CN115737897B - Preparation method of injectable hemostatic crystal gel for clotting disorder wound - Google Patents

Abstract

The invention discloses a method for preparing injectable hemostatic gelatin for wounds with coagulation disorders, which specifically includes: mixing an acetic acid solution containing drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan with oxidized dextran. After the solution is mixed and frozen, an injectable hemostatic gel for wounds with coagulopathy is obtained. The present invention cross-links alkylated chitosan and oxidized dextran, and simultaneously cooperates with silver-doped bioactive glass and deferoxamine to specifically solve the problem of healing deep and narrow non-compressible wound exits. It has good biological activity and can inhibit Bacterial growth and rapid wound healing.

Description

Preparation method of injectable hemostatic gel for wounds with coagulopathy

Technical field

The invention belongs to the technical field of biomedical materials, and specifically relates to a preparation method of injectable hemostatic gel for wounds with coagulation disorders.

Background technique

Coagulation disorders refer to blood coagulation disorders caused by various reasons, such as lack of coagulation factors in the body, abnormality of the blood vessel wall, genetics, increase in anticoagulant substances, massive bleeding, and excessive activation of the fibrinolytic system. The general symptoms include difficulty in stopping bleeding and slow wound healing. At the same time, bacterial infection during hemostasis and wound healing may also trigger a series of bacteremia reactions, which greatly threatens human life.

Blind leg injuries and penetrating injuries caused by firearms, natural disasters, traffic accidents, etc. are often irregular and uncompressible, and ordinary hemostatic agents (such as gauze, etc.) cannot prevent severe bleeding from such wounds in time. For non-compressible wound bleeding, packing is generally used to stop bleeding. However, the commercially available XstatTM will cause secondary damage to the wound when it is removed from the wound.

Providing a biomaterial for bleeding with coagulation disorders, especially non-compressible bleeding, is one of the main ways to solve the problem of clinical coagulation disorders.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing injectable hemostatic gel for wounds with coagulation disorders in view of the above-mentioned deficiencies in the prior art. The present invention cross-links alkylated chitosan and oxidized dextran, and simultaneously cooperates with silver-doped bioactive glass and deferoxamine to specifically solve the problem of healing deep and narrow non-compressible wound exits. It has good biological activity and can inhibit Bacterial growth and rapid wound healing.

In order to solve the above technical problems, the technical solution adopted by the present invention is: a preparation method of injectable hemostatic gel for wounds with coagulation disorders, which is characterized in that it specifically includes: containing drug-loaded silver-doped mesoporous bioactive glass and The acetic acid solution of alkylated chitosan and the oxidized dextran solution are mixed and subjected to a freezing reaction to obtain an injectable hemostatic crystal glue for wounds with coagulation disorders.

The above-mentioned method for preparing injectable hemostatic gel for wounds with coagulopathy is characterized in that the freezing reaction temperature is -18°C and the time is 18 hours.

The above-mentioned method for preparing injectable hemostatic gelatin for wounds with coagulopathy, characterized in that the volume of the oxidized dextran solution contains drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan. 0.02 times the volume of acetic acid solution.

The above-mentioned method for preparing injectable hemostatic gel for wounds with coagulation disorders is characterized in that the acetic acid solution containing drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan is an alkylated shell. A mixed solution of polysaccharide, drug-loaded silver-doped mesoporous bioactive glass, deionized water and acetic acid solution. In the acetic acid solution containing drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan, alkylation The mass of chitosan is 2 to 6 times the mass of the drug-loaded silver-doped mesoporous bioactive glass, and the volume of deionized water is 0.17 to 0.5 times the mass of the drug-loaded silver-doped mesoporous bioactive glass. The volume of the deionized water The unit is mL. The unit of the mass of the drug-loaded silver-doped mesoporous bioactive glass is mg. The volume of the acetic acid solution is 1 to 3 times the mass of the drug-loaded silver-doped mesoporous bioactive glass. The unit of the volume of the acetic acid solution is μL. The unit of mass of silver-doped mesoporous bioactive glass is mg.

The above-mentioned method for preparing injectable hemostatic gel for wounds with coagulopathy is characterized in that the preparation method of alkylated chitosan includes:

Step 1: Place the acetic acid solution into the chitosan solution, stir the reaction, and obtain system A;

Step 2: Add lauric aldehyde to the system A, and adjust the pH to 5 to 5.1 after the reaction to obtain system B;

Step 3: Add sodium cyanoborohydride to the system B, and adjust the pH to 9-10 after the reaction to obtain system C;

Step 4: Perform gradient centrifugation of the system C and freeze-dry to obtain alkylated chitosan;

The above-mentioned method for preparing injectable hemostatic gel for wounds with coagulation disorders is characterized in that in step one, the molecular weight of the chitosan is 70,000, and the chitosan solution is deionized chitosan. Aqueous solution, the volume of the deionized water is 100 times the mass of chitosan, the volume unit of the deionized water is mL, and the mass unit of chitosan is g; the stirring reaction described in step 1 is stirred at room temperature for 1.5h ; The volume of the acetic acid solution in step one is 1 times the mass of chitosan, the volume unit of the acetic acid solution is mL, the mass unit of chitosan is g, and the mass percentage of the acetic acid solution is 99.5%.

The above-mentioned method for preparing injectable hemostatic gel for wounds with coagulation disorders is characterized in that in step two, adjusting the pH is with sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 3 mol/L~ 5 mol/L, the volume of lauric aldehyde is 0.6 times the mass of chitosan in step one, the volume unit of lauric aldehyde is mL, and the mass unit of chitosan is g; the reaction in step two is 35°C for 5 hours;

In step three, the mass of sodium cyanoborohydride is 1.2 times the mass of chitosan in step one, the reaction is 45°C for 18 hours, the pH is adjusted with sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 3mol/L～5mol/L;

In step 4, the gradient centrifugation is performed sequentially using ethanol solutions with volume percentages of 70%, 80%, 90% and 100%. In the gradient centrifugation, the speed of each centrifugation is 8000r/min to 10000r. /min, the time is 5min-7min, the freeze-drying temperature is -50°C, and the time is 3 days.

The above-mentioned method for preparing injectable hemostatic crystal glue for coagulopathy wounds is characterized in that the preparation method of drug-loaded silver-doped mesoporous bioactive glass includes: under stirring conditions, adding silver-doped mesoporous bioactive glass. The glass is added to the deferoxamine aqueous solution for stirring reaction, dried, and freeze-dried to obtain drug-loaded silver-doped mesoporous bioactive glass.

The above-mentioned method for preparing injectable hemostatic gel for wounds with coagulation disorders is characterized in that the method for preparing silver-doped mesoporous bioactive glass includes:

Step 1. Stir and react the cetyltrimethylammonium bromide solution and ethyl acetate, then add ammonia aqueous solution and continue the stirring reaction to obtain mixed system A; the concentration of the ammonia aqueous solution is 5 mol/L to 7 mol/L;

Step 2: Under stirring conditions of 300r/min~400r/min, add tetraethyl orthosilicate to the mixed system A, continue stirring at 300r/min~400r/min, add triethyl phosphate, and continue stirring at 300r/min. /min～400r/min, stir, add calcium nitrate tetrahydrate, stir at 550r/min～600r/min, add silver nitrate, stir at 550r/min～600r/min, centrifuge to obtain the precipitate;

Step 3: The precipitate is washed alternately with ethanol and water, dried, and calcined to obtain silver-doped mesoporous bioactive glass.

The above-mentioned method for preparing injectable hemostatic gelatin for wounds with coagulation disorders is characterized in that the preparation method of oxidized dextran includes: mixing dextran and The deionized aqueous solution of sodium periodate reacts in the dark, adds ethylene glycol to terminate the reaction, dialyzes, and freeze-dries to obtain oxidized dextran; the molecular weight cutoff of the dialysis bag for dialysis is 8000 to 14000 Da; the molecular weight of the dextran is 7000Da.

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

1. The present invention’s method for preparing injectable hemostatic gelatin for wounds with coagulation disorders utilizes cross-linking of alkylated chitosan and oxidized dextran, and at the same time cooperates with silver-doped bioactive glass and deferoxamine to specifically solve the problem of deep-seated hemostatic gel. and narrow non-compressible wound exit problems. It has the characteristics of good biological activity, inhibiting bacterial growth and rapid wound healing.

2. The preparation method of the injectable hemostatic gel for coagulation disorder wounds of the present invention includes preparing alkylated chitosan from chitosan, acetic acid solution, lauric aldehyde, sodium cyanoborohydride, etc., and The alkane chains on the surface of sylated chitosan have the characteristics of being able to insert into the blood cell membrane and form a clot with the blood, which can improve the hemostatic efficiency and avoid dependence on the coagulation cascade.

3. The preparation method of the injectable hemostatic gel for coagulopathy wounds of the present invention includes cetyltrimethylammonium bromide, ethyl acetate, tetraethyl orthosilicate, triethyl phosphate, and tetraethyl phosphate. A method of preparing drug-loaded silver-doped mesoporous bioactive glass using hydrated calcium nitrate, silver nitrate, deferoxamine and other raw materials. This method successfully achieved drug-loading on silver-doped mesoporous bioactive glass, which can effectively promote the sustained release of drugs in wounds. Achieve wound bacteriostasis and healing.

4. The preparation method of the injectable hemostatic gel for coagulation disorder wounds of the present invention includes using dextran and sodium periodate as main raw materials to prepare oxidized dextran through a light-proof reaction, which can make full use of oxidized dextran. The polyaldehyde groups on the sugar surface form a hydrogel in situ with alkylated chitosan.

The technical solution of the present invention will be described in further detail below with reference to the accompanying drawings and examples.

Instructions with pictures

Figure 1 is an FTIR pattern of alkylated chitosan in Example 1-1.

Figure 2 is the Fourier transform infrared absorption spectrum of the oxidized dextran in Example 4-1.

Figure 3 is an XRD pattern of the silver-doped mesoporous bioactive glass of Example 2-1.

Figure 4 is an SEM image of the crystal glue described in Examples 5 to 8.

Figure 5 shows the swelling ratio of the crystal glue described in Examples 5 to 8.

Figure 6 shows the liquid absorption rate of the crystal glue described in Examples 5 to 8.

Figure 7 shows the hemolysis rate of the crystalline colloids described in Examples 5 to 8.

Figure 8 is a schematic diagram of the antibacterial effect of the crystalline colloids described in Examples 5 to 8.

Figure 9 is a schematic diagram of the cytotoxicity MTT analysis results of the crystalline colloids described in Examples 5 to 8.

Figure 10 is a photograph of the crystal gel described in Examples 5 to 8 after freeze-drying.

Figure 11 is a schematic diagram of the injectable performance of the crystalline gel described in Examples 5 to 8.

Figure 12 is a schematic diagram of liver hemostasis using the crystalline gel described in Examples 5 to 8.

Detailed ways

Example 1-1

This embodiment provides a method for preparing alkylated chitosan, which specifically includes:

Step 1. Dissolve 4g of chitosan in 400mL of deionized water to obtain a chitosan solution; the molecular weight of the chitosan is 70,000;

Step 2: Add 4 mL of acetic acid solution to the chitosan solution and stir at room temperature for 1.5 hours to obtain system A; the mass percentage of the acetic acid solution is 99.5%;

Step 3: Add 2.4 mL of lauric aldehyde to system A described in step 2, react at 35°C for 5 hours, adjust the pH to 5-5.1 with sodium hydroxide solution, and obtain system B; the concentration of the sodium hydroxide solution is 4 mol/ L;

Step 4: Add 4.8g sodium cyanoborohydride to the system B, react at 45°C for 18 hours, adjust the pH to 9-10 with sodium hydroxide solution, and obtain system C; the concentration of the sodium hydroxide solution is 4 mol /L;

Step 5: The system C is subjected to gradient centrifugation and freeze-drying to obtain alkylated chitosan, labeled as NACS; the gradient centrifugation is performed with volume percentages of 70%, 80%, 90% and 100%. The ethanol solution was centrifuged in sequence; in the gradient centrifugation, the speed of each centrifugation was 8000 r/min and the time was 7 min; the freeze-drying temperature was -50°C and the time was 3 days.

Figure 1 is an FTIR pattern of alkylated chitosan in Example 1-1. According to Figure 1, it can be seen that the peak positions at 2800-2900cm ^-1 , 2900-3000cm ^-1 , 1560cm ^-1 and 720cm ^-1 belong to the alkyl peak, indicating that the alkyl group is connected to the amino group in chitosan. Alkylation modification was successful.

Example 1-2

This embodiment provides a method for preparing alkylated chitosan, which specifically includes:

Step 1. Dissolve 4g of chitosan in 400mL of deionized water to obtain a chitosan solution; the molecular weight of the chitosan is 70,000;

Step 2: Add 4 mL of acetic acid solution to the chitosan solution and stir at room temperature for 1.5 hours to obtain system A; the mass percentage of the acetic acid solution is 99.5%;

Step 3. Add 2.4 mL of lauric aldehyde to system A described in step 2, react at 35°C for 5 hours, and adjust the pH to 5 to 5.1 with sodium hydroxide solution to obtain system B; the concentration of the sodium hydroxide solution can be 3 mol. /L;

Step 4: Add 4.8g sodium cyanoborohydride to the system B, react at 45°C for 18 hours, adjust the pH to 9-10 with sodium hydroxide solution, and obtain system C; the concentration of the sodium hydroxide solution can be 3mol/L;

Step 5: The system C is subjected to gradient centrifugation and freeze-drying to obtain alkylated chitosan, labeled as NACS; the gradient centrifugation is performed with volume percentages of 70%, 80%, 90% and 100%. The ethanol solution was centrifuged in sequence; in the gradient centrifugation, the speed of each centrifugation was 9000 r/min and the time was 6 min; the freeze-drying temperature was -50°C and the time was 3 days.

The alkylated chitosan in this example is consistent with Example 1-1.

Example 1-3

This embodiment provides a method for preparing alkylated chitosan, which specifically includes:

Step 1. Dissolve 4g of chitosan in 400mL of deionized water to obtain a chitosan solution; the molecular weight of the chitosan is 70,000;

Step 2: Add 4 mL of acetic acid solution to the chitosan solution and stir at room temperature for 1.5 hours to obtain system A; the mass percentage of the acetic acid solution is 99.5%;

Step 3. Add 2.4 mL of lauric aldehyde to system A described in step 2, react at 35°C for 5 hours, and adjust the pH to 5 to 5.1 with sodium hydroxide solution to obtain system B; the concentration of the sodium hydroxide solution can be 5 mol. /L;

Step 4: Add 4.8g sodium cyanoborohydride to the system B, react at 45°C for 18 hours, adjust the pH to 9-10 with sodium hydroxide solution, and obtain system C; the concentration of the sodium hydroxide solution can be 5mol/L;

Step 5: The system C is subjected to gradient centrifugation and freeze-drying to obtain alkylated chitosan, labeled as NACS; the gradient centrifugation is performed with volume percentages of 70%, 80%, 90% and 100%. The ethanol solution was centrifuged in sequence; in the gradient centrifugation, the speed of each centrifugation was 10,000 r/min and the time was 5 min; the freeze-drying temperature was -50°C and the time was 3 days.

The alkylated chitosan in this example is consistent with Example 1-1.

Example 2-1

This embodiment provides a method for preparing silver-doped mesoporous bioactive glass, which specifically includes:

Step 1. Place 1.4g cetyltrimethylammonium bromide in 66mL deionized water and stir at room temperature for 1 hour to obtain a cetyltrimethylammonium bromide solution;

Step 2: Add 20 mL of ethyl acetate to the cetyltrimethylammonium bromide solution described in step 1, stir for 30 min, add 14 mL of ammonia aqueous solution, and stir for 15 h to obtain mixed system A; the concentration of the ammonia aqueous solution is 6 mol /L;

Step 3. Under stirring conditions of 350r/min, add 7.2mL tetraethyl orthosilicate to the mixed system A, continue stirring at 350r/min for 30min, add 0.72mL triethyl phosphate, and continue stirring at 350r/min for 30min. , add 4.54g calcium nitrate tetrahydrate, stir at 580r/min for 30min, add 0.4g silver nitrate, stir at 580r/min for 4 hours, centrifuge to obtain a precipitate; the color of the precipitate is white;

Step 4: Wash the precipitate 3 times with ethanol and water alternately, dry at 60°C for 24 hours, and calcine in a muffle furnace at 650°C for 4 hours to remove organic matter and other impurities to obtain silver-doped mesoporous bioactive glass.

Example 2-2

This embodiment provides a method for preparing silver-doped mesoporous bioactive glass, which specifically includes:

Step 1. Place 1.4g cetyltrimethylammonium bromide in 66mL deionized water and stir at room temperature for 1 hour to obtain a cetyltrimethylammonium bromide solution;

Step 2. Add 20 mL of ethyl acetate to the cetyltrimethylammonium bromide solution described in step 1, stir for 30 min, add 14 mL of ammonia aqueous solution, and stir for 15 h to obtain mixed system A; the concentration of the ammonia aqueous solution is 5 mol. /L;

Step 3. Under stirring conditions of 300r/min, add 7.2mL tetraethyl orthosilicate to the mixed system A, continue stirring at 300r/min for 30min, add 0.72mL triethyl phosphate, and continue stirring at 300r/min for 30min. , add 4.54g calcium nitrate tetrahydrate, stir at 550r/min for 30min, add 0.4g silver nitrate, stir at 550r/min for 4 hours, centrifuge to obtain a precipitate; the color of the precipitate is white;

Step 4: Wash the precipitate 3 times with ethanol and water alternately, dry at 60°C for 24 hours, and calcine in a muffle furnace at 650°C for 4 hours to remove organic matter and other impurities to obtain silver-doped mesoporous bioactive glass.

The properties of the silver-doped mesoporous bioactive glass in this example are basically consistent with those in Example 2-1.

Example 2-3

This embodiment provides a method for preparing silver-doped mesoporous bioactive glass, which specifically includes:

Step 1. Place 1.4g cetyltrimethylammonium bromide in 66mL deionized water and stir at room temperature for 1 hour to obtain a cetyltrimethylammonium bromide solution;

Step 2: Add 20 mL of ethyl acetate to the cetyltrimethylammonium bromide solution described in step 1, stir for 30 min, add 14 mL of ammonia aqueous solution, and stir for 15 h to obtain mixed system A; the concentration of the ammonia aqueous solution is 7 mol /L;

Step 3. Under stirring conditions of 400r/min, add 7.2mL tetraethyl orthosilicate to the mixed system A, continue stirring at 400r/min for 30min, add 0.72mL triethyl phosphate, and continue stirring at 400r/min for 30min. , add 4.54g calcium nitrate tetrahydrate, stir at 600r/min for 30min, add 0.4g silver nitrate, stir at 600r/min for 4 hours, centrifuge to obtain a precipitate; the color of the precipitate is white;

Step 4: Wash the precipitate 3 times with ethanol and water alternately, dry at 60°C for 24 hours, and calcine in a muffle furnace at 650°C for 4 hours to remove organic matter and other impurities to obtain silver-doped mesoporous bioactive glass.

The properties of the silver-doped mesoporous bioactive glass in this example are basically consistent with those in Example 2-1.

Example 3

This embodiment provides a method for preparing drug-loaded silver-doped mesoporous bioactive glass, including:

Step 1. Dissolve 120 μm deferoxamine in 100 mL deionized water, stir for 30 hours at room temperature, add 0.1g of the silver-doped mesoporous bioactive glass described in Example 2-1, and stir for 24 hours at room temperature;

Step 2: Dry the system after the reaction in Step 1 and freeze-dry for 72 hours to obtain drug-loaded silver-doped mesoporous bioactive glass; the drying temperature is 50°C and the time is 24 hours.

Example 4-1

This embodiment provides a method for preparing oxidized dextran, including:

Step 1. Dissolve 4g dextran and 3.4g sodium periodate in 50mL deionized water under magnetic stirring to obtain a dextran solution; the molecular weight of the dextran is 7000Da;

Step 2: Under stirring conditions of 580r/min, react the dextran solution described in step 1 at room temperature in the dark for 24h, add 1g of ethylene glycol, and continue stirring for 2h to terminate further oxidation of dextran, and obtain the solution after terminating the reaction. system; after the reaction in the dark, the system is a light yellow solution; the room temperature is 20°C to 25°C;

Step 3: dialyze the system after terminating the reaction with deionized water for 3 days, replace the dialysis water twice a day, freeze-dry the dialyzed system to obtain oxidized dextran; the molecular weight cutoff of the dialysis bag is 8000 to 14000 Da; so The freeze-drying temperature was -50°C and the freeze-drying time was 3 days.

Figure 2 is the Fourier transform infrared absorption spectrum of the oxidized dextran in this example. According to the spectrum, a peak position attributed to the carbonyl stretching peak appears at 1730 cm ^-1 , indicating that dextran has been successfully oxidatively modified.

Example 4-2

This embodiment provides a method for preparing oxidized dextran, including:

Step 1. Dissolve 4g dextran and 3.4g sodium periodate in 50mL deionized water under magnetic stirring to obtain a dextran solution; the molecular weight of the dextran is 7000Da;

Step 2: Under stirring conditions of 600r/min, react the dextran solution described in step 1 at room temperature in the dark for 24h, add 1g of ethylene glycol, and continue stirring for 2h to terminate further oxidation of dextran, and obtain the result after terminating the reaction. system; after the reaction in the dark, the system is a light yellow solution; the room temperature is 20°C to 25°C;

Step 3: dialyze the system after terminating the reaction with deionized water for 3 days, replace the dialysis water twice a day, freeze-dry the dialyzed system to obtain oxidized dextran; the molecular weight cutoff of the dialysis bag is 8000 to 14000 Da; so The freeze-drying temperature was -50°C and the freeze-drying time was 3 days.

The performance of oxidized dextran in this example is basically consistent with that in Example 4-1.

Example 4-3

This embodiment provides a method for preparing oxidized dextran, including:

Step 1. Dissolve 4g dextran and 3.4g sodium periodate in 50mL deionized water under magnetic stirring to obtain a dextran solution; the molecular weight of the dextran is 7000Da;

Step 2: Under stirring conditions of 550r/min, react the dextran solution described in step 1 at room temperature in the dark for 24 hours, add 1g of ethylene glycol, and continue stirring for 2 hours to terminate further oxidation of the glucan, and obtain the solution after terminating the reaction. system; after the reaction in the dark, the system is a light yellow solution; the room temperature is 20°C to 25°C;

Step 3: dialyze the system after terminating the reaction with deionized water for 3 days, replace the dialysis water twice a day, freeze-dry the dialyzed system to obtain oxidized dextran; the molecular weight cutoff of the dialysis bag is 8000 to 14000 Da; so The freeze-drying temperature was -50°C and the freeze-drying time was 3 days.

The performance of oxidized dextran in this example is basically consistent with that in Example 4-1.

Example 5

This embodiment provides a method for preparing crystal glue, which specifically includes:

Step 1. Place 0.12g of the alkylated chitosan described in Example 1-1 in 10 mL of deionized water, add 60 μL of acetic acid solution, and stir until all the alkylated chitosan is dissolved to obtain a mixed solution; the acetic acid solution The mass percentage is 99.5%;

Step 2: Dissolve 0.5g of the oxidized dextran described in Example 4-1 in 10 mL of deionized water to obtain an oxidized dextran solution;

Step 3: Under vigorous stirring conditions, add 200 μL of the oxidized dextran solution to 10 mL of the mixed solution described in Step 1, react at -18°C for 18 hours, and thaw at room temperature to obtain a crystalline gel; labeled AC/ODEX.

Example 6

This embodiment provides a method for preparing injectable hemostatic gel for wounds with coagulation disorders, which specifically includes:

Step 1. Place 0.12g of the alkylated chitosan described in Example 1-1 in 10 mL of deionized water, add 60 μL of acetic acid solution, and stir until all the alkylated chitosan is dissolved to obtain a mixed solution; the acetic acid solution The mass percentage is 99.5%;

Step 2: Add 20 mg of the drug-loaded silver-doped mesoporous bioactive glass described in Example 3 into the mixed solution of step 1, and stir evenly to obtain a drug-loaded mixed solution;

Step 3: Dissolve 0.5g of the oxidized dextran described in Example 4-1 in 10 mL of deionized water to obtain an oxidized dextran solution;

Step 4: Under vigorous stirring conditions, add 200 μL of the oxidized dextran solution to 10 mL of the drug-loaded mixed solution described in Step 2, react at -18°C for 18 hours, and thaw at room temperature to obtain an injectable hemostatic gel for wounds with coagulation disorders; Labeled AC/ODEX/Ag-MBG2.

Example 7

This embodiment provides a method for preparing injectable hemostatic gel for wounds with coagulation disorders, which specifically includes:

Step 1. Place 0.12g of the alkylated chitosan described in Example 1-1 in 10 mL of deionized water, add 60 μL of acetic acid solution, and stir until all the alkylated chitosan is dissolved to obtain a mixed solution; the acetic acid solution The mass percentage is 99.5%;

Step 2: Add 40 mg of the drug-loaded silver-doped mesoporous bioactive glass described in Example 3 into the mixed solution of step 1, and stir evenly to obtain a drug-loaded mixed solution;

Step 3: Dissolve 0.5g of the oxidized dextran described in Example 4-1 in 10 mL of deionized water to obtain an oxidized dextran solution;

Step 4: Under vigorous stirring conditions, add 200 μL of the oxidized dextran solution to 10 mL of the drug-loaded mixed solution described in Step 2, react at -18°C for 18 hours, and thaw at room temperature to obtain an injectable hemostatic gel for wounds with coagulation disorders. ;Tagged AC/ODEX/Ag-MBG4.

Example 8

This embodiment provides a method for preparing injectable hemostatic gel for wounds with coagulation disorders, which specifically includes:

Step 1. Place 0.12g of the alkylated chitosan described in Example 1-1 in 10 mL of deionized water, add 60 μL of acetic acid solution, and stir until all the alkylated chitosan is dissolved to obtain a mixed solution; the acetic acid solution The mass percentage is 99.5%;

Step 2: Add 60 mg of the drug-loaded silver-doped mesoporous bioactive glass described in Example 3 into the mixed solution of step 1, and stir evenly to obtain a drug-loaded mixed solution;

Step 3: Dissolve 0.5g of the oxidized dextran described in Example 4-1 in 10 mL of deionized water to obtain an oxidized dextran solution;

Step 4. Under vigorous stirring conditions, add 200 μL of the oxidized dextran solution to 10 mL of the drug-loaded mixed solution described in Step 2, react at -18°C for 18 hours, and thaw at room temperature to obtain an injectable hemostasis for wounds with coagulation disorders. Crystal gel; labeled AC/ODEX/Ag-MBG6.

Performance evaluation:

Figure 3 is an XRD pattern of the silver-doped mesoporous bioactive glass of Example 2-1. As shown in the figure, a peak attributed to Ag can be observed at 44°, indicating that silver ions are successfully doped.

Figure 4 is an SEM image of the crystal glue described in Examples 5 to 8. According to Figure 4, it can be seen that the crystal gel of the present invention has a pore diameter of 50 μm to 200 μm and has an interconnected macroporous structure.

Figure 5 shows the swelling ratio of the crystal glue described in Examples 5 to 8. The test method includes: soaking the freeze-dried crystal gel in ultrapure water at 37°C. After 24 hours, use filter paper to remove the liquid on the surface, weigh and record, and use the following equation to calculate the swelling ratio of the crystal gel:

Swelling rate (%) = (M ₁ -M ₀ )/M ₀ ×100%

M ₀ is the mass of the freeze-dried gelatin, and M ₁ is the mass of the gelatin after swelling.

According to Figure 5, it can be seen that the swelling rates of each crystal glue in Examples 6 to 8 are 3818.15%, 3305.01%, and 2924.55% respectively, indicating that the crystal glue of the present invention can absorb blood faster and effectively promote the blocking of bleeding wounds. Form a physical barrier to achieve preliminary hemostasis.

Figure 6 shows the liquid absorption rate of the crystalline gel described in Examples 5 to 8. The test method includes: immersing the frozen crystalline gel in PBS or blood, taking it out and weighing it after 2 hours. The liquid absorption rate is calculated by the following formula:

Liquid absorption rate (%) = (M ₁ -M ₀ )/M ₀ ×100%

The W ₀ is the mass of gelatin after freezing, and W ₁ is the mass of gelatin taken out from PBS or blood.

The absorption rates of the four groups of crystal gels for PBS were 3866.15%, 3782.56%, 3380.76% and 2972.55% respectively. The ability of the crystal gel to absorb blood was slightly lower than the ability to absorb PBS, which may be due to the higher blood viscosity. It shows that the crystalline gel of the present invention has high liquid absorbency, helps to quickly absorb blood, gather blood cells, coagulation factors and fibrinogen, and achieve accelerated hemostasis.

Figure 7 shows the hemolysis rate of the crystalline colloids described in Examples 5 to 8. The test method includes: soaking the crystalline gel in physiological saline at 37°C for 72 hours to obtain an extract with a concentration of 0.1g/mL, collecting fresh blood (5mL) from the abdominal cavity of rats, separating red blood cells at 2000rpm for 5min, and then using physiological Wash the separated red blood cells with saline until the supernatant is colorless and transparent, and then dilute it to a final concentration of 2% (v/v). In a 1mL centrifuge tube, mix 0.5mL of the extract and an equal amount of 2% ( v/v) RBC suspension was mixed in physiological saline and incubated at 37°C for 1 h. After centrifugation, the absorbance of the supernatant was measured at 545 nm, where physiological saline and deionized water were used as negative and positive controls respectively, followed by The in vitro hemolysis test was used for evaluation, and the results are shown in Figure 7. According to Figure 7, it can be seen that the hemolysis rates of the crystal gels are all less than 5%, which is considered to be no hemolysis. It shows that the crystal glue of the present invention does not cause red blood cells to rupture and has good blood compatibility.

Figure 8 is a schematic diagram of the antibacterial effect of the crystalline colloids described in Examples 5 to 8. The test method is to use Staphylococcus aureus (Staphylococcus aureus, ATCC 25923) and Escherichia coli (E.coli, ATCC 25922) as Gram-positive bacteria. bacteria and Gram-negative bacteria to test the antibacterial activity of the crystal gel. The details include: first sterilizing the crystal gel with a diameter of 10 mm and a thickness of about 1 mm in 75% alcohol, and then purifying it in sterilized PBS for one day. Remove the residual alcohol to obtain purified crystal gel. Incubate the purified crystal gel, culture medium and bacteria together for 12 hours to obtain a suspension. Add the suspension (10 ⁵ CFU/mL) to a 96-well culture plate. The optical density of the resuspended bacteria was measured at 600 nm, and the suspension was diluted and spread on a solid medium plate, placed at 37°C for 12 hours, and bacterial colonies were counted. According to Figure 8, it can be seen that as the co-culture time is prolonged, the antibacterial effect becomes more and more obvious. The antibacterial rate of the crystal gel of Example 7 corresponding to 24 hours reaches 92.8%, indicating that the crystal gel of the present invention has a good antibacterial effect.

Figure 9 is a schematic diagram of the cytotoxicity MTT analysis results of the crystalline colloids described in Examples 5 to 8. The test method includes: soaking the crystalline gel in RPM1-1640 medium at 37°C for 72 hours to obtain an extract with a concentration of 0.1g/mL, and then culture the mouse fibroblasts (L929) in the RPMI-1640 medium for 24 hours. cells) were replaced with the extract solution, and cultured for 24 hours, 48 hours and 72 hours respectively. The absorbance was measured using the MTT method and the survival rate of L929 cells was calculated. Each test was repeated in 6 parallel groups. The results are shown in Figure 9 Show. As shown in Figure 9, the cell survival rates at 24 hours, 48 hours and 72 hours were all above 90%, indicating that the gelatin of the present invention has good biocompatibility, is safe and non-toxic.

Figure 10 is a photograph of the crystal gel described in Examples 5 to 8 after freeze-drying. AC/ODEX crystal gel (Example 5) is white. As the concentration of silver-doped drug-loaded mesoporous bioactive glass increases from 0.2% to 0.6%, the color of the crystal gel changes from light brown to brown.

Figure 11 is a schematic diagram of the injectable performance of the crystalline gel described in Examples 5 to 8. As can be seen from Figure 11, the crystal glue can be put into a syringe and injected into the wound.

Figure 12 is a schematic diagram of liver hemostasis using crystalline gel as described in Examples 5 to 8. A direct 5mm circular wound is made in the liver through a punch, and crystalline gel is injected into the wound site through a syringe. Filter paper is placed under the liver and observed. Bleeding in the liver, until the bleeding stops, can be seen from observation, there is a small area of blood stains on the surface of the filter paper under the liver, indicating that the crystal gel has a better hemostatic effect.

Table 1 shows the recovery time of the crystal glue in water described in Examples 5 to 8. The test method includes: compressing the crystal glue to 80% of the initial height, then immersing it in PBS or blood, and recording the time required to restore the shape and the shape after recovery. length.

Shape recovery rate (%)=H ₁ /H ₀ ×100%

Where H ₀ represents the initial height of the frozen gel, and H ₁ represents the height of the frozen gel after shape recovery.

According to Table 1, it can be seen that the crystal glue prepared by the present invention has good compression resilience.

Table 1 Recovery time of crystal glue in water according to Examples 5 to 8

The above are only preferred embodiments of the present invention and do not limit the present invention in any way. Any simple modifications, changes and equivalent structural changes made to the above embodiments based on the technical essence of the invention still belong to the technical solutions of the present invention. within the scope of protection.

Claims (5)

1. The preparation method of the injectable hemostatic crystal gel for the clotting disorder wound is characterized by comprising the following steps of: mixing acetic acid solution containing drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan with oxidized dextran solution, and performing freezing reaction to obtain injectable hemostatic crystal gel for clotting disorder wound; the freezing reaction temperature is-18 ℃ and the freezing reaction time is 18 hours; the acetic acid solution containing the drug-loaded silver-doped mesoporous bioactive glass and the alkylated chitosan is a mixed solution of the alkylated chitosan, the drug-loaded silver-doped mesoporous bioactive glass, deionized water and the acetic acid solution;

the preparation method of the alkylated chitosan comprises the following steps:

step one, placing an acetic acid solution into a chitosan solution, and stirring for reaction to obtain a system A;

step two, adding lauraldehyde into the system A, and adjusting the pH value to 5-5.1 after the reaction to obtain a system B;

adding sodium cyanoborohydride into the system B, and adjusting the pH value to 9-10 after the reaction to obtain a system C;

step four, carrying out gradient centrifugation and freeze drying on the system C to obtain alkylated chitosan;

the preparation method of the drug-loaded silver-doped mesoporous bioactive glass comprises the following steps: adding silver-doped mesoporous bioactive glass into a deferoxamine water solution under the stirring condition, stirring for reaction, drying, and freeze-drying to obtain the drug-loaded silver-doped mesoporous bioactive glass;

the preparation method of the silver-doped mesoporous bioactive glass comprises the following steps:

step 101, adding ammonia water solution for continuous stirring reaction after stirring reaction of hexadecyl trimethyl ammonium bromide solution and ethyl acetate to obtain a mixed system A; the concentration of the ammonia water solution is 5 mol/L-7 mol/L;

102, adding tetraethyl orthosilicate into the mixed system A under the stirring condition of 300 r/min-400 r/min, continuously stirring at 300 r/min-400 r/min, adding triethyl phosphate, continuously stirring at 300 r/min-400 r/min, adding calcium nitrate tetrahydrate, stirring at 550 r/min-600 r/min, adding silver nitrate, stirring at 550 r/min-600 r/min, and centrifuging to obtain a precipitate;

and 103, alternately washing the precipitate with ethanol and water, drying, and calcining to obtain the silver-doped mesoporous bioactive glass.

2. The method for preparing injectable hemostatic crystal gum for blood coagulation disorder wound according to claim 1, wherein the volume of the oxidized dextran solution is 0.02 times of the volume of the acetic acid solution containing drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan.

3. The preparation method of the injectable hemostatic crystal gel for the clotting disorder wound of claim 1, wherein in the acetic acid solution containing the silver-doped mesoporous bioactive glass and the alkylated chitosan, the mass of the alkylated chitosan is 2-6 times of that of the silver-doped mesoporous bioactive glass, the volume of deionized water is 0.17-0.5 times of that of the silver-doped mesoporous bioactive glass, the volume of deionized water is mL, the volume of the silver-doped mesoporous bioactive glass is mg, the volume of the acetic acid solution is 1-3 times of that of the silver-doped mesoporous bioactive glass, the volume of the acetic acid solution is μL, and the volume of the silver-doped mesoporous bioactive glass is mg.

4. The method for preparing injectable hemostatic crystal gum for a blood coagulation disorder wound according to claim 1, wherein in the first step, the molecular weight of chitosan is 70000, the chitosan solution is deionized water solution of chitosan, the volume of deionized water is 100 times of the mass of chitosan, the volume unit of deionized water is mL, and the mass unit of chitosan is g; stirring the mixture for 1.5h at room temperature; in the first step, the volume of the acetic acid solution is 1 time of the mass of chitosan, the volume unit of the acetic acid solution is mL, the mass unit of chitosan is g, and the mass percentage of the acetic acid solution is 99.5%.

5. The method for preparing injectable hemostatic crystal gum for clotting wound as claimed in claim 1, wherein in the second step, the pH is adjusted by sodium hydroxide solution, the concentration of the sodium hydroxide solution is 3mol/L to 5mol/L, the laural volume is 0.6 times of the mass of chitosan in the first step, the laural volume unit is mL, and the mass unit of chitosan is g; step two, the reaction is carried out for 5 hours at 35 ℃;

in the third step, the mass of the cyano sodium borohydride is 1.2 times that of the chitosan in the first step, the reaction is carried out for 18 hours at 45 ℃, the pH is regulated by a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 3 mol/L-5 mol/L;

and step four, sequentially centrifuging by using ethanol solution with the volume percentage of 70%, 80%, 90% and 100%, wherein the speed of each centrifugation in the gradient centrifugation is 8000 r-10000 r/min, the time is 5-7 min, and the freeze-drying temperature is-50 ℃ for 3 days.