Preparation method of recombinant collagen-based antibacterial hemostatic powder

Abstract

The invention discloses a preparation method of recombinant collagen-based antibacterial hemostatic powder, which comprises the following steps: mixing a recombinant collagen solution, a sodium polyacrylate solution and a quaternized chitosan solution, freeze-drying, and grinding. Based on the principle that molecular chains are wound to form gel through electrostatic interaction between negative charges of sodium polyacrylate and positive charges of quaternized chitosan, a novel hemostatic material design is inspired, recombinant collagen, sodium polyacrylate and a quaternized chitosan solution are blended, freeze-dried and ground, and the hydrogel is prepared. The recombinant collagen-based styptic powder is prepared. The styptic powder does not contain a cross-linking agent, avoids the risk of residual toxicity, and combines the high water absorption of sodium polyacrylate, the hemostatic and antibacterial characteristics of quaternized chitosan and the excellent biocompatibility of recombinant collagen. The material not only can realize instantaneous hemostasis of a bleeding part, but also can remarkably reduce the infection risk and accelerate wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of hemostatic materials, and in particular relates to a method for preparing recombinant collagen-based antibacterial hemostatic powder. Background Art

[0002] Uncontrollable bleeding refers to bleeding that cannot be effectively controlled by conventional hemostatic means (such as compression, suturing or drugs), which usually occurs in cases of major trauma, surgery or certain diseases (such as coagulation disorders). It can quickly lead to hemorrhagic shock and organ failure, and has a high mortality rate. Uncontrollable bleeding is more common in cases of traumatic brain injury, spleen rupture, etc. Traditional hemostatic methods such as pressure, suturing or drugs are sometimes difficult to stop bleeding quickly, especially in complex clinical environments. In order to meet this challenge, the development of fast and effective hemostatic materials and technologies, such as biological hemostatic agents and surgical colloids, has become an important direction of current medical research.

[0003] At the current stage, hemostatic powder is mainly used to quickly control external bleeding. Its principle is to absorb water or promote coagulation reaction, form a physical barrier or accelerate the aggregation of coagulation factors. Common hemostatic powders include biomaterials such as chitosan and fibrinogen. Such products are widely used in battlefield first aid, trauma care and surgical operations. However, its disadvantages are: limited effect on large-area bleeding, arterial bleeding or internal bleeding; may cause local tissue inflammation or allergic reaction; reduced hemostatic effect in high humidity environment; and some products degrade slowly in the body, which may cause foreign body reaction. Therefore, the development of more efficient, safe and complex environment adaptable hemostatic materials has become the focus of current research.

[0004] Therefore, in view of the above situation, there is an urgent need to develop a hemostatic powder with good biocompatibility and good hemostatic effect that can adapt to various irregular bleeding conditions to overcome the shortcomings in current practical applications. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a recombinant collagen-based antibacterial hemostatic powder in view of the deficiencies of the above-mentioned prior art. The method is based on the principle that the molecular chains are entangled to form a gel by the electrostatic interaction between the negative charge of sodium polyacrylate and the positive charge of quaternized chitosan, which inspires the design of a new hemostatic material. Recombinant collagen, sodium polyacrylate and quaternized chitosan solution are blended and freeze-dried and ground to prepare a recombinant collagen-based hemostatic powder. The prepared hemostatic powder does not contain a cross-linking agent, thereby avoiding the risk of residual toxicity. At the same time, it combines the high water absorption of sodium polyacrylate, the hemostatic and antibacterial properties of quaternized chitosan, and the excellent biocompatibility of recombinant collagen, which can not only achieve instantaneous hemostasis at the bleeding site, but also significantly reduce the risk of infection and accelerate wound healing.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a recombinant collagen-based antibacterial hemostatic powder, characterized in that it includes: mixing a recombinant collagen solution, a sodium polyacrylate solution and a quaternary ammonium chitosan solution, freeze-drying and then grinding.

[0007] The above-mentioned method for preparing a recombinant collagen-based antibacterial hemostatic powder is characterized in that the recombinant collagen is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen.

[0008] The above-mentioned method for preparing a recombinant collagen-based antibacterial hemostatic powder is characterized in that the mixing method includes: uniformly mixing the recombinant collagen solution and the sodium polyacrylate solution to obtain a mixed solution, and then dripping the quaternized chitosan solution into the mixed solution under stirring conditions;

[0009] Or it includes: uniformly mixing the recombinant collagen solution and the quaternized chitosan solution to obtain a mixed solution, and then dripping the sodium polyacrylate solution into the mixed solution under stirring conditions.

[0010] The method for preparing the above-mentioned recombinant collagen-based antibacterial hemostatic powder is characterized in that the concentration of the recombinant collagen solution is not greater than 200 mg / mL.

[0011] The method for preparing the above-mentioned recombinant collagen-based antibacterial hemostatic powder is characterized in that the concentration of the sodium polyacrylate solution is 5wt% to 15wt%, and the molecular weight of the sodium polyacrylate is 100000 to 300000Da.

[0012] The method for preparing the above-mentioned recombinant collagen-based antibacterial hemostatic powder is characterized in that the concentration of the quaternary ammonium chitosan solution is 5wt% to 15wt%, the molecular weight of the quaternary ammonium chitosan is 100000 to 500000Da, and the degree of substitution is 40% to 98%.

[0013] The method for preparing the above-mentioned recombinant collagen-based antibacterial hemostatic powder is characterized in that the mass ratio of the recombinant collagen, quaternized chitosan and sodium polyacrylate is (0.05-2):5:5.

[0014] Traditional collagen hemostatic materials usually use crosslinking agents, but their residues may cause toxicity problems. The present invention is based on the principle that the electrostatic interaction between the negative charge of sodium polyacrylate and the positive charge of quaternized chitosan causes the molecular chains to entangle to form a gel, which inspires the design of a new hemostatic material. Recombinant collagen-based hemostatic powder is prepared by blending recombinant collagen, sodium polyacrylate and quaternized chitosan solution and then freeze-drying and grinding them.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The hemostatic powder of the present invention does not contain a cross-linking agent, thus avoiding the risk of residual toxicity, and at the same time combines the high water absorption of sodium polyacrylate, the hemostatic and antibacterial properties of quaternized chitosan, and the excellent biocompatibility of recombinant collagen; the hemostatic powder can not only achieve instantaneous hemostasis at the bleeding site, but also significantly reduce the risk of infection and accelerate wound healing.

[0017] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the Zeta potential of the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 of the present invention.

[0019] Figure 2 This is the gel storage modulus of the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 of the present invention after absorbing blood.

[0020] Figure 3 The antibacterial properties of the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 of the present invention.

[0021] Figure 4 This is the hemostatic effect of the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 of the present invention.

[0022] Figure 5 This is the MTT test result of the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 of the present invention.

[0023] Figure 6 The red blood cell adhesion rate of the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 of the present invention

[0024] Figure 7 The platelet adhesion rate of the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0026] Example 1

[0027] Prepare 100mg / mL recombinant collagen solution, 10wt% sodium polyacrylate solution (molecular weight 150000Da) and 10wt% quaternized chitosan solution (molecular weight 400000Da, degree of substitution 95%); then add the prepared recombinant collagen solution to the sodium polyacrylate solution and mix evenly; under stirring, add the quaternized chitosan solution dropwise to the mixed solution, the mass ratio of recombinant collagen, quaternized chitosan and sodium polyacrylate is 1:5:5; freeze-dry and grind to powder to obtain recombinant collagen-based antibacterial hemostatic powder. The recombinant collagen is recombinant type I collagen, which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit and repeated multiple times, wherein the short amino acid sequence is: GAPGAPGSQGAPGL, repeated 52 times, the collagen has good water solubility, a molecular weight of 78862Da, and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.

[0028] Comparative Example

[0029] 10 wt% sodium polyacrylate (molecular weight 150000 Da) and 10 wt% quaternary ammonium chitosan solution (molecular weight 400000 Da, degree of substitution 95%) were mixed evenly, freeze-dried and ground into powder for later use. The final product was recorded as hemostatic powder.

[0030] Example 2

[0031] Prepare 200mg / mL recombinant collagen solution, 5wt% sodium polyacrylate solution (molecular weight 300000Da) and 15wt% quaternized chitosan solution (molecular weight 100000Da, degree of substitution 98%); then add the prepared recombinant collagen solution to the quaternized chitosan solution and mix evenly; under stirring, add the sodium polyacrylate solution dropwise to the mixed solution, the mass ratio of recombinant collagen, quaternized chitosan and sodium polyacrylate is 0.05:5:5; freeze-dry and grind to powder to obtain recombinant collagen-based antibacterial hemostatic powder. The recombinant collagen is recombinant type I collagen, which is composed of the full-length α1 chain sequence from human type II collagen. The collagen has good water solubility and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.

[0032] Example 3

[0033] Prepare 100mg / mL recombinant collagen solution, 15wt% sodium polyacrylate solution (molecular weight 100000Da) and 5wt% quaternized chitosan solution (molecular weight 500000Da, degree of substitution 40%); then add the prepared recombinant collagen solution to the sodium polyacrylate solution and mix evenly; under stirring, add the quaternized chitosan solution dropwise to the mixed solution, the mass ratio of recombinant collagen, quaternized chitosan and sodium polyacrylate is 2:5:5; freeze-dry and grind to powder to obtain recombinant collagen-based antibacterial hemostatic powder. The recombinant collagen is recombinant type III collagen, which is composed of a short amino acid sequence from natural human type III collagen as a repeating unit and repeated multiple times, wherein the short amino acid sequence (GERGAPGFRGPAGPNGIPGEKGPAGER) is repeated 30 times, and the collagen has good water solubility and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.

[0034] The above recombinant collagen may also be recombinant type IV collagen or recombinant human-like collagen.

[0035] The recombinant type IV collagen is composed of the full-length α1 chain sequence of human type IV collagen. The collagen has good water solubility and was purchased from Xi'an Juzi Biogene Technology Co., Ltd.

[0036] The recombinant human-like collagen is a large molecule type I recombinant collagen CF-5, which is composed of type I collagen from human. The collagen has good water solubility and was purchased from Xi'an Juzi Biogene Technology Co., Ltd.

[0037] Zeta potential measurement

[0038] The gel formation of recombinant collagen-based antibacterial hemostatic powder is based on the attraction of positive and negative charges. When the concentration of recombinant type I collagen solution is 100 mg / mL, when the Zeta potential is close to 0, the gelation effect is the best. Therefore, the ratio of sodium polyacrylate to quaternary ammonium chitosan is optimized to 5:5 ( Figure 1 ).

[0039] Storage modulus of recombinant collagen-based antibacterial hemostatic powder after absorbing blood gel

[0040] For storage modulus, strain amplitude test was performed on the hemostatic powder prepared in the comparative example and the recombinant collagen-based antibacterial hemostatic powder prepared in Example 1 using a rheometer. 0.5 g of hemostatic powder and recombinant collagen antibacterial hemostatic powder were weighed into a 10 mL sample bottle and 1 mL of deionized water was added to obtain recombinant collagen antibacterial hemostatic powder gel. The storage modulus of all prepared hemostatic powder gels and recombinant collagen hemostatic powder gels are as follows: Figure 2 As shown, the results showed that the modulus was higher after adding recombinant collagen.

[0041] Antibacterial effect of recombinant collagen-based antibacterial hemostatic powder

[0042] Using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as bacterial models, the bacteria were first activated. Single E. coli and Staphylococcus aureus colonies were taken, respectively, and placed in a 50-mL centrifuge tube containing 10 mL of liquid culture medium. The tube was activated at 37°C and 180 r / min for 10 to 14 hours. The activated bacterial solution was then diluted 1000 times, 100 μL of the diluted activated bacterial solution was added to 10 mL of liquid culture medium, and then 10 mg of the recombinant collagen-based antibacterial hemostatic powder sample prepared in Example 1 was added and co-cultured for 12 hours. During this period, the changes in the OD value of the bacterial solution were monitored as follows: Figure 3 As shown, it shows that recombinant collagen-based hemostatic powder has a strong antibacterial effect.

[0043] Hemostasis

[0044] The hemostatic effect of recombinant collagen-based antibacterial hemostatic powder was investigated through a rat liver hemostasis experiment. Figure 4 As shown in the figure, it can be seen that the recombinant collagen-based antibacterial hemostatic powder significantly reduced the amount of liver bleeding in rats. The amount of bleeding in the blank group was as high as 290 mg, while the amount of bleeding in the hemostatic powder group was only 33 mg.

[0045] Biocompatibility

[0046] The extract was prepared according to the method recommended by GB / T 16886.5-2017 for in vitro cell compatibility testing using MTT and cell scratch methods. Figure 5 The results showed that the recombinant collagen hemostatic powder extract prepared in Example 1 could effectively promote the proliferation of human epidermal fibroblasts (HSF), and the 24h cell proliferation rate was 131.25%, indicating that it had good biocompatibility.

[0047] Red blood cell adhesion rate

[0048] Fresh citrate whole blood of SD rats was centrifuged at 400g for 10min to obtain red blood cell suspension (diluted with PBS, 10% v / v PBS, 10% v / v) and platelet-rich plasma (PRP). When performing the RBCs adhesion test, 100μL was dropped on 10 mg of hemostatic powder, incubated at 37°C for 30min, and then the sample was rinsed with PBS to remove non-adherent RBCs, and then deionized water (4mL) was added to dissolve the attached RBCs, and the absorbance value of the supernatant at a wavelength of 540nm was measured. Compared with the control example, the recombinant collagen antibacterial hemostatic powder significantly increased the red blood cell adhesion rate, and its red blood cell adhesion rate was 78.2% ( Figure 6 ).

[0049] Platelet adhesion rate

[0050] 40 μL of PRP was dropped on 10 mg of sample and then incubated at 37°C for 5 min. Unattached platelets were removed by rinsing with PBS, and then the sample was immersed in 1 mL of 1% Triton X-100 to completely dissolve attached platelets (37°C, 1 h). The released lactate dehydrogenase (LDH) content was measured using an LDH kit. The results showed that recombinant collagen significantly increased the platelet adhesion rate of hemostatic powder. The platelet adhesion rate of recombinant collagen hemostatic powder was 82.1% ( Figure 7 ).

[0051] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a recombinant collagen-based antibacterial hemostatic powder, characterized in that: The method comprises: mixing a recombinant collagen solution, a sodium polyacrylate solution and a quaternary ammonium chitosan solution, and then grinding the mixture after freeze-drying.

2. The method for preparing a recombinant collagen-based antibacterial hemostatic powder according to claim 1, characterized in that: The recombinant collagen is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen.

3. The method for preparing a recombinant collagen-based antibacterial hemostatic powder according to claim 1, characterized in that: The mixing method includes: uniformly mixing the recombinant collagen solution and the sodium polyacrylate solution to obtain a mixed solution, and then dropping the quaternized chitosan solution into the mixed solution under stirring conditions; Or it includes: mixing the recombinant collagen solution and the quaternized chitosan solution evenly to obtain a mixed solution, and then dropping the sodium polyacrylate solution into the mixed solution under stirring conditions.

4. The method for preparing a recombinant collagen-based antibacterial hemostatic powder according to claim 1, characterized in that: The concentration of the recombinant collagen solution is not greater than 200 mg / mL.

5. The method for preparing a recombinant collagen-based antibacterial hemostatic powder according to claim 1, characterized in that: The concentration of the sodium polyacrylate solution is 5wt%-15wt%, and the molecular weight of the sodium polyacrylate is 100000-300000Da.

6. The method for preparing a recombinant collagen-based antibacterial hemostatic powder according to claim 1, characterized in that: The concentration of the quaternary ammonium chitosan solution is 5wt%-15wt%, the molecular weight of the quaternary ammonium chitosan is 100000-500000Da, and the degree of substitution is 40%-98%.

7. The method for preparing a recombinant collagen-based antibacterial hemostatic powder according to claim 1, characterized in that: The mass ratio of recombinant collagen, quaternary ammonium chitosan and sodium polyacrylate is (0.05~2):5:5.

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