Hemostatic powder with rapid imbibition and antibacterial functions and preparation method thereof

Abstract

The invention discloses hemostatic powder with rapid high-strength imbibition capacity and an antibacterial function and a preparation method thereof.The preparation method includes the steps that chitosan derivatives and deionized water are mixed to be uniform, and a chitosan derivative solution is prepared; the preparation method comprises the following steps: adding acrylic acid and methacryloyloxyethyl trimethyl ammonium chloride into a chitosan derivative solution, uniformly mixing, adding a cross-linking agent and a photoinitiator, carrying out polymerization reaction to obtain a cross-linked product, freeze-drying, and grinding to obtain the hemostatic powder with rapid liquid absorption and antibacterial functions. According to the hemostatic powder disclosed by the invention, a material which is good in biocompatibility, relatively strong in antibacterial function, rapid in imbibition and capable of stopping bleeding in time is obtained by regulating and controlling the proportion of polycations and anions, and the problems that a cationic material is poor in biocompatibility, and the hemostatic powder is poor in imbibition capacity are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and in particular relates to a hemostatic powder with rapid liquid absorption and antibacterial functions and a preparation method thereof. Background Art

[0002] Wound bleeding control, especially non-compressible wound bleeding, accounts for 30% to 40% of preventable deaths, ranking first and is a huge challenge faced by military injuries and accident trauma worldwide. Developing strategies to control non-compressible bleeding is urgent.

[0003] In response to non-compressible bleeding, researchers focus on developing more effective hemostatic materials and have developed different forms of hemostatic materials, such as hemostatic sponges, adhesives, and powders. For irregularly shaped wounds, powders can be directly applied to the wound to fill the irregular shape and absorb blood to concentrate coagulation factors to achieve the purpose of hemostasis. However, most powders have insufficient liquid absorption capacity, which affects the hemostatic effect. Therefore, there is an urgent need to develop hemostatic powders with efficient liquid absorption capacity.

[0004] Chitosan is a natural cationic polymer with strong biocompatibility, biodegradability, antibacterial and hemostatic properties, which can promote the whole process of wound healing. However, most chitosan hemostatic powders have poor water solubility, weak tissue adhesion and mechanical properties, and it is difficult to form an effective physical blockage. Chitosan modified with quaternary ammonium salts (such as HTCC) has good water solubility, strong antibacterial activity and can enhance hemostatic properties. However, the cytotoxicity of HTCC limits its application in biomedicine. The molecular chain of polyacrylic acid (PAA) contains a large number of carboxylic acid groups and has good hydrophilicity. In the solution, it can form a network structure with positively charged polymers through electrostatic interaction and hydrogen bonds. [2-(Methacryloyloxy)ethyl]trimethylammonium chloride (MTAC) contains a cationic unit -N(CH3)3, and there is electrostatic repulsion generated by ionic charges in its polymer network, which can lock more water. Therefore, the introduction of MTAC is expected to prepare a new type of hemostatic material with super liquid absorption ability.

[0005] Therefore, there is an urgent need in the art for a rapid hemostatic material with good biosafety, strong liquid absorption capacity, certain tissue adhesion and antibacterial function. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a hemostatic powder having rapid and high-strength liquid absorption capacity and antibacterial function.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for preparing a hemostatic powder with rapid and high-strength liquid absorption ability and antibacterial function, comprising:

[0010] The chitosan derivative is mixed evenly with deionized water to prepare a chitosan derivative solution;

[0011] Acrylic acid and methacryloyloxyethyl trimethyl ammonium chloride are added to a chitosan derivative solution, mixed evenly, a crosslinking agent and a photoinitiator are added to carry out polymerization reaction to obtain a crosslinked product, which is freeze-dried and ground to obtain a hemostatic powder with rapid liquid absorption and antibacterial functions.

[0012] As a preferred embodiment of the preparation method of the present invention, the chitosan derivative comprises one or more of chitosan quaternary ammonium salt and carboxymethyl chitosan.

[0013] As a preferred solution of the preparation method of the present invention, the mass concentration of the chitosan derivative solution is 5-10%.

[0014] As a preferred embodiment of the preparation method of the present invention, the molar ratio of methacryloyloxyethyl trimethyl ammonium chloride to acrylic acid is 0.01-3:0.01-3, and the molar ratio of methacryloyloxyethyl trimethyl ammonium chloride to acrylic acid is preferably 1:2.

[0015] As a preferred embodiment of the preparation method of the present invention, the cross-linking agent includes polyethylene glycol diacrylate, and the photoinitiator includes 2-hydroxy-2-methyl-1-phenyl-1-propanone.

[0016] As a preferred embodiment of the preparation method of the present invention, the crosslinking agent accounts for 2% to 4% of the total molar mass of methacryloyloxyethyl trimethyl ammonium chloride and acrylic acid, and the photoinitiator accounts for 2% to 4% of the total molar mass of methacryloyloxyethyl trimethyl ammonium chloride and acrylic acid.

[0017] As a preferred embodiment of the preparation method of the present invention, the polymerization reaction includes photopolymerization under ultraviolet 365nm, and the photopolymerization reaction time is 50 to 70 minutes.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide a hemostatic powder with rapid and high-strength liquid absorption capacity and antibacterial function.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a hemostatic powder for use in preparing medical hemostatic materials.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention provides a method for preparing a hemostatic powder having rapid and high-strength liquid absorption capacity and antibacterial function. By adjusting the ratio of polycations and anions, a material having good biocompatibility, strong antibacterial function, rapid liquid absorption and timely hemostasis is obtained, thereby solving technical problems such as poor biocompatibility of cationic materials and poor liquid absorption capacity of hemostatic powders.

[0022] (2) The hemostatic powder of the present invention can quickly absorb blood and achieve rapid hemostasis by adsorbing and aggregating red blood cells and platelets through positive charge; in the tail vein bleeding model and the femoral artery puncture bleeding model, the hemostatic powder has less blood loss and shorter hemostasis time than commercially available hemostatic materials.

[0023] (3) The hemostatic powder of the present invention has a simple preparation process, high productivity, and is capable of large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0025] Figure 1 This is a diagram showing an experiment on the liquid absorption capacity of the hemostatic powder prepared in the present invention.

[0026] Figure 2 This is a diagram showing the cytotoxicity test of the hemostatic powder prepared in the present invention.

[0027] Figure 3 This is a graph showing the antibacterial performance test of the hemostatic powder prepared by the present invention.

[0028] Figure 4 This is a diagram showing the in vitro coagulation test of the hemostatic powder prepared by the present invention.

[0029] Figure 5 This is a diagram showing the in vivo coagulation test of the hemostatic powder prepared in the present invention. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0033] Chitosan quaternary ammonium salt (HTCC, degree of substitution>95%) in the examples of the present invention was purchased from Maclean (Shanghai, China);

[0034] Acrylic acid (AA, >99% by weight in water) was purchased from Aladdin (Shanghai, China);

[0035] Methacryloyloxyethyltrimethylammonium chloride solution (MTAC, 75% by weight in water) was purchased from Aladdin (Shanghai, China);

[0036] Polyethylene glycol diacrylate (PEGDA, molecular weight 600, weight percentage in water 98%) was purchased from Aladdin (Shanghai, China);

[0037] 2-Hydroxy-2-methylpropanone (1173, 97% by weight in water) was purchased from Aladdin (Shanghai, China).

[0038] Other raw materials were purchased from commercial sources and used directly without any special treatment.

[0039] Example 1

[0040] This embodiment provides a method for preparing a hemostatic powder with rapid liquid absorption and antibacterial functions, the main steps of which are:

[0041] (1) Add 0.2 g of chitosan quaternary ammonium salt into 2 mL of deionized water and stir evenly to prepare a 10% chitosan quaternary ammonium salt solution;

[0042] (2) adding 2.77 mL (1 mol) of methacryloyloxyethyl trimethyl ammonium chloride solution and 1.44 mL (2 mol) of acrylic acid solution to the chitosan quaternary ammonium salt solution prepared in (1), and finally adding water to 10 mL;

[0043] (3) After the mixed solution in step (2) is stirred evenly, 10.5 mg of PEGDA and 10.5 mg of photocrosslinker 1173 are added, stirred evenly and injected into the mold, and placed under 365 nm ultraviolet light for polymerization for 60 min;

[0044] (4) After the reaction is completed, the hydrogel is taken out, freeze-dried, and ground into powder, which is named HTCC-pM1A2.

[0045] Example 2

[0046] This embodiment provides a method for preparing a hemostatic powder with rapid liquid absorption and antibacterial functions, the main steps of which are:

[0047] (1) Add 0.2 g of chitosan quaternary ammonium salt into 2 mL of deionized water and stir evenly to prepare a 10% chitosan quaternary ammonium salt solution;

[0048] (2) adding 4.155 mL (1.5 mol) of methacryloyloxyethyl trimethyl ammonium chloride solution and 1.08 mL (1.5 mol) of acrylic acid solution to the chitosan quaternary ammonium salt solution prepared in (1), and finally adding water to 10 mL;

[0049] (3) After the mixed solution in step (2) is stirred evenly, 12.6 mg of PEGDA and 12.6 mg of photocrosslinker 1173 are added, stirred evenly and injected into the mold, and placed under 365 nm ultraviolet light for polymerization for 50 min;

[0050] (4) After the reaction is completed, the hydrogel is taken out, freeze-dried, and ground into powder, which is named HTCC-pM 1.5 A 1.5 .

[0051] Example 3

[0052] This embodiment provides a method for preparing a hemostatic powder with rapid liquid absorption and antibacterial functions, the main steps of which are:

[0053] (1) Add 0.2 g of chitosan quaternary ammonium salt into 2 mL of deionized water and stir evenly to prepare a 10% chitosan quaternary ammonium salt solution;

[0054] (2) adding 5.54 mL (2 mol) of methacryloyloxyethyl trimethyl ammonium chloride solution and 0.72 mL (1 mol) of acrylic acid solution to the chitosan quaternary ammonium salt solution prepared in (1), and finally adding water to 10 mL;

[0055] (3) After the mixed solution in step (2) is stirred evenly, 14.6 mg of PEGDA and 14.6 mg of photocrosslinker 1173 are added, stirred evenly and injected into the mold, and placed under 365 nm ultraviolet light for polymerization for 40 min;

[0056] (4) After the reaction is completed, the hydrogel is taken out, freeze-dried, and ground into powder, which is named HTCC-pM2A1.

[0057] Comparative Example 1

[0058] (1) Add 0.2 g of chitosan quaternary ammonium salt into 2 mL of deionized water and stir evenly to prepare a 10% chitosan quaternary ammonium salt solution;

[0059] (2) adding 2.16 mL (3 mol) of acrylic acid solution to the chitosan quaternary ammonium salt solution prepared in (1), and finally adding water to 10 mL;

[0060] (3) After the mixed solution in step (2) is stirred evenly, 6.48 mg of PEGDA and 6.48 mg of photocrosslinker 1173 are added, stirred evenly and injected into the mold, and placed under 365 nm ultraviolet light for polymerization for 90 min;

[0061] (4) After the reaction is completed, the hydrogel is taken out, freeze-dried, and ground into powder, which is named HTCC-pA.

[0062] Comparative Example 2

[0063] (1) Add 0.2 g of chitosan quaternary ammonium salt into 2 mL of deionized water and stir evenly to prepare a 10% chitosan quaternary ammonium salt solution;

[0064] (2) adding 8.310 mL (3 mol) of methacryloyloxyethyl trimethyl ammonium chloride solution to the chitosan quaternary ammonium salt solution prepared in (1);

[0065] (3) After the mixed solution in step (2) is stirred evenly, 19 mg of PEGDA and 19 mg of photocrosslinker 1173 are added, stirred evenly and injected into the mold, and placed under 365 nm ultraviolet light for polymerization for 30 minutes.

[0066] (4) After the reaction is completed, the hydrogel is taken out and the prepared hydrogel is freeze-dried and ground into powder, which is named pM.

[0067] Figure 1 For the liquid absorption experiment of Examples 1 to 3 and Comparative Examples 1 to 2, the blood absorption amount was measured as follows:

[0068] Deionized water was added to 100 mg of the hemostatic powder until the hemostatic powder no longer absorbed the liquid.

[0069] Using commercial hemostatic powder BYC as control, record the mass of hemostatic powder as m mg and calculate the liquid absorption ratio;

[0070] Liquid absorption ratio = (m-100) / 100×100%;

[0071] Repeat the test 3 times and take the average value.

[0072] from Figure 1 It can be seen that as the content of methacryloyloxyethyltrimethylammonium chloride (MTAC) increases, the water absorption increases. The results show that HTCC-pMA has a strong ability to absorb liquid.

[0073] Figure 2 The cytotoxicity test of Examples 1 to 5 is performed as follows:

[0074] Rat fibroblast (RS1) cells were seeded in 96-well plates in medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a density of 10,000 cells per well;

[0075] After growing for 24 h in a humidified 5% CO incubator at 37 °C, the culture medium was replaced with PBS extracts (1:10) containing different hemostatic powders (commercial hemostatic powders BYC, pM, HTCC-pA, and HTCC-pMA) and DMEM medium at a ratio of 1:9;

[0076] After incubating the cells for 24 h, the viability of the cells was tested by using a cell proliferation-toxicity assay (CCK-8) and measured by a microplate reader at 450 nm;

[0077] Complete DMEM medium containing PBS and medium containing 10 mg / mL zinc diethyldithiocarbamate were used as negative and positive controls.

[0078] from Figure 2 It can be seen that the cell survival rate of commercial hemostatic powder BYC and HTCC-pA powder is greater than 80%, while the cell survival rate of HTCC-pMA is related to the content of MTAC. When the molar ratio of MTAC to AA is 1:2, the cell survival rate of powder is greater than 80%, while that of pM and HTCC-pMA is 1.5 A 1.5 Due to the high MTAC content, the cell survival rate of HTCC-pM2A1 is less than 80%, and it has high cytotoxicity; through balance, the optimal ratio of HTCC-pMA powder is selected as 1:2.

[0079] Good antibacterial ability is also an aspect that should be considered when applied to wound hemostasis. Therefore, the antibacterial effect of hemostatic powder on Escherichia coli and Staphylococcus aureus was explored. The test method is as follows:

[0080] 10 mg of different hemostatic powders (BYC, HTCC-pA and HTCC-pM1A2) were mixed with 1 mL of diluted E. coli suspension (10 5 CFU / mL) were co-cultured in a 37°C incubator for 4 h, and PBS was used as a blank control. 100 μL of the above suspension was spread on an agar plate and incubated at 37°C for 12 h.

[0081] The results are as follows Figure 3 The results showed that HTCC-pM1A2 hemostatic powder can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus, provide a sterile environment for wound hemostasis, and reduce bacterial infection.

[0082] Figure 4 For in vitro hemostatic performance experiments, the test method is as follows:

[0083] 100 μL of 0.1 mol / L calcium chloride aqueous solution was added to 900 μL of citrated blood and vortexed for 10 s. 10 mg of different hemostatic powders (BYC, HTCC-pA, and HTCC-pM1A2) were placed in consecutive wells on a 96-well plate, and 100 μL of recalcified blood was dripped onto the different hemostatic powders;

[0084] Then, at the preset time point, the coagulated part was removed with PBS;

[0085] The time required for uniform and stable clots to form in the wells after washing was defined as the clotting time.

[0086] from Figure 4 It can be seen that the clotting time of HTCC-pM1A2 powder is 30s, which is shorter than other materials. The results show that HTCC-pM1A2 powder can achieve a good hemostatic effect in vitro.

[0087] In order to demonstrate the hemostatic performance of the hemostatic powder obtained in this example in vivo, this experiment evaluated the hemostatic effects of different powders (BYC, HTCC-pA and HTCC-pM1A2) using a rat tail-broken bleeding model:

[0088] The rats were randomly divided into 4 groups (n=3), and the rats were anesthetized with 2% sodium phenobarbital before surgery;

[0089] Place a pre-weighed filter paper on the rat's tail, cut the tail 8 cm from the tail root with surgical scissors, and quickly place 100 mg of the powder on the rat's tail;

[0090] The amount of bleeding and bleeding time were recorded, and rats without hemostatic treatment were used as the control group.

[0091] The results are as follows Figure 5 Compared with the blank group, BYC powder and HTCC-pA powder, HTCC-pM1A2 powder had less blood loss. This result showed that HTCC-pM1A2 powder could effectively achieve hemostasis in vivo and had great potential value in clinical application.

[0092] 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 preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing a hemostatic powder having rapid and high-strength liquid absorption capacity, antibacterial function and good biocompatibility, characterized in that: include, The chitosan derivative is mixed evenly with deionized water to prepare a chitosan derivative solution; Acrylic acid and methacryloyloxyethyl trimethyl ammonium chloride are added to the chitosan derivative solution, mixed evenly, and a crosslinking agent and a photoinitiator are added to carry out polymerization reaction to obtain a crosslinked product, which is freeze-dried and ground to obtain a hemostatic powder with rapid and high liquid absorption capacity, antibacterial function and biocompatibility.

2. The preparation method according to claim 1, characterized in that: The chitosan derivatives include one or more of chitosan quaternary ammonium salt and carboxymethyl chitosan.

3. The preparation method according to claim 1 or 2, characterized in that: The mass concentration of the chitosan derivative solution is 5-10%.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the methacryloyloxyethyl trimethylammonium chloride to acrylic acid is 0.01-3:0.01-3.

5. The preparation method according to claim 4, characterized in that: The crosslinking agent includes polyethylene glycol diacrylate, and the photoinitiator includes 2-hydroxy-2-methyl-1-phenyl-1-propanone.

6. The preparation method according to claim 5, characterized in that: The crosslinking agent accounts for 2% to 4% of the total molar mass of methacryloyloxyethyl trimethyl ammonium chloride and acrylic acid, and the photoinitiator accounts for 2% to 4% of the total molar mass of methacryloyloxyethyl trimethyl ammonium chloride and acrylic acid.

7. The preparation method according to claim 1 or 6, characterized in that: The polymerization reaction includes a photopolymerization reaction under ultraviolet 365nm, and the reaction time is 50 to 70 minutes.

8. The hemostatic powder prepared by the preparation method according to any one of claims 1 to 7, which has rapid and high liquid absorption capacity, antibacterial function and good biocompatibility.

9. Use of the hemostatic powder as claimed in claim 8 in the preparation of medical hemostatic materials.

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