CN115737897B

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

Info

Publication number: CN115737897B
Application number: CN202211131545.7A
Authority: CN
Inventors: 赵婧; 雷栋; 田莉莉; 王凯歌; 米钰; 范代娣; 马沛
Original assignee: NORTHWEST UNIVERSITY
Current assignee: NORTHWEST UNIVERSITY
Priority date: 2022-09-16
Filing date: 2022-09-16
Publication date: 2024-01-09
Anticipated expiration: 2042-09-16
Also published as: CN115737897A

Abstract

The invention discloses a preparation method of an injectable hemostatic crystal gel for a coagulation disorder wound, which specifically comprises the following steps: 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 invention utilizes the cross-linking of the alkylated chitosan and the oxidized dextran, and simultaneously combines the silver-doped bioactive glass and the deferoxamine, thereby solving the difficult problem of healing the deep and narrow non-compressible wound outlet in a targeted manner.

Description

Preparation method of injectable hemostatic crystal gel for clotting disorder wound

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of an injectable hemostatic crystal gel for a blood coagulation disorder wound.

Background

Coagulation disorders refer to blood coagulation dysfunctional diseases caused by various causes, such as lack of blood coagulation factors in vivo, abnormality of blood vessel walls, inheritance, increase of anticoagulant substances, massive hemorrhage, excessive activation of fibrinolytic systems, and the like. It is generally manifested by bleeding, difficult hemostasis, and slow wound healing. At the same time, a series of bacteremia reactions may also be initiated due to bacterial infection during hemostasis and wound healing, which is a great threat to human life.

Blind and penetrating injuries caused by firearms, natural disasters, traffic accidents and the like are often irregular and incompressible, and common hemostatic agents (such as gauze and the like) cannot timely prevent severe bleeding of the wounds. For non-compressible wound hemorrhages, tamponade is typically used, but commercially available XstatTM can cause secondary injury to the wound when removed from the wound.

Provides a biological material for bleeding with coagulation disorder, in particular non-compressible bleeding, which is one of the main ways to solve the problem of coagulation disorder clinically.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method of injectable hemostatic crystal gum for a coagulation disorder wound aiming at the defects of the prior art. The invention utilizes the cross-linking of the alkylated chitosan and the oxidized dextran, and simultaneously combines the silver-doped bioactive glass and the deferoxamine, thereby solving the difficult problem of healing the deep and narrow non-compressible wound outlet in a targeted manner.

In order to solve the technical problems, the invention adopts the following technical scheme: 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 preparation method of the injectable hemostatic crystal gel for the blood coagulation disorder wound is characterized in that the freezing reaction temperature is-18 ℃ and the time is 18 hours.

The preparation method of the injectable hemostatic crystal gel for the blood coagulation disorder wound is characterized in that the volume of the oxidized dextran solution is 0.02 times of the volume of an acetic acid solution containing drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan.

The preparation method of the injectable hemostatic crystal gel for the blood coagulation disorder wound is characterized in that an acetic acid solution containing drug-loaded silver-doped mesoporous bioactive glass and alkylated chitosan is a mixed solution of alkylated chitosan, drug-loaded silver-doped mesoporous bioactive glass, deionized water and acetic acid solution, wherein the mass of the alkylated chitosan is 2-6 times of the mass of the drug-loaded silver-doped mesoporous bioactive glass, the volume of deionized water is 0.17-0.5 times of the mass of the drug-loaded silver-doped mesoporous bioactive glass, the volume of the deionized water is in mL, the mass of the drug-loaded silver-doped mesoporous bioactive glass is in mg, the volume of the acetic acid solution is 1-3 times of the mass of the drug-loaded silver-doped mesoporous bioactive glass, the volume of the acetic acid solution is in mu L, and the mass of the drug-loaded silver-doped mesoporous bioactive glass is in mg.

The preparation method of the injectable hemostatic crystal gel for the clotting disorder wound is characterized by comprising the following steps of:

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 regulating 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 injectable hemostatic crystal gel for the blood coagulation disorder wound is characterized by comprising the following steps that 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%.

The preparation method of the injectable hemostatic crystal gel for the blood coagulation disorder wound is characterized by comprising the following steps of adjusting the pH value by using a sodium hydroxide solution, wherein the concentration of the sodium hydroxide solution is 3-5 mol/L, the laural volume is 0.6 times of the mass of chitosan in the step one, the volume unit of the laural 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 8000-10000 r/min, the time is 5-7 min, the freeze-drying temperature is-50 ℃, and the time is 3 days.

The preparation method of the injectable hemostatic crystal gel for the clotting obstacle wound is characterized by comprising the following steps of: under the stirring condition, adding the silver-doped mesoporous bioactive glass into the deferoxamine water solution, stirring for reaction, drying, and freeze-drying to obtain the drug-loaded silver-doped mesoporous bioactive glass.

step one, 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 5mol/L to 7mol/L;

step two, under the stirring condition of 300 r/min-400 r/min, adding tetraethyl orthosilicate into the mixed system A, 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 step three, alternately washing the precipitate with ethanol and water, drying, and calcining to obtain the silver-doped mesoporous bioactive glass.

The preparation method of the injectable hemostatic crystal gel for the clotting disorder wound is characterized by comprising the following steps of: under the stirring condition of 550 r/min-600 r/min, the dextran and deionized water solution of sodium periodate are subjected to light-proof reaction, ethylene glycol is added to terminate the reaction, and the dextran oxide is obtained through dialysis and freeze drying; dialyzing the dialysis bag with the molecular weight cut-off of 8000-14000 Da; the molecular weight of the glucan is 7000Da.

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

1. the preparation method of the injectable hemostatic crystal gel for the clotting disorder wound utilizes the cross-linking of the alkylated chitosan and the oxidized dextran, and simultaneously combines the silver-doped bioactive glass and the deferoxamine, thereby solving the difficult problem of deep and narrow non-compressible wound outlet healing in a targeted manner, and having the characteristics of good bioactivity, capability of inhibiting bacterial growth and rapid wound healing.

2. The preparation method of the injectable hemostatic crystal gel for the blood coagulation disorder wound comprises the steps of preparing the alkylated chitosan by taking chitosan, acetic acid solution, laural, cyano sodium borohydride and the like as raw materials, wherein an alkane chain on the surface of the alkylated chitosan has the characteristic of being capable of being inserted into a blood cell membrane to form a clot with blood, thereby improving the hemostatic efficiency and avoiding dependence on blood coagulation cascade.

3. The preparation method of the injectable hemostatic crystal gel for the clotting disorder wound comprises the steps of preparing the silver-doped mesoporous bioactive glass with raw materials such as cetyl trimethyl ammonium bromide, ethyl acetate, tetraethyl orthosilicate, triethyl phosphate, calcium nitrate tetrahydrate, silver nitrate, deferoxamine and the like, and successfully realizing the silver-doped mesoporous bioactive glass drug loading, effectively promoting the slow release of the drug at the wound and realizing the bacteriostasis and healing of the wound.

4. The preparation method of the injectable hemostatic crystal gel for the clotting disorder wound comprises the steps of preparing oxidized dextran by taking dextran and sodium periodate as main raw materials through light-shielding reaction, and fully utilizing polyaldehyde groups on the surface of the oxidized dextran and alkylated chitosan to form hydrogel in situ.

The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and the examples.

Drawings

FIG. 1 is a FTIR plot of alkylated chitosan of example 1-1.

FIG. 2 is a Fourier transform infrared absorption spectrum of oxidized dextran of example 4-1.

FIG. 3 is an XRD pattern for the silver-doped mesoporous bioactive glass of example 2-1.

Fig. 4 is an SEM image of the crystal gels of examples 5 to 8.

FIG. 5 shows the swelling ratios of the crystal gums according to examples 5 to 8.

Fig. 6 shows the liquid absorption rate of the crystal gums according to examples 5 to 8.

FIG. 7 shows the hemolysis ratio of the crystal gels according to examples 5 to 8.

Fig. 8 is a schematic diagram showing the bacteriostatic effect of the crystal gums of examples 5 to 8.

FIG. 9 is a schematic diagram showing the results of the cytotoxicity MTT assay of the crystal gels described in examples 5 to 8.

Fig. 10 is a photograph of the crystal gels of examples 5 to 8 after lyophilization.

FIG. 11 is a schematic illustration of the injectability of the gels of examples 5-8.

Fig. 12 is a schematic view of liver hemostasis of the crystal gels of examples 5-8.

Detailed Description

Example 1-1

The embodiment provides a preparation method of alkylated chitosan, which specifically comprises the following steps:

step one, dissolving 4g of chitosan in 400mL of deionized water to obtain a chitosan solution; the molecular weight of the chitosan is 70000;

step two, adding 4mL of acetic acid solution into the chitosan solution, and stirring for 1.5h at room temperature to obtain a system A; the mass percentage of the acetic acid solution is 99.5%;

step three, adding 2.4mL of laural into the system A in the step two, reacting for 5 hours at 35 ℃, and regulating the pH to 5-5.1 by using sodium hydroxide solution to obtain a system B; the concentration of the sodium hydroxide solution is 4mol/L;

adding 4.8g of sodium cyanoborohydride into the system B, reacting for 18 hours at 45 ℃, and regulating the PH to 9-10 by using a sodium hydroxide solution to obtain a system C; the concentration of the sodium hydroxide solution is 4mol/L;

step five, carrying out gradient centrifugation and freeze drying on the system C to obtain alkylated chitosan, and marking the alkylated chitosan as NACS; the gradient centrifugation is to sequentially centrifuge with ethanol solutions with the volume percentage of 70%, 80%, 90% and 100%; the gradient centrifugation is carried out at a speed of 8000r/min for 7min each time; the freeze-drying temperature is-50 ℃ and the time is 3 days.

FIG. 1 is a FTIR plot of alkylated chitosan of example 1-1. As can be seen from FIG. 1, at 2800-2900cm ^-1 、2900-3000cm ^-1 、1560cm ^-1 720cm ^-1 The peak position is assigned to an alkyl peak, which indicates that the alkyl is connected with the amino in the chitosan, and the alkylation modification of the chitosan is successful.

Examples 1 to 2

step three, adding 2.4mL of laural into the system A in the step two, reacting for 5 hours at 35 ℃, and regulating the pH to 5-5.1 by using sodium hydroxide solution to obtain a system B; the concentration of the sodium hydroxide solution may be 3mol/L;

adding 4.8g of sodium cyanoborohydride into the system B, reacting for 18 hours at 45 ℃, and regulating the PH to 9-10 by using a sodium hydroxide solution to obtain a system C; the concentration of the sodium hydroxide solution may be 3mol/L;

step five, carrying out gradient centrifugation and freeze drying on the system C to obtain alkylated chitosan, and marking the alkylated chitosan as NACS; the gradient centrifugation is to sequentially centrifuge with ethanol solutions with the volume percentage of 70%, 80%, 90% and 100%; the gradient centrifugation is carried out at a speed of 9000r/min for 6min each time; the freeze-drying temperature is-50 ℃ and the time is 3 days.

The alkylated chitosan of this example is identical to example 1-1.

Examples 1 to 3

step three, adding 2.4mL of laural into the system A in the step two, reacting for 5 hours at 35 ℃, and regulating the pH to 5-5.1 by using sodium hydroxide solution to obtain a system B; the concentration of the sodium hydroxide solution may be 5mol/L;

adding 4.8g of sodium cyanoborohydride into the system B, reacting for 18 hours at 45 ℃, and regulating the PH to 9-10 by using a sodium hydroxide solution to obtain a system C; the concentration of the sodium hydroxide solution may be 5mol/L;

step five, carrying out gradient centrifugation and freeze drying on the system C to obtain alkylated chitosan, and marking the alkylated chitosan as NACS; the gradient centrifugation is to sequentially centrifuge with ethanol solutions with the volume percentage of 70%, 80%, 90% and 100%; the gradient centrifugation is carried out at a speed of 10000r/min for 5min each time; the freeze-drying temperature is-50 ℃ and the time is 3 days.

The alkylated chitosan of this example is identical to example 1-1.

Example 2-1

The embodiment provides a preparation method of silver-doped mesoporous bioactive glass, which specifically comprises the following steps:

step one, 1.4g of cetyltrimethylammonium bromide is placed in 66mL of deionized water, and stirred for 1h at room temperature to obtain cetyltrimethylammonium bromide solution;

step two, adding 20mL of ethyl acetate into the cetyl trimethyl ammonium bromide solution in the step one, stirring for 30min, adding 14mL of ammonia water solution, and stirring for 15h to obtain a mixed system A; the concentration of the ammonia water solution is 6mol/L;

adding 7.2mL of tetraethyl orthosilicate into the mixed system A under the stirring condition of 350r/min, continuously stirring for 30min at 350r/min, adding 0.72mL of triethyl phosphate, continuously stirring for 30min at 350r/min, adding 4.54g of calcium nitrate tetrahydrate, stirring for 30min at 580r/min, adding 0.4g of silver nitrate, stirring for 4h at 580r/min, and centrifuging to obtain a precipitate; the color of the precipitate is white;

and fourthly, alternately washing the precipitate with ethanol and water for 3 times, drying at 60 ℃ for 24 hours, and calcining in a muffle furnace at 650 ℃ for 4 hours to remove organic matters and other impurities, thereby obtaining the silver-doped mesoporous bioactive glass.

Example 2-2

step two, adding 20mL of ethyl acetate into the cetyl trimethyl ammonium bromide solution in the step one, stirring for 30min, adding 14mL of ammonia water solution, and stirring for 15h to obtain a mixed system A; the concentration of the ammonia water solution is 5mol/L;

adding 7.2mL of tetraethyl orthosilicate into the mixed system A under the stirring condition of 300r/min, continuously stirring for 30min at 300r/min, adding 0.72mL of triethyl phosphate, continuously stirring for 30min at 300r/min, adding 4.54g of calcium nitrate tetrahydrate, stirring for 30min at 550r/min, adding 0.4g of silver nitrate, stirring for 4h at 550r/min, and centrifuging to obtain a precipitate; the color of the precipitate is white;

The properties of the silver-doped mesoporous bioactive glass of this example are substantially the same as those of example 2-1.

Examples 2 to 3

step two, adding 20mL of ethyl acetate into the cetyl trimethyl ammonium bromide solution in the step one, stirring for 30min, adding 14mL of ammonia water solution, and stirring for 15h to obtain a mixed system A; the concentration of the ammonia water solution is 7mol/L;

adding 7.2mL of tetraethyl orthosilicate into the mixed system A under the stirring condition of 400r/min, continuously stirring for 30min at 400r/min, adding 0.72mL of triethyl phosphate, continuously stirring for 30min at 400r/min, adding 4.54g of calcium nitrate tetrahydrate, stirring for 30min at 600r/min, adding 0.4g of silver nitrate, stirring for 4 hours at 600r/min, and centrifuging to obtain a precipitate; the color of the precipitate is white;

Example 3

The embodiment provides a preparation method of silver-doped mesoporous bioactive glass for drug loading, which comprises the following steps:

dissolving 120 mu m deferoxamine in 100mL deionized water, stirring for 30 hours at room temperature, adding 0.1g of silver-doped mesoporous bioactive glass described in the example 2-1, and stirring for reaction for 24 hours at room temperature;

step two, drying the system after the reaction in the step one, and freeze-drying for 72 hours to obtain the silver-doped mesoporous bioactive glass carrying the medicine; the temperature of the drying is 50 ℃ and the time is 24 hours.

Example 4-1

The embodiment provides a method for preparing oxidized dextran, which comprises the following steps:

step one, under magnetic stirring, dissolving 4g of glucan and 3.4g of sodium periodate in 50mL of deionized water to obtain a glucan solution; the molecular weight of the glucan is 7000Da;

step two, under the stirring condition of 580r/min, the glucan solution in the step one reacts for 24 hours at room temperature under the dark condition, 1g of ethylene glycol is added, and stirring is continued for 2 hours to terminate further oxidation of glucan, so that a system after termination reaction is obtained; the system is a pale yellow solution after the light-shielding reaction; the room temperature is 20-25 ℃;

continuously dialyzing the system after termination of the reaction with deionized water for 3 days, replacing dialysis water for 2 times per day, and freeze-drying the system after dialysis to obtain oxidized dextran; dialyzing the dialysis bag with the molecular weight cut-off of 8000-14000 Da; the freeze-drying temperature is-50 ℃ and the time is 3 days.

FIG. 2 is a Fourier transform infrared absorption spectrum of oxidized dextran of the present example. 1730cm according to the spectrogram ^-1 Peak positions belonging to carbonyl stretching peaks appear at the positions, which indicate that the dextran is successfully oxidized and modified.

Example 4-2

step two, under the stirring condition of 600r/min, the glucan solution in the step one reacts for 24 hours at room temperature under the dark condition, 1g of ethylene glycol is added, and stirring is continued for 2 hours to terminate further oxidation of glucan, so that a system after termination reaction is obtained; the system is a pale yellow solution after the light-shielding reaction; the room temperature is 20-25 ℃;

The oxidized dextran properties of this example were substantially identical to those of example 4-1.

Examples 4 to 3

step two, under the stirring condition of 550r/min, the glucan solution in the step one reacts for 24 hours at room temperature under the dark condition, 1g of ethylene glycol is added, and stirring is continued for 2 hours to terminate further oxidation of glucan, so that a system after termination reaction is obtained; the system is a pale yellow solution after the light-shielding reaction; the room temperature is 20-25 ℃;

Example 5

The embodiment provides a preparation method of a crystal glue, which specifically comprises the following steps:

step one, placing 0.12g of the alkylated chitosan described in the embodiment 1-1 into 10mL of deionized water, adding 60 mu L of acetic acid solution, and stirring until the alkylated chitosan is completely dissolved to obtain a mixed solution; the mass percentage of the acetic acid solution is 99.5%;

step two, dissolving 0.5g of oxidized dextran described in the example 4-1 in 10mL of deionized water to obtain oxidized dextran solution;

step three, adding 200 mu L of the oxidized dextran solution into 10mL of the mixed solution obtained in the step one under the condition of intense stirring, reacting for 18 hours at the temperature of minus 18 ℃, and thawing at normal temperature to obtain crystal gum; labeled AC/ODEX.

Example 6

The embodiment provides a preparation method of an injectable hemostatic crystal gel for a blood coagulation disorder wound, which specifically comprises the following steps:

step two, adding 20mg of the drug-loaded silver-doped mesoporous bioactive glass described in the embodiment 3 into the mixed solution in the step one, and uniformly stirring to obtain a drug-loaded mixed solution;

step three, dissolving 0.5g of oxidized dextran described in the example 4-1 in 10mL of deionized water to obtain oxidized dextran solution;

step four, under the condition of intense stirring, adding 200 mu L of oxidized dextran solution into 10mL of the drug-loaded mixed solution in the step two, reacting for 18 hours at the temperature of minus 18 ℃, and thawing at normal temperature to obtain the injectable hemostatic crystal gel for the clotting disorder wound; labeled AC/ODEX/Ag-MBG2.

Example 7

step two, adding 40mg of the drug-loaded silver-doped mesoporous bioactive glass described in the embodiment 3 into the mixed solution in the step one, and uniformly stirring to obtain a drug-loaded mixed solution;

step four, under the condition of intense stirring, adding 200 mu L of oxidized dextran solution into 10mL of the drug-loaded mixed solution in the step two, reacting for 18 hours at the temperature of minus 18 ℃, and thawing at normal temperature to obtain the injectable hemostatic crystal gel for the clotting disorder wound; labeled AC/ODEX/Ag-MBG4.

Example 8

step two, adding 60mg of the drug-loaded silver-doped mesoporous bioactive glass described in the embodiment 3 into the mixed solution in the step one, and uniformly stirring to obtain a drug-loaded mixed solution;

step four, under the condition of intense stirring, adding 200 mu L of the oxidized dextran solution into 10mL of the drug-carrying mixed solution in the step two, reacting for 18 hours at the temperature of minus 18 ℃, and thawing at normal temperature to obtain the injectable hemostatic crystal gel for the clotting disorder wound; labeled AC/ODEX/Ag-MBG6.

Evaluation of performance:

FIG. 3 is an XRD pattern of the silver-doped mesoporous bioactive glass of example 2-1, in which peaks ascribed to Ag can be observed at 44℃as shown, indicating successful silver ion doping.

Fig. 4 is an SEM image of the crystal gels of examples 5 to 8. As can be seen from FIG. 4, the crystal gel of the present invention has a pore size of 50 μm to 200 μm and a macroporous structure communicating with each other.

FIG. 5 shows the swelling ratios of the crystal gums according to examples 5 to 8. The testing method comprises the following steps: the freeze-dried gel was immersed in ultrapure water at 37℃and after 24 hours, the surface liquid was removed with filter paper, and then weighed and recorded, and the swelling ratio of the gel was calculated using the following equation:

swelling ratio (%)＝(M ₁ -M ₀ )/M ₀ ×100％

M ₀ For the quality of the freeze-dried crystal gum, M ₁ The mass of the crystal glue after swelling.

According to fig. 5, the swelling rates of the crystal gels in examples 6 to 8 are 3818.15%, 3305.01% and 2924.55%, respectively, which shows that the crystal gel of the present invention can achieve faster absorption of blood, and can effectively promote blockage of bleeding wounds, form a physical barrier, and achieve the effect of primary hemostasis.

Fig. 6 shows the liquid absorptivity of the crystal glue according to examples 5 to 8, and the test method includes: the frozen gel was immersed in PBS or blood, taken out after 2 hours and weighed, and the liquid absorption rate was calculated by the following formula:

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

The W is ₀ Is the quality of frozen crystal glue, W ₁ Is the mass of the crystal gel taken out of PBS or blood.

The absorption rates of the four groups of gels for PBS were 3866.15%, 3782.56%, 3380.76% and 2972.55%, respectively, with the gels having a slightly lower ability to absorb blood than the PBS, possibly due to the higher viscosity of the blood. The crystal gel has high liquid absorptivity, is favorable for quickly absorbing blood, aggregated blood cells, coagulation factors and fibrinogen, and realizes acceleration of hemostasis.

FIG. 7 shows the hemolysis ratio of the crystal gels according to examples 5 to 8. The testing method comprises the following steps: the gel was soaked in physiological saline at 37 ℃ for 72 hours to obtain a leaching solution with a concentration of 0.1g/mL, fresh blood (5 mL) was collected from the abdominal cavity of the rat, red blood cells were separated at 2000rpm for 5min, then the separated red blood cells were washed to be colorless and transparent to the supernatant using physiological saline, and then diluted to a final concentration of 2% (v/v), 0.5mL of the leaching solution and an equivalent amount of 2% (v/v) RBC suspension were mixed in physiological saline in a 1mL centrifuge tube, and cultured at 37 ℃ for 1 hour, and after centrifugation, absorbance of the supernatant was measured at 545nm, wherein physiological saline and deionized water were used as negative and positive controls, respectively, and then evaluated using an in vitro hemolysis experiment, the results of which are shown in fig. 7. As can be seen from fig. 7, the hemolysis rate of the crystal gum was lower than 5%, and no hemolysis was observed. The crystal gel of the invention has no red blood cell rupture and good blood compatibility.

Fig. 8 is a schematic diagram showing the antibacterial effect of the crystal glue according to examples 5 to 8, wherein the test method is to test the antibacterial activity of the crystal glue by using staphylococcus aureus (staphylococcus aureus, ATCC 25923) and escherichia coli (e.coli, ATCC 25922) as gram-positive bacteria and gram-negative bacteria, and specifically comprises: the method comprises sterilizing a gel having a diameter of 10mm and a thickness of about 1mm in 75% alcohol, purifying in sterilized PBS for one day to remove residual alcohol to obtain purified gel, incubating the purified gel, culture medium and bacteria for 12 hr to obtain a suspension, and separating the suspension (10 ⁵ CFU/mL) was added to 96-well plates, the optical density of bacterial resuspension was measured at 600nm, the suspension was diluted and spread on solid medium plates, and colony counts were performed by standing at 37 ℃ for 12 h. According to fig. 8, as the co-cultivation time is prolonged, the bacteriostasis effect is more and more obvious, and the bacteriostasis rate of the crystal gum of the example 7 corresponding to 24 hours reaches 92.8%, which indicates that the crystal gum has good bacteriostasis effect.

FIG. 9 is a schematic diagram showing the results of the cytotoxicity MTT assay of the crystal gels described in examples 5 to 8. The testing method comprises the following steps: the gel was immersed in a medium of RPM1-1640 at 37℃for 72 hours to obtain an extract with a concentration of 0.1g/mL, the medium of mouse fibroblasts (L929 cells) after normal culture in RPMI-1640 medium for 24 hours was replaced with the extract, and the absorbance was measured by MTT method for 24 hours, 48 hours and 72 hours, and the survival rate of L929 cells was calculated, and 6 replicates were repeated for each test, and the results are shown in FIG. 9. According to the results shown in FIG. 9, the cell viability of 24 hours, 48 hours and 72 hours is over 90%, which shows that the crystal gel of the invention has good biocompatibility, safety and no toxicity.

Fig. 10 is a photograph of the crystal gels of examples 5 to 8 after lyophilization. The AC/ODEX gel (example 5) was white, and the color of the gel changed from light brown to brown as the concentration of the silver-doped drug-loaded mesoporous bioactive glass increased from 0.2% to 0.6%.

FIG. 11 is a schematic illustration of the injectability of the gels of examples 5-8. As can be seen from fig. 11, the gel may be placed in a syringe and introduced into the wound by injection.

Fig. 12 is a schematic diagram of liver hemostasis of the crystal gel in examples 5-8, wherein a circular wound of 5mm is formed at the liver part by a puncher, the crystal gel is injected into the wound part by a syringe, filter paper is placed under the liver, the bleeding condition of the liver is observed until the bleeding is stopped, and the observation shows that the surface of the filter paper under the liver has a small-area blood trace, which indicates that the crystal gel has a better hemostasis effect.

Table 1 shows the recovery time of the crystal gum in water according to examples 5 to 8, and the test method comprises: the gel was compressed to 80% of the original height and then immersed in PBS or blood, and the time required to recover the shape and the length after recovery were recorded.

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

Wherein H is ₀ Represents the initial height of the frozen gel, H ₁ The height of the frozen gel after shape recovery is shown.

As can be seen from Table 1, the crystal gum prepared by the invention has good compression rebound resilience.

Table 1 recovery time of the Crystal gums in Water as described in examples 5 to 8

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent structural changes of the above embodiment according to the technical matter of the present invention still fall within the scope of the technical solution of the present invention.

Claims

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:

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

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 ℃;

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.