A alginate macroporous hemostatic material constructed with calcium carbonate as a sacrificial template

Abstract

The application discloses a kind of alginate macroporous hemostatic material constructed with calcium carbonate as sacrifice template, comprising the following steps: (1) sodium alginate is dissolved with calcium carbonate powder in ultrapure water, and stirring is carried out until the mixture becomes uniform paste;(2) L-galactonic acid-1, 4-lactone is added to the paste, and stirring is uniformly carried out;(3) the mixture is placed at 4 DEG C to form hydrogel, and then immersed in hydrochloric acid solution to obtain macroporous hydrogel.By using calcium carbonate as a sacrificial template to prepare a macroporous system, the uniform pore structure can be used to load bioactive clotting factors, which can accelerate platelet aggregation and initiate the coagulation cascade, ensuring excellent hemostatic performance of the material. The gel material is soft, and can fully meet the requirements of complex wound environment for the form of hemostatic material.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of alginate macroporous hemostatic material constructed with calcium carbonate as sacrificial template, mainly applied to sudden situation in wound bleeding out of control management. BACKGROUND

[0002] In some emergencies (traffic accidents, natural disasters), about one-third of pre-hospital deaths are caused by uncontrolled bleeding. In addition, more than half of the preventable battlefield deaths are caused by limb injuries and blood loss. There are also high-risk severe bleeding in the medical field, especially in spinal and liver surgery. In the above-mentioned situations, if handled properly, mortality can be effectively reduced, so effective hemostatic control is crucial, not only in battlefield and emergency civilian environment, but also during trauma surgery.

[0003] Current hemostatic materials are in various forms, such as gauze, glue, powder, and hydrogel, etc. Although hemostatic materials are diverse in form, hydrogel is favored by the majority of researchers due to its excellent swelling performance and the advantage of fitting the wound environment. For example, patent CN2015800393592 discloses a preparation method of macroporous aerogel based on alginate; patent CN2020111932823 discloses a hydrogel sheet of alginate particles and a preparation method thereof; patent CN2020108740940 discloses a preparation method of high-strength self-healing multilayer hydrogel based on alginate. As above, alginate hydrogel is diverse in form and widely used, but there are few studies on macroporous hydrogel at present. Macroporous hydrogel is a heterogeneous hydrogel with pore size in the micron range, which greatly exceeds the grid size of cross-linked polymer, and has become an important and novel branch of hydrogel materials. The main advantages of macroporous hydrogel compared to ordinary gel materials are: the explicit internal structure (porosity) can effectively reduce the diffusion resistance, and then can have other functions by loading drugs; the macroporous structure of the gel can increase the contact area with red blood cells and platelets, and accelerate the coagulation process. SUMMARY

[0004] The purpose of the present application is to construct a kind of alginate macroporous hemostatic material constructed with calcium carbonate as sacrificial template, which is a macroporous system prepared by calcium carbonate as sacrificial template. The present application provides a method for effectively managing bleeding out of control by the macroporous hemostatic material.

[0005] In order to achieve the above technical scheme, the present application relates to a kind of alginate macroporous hemostatic material constructed with calcium carbonate as sacrificial template, and the preparation method comprises the following steps:

[0006] (1) Dissolve sodium alginate and calcium carbonate powder in ultrapure water, and stir until the mixture becomes a uniform sticky paste.

[0007] (2) Add L-galacton-1,4-lactone to the paste and stir until well mixed.

[0008] (3) The mixture was placed at 4°C to form a hydrogel, and then immersed in hydrochloric acid solution to obtain a macroporous hydrogel.

[0009] The present invention relates to step (1) where the viscosity of sodium alginate is 350 mPa·s, the size of calcium carbonate powder is 20-60 mesh, and the mass ratio of sodium alginate to calcium carbonate is 10:1, 10:2, 10:3, 10:4 and 10:5.

[0010] In step (2) of this invention, the mass ratio of L-galacton-1,4-lactone to sodium alginate is 1:40.

[0011] The present invention relates to step (3) of storing at 4°C for at least 12 hours, with a hydrochloric acid solution concentration of 1% and a soaking time of 15 minutes.

[0012] Compared with the prior art, the present invention has the following advantages: (1) low cost, simple preparation, easy operation, easy large-scale production and storage; (2) good biocompatibility of alginate materials; (3) the prepared macroporous structure can increase the contact area between the material and red blood cells and platelets; (4) the hydrogel is soft and easy to fit the complex wound environment. Attached Figure Description

[0013] Appendix Figure 1 This is a SEM image of the external structure of Embodiment 1 of the present invention;

[0014] Appendix Figure 2 The porosity and swelling properties of Example 1 of this invention;

[0015] Appendix Figure 3 The rheological behavior of Embodiment 1 of the present invention;

[0016] Appendix Figure 4 This invention relates to Example 1, which describes the adhesion of red blood cells and platelets.

[0017] Appendix Figure 5 This is Example 1 of the in vivo experiment involved in the present invention.

[0018] Appendix Figure 6 Example 1 of the present invention is a cytotoxicity experiment.

[0019] DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0021] Example 1:

[0022] (1) Gel SEM characterization

[0023] SEM imaging of lyophilized macroporous sodium alginate hydrogels was performed using a Hitachi SU3500 microscope with an accelerating voltage of 5 kV. (See attached image.) Figure 1 As shown in the figure, the gel has a uniform macroporous structure.

[0024] (2) Determination of porosity and swelling properties

[0025] First, the alginate gel was freeze-dried. Then, a pre-weighed sample was immersed in 10 mL of anhydrous ethanol. After 5 minutes, the sample was removed, residual ethanol was removed from the surface, and the sample was weighed again. The porosity of the material was calculated as follows: Porosity = (N1 - N0) / ρV0, where N0 and N1 represent the mass of the material before and after immersion in ethanol, respectively. V0 represents the volume of the freeze-dried sample, and ρ represents the density of anhydrous ethanol (0.789 g / cm³). -3 ). As attached Figure 2 As shown in Figure A, the porosity can reach up to 50%, indicating that the material has a high porosity.

[0026] The macroporous sodium alginate gel sample was weighed, and then 200 μL of fresh New Zealand rabbit heart blood was injected into the hydrogel using a 1 mL syringe. After wiping off any remaining blood, the sample was weighed again. The blood absorption capacity of the material was calculated as follows: Blood absorption rate = (M1 - M0) / M b Where M0 and M1 represent the mass of the material before and after blood injection, respectively, M b Indicates blood quality. (See attached image) Figure 2 As shown in B, the gel has an absorption rate of up to 47% for blood, indicating that the material has good swelling properties.

[0027] (3) Study on rheological behavior

[0028] The rheological behavior of hydrogels was characterized using a cone-plate geometry on a Thermo Scientific Haake MARS III modular rheometer. (See attached image.) Figure 3 As shown in Figure A, at a fixed temperature but different frequencies, alginate materials exhibit stable typical hydrogel properties and possess strong mechanical strength. (Appendix) Figure 3 As shown in Figure B, at a fixed frequency and different temperatures, the mechanical strength of alginate gel changes with increasing temperature, and the two are directly proportional.

[0029] (4) Adhesion of red blood cells and platelets

[0030] Freeze-dried macroporous hydrogels with different porosities were cut into 1×1×0.5cm pieces. 3The hydrogel was placed in a 5 mL beaker containing 3 mL of New Zealand rabbit heart blood, diluted with PBS (pH = 7.4) to four times its volume, and incubated at 37 °C for 1 h. The hydrogel was then washed three times with PBS, fixed with 2.5% (v / v) glutaraldehyde / PBS for 1.5 h, and washed three times with ultrapure water. Next, it was dehydrated sequentially with 25%, 50%, 75%, and 100% (v / v) ethanol / PBS solutions, and then air-dried at 37 °C. Characterization was performed by SEM, and the results are attached. Figure 4 As shown, the material's macroporous structure can effectively accumulate platelets and red blood cells on its surface, accelerating the coagulation process.

[0031] (5) Live animal experiments

[0032] Male rats (approximately 250g) were first anesthetized by intraperitoneal injection of chloral hydrate and fixed in a supine position on a surgical cork board throughout the experiment. A 4-8mm incision was made in the right lobe of the liver to induce hepatic hemorrhage. 1mL of macroporous hydrogel (pre-embedded with TF) was rapidly applied to the bleeding wound, and clotting time was recorded. The amount of blood loss was also recorded by weighing the filter paper after the blood loss. (See attached image.) Figure 5 As shown, this macroporous material can carry bioactive hemostatic drugs for sustained release, further accelerating the coagulation process and demonstrating the material's excellent hemostatic properties.

[0033] (6) Cytotoxicity test

[0034] The cell compatibility of the macroporous hydrogel was assessed by co-culturing NIH-3T3 mouse fibroblasts with macroporous hydrogel extract. 3 mL of macroporous hydrogel sample was immersed in 10 mL of DMEM complete medium at 37°C for 16 h to obtain the extract. NIH-3T3 cells were then cultured in the macroporous hydrogel extract medium in a humidified incubator containing 5% CO2 for 24 h. After culturing, the medium was removed, and the cells were washed twice with sterile Tris-HCl buffer (pH 7.4), stained with calcein and propidium iodide, and observed under an inverted fluorescence microscope. (See attached image.) Figure 6 As shown in the image, no obvious red fluorescence is observed, indicating that the macroporous gel has good biocompatibility.

Claims

1. A method for preparing alginate macroporous hemostatic material constructed with calcium carbonate as a sacrificial template, characterized by, Specifically comprising the following steps: (1) Dissolve sodium alginate with viscosity of 350 mPa.s and calcium carbonate powder with size of 20-60 mesh in ultrapure water, and stir until the mixture becomes a uniform sticky paste; (2) Add L-galactonic acid-1,4-lactone and sodium alginate of step (1) into the paste according to the mass ratio of 1:40, and stir uniformly; (3) Place the mixture at 4℃ to form a hydrogel, and then immerse it in a hydrochloric acid solution to obtain a macroporous hydrogel.

2. The method for preparing alginate macroporous hemostatic material using calcium carbonate as a sacrificial template according to claim 1, characterized in that, In step (1), the mass ratio of sodium alginate to calcium carbonate is 10:1, 10:2, 10:3, 10:4 and 10:

5.

3. A method for preparing alginate macroporous hemostatic material constructed using calcium carbonate as a sacrificial template according to claim 2, characterized in that, In step (3), the storage time at 4℃ is at least 12 h, the concentration of the hydrochloric acid solution is 1%, and the soaking time is 15 min.

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