Kaolin / oxidized microcrystalline cellulose hemostatic antibacterial hydrogel bandage and preparation method thereof

Abstract

The invention provides a kaolin / oxidized microcrystalline cellulose hemostatic antibacterial hydrogel bandage preparation method, which comprises: A) mixing an acrylic acid solution, kaolin, oxidized microcrystalline cellulose grafted N-hydroxysuccinimide and deionized water to obtain a mixture solution; and B) adding a free radical initiator and a cross-linking agent into the mixture solution, putting the mixture solution into a mold, and reacting to obtain the hemostatic antibacterial hydrogel bandage. By adding uniformly dispersed kaolin and oxidized microcrystalline cellulose into the gel precursor solution, the tensile strength and elongation can be improved. The hydrogel bandage prepared by the invention has relatively high strength and toughness, and shows good tensile property. The gel bandage has good adhesion to tissues, and has good adhesion strength through a shear tensile test (ASTM F2255). The adhesive can be quickly adhered to the surface of bleeding tissue, and continuous exudation of tissue blood is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of materials, and in particular to a kaolin / oxidized microcrystalline cellulose hemostatic and antibacterial hydrogel bandage and a preparation method thereof. Background Art

[0002] Emergency bleeding often faces the problem of large blood flow and fast blood flow rate. Uncontrollable bleeding may cause a significant drop in blood pressure, severe anemia, organ failure, and even shock. These symptoms often pose a great threat to life and require immediate emergency bleeding intervention. However, in the event of emergency bleeding, the blood flow is often large and the flow rate is fast, and platelets cannot quickly form a stable thrombus. Due to the complexity of the hemostasis process, conventional surgical procedures are often accompanied by the occurrence of various problems, such as vascular damage, wound infection, and wound adhesion, which cause pain and inconvenience to patients. At the same time, emergency hemostatic agents are different from clinical hemostatic materials. Their operation is often not performed by professionals, and the requirements for their portability, ease of use, and storage are more stringent. The ideal first aid hemostatic material should have the following characteristics: (1) good biocompatibility, no hemolytic reaction and organ toxicity; (2) able to quickly stop bleeding from arterial and venous bleeding within 2 minutes; (3) simple and easy to use, no need for additional training, and able to meet the requirements of personnel for rapid hemostasis in complex trauma situations; (4) excellent mechanical properties, able to match various wound tissues, and not affecting the movement of the injured; (5) anti-bacterial infection, easy to store, so as to be used under harsh conditions. (6) simple preparation, so as to facilitate large-scale manufacturing.

[0003] At present, the hemostatic materials on the market usually include chitosan hemostatic powder, fibrin sealants (such as Tisseel), α-cyanoacrylate tissue adhesives, gelatin hemostatic sponges, etc. Although they show good hemostatic effects, they may have various disadvantages in emergency bleeding scenarios. For example, chitosan hemostatic powder may cause allergic reactions in people who are allergic to shellfish; fibrin sealants often need to be refrigerated and are not suitable for use in outdoor emergency scenarios; α-cyanoacrylate has potential cytotoxicity, and gelatin hemostatic sponges have poor mechanical strength and may swell and compress nearby tissues.

[0004] Hemostatic bandages are widely used in emergency and complex scenarios because they are light, easy to use, and can quickly seal bleeding wounds. At present, cotton gauze bandages are still the most widely used emergency hemostatic materials in clinical practice, but they cannot effectively promote platelet aggregation and activation of coagulation reactions, have low hemostatic efficiency, and lack tissue adhesion and antibacterial properties. Specifically, they have the following disadvantages: (1) They cannot effectively promote platelet aggregation and activation of coagulation reactions, and only rely on blood absorption to stop bleeding, with low hemostatic efficiency; (2) They have poor elasticity, which may limit the movement of joints and cause inconvenience to patients; (3) They do not have tissue adhesion and often require additional medical tape for fixation; (4) They do not have antibacterial properties, which brings inconvenience to storage and use.

[0005] Therefore, in order to avoid secondary damage to surrounding tissues and seamlessly match skin tissue, higher requirements are placed on the elasticity and toughness of the hemostatic bandage, and it is particularly important to provide a new type of hemostatic bandage. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a kaolin / oxidized microcrystalline cellulose hemostatic and antibacterial hydrogel bandage. The bandage provided by the present invention has high strength, elasticity, fatigue resistance, strong adhesion performance and good antibacterial effect.

[0007] The present invention provides a method for preparing a kaolin / oxidized microcrystalline cellulose hemostatic and antibacterial hydrogel bandage, comprising the following steps: A) mixing an acrylic acid solution, kaolin, oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide and deionized water to obtain a mixture solution; B) adding a free radical initiator and a cross-linking agent into the mixture solution, placing the mixture in a mold, and reacting the mixture to obtain a hemostatic and antibacterial hydrogel bandage.

[0008] The invention firstly prepares oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide.

[0009] According to the present invention, the preparation method of oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide comprises: S1) Avicel, TEMPO, NaBr and NaClO initiate an oxidation reaction, and the product is washed to obtain oxidized Avicel; S2) The oxidized microcrystalline cellulose, EDC and NHS are mixed for reaction, and the product is dialyzed with water to obtain the oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide.

[0010] The preparation method of oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide provided by the invention firstly initiates oxidation reaction of microcrystalline cellulose, TEMPO, NaBr and NaClO.

[0011] In some embodiments, microcrystalline cellulose is dissolved in water, and then TEMPO, NaBr and NaClO are added to initiate an oxidation reaction.

[0012] In one specific embodiment, the mass ratio of microcrystalline cellulose, TEMPO, NaBr and NaClO is 5:0.1:1.5:5-10; In one specific embodiment, the mass ratio of microcrystalline cellulose, TEMPO, NaBr and NaClO is 5:0.1:1.5:6; according to the present invention, the reaction time is 5.5-6.5h, preferably 6h; the reaction temperature is 20-30°C; the reaction pH is 9.5-10.5, preferably 10.

[0013] After the reaction was completed, the product was washed with anhydrous ethanol and centrifuged at least 3 times to obtain oxidized microcrystalline cellulose.

[0014] The inventors have found that the above-mentioned oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide can improve the adhesion performance of the hydrogel bandage to tissues.

[0015] The oxidized microcrystalline cellulose, EDC and NHS are mixed for reaction; In some embodiments, the molar ratio of the oxidized microcrystalline cellulose, EDC and NHS is 3:3:(1-15); The reaction temperature is 20~30℃; the reaction time is 1.5~2.5h; The reaction product is preferably dialyzed with distilled water for 3 days, and finally centrifuged and dried to obtain OMCC-NHS.

[0016] The invention firstly mixes acrylic acid solution, kaolin, oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide and deionized water to obtain a mixture solution.

[0017] The concentration of the acrylic acid solution of the present invention is preferably 10% to 60%; most preferably 50%; According to the present invention, the mass ratio of the volume mL of pure acrylic acid, g of kaolin and g of oxidized microcrystalline cellulose grafted N-hydroxysuccinimide is 30:(10-16):(1-5); In some specific embodiments, the mass ratio of the pure acrylic acid, kaolin and oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide is 30:(11-15):(2-4) In some specific embodiments, the volume ratio of the acrylic acid, kaolin and oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide is 30:14:4; According to the present invention, the mass percentage of the oxidized microcrystalline cellulose grafted N-hydroxysuccinimide in the hemostatic antibacterial hydrogel bandage is 1% to 5%; specifically, it can be 1%, 2%, 3%, 4% or 5%; or a range between any two of the above.

[0018] According to the present invention, the mass percentage of kaolin in the hemostatic antibacterial hydrogel bandage is 10% to 16%; specifically, it can be 10%, 11%, 12%, 13%, 14%, 15% or 16%; or a range value between any two of the above.

[0019] Kaolin is a nano-clay with a negatively charged surface, a large specific surface area and a flaky crystal structure. Under the action of the negative charge on the surface of kaolin, it can quickly activate coagulation factor XII, thereby promoting the endogenous coagulation cascade reaction. The nano-sheet structure of kaolin enables it to bind platelets and promote platelet aggregation. The large specific surface area further enhances the adhesion of red blood cells, thereby promoting hemostasis. In addition, its loose structure helps to concentrate blood, further enhancing its hemostatic properties.

[0020] The inventors creatively discovered that the tensile strength and elongation can be improved by adding uniformly dispersed kaolin and oxidized microcrystalline cellulose to the gel precursor solution.

[0021] Kaolin or oxidized cellulose can bring about changes in the morphology and structure of polyacrylic acid hydrogels. Multi-crosslinked hydrogel bandages can promote the adhesion of red blood cells and platelets and promote the intrinsic coagulation cascade reaction.

[0022] According to the present invention, the mass percentage of the acrylic acid solution in the hemostatic antibacterial hydrogel bandage is preferably 30%.

[0023] The free radical initiator and the crosslinking agent are added to the mixture solution, and then stirred vigorously to form a homogeneous solution.

[0024] In some embodiments, the free radical initiator is ammonium persulfate or potassium persulfate; the mass percentage of the free radical initiator in the hemostatic antibacterial hydrogel bandage is 0.1% to 1%; specifically, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%; or a range between any two of the above.

[0025] According to the present invention, the mixing is performed by stirring and mixing and then ultrasonic treatment in an ice water bath; the mixing speed is 500rpm~700rpm; preferably 600rpm; the mixing time is 4~6min; preferably 5min; the power of the ultrasonic treatment is 300W, and the time is 9~11min; preferably 10min.

[0026] According to the present invention, the cross-linking agent is one or more of N'N-dimethylacrylamide (MBA), methacrylic anhydride-capped polyethylene glycol, acrylate-capped polyethylene glycol, maleic anhydride-capped polyethylene glycol, itaconic anhydride-capped polyethylene glycol, formaldehyde and glutaraldehyde; the mass percentage of the cross-linking agent in the hemostatic antibacterial hydrogel bandage is 0.01% to 0.1%; specifically, it can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%; or a range value between any two of the above.

[0027] The mixed solution is poured into a mold and reacted to obtain a hemostatic and antibacterial hydrogel bandage.

[0028] According to the present invention, the mold is a silicone mold.

[0029] In some embodiments, the reaction temperature is 60-80° C., and the reaction time is 0.5-1.5 h.

[0030] In some embodiments, the reaction temperature is 65-75° C., and the reaction time is 0.8-1.3 h.

[0031] In some embodiments, the reaction temperature is 70° C. and the reaction time is 1 h.

[0032] The present invention adopts Kaolin nano-components to synthesize a multifunctional hemostatic hydrogel bandage with negative charge, high strength, elasticity, fatigue resistance, strong adhesion and antibacterial properties through a one-pot method. This multifunctional hydrogel bandage is composed of a polymerized superabsorbent polymer as a skeleton, and a long-chain macromolecular polysaccharide is physically cross-linked to form another layer of network, in which kaolin is used as a physical cross-linking agent.

[0033] The hemostatic bandage of the present invention can be prepared by a one-pot method, which is conducive to large-scale production and convenient for quality control.

[0034] The present invention provides a kaolin / oxidized microcrystalline cellulose hemostatic and antibacterial hydrogel bandage, which is prepared by the preparation method described in any one of the above technical solutions.

[0035] The multifunctional hemostatic bandage developed by the present invention has the following advantages: (1) it has good elasticity and meets the requirements of high strength and high toughness at the same time, and can be used to bandage and tangle wounds; (2) it has good tissue adhesion and can quickly adhere to the surface of bleeding wounds to seal the bleeding wounds without the need for additional tape to fix them, and prevent secondary bleeding; (3) it can simultaneously complete multi-step hemostasis. After physically sealing the bleeding wound, it can quickly absorb blood, promote the adhesion of platelets and red blood cells, accelerate the endogenous coagulation cascade reaction, and achieve rapid hemostasis; (4) it can be painlessly removed by sodium bicarbonate and L-glutathione solution; (5) it has certain antibacterial properties against Staphylococcus aureus and Escherichia coli.

[0036] The hydrogel bandage prepared by the present invention has high strength and toughness, and exhibits good tensile properties (942% ultimate strain and 220 kPa tensile strength). Therefore, it can be used to bandage and tie a knot on the tissue. The gel bandage has good adhesion to the tissue, and has an adhesion strength of 55.02 ± 2.65 kPa through a shear tensile test (ASTM F2255). It can quickly adhere to the surface of the bleeding tissue to avoid continuous exudation of blood from the tissue.

[0037] The hydrogel bandage has good water absorption, can quickly concentrate blood, and promote coagulation. The unique pore structure and partially exposed kaolin of the hydrogel bandage enable it to have good adhesion and aggregation effects on red blood cells and platelets. At the same time, the partially exposed kaolin and negative surface structure enable the hydrogel bandage to activate the endogenous coagulation cascade reaction. These effects, combined with the wound closure brought by good adhesion, make it have excellent emergency hemostasis ability.

[0038] The hemostatic bandage has a good hemostatic effect on rat liver bleeding, femoral artery and tail bleeding, as well as New Zealand rabbit heart puncture and liver injury bleeding.

[0039] The gel bandage also has a certain antibacterial effect, which can seal the wound surface and prevent external bacteria from directly contacting the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a physical picture of the kaolin-polyacrylic acid / oxidized microcrystalline cellulose grafted N-hydroxysuccinimide hydrogel bandage prepared in Example 4 (capable of bandaging, knotting, stretching, adhering, and hanging heavy objects, resisting puncture, and resisting stretching); Figure 2 The following are scanning electron microscope images of the prepared samples; A: polyacrylic acid hydrogel; B: kaolin-polyacrylic acid hydrogel; C: polyacrylic acid / oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide hydrogel; D: kaolin-polyacrylic acid / oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide hydrogel; Figure 3 The photos show the antibacterial effect of the prepared kaolin-polyacrylic acid / oxidized microcrystalline cellulose grafted N-hydroxysuccinimide hydrogel on Escherichia coli and Staphylococcus aureus; Figure 4 The scanning electron micrographs of red blood cells adhered to different samples; Figure 5 Schematic diagram of the preparation process of the sample of Example 2 of the present invention. DETAILED DESCRIPTION

[0041] The present invention provides a method for preparing a kaolin / oxidized microcrystalline cellulose hemostatic antibacterial hydrogel bandage. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0042] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0043] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0044] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0045] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0046] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0047] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0048] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0049] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0050] To further illustrate the present invention, a method for preparing a kaolin / oxidized microcrystalline cellulose hemostatic and antibacterial hydrogel bandage provided by the present invention is described in detail below in conjunction with examples.

[0051] AA: acrylic acid; PAA: polyacrylic acid; Kaolin: kaolin; MCC: microcrystalline cellulose; OMCC: oxidized microcrystalline cellulose; EDC: 1-ethyl-(3-dimethylaminopropyl) carbodiimide; NHS: N-hydroxysuccinimide; OMCC-NHS: oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide; APS: ammonium persulfate; MBA: N'N-dimethylacrylamide Example 1 This embodiment provides a method for preparing an oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide (OMCC-NHS) macromolecular substance, and the method includes the following steps.

[0052] First, 5 g of microcrystalline cellulose powder was added to 500 mL of distilled water and stirred. Subsequently, 100 mg of TEMPO and 1.5 g of NaBr were added. 6 g of NaClO was added to initiate oxidation, and the mixture was continuously stirred for 6 hours at room temperature while maintaining the pH at around 10. After the reaction was completed, the product was washed with anhydrous ethanol and centrifuged at least 3 times to obtain oxidized microcrystalline cellulose. Then, oxidized microcrystalline cellulose, EDC, and NHS (molar ratio of 3:3:1) were added to a round-bottom flask. After 2 hours of reaction, the product was dialyzed with distilled water for 3 days. Finally, OMCC-NHS was obtained by centrifugation and drying.

[0053] Example 2 On the other hand, the present invention provides a method for preparing a Kaolin-PAA / OMCC-NHS hydrogel hemostatic bandage, which comprises the following steps.

[0054] First, the acrylic acid solution was diluted to 50% (v / v). Next, 3 mL of the diluted acrylic acid solution, 0.6 g NaOH, 0.7 g kaolin, 0.2 g OMCC-NHS, and 2 mL DI water were added. The mixture was stirred (600 rpm, 5 min) and then sonicated in an ice-water bath (300 W, 10 min) to obtain a uniform initial solution. Next, 20 mg APS and 2.5 mg MBA were added to the mixture, which was then stirred vigorously to form a uniform solution. The mixture was poured into a silicone mold and kept at 70 °C for 1 h to synthesize Kaolin-PAA / OMCC-NHS (KPON).

[0055] The hydrogel bandage prepared by the present invention has high strength and toughness, and exhibits good tensile properties (942% ultimate strain and 220 kPa tensile strength), so it can be used to bandage and tie tissues.

[0056] The gel bandage has good adhesion to tissues, and has an adhesion strength of 55.02 ± 2.65 kPa through shear tensile test (ASTM F2255). It can quickly adhere to the surface of bleeding tissues to avoid continuous blood seepage from tissues.

[0057] Comparative Example 1 A polyacrylic acid hydrogel was synthesized as a control group. Compared with Example 2, no kaolin and oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide were added. The specific method is as follows: First, a 50% (v / v) deionized (DI) solution of acrylic acid (AA) was prepared. Next, 3 mL of the prepared AA solution, 0.6 g of NaOH, and 2 mL of DI water were added. The mixture was stirred (600 rpm, 5 min), and then 20 mg of APS and 2.5 mg of MBA were added to the mixture and stirred vigorously to form a homogeneous solution. The mixture was poured into a silicone mold and kept at 70 °C for 1 h to synthesize the PAA hydrogel.

[0058] Comparative Example 2 Kaolin-polyacrylic acid hydrogel was synthesized as a control group. Compared with Example 2, OMCC-NHS was not added. The specific method is as follows: First, a 50% (v / v) deionized (DI) solution of acrylic acid (AA) was prepared. Next, 3 mL of the prepared AA solution, 0.6 g of NaOH, 0.7 g of kaolin, and 2 mL of DI water were added. The mixture was stirred (600 rpm, 5 min) and then sonicated in an ice-water bath (300 W, 10 min) to obtain a homogeneous initial solution. Next, 20 mg of APS and 2.5 mg of MBA were added to the mixture, which was then stirred vigorously to form a homogeneous solution. The mixture was poured into a silica gel mold and kept at 70 °C for 1 h to synthesize Kaolin-PAA.

[0059] Comparative Example 3 A polyacrylic acid / oxidized microcrystalline cellulose grafted N-hydroxysuccinimide hydrogel was synthesized as a control group. Compared with Example 2, no OMCC-NHS was added. The specific method is as follows: First, a 50% (v / v) deionized (DI) solution of acrylic acid (AA) was prepared. Next, 3 mL of the prepared AA solution, 0.6 g of NaOH, 0.2 g of OMCC-NHS, and 2 mL of DI water were added. The mixture was stirred (600 rpm, 5 min) and then sonicated in an ice-water bath (300 W, 10 min) to obtain a homogeneous initial solution. Next, 20 mg of APS and 2.5 mg of MBA were added to the mixture, which was then stirred vigorously to form a homogeneous solution. The mixture was poured into a silicone mold and kept at 70 °C for 1 h to synthesize PAA / OMCC-NHS.

[0060] Application Examples Tensile strength test: The tensile test of the hydrogel was carried out on a mechanical testing machine (WDW-500N, Weidu Electronic, China) with a tensile force of 50 N and a tensile speed of 50 mm min -1 The sample size is approximately 20 × 20 × 2 mm. 3 The hydrogel was subjected to cyclic stretching-unloading tests using a universal testing machine (CTM2500, Xie Qiang Instrument, China) at a stretching speed of 10 mm min -1 The rheological properties of the hydrogels were analyzed using a rheometer (HR-10, TA Instruments, USA). The hydrogel samples were loaded between parallel plates with a diameter of 40 mm and a spacing of ≈1 mm. The rheological properties of the hydrogels were analyzed using a rheometer (HR-10, TA Instruments, USA). The hydrogel samples were loaded between parallel plates with a diameter of 40 mm and a spacing of ≈1 mm. -1Frequency sweep tests were performed at a constant strain of 1% over a frequency range of 20 Å. To characterize the stress relaxation behavior of the hydrogels, their stress evolution over time was measured when a constant strain of 20% was applied.

[0061] Table 1 Case Material Tensile strength (kPa) Ultimate strain(%) Elastic modulus (kPa) Comparative Example 1 PAA 25 ± 5 545 ± 48 4 ± 1 Comparative Example 2 Kaolin-PAA 62 ± 5 723 ± 104 21 ± 3 Comparative Example 3 PAA / OMCC-NHS 152 ± 2 673 ± 49 9 ± 1 Example 2 Kaolin-PAA / OMCC-NHS 210 ± 10 1053 ± 111 22 ± 2 As shown in Table 1, Kaolin-PAA / OMCC-NHS has the highest tensile strength, ultimate strain, and elastic modulus, with a maximum tensile strength of 210 ± 10 kPa, an ultimate strain of 1053 ± 111%, and an elastic modulus of 22 ± 2 kPa.

[0062] Adhesion performance test: The adhesion strength of the hydrogels to various substrates was evaluated by lap shear test (ASTM F2255). The test was performed using a universal testing machine (CTM2500, Xieqiang Instrument, China). In this test, the candidate hydrogel was sandwiched between two skin substrates with an adhesion area of ​​20 × 20 mm2. Then, the adhesion was measured at 10 mm min. -1 The tensile test was performed at a speed of 1.5 %. The maximum force measured was divided by the bond area to calculate the shear strength.

[0063] Table 2 Case Material Shear strength(kPa) Comparative Example 1 PAA 10.2 ± 3.5 Comparative Example 2 Kaolin-PAA 6.8 ± 1.9 Example 2 Kaolin-PAA / OMCC-NHS 55.0 ± 2.6 Positive Control Wound Closure Strips 14.4 ± 3.0 As can be seen from Table 2, the shear strengths of PAA, Kaolin-PAA, Kaolin-PAA / OMCC-NHS and commercial anti-slip stickers are 10.2 ± 3.5 kPa, 6.8 ± 1.9 kPa, 55.0 ± 2.6 kPa, and 14.4 ± 3.0 kPa, respectively. Kaolin-PAA / OMCC-NHS in Example 2 has the highest shear strength and has the best adhesion effect on pig skin.

[0064] In vitro coagulation index test: First, different samples (10 mg) were placed in a centrifuge tube, and then 200 μL of blood was inoculated onto each surface of the sample, and 10 μL of CaCl2 solution (0.2M) was immediately added. After 5 minutes, 1 mL of deionized (DI) water was added to the centrifuge tube to dissolve any free red blood cells by gently shaking, and the centrifuge tube was inverted to observe the blood clot. The absorbance of the supernatant was measured at a wavelength of 540 nm using an enzyme label. All experiments were repeated three times. The BCI coagulation index is shown in the formula:

[0065] Table 3 Case Material BCI Blank control none 100.0 ± 0.0 Positive Control bandage 92.7 ± 4.2 Comparative Example 1 PAA 52.8 ± 10.7 Comparative Example 2 Kaolin-PAA 45.8 ± 1.5 Comparative Example 3 PAA / OMCC-NHS 24.4 ± 1.3 Example 2 Kaolin-PAA / OMCC-NHS 13.9 ± 0.3 As shown in Table 3, compared with other comparative examples and the positive control group, Kaolin-PAA / OMCC-NHS has the smallest BCI value and has excellent in vitro hemostatic effect.

[0066] In vitro coagulation time test: The clotting time of the hydrogels was investigated by exposing them to fresh blood and monitoring clot formation over time.

[0067] Table 4 Case Material Coagulation time Blank control none 10.9 ± 0.4 Positive Control bandage 10.5 ± 0.4 Comparative Example 1 PAA 8.1 ± 0.6 Comparative Example 2 Kaolin-PAA 6.7 ± 0.8 Comparative Example 3 PAA / OMCC-NHS 4.4 ± 0.3 Example 2 Kaolin-PAA / OMCC-NHS 3.1 ± 0.3 As shown in Table 4, compared with other comparative examples and the control group, Kaolin-PAA / OMCC-NHS has the shortest in vitro coagulation time, so we speculate that it has excellent in vitro hemostatic effect.

[0068] Rat liver hemostasis test: Animals (male SD rats (250–300 g)) were monitored daily for signs of pain or discomfort during the experimental period. After general anesthesia, wounds of 10 mm in length and 5 mm in depth were cut with a scalpel. A sterile sample was immediately applied to the bleeding site. Five experimental groups were selected for this study, including an untreated group as a blank control, PAA hydrogel, KPON hydrogel, and two positive control groups using commercial gauze and elastic bandages (Coban™, 3M™, USA). When bleeding stopped and no further bleeding occurred after the dressing was removed, filter paper was used to collect blood. The amount of bleeding was determined by weighing the filter paper. The amount of bleeding and the time to hemostasis in each case are shown in the table.

[0069] Table 5 Case Material Amount of bleeding (mg) Hemostasis time (s) Blank control group none 633 ± 160 417 ± 15 Positive control group bandage 550 ± 130 327 ± 31 Positive control group gauze 336 ± 111 281 ± 27 Comparative Example 1 PAA 320 ± 10 225 ± 51 Example 2 Kaolin-PAA / OMCC-NHS 106 ± 25 101 ± 18 As shown in Table 5, the experimental group using Kaolin-PAA / OMCC-NHS of Example 2 had the shortest hemostatic time and the least amount of bleeding, and had the best hemostatic effect.

[0070] Red blood cell adhesion test: The red blood cell adhesion test was performed by incubating 10 mg of the candidate gel with 200 μL of 10% RBCs (v / v) at 37°C for 30 minutes, adding 1 mL of ddH2O, and measuring the absorbance of the supernatant. SEM was used to qualitatively detect the adhesion amount and morphology of red blood cells on the surface of the material.

[0071] Table 6 Case Material Relative OD Value (au) Positive control group bandage 0.85 ± 0.08 Positive control group gauze 0.80 ± 0.05 Comparative Example 1 PAA 0.07 ± 0.00 Comparative Example 2 Kaolin-PAA 0.12 ± 0.01 Comparative Example 3 PAA / OMCC-NHS 0.07 ± 0.01 Example 2 Kaolin-PAA / OMCC-NHS 0.05 ± 0.00 It can be seen from Table 6 that Example 2 Kaolin-PAA / OMCC-NHS has the best red blood cell adhesion effect.

[0072] Platelet Adhesion Test: 200 μL of platelet-rich plasma was added to the sample surface and incubated at 37°C for 45 min. After washing with PBS, 150 μL of 0.1% Triton solution (Triton X-100, Sigma) was added and lysed at room temperature for 0.5 h. 20 μL of lysate was transferred to a 96-well plate from each group, and the lactate dehydrogenase content was determined using an LDH kit.

[0073] Table 7 Case Material LDH (U / mL) Positive Control bandage 0.29 ± 0.12 Comparative Example 1 PAA 0.24 ± 0.04 Comparative Example 2 Kaolin-PAA 0.23 ± 0.14 Comparative Example 3 PAA / OMCC-NHS 0.29 ± 0.02 Example 2 Kaolin-PAA / OMCC-NHS 1.11 ± 0.19 It can be seen from Table 7 that Example 2 Kaolin-PAA / OMCC-NHS has the best platelet adhesion effect.

[0074] Antibacterial performance test: Liquid antibacterial test was performed by mixing the bacterial suspension at 1×10 6 CFU / mL density was added to a 24-well plate. PAA, Kaolin-PAA, PAA / OMCC-NHS or Kaolin-PAA / OMCC-NHS gel samples were added to a 24-well plate and incubated with bacteria at 37°C. After 12 h of co-culture, 10 μL of bacterial suspension in the well was diluted to 1 mL and then spread on LB medium to determine the number of bacteria. The survival rate of bacteria was calculated according to Formula 2: "Survival rate" (%) = CFUs / CFUc Where CFUs and CFUc are the colony counts of the sample group and the control group.

[0075] Table 8 Case Material E. coli survival rate S. aureus survival rate Comparative Example 1 PAA 24.5 ± 0.3 73.3 ± 0.04 Comparative Example 2 Kaolin-PAA 8.0 ± 0.08 52.8 ± 0.28 Comparative Example 3 PAA / OMCC-NHS 3.6 ± 0.01 3.8 ± 0.02 Example 2 Kaolin-PAA / OMCC-NHS 4.1 ± 0.02 4.0 ± 0.01 As shown in Table 8, the bacterial survival rates of Escherichia coli and Staphylococcus aureus in the Kaolin-PAA / OMCC-NHS group were the lowest, and the bacteria were basically inhibited. This hydrogel had the best antibacterial effect.

[0076] Cytocompatibility test: The CCK-8 method was used to quantitatively detect the cell viability of mouse fibroblasts (L929). Specifically, L929 cells were cultured at 2 × 10 5 The cells were seeded in a 96-well plate at a density of 1.50 cells / well and incubated for 24 h at 37 °C and 5% CO2 to allow them to adhere to the plate. The original culture medium was removed, and sample extracts of different concentrations were added and incubated for 24, 48, and 72 h, respectively. Afterwards, CCK-8 cytotoxicity assay was performed, and the absorbance was measured using an ELISA reader.

[0077] Table 9 Concentration (mg / mL) 24 h cell viability 48 h cell viability 72 h cell viability 1.25 105.0 ± 1.3 94.8 ± 11.4 100.0 ± 2.9 2.5 107.3 ± 3.9 94.9 ± 7.4 110.0 ± 11.6 5 114.6 ± 3.3 109.6 ± 9.9 123.0 ± 4.8 10 102.7 ± 4.4 99.5 ± 1.8 95.5 ± 1.2 The data in Table 9 show that Kaolin-PAA / OMCC-NHS in Example 2 has good cell compatibility.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a kaolin / oxidized microcrystalline cellulose hemostatic and antibacterial hydrogel bandage, characterized in that: The steps include: A) mixing an acrylic acid solution, kaolin, oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide and deionized water to obtain a mixture solution; B) adding a free radical initiator and a cross-linking agent into the mixture solution, placing the mixture in a mold, and reacting the mixture to obtain a hemostatic and antibacterial hydrogel bandage.

2. The preparation method according to claim 1, characterized in that: The preparation method of the oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide comprises: S1) Avicel, TEMPO, NaBr and NaClO initiate an oxidation reaction, and the product is washed to obtain oxidized Avicel; S2) The oxidized microcrystalline cellulose, EDC and NHS are mixed for reaction, and the product is dialyzed with water to obtain the oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide.

3. The preparation method according to claim 2, characterized in that: The mass ratio of microcrystalline cellulose, TEMPO, NaBr and NaClO in S1) is 5:0.1:1.5:6; the reaction time is 5.5-6.5h; the reaction temperature is 20-30°C; the pH value of the reaction is 9.5-10.5; In S2), the molar ratio of oxidized microcrystalline cellulose, EDC and NHS is 3:3:1; the reaction temperature is 20-30°C; the time is 1.5-2.5h; and the dialysis time is 3 days.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the acrylic acid solution, kaolin and oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide is 30:(10-16):(1-5); The mass percentage of the oxidized microcrystalline cellulose grafted with N-hydroxysuccinimide in the hemostatic and antibacterial hydrogel bandage is 1% to 5%.

5. The preparation method according to claim 1, characterized in that: The mass percentage of the kaolin in the hemostatic antibacterial hydrogel bandage is 10% to 16%; The mass percentage of the acrylic acid solution in the hemostatic antibacterial hydrogel bandage is 30%.

6. The preparation method according to claim 1, characterized in that: The free radical initiator is ammonium persulfate or potassium persulfate; the mass percentage of the free radical initiator in the hemostatic antibacterial hydrogel bandage is 0.1% to 1%.

7. The preparation method according to claim 1, characterized in that: The cross-linking agent is one or more of N'N-dimethylacrylamide (MBA), methacrylic anhydride-capped polyethylene glycol, acrylate-capped polyethylene glycol, maleic anhydride-capped polyethylene glycol, itaconic anhydride-capped polyethylene glycol, formaldehyde and glutaraldehyde; the mass percentage of the cross-linking agent in the hemostatic antibacterial hydrogel bandage is 0.01% to 0.1%.

8. The preparation method according to claim 1, characterized in that: Step A) The mixing is performed by stirring and mixing and then ultrasonic treatment in an ice water bath; the mixing speed is 500 rpm to 700 rpm; the mixing time is 4 to 6 minutes; the power of the ultrasonic treatment is 300 W, and the time is 9 to 11 minutes.

9. The preparation method according to claim 1, characterized in that: Step B) The mold is a silicone mold; the reaction temperature is 60-80°C, and the reaction time is 0.5-1.5h.

10. A kaolin / oxidized microcrystalline cellulose hemostatic and antibacterial hydrogel bandage, characterized in that: The method is prepared according to any one of claims 1 to 9.

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