein the polyglutamic acid is γ-polyglutamic acid, and the molecular weight is 50,000 to 100,000. 3 . 3. a kind of multi-component wound repair hemostatic dressing based on polymeric amino acid according to claim 1, is characterized in that: described solvent is composed of water and N,N-dimethylformamide according to mass ratio of 1～3:2 ~3 is obtained by mixing. 4. a kind of multi-component wound repair hemostatic dressing based on polymeric amino acid according to claim 1, is characterized in that: described condensing agent is 1-ethyl-(3-dimethylaminopropyl) carbonyl two imine hydrochloride. 5 . The multi-component wound repair hemostatic dressing based on polymeric amino acids according to claim 1 , wherein the average molecular weight of the chitosan is 150,000 to 250,000, and the degree of deacetylation is greater than 90%. 6 . 6 . The application of the polyamino acid-based multi-component wound repair hemostatic dressing according to claim 1 , wherein the application is in the preparation of wound dressings or wound repair materials. 7 .

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