Preparation method of in-situ curing hydrogel, hydrogel and application thereof

Abstract

The invention provides a preparation method of in-situ curing hydrogel, hydrogel and application thereof, and relates to the technical field of biomaterials.The preparation method comprises the steps that a first precursor solution and a second precursor solution are mixed, and the in-situ curing hydrogel is obtained; the first precursor solution contains chitosan or a derivative thereof and a medicine, and the concentration of the chitosan or the derivative thereof is 6-12% w / v; the second precursor solution contains DF-PEG and Cu < 2 + >, the concentration of the DF-PEG is 10-15% w / v, and the concentration of the Cu < 2 + > is 1.9-5.6 mmol / L. The in-situ curing hydrogel obtained by the preparation method is short in curing time, the gel strength after curing is high, medullary cavity hemostasis can be simply, conveniently and efficiently realized, and the in-situ curing hydrogel has important significance for solving the problem of lacuna bone bleeding.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a preparation method of an in-situ solidified hydrogel, the hydrogel and applications thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Bleeding from cancellous bone wounds is difficult to control because of its loose structure, rich blood supply, and the difficulty of blood vessels contracting and blood clot formation. At present, the most widely used treatment for cancellous bone bleeding is smear-type bone wax. However, when traumatic bleeding occurs in the medullary cavity, it is not easy to block the cavity with smear-type bone wax to achieve the purpose of hemostasis. Therefore, injectable hydrogel materials can meet the needs of medullary cavity hemostasis.

[0004] The principle of in situ curing hydrogel is mainly based on the in situ curing mechanism of its carrier material in vivo. As reported in the literature, in situ curing hydrogels mainly include temperature-sensitive, pH-sensitive, ion-sensitive and in situ polymer precipitation systems. After these hydrogels are administered in a solution state, a phase transition occurs at the medication site, and the liquid solidifies to form a semisolid gel. Specifically, the gelation process of most injectable hydrogels needs to be completed under invasive external stimuli, such as pH, light, temperature or organic cross-linking agents. This raises concerns about the potential safety of unreacted initiators or monomers. In contrast, injectable hydrogels (such as Schiff bases) that are spontaneously formed through phase transitions or dynamic reactions have higher advantages.

[0005] At present, in situ curing hydrogels based on Schiff base reaction, as reported in the literature, use the "A+B" in situ molding method to prepare a gel precursor solution of DF-PEG and carboxymethyl chitosan, mix the two precursors before injection, and then inject them. The in situ curing in vivo by chemical reaction can show good tissue adhesion and rapid degradation ability. However, the previous in situ curing hydrogels based on DF-PEG and carboxymethyl chitosan are not suitable for medullary cavity hemostasis. The main deficiencies are: the in situ curing time is too long, the precursor solution is dispersed by the blood; and the strength of the cured hydrogel is not enough to achieve hemostasis. Therefore, how to improve the in situ curing hydrogel to shorten its curing time and increase the strength of the hydrogel is a problem that needs to be solved.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing an in-situ curing hydrogel, so as to alleviate the technical problems that the existing in-situ curing hydrogel based on DF-PEG and chitosan or its derivatives has a long curing time and poor strength after curing.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In a first aspect, a method for preparing an in-situ curing hydrogel is provided, the method comprising: mixing a first precursor solution and a second precursor solution to obtain the in-situ curing hydrogel;

[0010] The first precursor solution contains chitosan or its derivatives and drugs, and the concentration of the chitosan or its derivatives is 6-12% w / v;

[0011] The second precursor solution contains DF-PEG and Cu 2+ , the concentration of DF-PEG is 10-15% w / v, Cu 2+ The concentration is 1.9~5.6mmol / L.

[0012] In a second aspect, a hydrogel prepared by the method for preparing the in-situ curing hydrogel described in the first aspect is also provided.

[0013] In a third aspect, a composition for preparing an in-situ curing hydrogel is provided, the composition comprising the first precursor solution described in the first aspect and the second precursor solution described in the second aspect, which are separately packaged.

[0014] In the fourth aspect, a method for preparing the in situ curing hydrogel described in the first aspect, the hydrogel described in the second aspect, or the composition described in the third aspect in the preparation of wound hemostatic materials, cell scaffold materials, biological tissue engineering materials or biological inks is also provided.

[0015] In a fifth aspect, a wound hemostasis product is also provided, wherein the wound hemostasis product comprises the in situ curing hydrogel composition described in the third aspect and an injection device.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The in-situ cured hydrogel obtained by the preparation method provided by the present invention is prepared by adjusting the concentrations of chitosan or its derivatives and DF-PEG in the precursor solution so that the cross-linking strength of the hydrogel after curing can meet a good blocking effect. After simulation of the in vitro liquid sealing experiment, the blocking time can reach more than 7 days, and the blocking effect is very stable. By loading drugs, the hydrogel matrix is ​​endowed with potential therapeutic effects. For example, in some embodiments, loading thrombin further improves the coagulation effect. The in-situ cured hydrogel obtained by the preparation method is also loaded with copper ions (Cu 2+), copper ions can form coordination bonds with the aminocarboxyl groups of chitosan to improve the mechanical properties of the hydrogel, and copper ions also have antibacterial functions and promote angiogenesis. The in-situ cured hydrogel obtained by the preparation method provided by the present invention can be injected into the position of medullary cavity bleeding, has a simple method of use, and has a fast in-situ curing speed, and can best achieve medullary cavity blocking within 1 minute. Therefore, the in-situ cured hydrogel provided by the present invention can be used to simply and efficiently achieve medullary cavity hemostasis, which is of great significance for solving the problem of lacunar bone bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The storage modulus (G') and loss modulus (G") of the hydrogel matrix in Example 2 as a function of time;

[0020] Figure 2 The SEM morphology of the freeze-dried hydrogel prepared in Example 2, scale bar 200 μm;

[0021] Figure 3 The SEM morphology of the freeze-dried hydrogel prepared in Example 2, scale bar 400 μm;

[0022] Figure 4 The pore size distribution of the hydrogel prepared in Example 2 after freeze-drying;

[0023] Figure 5 The infrared structure of the hydrogel prepared in Example 2 is shown in the figure;

[0024] Figure 6 is the curve of BCI value change over time in each experimental group in Example 3;

[0025] Figure 7 The curve showing the change of BCI value of the hydrogel loaded with different concentrations of thrombin over time in Example 4;

[0026] Figure 8 is the hemolysis rate of the hydrogel loaded with different concentrations of thrombin in Example 4;

[0027] Fig. 9 Example 5 contains Cu 2+ Stress loss of hydrogel;

[0028] Fig.10 For Example 5, there is no Cu2+ Stress loss of hydrogel. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] It should be noted that:

[0031] In the present invention, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution.

[0032] In the present invention, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0033] In the present invention, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution.

[0034] In the present invention, unless otherwise specified, the numerical range "a-b" represents an abbreviation of any real number combination between a and b, where a and b are real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" have been listed in this document, and "6-22" is just an abbreviation of these numerical combinations.

[0035] The “range” disclosed in the present invention is in the form of a lower limit and an upper limit, which can be one or more lower limits, and one or more upper limits, respectively.

[0036] In the present invention, unless otherwise specified, each reaction or operation step can be carried out in sequence or not. Preferably, the reaction method herein is carried out in sequence.

[0037] In the present invention, unless otherwise specified, arbitrary numbering is used to distinguish one entity or behavior from another entity or behavior, and does not necessarily require or imply any actual relationship, order or importance between these entities or behaviors, such as numbering first or second.

[0038] In the present invention, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or environment can but need not occur, and the description includes occasions where the event or environment occurs or does not occur.

[0039] In the present invention, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but do not exclude any other elements, integers or steps.

[0040] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0041] In a first aspect, a method for preparing an in-situ curing hydrogel is provided, the method comprising mixing a first precursor solution and a second precursor solution to obtain the in-situ curing hydrogel.

[0042] The first precursor solution contains chitosan or its derivatives and drugs, and the concentration of chitosan or its derivatives is 6-12% w / v, for example, but not limited to 6, 7, 8, 9, 10, 11 or 12 w / v.

[0043] In an optional embodiment, the drug includes a procoagulant drug. Optionally, the procoagulant drug includes but is not limited to one or more of thrombin, tranexamic acid, epinephrine, coagulation factor VIII concentrate, prothrombin complex and phenethylamine.

[0044] In an optional embodiment, the drug in the first precursor solution includes thrombin. Thrombin contains amino groups and does not undergo a cross-linking reaction when mixed with chitosan or a derivative thereof, so thrombin is distributed into the first precursor solution.

[0045] In an optional embodiment, the first precursor solution contains 50-200 U / mL thrombin, and the concentration of thrombin can be, for example, but not limited to, 50, 100, 150 or 200 U / mL thrombin.

[0046] In an optional embodiment, the chitosan derivative includes one or more of carboxymethyl chitosan, hydroxymethyl chitosan and hydroxyethyl chitosan.

[0047] The second precursor solution contains DF-PEG and Cu 2+ , the concentration of DF-PEG is 10-15% w / v, Cu 2+ The concentration of Cu is 1.9~5.6mmol / L. 2+ It can be rapidly cross-linked with chitosan or its derivatives to form a solid gel, but the concentration should be controlled in trace amounts, otherwise the cross-linking speed will be too fast. 2+ This concentration range does not affect the speed of in situ curing of the hydrogel.

[0048] DF-PEG is a polyethylene glycol modified with double-terminal aldehyde groups. In an optional embodiment, the molecular weight of DF-PEG is 2000.

[0049] In an optional embodiment, the Cu 2+ It is provided by one or more salts of the halide, sulfate, nitrate and phosphate species, preferably by cupric chloride.

[0050] In an optional embodiment, the first precursor solution and the second precursor solution are mixed in equal volumes.

[0051] In an optional embodiment, the first precursor solution contains 8-10% w / v carboxymethyl chitosan; the second precursor solution contains 15% w / v DF-PEG. In this embodiment, the curing time of the in-situ cured hydrogel does not exceed 1 minute, and has good plugging ability.

[0052] In an optional embodiment, the first precursor solution contains 6-12% w / v carboxymethyl chitosan and 50-200 U / mL thrombin; the second precursor solution contains 10-15% w / v DF-PEG and 1.9-5.6 mmol / L copper chloride; the first precursor solution and the second precursor solution are mixed in equal volumes to obtain the in-situ cured hydrogel. This embodiment contains both thrombin and copper chloride, which can achieve better hemostasis, antibacterial, and angiogenesis-promoting effects.

[0053] In a second aspect, a hydrogel prepared by the method for preparing the in-situ curing hydrogel described in the first aspect is also provided.

[0054] In a third aspect, a composition for preparing an in-situ curing hydrogel is provided, the composition comprising the first precursor solution described in the first aspect and the second precursor solution described in the second aspect, which are separately packaged. When used, the two are mixed to obtain the in-situ curing hydrogel.

[0055] In the fourth aspect, a method for preparing the in situ curing hydrogel described in the first aspect, the hydrogel described in the second aspect, or the composition described in the third aspect in the preparation of wound hemostatic materials, cell scaffold materials, biological tissue engineering materials or biological inks is also provided.

[0056] In an optional embodiment, the wound hemostatic material is used for bone wound bleeding.

[0057] In an optional embodiment, the wound hemostatic material is used for cancellous bone wound bleeding and / or medullary cavity bleeding.

[0058] In a fifth aspect, a wound hemostasis product is also provided, the wound hemostasis product comprising the composition of the in situ curing hydrogel described in the third aspect and an injection device. The injection device is used to mix the first precursor solution and the second precursor solution in the product and inject them into the wound.

[0059] In an optional embodiment, the injection device is a syringe.

[0060] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0061] Example 1

[0062] Screening and preparation of hydrogel matrix:

[0063] According to the concentrations in Table 1, DF-PEG solution and carboxymethyl chitosan solution were added. At room temperature, the two solutions were drawn separately with a syringe and mixed in a volume ratio of 1:1. The gelation time of the hydrogel was recorded using the vial tilting method, and the plugging time of the cured hydrogel was recorded using an in vitro liquid seal. The results are shown in Table 1. It can be seen from Table 1 that the concentrations of DF-PEG and carboxymethyl chitosan solutions as precursors can shorten the gelation time and prolong the plugging time. When the DF-PEG concentration in the precursor solution is 15w / v%, and the carboxymethyl chitosan concentration is 8-10w / v%, the gelation time is less than 1min, and the plugging time is greater than seven days, which is better than other solutions.

[0064] Table 1

[0065]

[0066] Example 2

[0067] The performance of the hydrogel matrix with a DF-PEG concentration of 15 w / v% and a carboxymethyl chitosan concentration of 10 w / v% in Example 2 was investigated:

[0068] 15% (w / v%) DF-PEG solution and 10% (w / v%) carboxymethyl chitosan solution were respectively drawn with a syringe at room temperature and mixed in a volume ratio of 1:1. The gelation time of the hydrogel was recorded using the vial tilting method, the plugging time of the cured hydrogel was recorded using an in vitro liquid seal, rheological analysis was performed using a rheometer, and the porous morphology was observed using a SEM. The results are as follows:

[0069] (1) Gel time: 30s~50s;

[0070] (2) Blockage time: more than seven days.

[0071] (3) Rheological analysis: As a typical operation, 0.2 mL of chitosan solution was coated on the parallel plates of the rheometer. (The chitosan solution has high viscosity and can be stirred evenly before use.) Then, 0.2 mL of DF-PEG aqueous solution was evenly dropped onto the surface of the chitosan solution and quickly mixed by stirring with a needle. The analysis of the change of storage modulus and loss modulus with frequency was carried out using a steel plate (diameter: 20 mm) at 1% strain and 6.3 rad s. The storage modulus (G') and loss modulus (G") were then recorded as a function of time (n = 3). The results are shown in Figure 2. Figure 1 As shown, the results show that the storage modulus (G') and the loss modulus (G") reach an intersection point (gel point) at 40s, corresponding to the gel time. As time goes by, the storage modulus (G') continues to increase, indicating that the strength of the hydrogel is enhanced and the plugging is more stable.

[0072] (4) Morphology analysis: The hydrogel was freeze-dried and its morphology was observed under a scanning electron microscope (SEM) at 5 kV. Figure 2-3 As shown, the hydrogel has a porous morphology; the pore size distribution of the freeze-dried hydrogel is shown in Figure 4 As shown, it can be seen that the pore size is mainly concentrated in the range of 80 to 100 μm. The larger pore size is conducive to blood aggregation and cell passage.

[0073] (5) Infrared structure such as Figure 5 As shown: Through infrared analysis, it can be seen that there are imine bonds in the hydrogel, proving the occurrence of in situ cured Schiff base reaction.

[0074] Example 3

[0075] The hydrogel matrix with a DF-PEG concentration of 15 w / v% and a carboxymethyl chitosan concentration of 10 w / v% in Example 2 was used as a drug-carrying group to load thrombin and examine its performance:

[0076] 1. Preparation of in situ curing hydrogel:

[0077] Add 0.5g of carboxymethyl chitosan powder to 4.5mL of aqueous solution and stir evenly. Prepare 1000U / mL solution of thrombin, draw 0.5mL of thrombin solution and add it to the stirred carboxymethyl chitosan solution, and continue to stir evenly. In this way, a 10% (w / v%) carboxymethyl chitosan solution containing 100U / mL of thrombin is obtained. At the same time, prepare 15% (w / v%) DF-PEG solution. At room temperature, use a syringe to draw these two solutions separately, mix the solutions in a volume ratio of 1:1, and perform a coagulation test.

[0078] 2. Coagulation test:

[0079] 1. Randomly recruit a healthy volunteer (male, age 37 years old) and collect 2 mL of peripheral blood sample through vacuum vein.

[0080] 2. Cut the sample into appropriate size and place it at the bottom of a 24-well plate with the test surface facing upward. Cut the medical gauze into appropriate size and place it at the bottom of a 24-well plate as a control.

[0081] 3. Take 20μL 0.1M CaCl2 and 200μL anticoagulated whole blood and add them to a 1.5mL centrifuge tube. Gently blow and mix with a pipette 5 times. Use a pipette to draw 10μL of calcium-recombined anticoagulated whole blood and add it to the surface of the sample and gauze, and try to keep the blood in the middle of the sample. Draw 10μL of calcium-recombined anticoagulated whole blood and add it to the bottom of the 24-well plate as a control. Incubate the samples and blank controls in a 37℃ incubator (under saturated humidity conditions) for 3, 10, 20, 40 and 60 minutes; after the incubation time is reached, add 1mL of deionized water to the 24-well plate along the edge and incubate it in a constant temperature incubator at 37℃ for 5 minutes to cause hemolysis of the uncoagulated red blood cells.

[0082] 4. Add 10 μL of calcium-replenished anticoagulated whole blood used at the corresponding incubation time point directly into 1 mL of deionized water and incubate at 37°C for 5 min as a reference.

[0083] 5. After incubation, use a pipette to draw the supernatant and add it to a new 1.5mL centrifuge tube. Centrifuge at 3000rmp for 10min in a high-speed centrifuge. Draw 200μL of the last supernatant from the centrifuge tube and add it to a 96-well plate. Use an ELISA reader to measure the absorbance of the solution at a wavelength of 545nm. Repeat the test 3 times for each group of sample supernatant solutions and take the average value. The larger the absorbance value of the solution, the higher the hemoglobin concentration in the solution, the more red blood cells are dissolved, and the lower the degree of blood coagulation. Vice versa.

[0084] 6. The blood anticoagulation degree (BCI) of the sample is calculated according to the following formula:

[0085] BCI = Ia / Iw×100%, where Ia represents the absorbance of the solution after calcium-recombined anticoagulated whole blood contacts the sample for a set time; Iw represents the absorbance of the solution after anticoagulated whole blood is mixed with deionized water (reference). The smaller the BCI value, the higher the degree of blood coagulation, indicating that the material has a higher degree of blood coagulation. Vice versa.

[0086] The experimental results are as follows Figure 6 As shown in the figure, under the experimental conditions, the blank control is the positive control group, and the calcium-recombined whole blood can coagulate on the surface of the culture dish, indicating that the coagulation function of the experimental blood sample is normal. Compared with the non-drug-loaded hydrogel sample, the BCI curve of the 100U / mL thrombin-loaded hydrogel sample moves downward, indicating that the blood coagulates faster in the drug-loaded hydrogel sample over time. For the gauze sample, the blood sample is completely dispersed in the gauze, and the degree of blood coagulation does not change significantly over time.

[0087] Example 4

[0088] 1. Using the hydrogel matrix with a DF-PEG concentration of 15 w / v% and a carboxymethyl chitosan concentration of 10 w / v% in Example 2 as the drug-carrying group, different concentrations of thrombin were loaded:

[0089] Add 0.5g carboxymethyl chitosan powder to 4.75mL of aqueous solution and stir evenly. Prepare 1000U / mL thrombin solution, draw 0.25mL thrombin solution and add it to the stirred carboxymethyl chitosan solution, and continue to stir evenly. In this way, a 10% (w / v%) carboxymethyl chitosan solution containing 50U / mL thrombin is obtained. At the same time, prepare a 15% (w / v%) DF-PEG solution. At room temperature, use a syringe to draw these two solutions separately and mix the solutions in a volume ratio of 1:1.

[0090] Add 0.5g of carboxymethyl chitosan powder to 4.75mL of aqueous solution and stir evenly. Prepare 1000U / mL solution of thrombin, draw 0.5mL of thrombin solution and add it to the stirred carboxymethyl chitosan solution, and continue to stir evenly. In this way, a 10% (w / v%) carboxymethyl chitosan solution containing 100U / mL of thrombin is obtained. At the same time, prepare 15% (w / v%) DF-PEG solution. At room temperature, use a syringe to draw these two solutions separately and mix the solutions in a volume ratio of 1:1.

[0091] Add 0.5g of carboxymethyl chitosan powder to 4.25mL of aqueous solution and stir evenly. Prepare 1000U / mL of thrombin solution, draw 0.75mL of thrombin solution and add it to the stirred carboxymethyl chitosan solution, and continue to stir evenly. In this way, a 10% (w / v%) carboxymethyl chitosan solution containing 150U / mL of thrombin is obtained. At the same time, prepare a 15% (w / v%) DF-PEG solution. At room temperature, use a syringe to draw these two solutions separately and mix the solutions in a volume ratio of 1:1.

[0092] Add 0.5g carboxymethyl chitosan powder to 4mL of aqueous solution and stir evenly. Prepare 1000U / mL thrombin solution, draw 1mL thrombin solution and add it to the evenly stirred carboxymethyl chitosan solution, and continue to stir evenly. In this way, a 10% (w / v%) carboxymethyl chitosan solution containing 200U / mL thrombin is obtained. At the same time, prepare 15% (w / v%) DF-PEG solution. At room temperature, use a syringe to draw these two solutions separately and mix the solutions in a volume ratio of 1:1.

[0093] 2. Performance test of thrombin-loaded hydrogel:

[0094] For the above hydrogel matrix and the thrombin hydrogel loaded with four concentrations, coagulation test and hemolysis test were performed. The method of coagulation test is consistent with that of Example 3, which is a test method for dynamic coagulation of whole blood. The method of hemolysis test is as follows:

[0095] A healthy volunteer (male, 41 years old) was randomly recruited, and 2 mL of peripheral blood specimens were collected by vacuum vein; the anticoagulated whole blood was centrifuged at 1000 rpm for 10 min, 0.2 mL of the lower red blood cell sediment was taken and added to a 1.5 mL centrifuge tube, 1.0 mL of normal saline was added, and the mixture was gently inverted and centrifuged at 1000 rpm for 10 min. The supernatant in the centrifuge tube was carefully aspirated, and then 0.25 mL of normal saline was added to the centrifuge tube to dilute the red blood cells and prepare a red blood cell suspension for use. 0.4 g of each of the #1 to #5 samples was accurately weighed and placed in a 5 mL centrifuge tube, irradiated with ultraviolet light for 15 min, and 4.0 mL of PBS (extract concentration was 0.1 g / mL) was added with a pipette, and the extract was prepared by constant temperature incubation at 37 °C for 24 h. Take 1mL of sample extract from #1 to #5, incubate at 37℃ for 30min, then add 20μL of red blood cell suspension, and incubate at 37℃ for 1h; take 2.5mg of sample #6 and #7 with precision weighing and add them to 1.5mL centrifuge tubes, irradiate with UV light for 15min, add 1.0mL PBS with a pipette, incubate at 37℃ for 30min, then add 20μL of red blood cell suspension, and incubate at 37℃ for 1h. Add 20μL of red blood cell suspension to 1mL of PBS as a negative control, and add 20μL of red blood cell suspension to 1mL of deionized water as a positive control, and incubate at 37℃ for 1h. Both the experiment and the control were repeated 3 times. After incubation, the sample was centrifuged at 2000g for 10min, and 0.2mL of supernatant was aspirated and transferred to a 96-well plate. The absorbance at 545nm was measured with an ELISA reader, and photos of the centrifuge tubes were taken at the same time. The relative hemolysis rate of red blood cells in the sample is calculated by the formula: (D t -D nc ) / (D pc -D nc )×100%Wherein, D t is the absorbance of the experimental sample; D nc is the absorbance of the negative control group; D pc It is the absorbance of the positive control group. (The red blood cell hemolysis rate is ≤5%, indicating that the material meets the hemolysis rate requirements of medical materials and will not cause severe hemolysis reaction; the red blood cell hemolysis rate is >5%, indicating that the material will have hemolysis reaction)

[0096] Experimental results Figure 7 and Figure 8As shown. The results of the whole blood dynamic coagulation test showed that the coagulation effect of the drug-loaded hydrogel was significantly improved compared with the non-drug-loaded hydrogel, and the ability to promote coagulation increased with the increase of thrombin content. The hemolysis results showed that the above hydrogels all met the national standard requirements (hemolysis rate less than 5%) and were blood compatible. According to the above test results, the hemostatic effect of 200U / mL of thrombin was the best.

[0097] Example 5

[0098] The hydrogel loaded with thrombin in Example 3 was further loaded with copper chloride, and its performance was examined:

[0099] 1. Add 0.5g carboxymethyl chitosan powder to 4.5mL aqueous solution and stir evenly. Mix 0.1g, 0.01g and 0.005g of copper chloride directly with chitosan. It is found that the carboxymethyl chitosan solution immediately forms a water-insoluble solid after the addition of copper chloride. Therefore, the experiment confirms that copper chloride cannot be directly added to the carboxymethyl chitosan solution.

[0100] 2. Add 0.5g of carboxymethyl chitosan powder to 4.5mL of aqueous solution and stir evenly. Then prepare thrombin into a 1000U / mL solution, draw 1mL of thrombin solution and add it to the stirred carboxymethyl chitosan solution, continue to stir evenly, and obtain a 10% (w / v%) carboxymethyl chitosan solution containing 200U / mL of thrombin. At the same time, prepare a 15% (w / v%) DF-PEG solution, that is, dissolve 0.6g DF-PEG in 4mL of aqueous solution, and then add 3mg of copper chloride. At room temperature, use a syringe to draw these two solutions separately, and mix the solutions in a volume ratio of 1:1. The gel time and plugging time were tested according to the method of Example 1. The results showed that the gel time was about 30s and the plugging time was greater than 7 days. It shows that dissolving copper chloride in DF-PEG solution first and then preparing in situ cured hydrogel does not affect the curing and curing performance of the hydrogel.

[0101] 3. Performance test of in-situ cured hydrogel loaded with thrombin and copper chloride:

[0102] Mechanical properties characterization: A compression test was performed using a universal testing machine to compare the stress loss after loading copper ions. The greater the stress loss, the worse the mechanical properties of the material will be. The parameters were set to: speed 13, deformation 40%, and compression test. Sample preparation: The hydrogel was cured and demolded in a cylindrical mold with a height of 6mm and a smooth surface.

[0103] The experimental results are as follows Fig. 9 and Fig.10 As shown, under the experimental conditions, compared with the 2+ The drug-loaded hydrogel samples, containing Cu2+ The stress loss of the drug-loaded hydrogel sample is smaller, indicating that the mechanical properties of the material are improved after the addition of copper ions. It has more stable mechanical properties and improved hardness, so it will have a better blocking effect as a hemostatic material.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an in-situ cured hydrogel, characterized in that: include: Mixing the first precursor solution and the second precursor solution to obtain the in-situ cured hydrogel; The first precursor solution contains chitosan or its derivatives and drugs, and the concentration of the chitosan or its derivatives is 6-12% w / v; The second precursor solution contains DF-PEG and Cu 2+ , the concentration of DF-PEG is 10-15% w / v, Cu 2+ The concentration is 1.9~5.6mmol / L.

2. The preparation method according to claim 1, characterized in that: The first precursor solution and the second precursor solution are mixed in equal volumes.

3. The preparation method according to claim 1, characterized in that: The chitosan derivatives include one or more of carboxymethyl chitosan, hydroxymethyl chitosan and hydroxyethyl chitosan; Optionally, the molecular weight of DF-PEG is 2000; Optionally, the Cu 2+ provided by one or more salts of the halides, sulfates, nitrates and phosphates; Optionally, the Cu 2+ Provided by cupric chloride.

4. The method for preparing an in-situ curing hydrogel according to claim 1, characterized in that: The drugs include procoagulant drugs: Optionally, the procoagulant drug includes one or more of thrombin, tranexamic acid, adrenaline, coagulation factor VIII concentrate, prothrombin complex and phenethylamine; Optionally, the first precursor solution contains 50-200 U / mL thrombin.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The first precursor solution contains 8-10% w / v carboxymethyl chitosan; the second precursor solution contains 15% w / v DF-PEG; Optionally, the first precursor solution contains 6-12% w / v carboxymethyl chitosan and 50-200 U / mL thrombin; the second precursor solution contains 10-15% w / v DF-PEG and 1.9-5.6 mmol / L copper chloride; the first precursor solution and the second precursor solution are mixed in equal volumes to obtain the in situ cured hydrogel.

6. The hydrogel prepared by the method for preparing an in-situ curing hydrogel according to any one of claims 1 to 5.

7. A composition for preparing an in-situ curing hydrogel, characterized in that: The method comprises independently packaging the first precursor solution described in any one of claims 1 to 5 and the second precursor solution described in any one of claims 1 to 5.

8. Use of the method for preparing the in situ curing hydrogel according to any one of claims 1 to 5, the hydrogel according to claim 6, or the composition according to claim 7 in preparing wound hemostatic materials, cell scaffold materials, biological tissue engineering materials or biological inks.

9. The use according to claim 8, characterized in that: The wound hemostatic material is used for bleeding of bone wounds; Optionally, the wound surface hemostatic material is used for cancellous bone wound surface bleeding and / or medullary cavity bleeding.

10. A wound hemostasis product, characterized in that: Comprising the composition of claim 7 and an injection device.

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