A thrombin-modified hemostatic membrane and preparation method thereof

Abstract

The invention discloses a thrombin-modified hemostatic membrane and a preparation method thereof, and belongs to the technical field of hemostasis. The invention prepares a PAN-based PAN fiber membrane, and then performs carboxylation, amination, and itaconic acid cross-linking and activation on the PAN fiber membrane to obtain a surface-modified PAN fiber membrane with stable structure, and the surface-modified PAN fiber membrane can expose more active sites and bind with more thrombin. Thrombin is loaded on the surface-modified fiber membrane to obtain a thrombin-modified hemostatic membrane, and the thrombin-modified hemostatic membrane has good biocompatibility, can absorb blood or exudate in a short time, achieve the effect of rapid and long-term hemostasis, and can promote the growth and reproduction of adipose stem cells and human skin fibroblasts, promote cell regeneration, accelerate the healing of wounds, prevent secondary bleeding, and reduce the amount of bleeding.

Description

Technical Field

[0001] The invention relates to the technical field of hemostasis, in particular to a thrombin-modified hemostatic membrane and a preparation method thereof. Background Art

[0002] Bleeding incidents not only occur on the battlefield, but also in daily life, and uncontrollable bleeding is a major factor leading to death. Deaths caused by untimely hemostasis account for 30% to 40% of traumatic deaths. In the current medical technology, hemostatic gauze is usually used to stop bleeding. However, traditional hemostatic gauze still remains in the stage of non-woven gauze, with poor surface texture, toughness and strength, large microscopic size, which is not conducive to the rapid absorption of blood, slow coagulation speed, and usually takes 2 to 8 minutes to stop bleeding. The hemostatic effect is not ideal, and it is easy to cause secondary bleeding to the wound due to the rough texture of the gauze, resulting in more blood loss. In addition, the healing effect on the wound is very small, and it is impossible to accelerate the healing of the wound while stopping bleeding. Therefore, the research and development of composite hemostatic products is of great significance to saving lives. Summary of the invention

[0003] The main purpose of the present invention is to provide a thrombin-modified hemostatic membrane and a preparation method thereof, aiming to solve the technical problems that traditional hemostatic gauze has poor surface texture, toughness and strength, large microscopic size, is not conducive to blood absorption, has a slow coagulation speed, is easy to cause secondary bleeding to the wound surface, resulting in increased blood loss, and cannot accelerate wound healing while stopping bleeding.

[0004] To achieve the above object, the present invention provides a thrombin-modified hemostatic membrane.

[0005] In addition, to achieve the above object, the present invention also provides a method for preparing a thrombin-modified hemostatic membrane, the preparation method comprising the following steps:

[0006] S10, preparing PAN nanofiber membrane: dissolving PAN in an organic solvent to obtain a spinning solution, and then preparing a PAN fiber membrane by an electrospinning method, and drying it for later use;

[0007] S20, surface modification:

[0008] S21, carboxylation: soaking the dried PAN fiber membrane in an alkaline solution, reacting at a constant temperature of 50-60°C, then soaking in an acid solution for pickling, and then rinsing with deionized water until neutral, to obtain a surface carboxylated PAN fiber membrane;

[0009] S22, amination: soaking the surface hydroxylated PAN fiber membrane in a mixed solution of ethylenediamine and ethanol, reacting at a constant temperature of 30-45°C, and then rinsing with deionized water until neutral, to obtain a surface aminated PAN fiber membrane;

[0010] S23, itaconic acid crosslinking and activation: soaking the surface aminated PAN fiber membrane in an itaconic acid aqueous solution, reacting at a constant temperature of 50-65° C. to obtain a crosslinked PAN fiber membrane, and transferring the crosslinked PAN fiber membrane into an 80% ethanol mixture of EDC / NHS for immersion activation to obtain a surface modified PAN fiber membrane;

[0011] S30, loading thrombin: transferring the surface modified PAN fiber membrane into a thrombin solution, incubating it at a constant temperature of 30-50° C., and then washing it with deionized water to obtain a thrombin-modified hemostatic membrane.

[0012] Optionally, in the step of preparing the PAN nanofiber membrane in S10, the organic solvent is at least one of N,N-dimethylformamide and dimethyl sulfoxide.

[0013] Optionally, in the step of preparing the PAN nanofiber membrane in S10, the mass concentration of the spinning solution is 8% to 10%.

[0014] Optionally, in the carboxylation step S21, the alkaline solution is at least one of a sodium hydroxide solution, a potassium hydroxide solution, a mixture of potassium hydroxide and ammonia water, and a mixture of sodium hydroxide and ammonia water.

[0015] Optionally, in the carboxylation step S21, the acidic solution is at least one of a hydrochloric acid solution, a nitric acid solution or a sulfuric acid solution.

[0016] Optionally, in the carboxylation step S21, the immersion and acid washing time is 3 to 5 hours.

[0017] Further optionally, the molar concentration of the sodium hydroxide is 1 to 3 mol / L; and the molar concentration of the hydrochloric acid solution is 0.5 to 2 mol / L.

[0018] Optionally, in the amination step S22, the volume ratio of ethylenediamine to ethanol is 4:5-8.

[0019] Optionally, in the itaconic acid cross-linking and activation step S23, the mass fraction of the itaconic acid solution is 8% to 10%.

[0020] Optionally, in the itaconic acid cross-linking and activation step S23, the activation is performed by immersion for 2 to 4 times, with each immersion for 6 to 8 hours.

[0021] Optionally, in the S23 itaconic acid cross-linking and activation step, the mass ratio of EDC to NHS is 5:1-3.

[0022] Optionally, in the step of loading thrombin in S30, the concentration of the thrombin solution is 10 to 30 NIH units / ml, and the incubation time is 4 to 8 hours.

[0023] The beneficial effects that can be achieved by the present invention are:

[0024] The thrombin-modified hemostatic membrane of the present invention is based on a PAN fiber membrane, which is a high-strength and high-model fiber with a good surface texture, excellent tensile strength and wear resistance.

[0025] The surface-modified PAN fiber membrane obtained by sequentially performing a series of surface modifications such as carboxylation, amination, itaconic acid cross-linking and activation has a stable structure, exposes more active sites that can bind to thrombin, can load more thrombin, and significantly improves the hemostatic effect of the hemostatic membrane.

[0026] The thrombin-modified hemostatic membrane prepared by loading thrombin on the surface-modified PAN fiber membrane has good biocompatibility, can absorb blood or exudate in a short time, quickly adhere to the wound surface, promote platelet adhesion to form thrombus, thereby achieving the effect of rapid hemostasis, and can promote the growth and reproduction of adipose stem cells and human skin fibroblasts, accelerating the healing of the wound surface. Compared with the existing hemostatic gauze, the hemostatic membrane of the present invention has better skin feel and significant hemostatic effect, and can promote wound healing, prevent secondary bleeding, and effectively reduce blood loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 the structures shown in these drawings without paying creative work.

[0028] Figure 1 These are the results of the morphology test of performance test 1, and the SEM images of PAN-DIA-ITA-Th, PAN, PAN-COOH, PAN-DIA, PAN-DIA-ITA, and PAN-COOH-Th.

[0029] Figure 2 This is the result diagram of the structural analysis of performance test 2, where:

[0030] Figure 2 Area A is an infrared spectrum, in which a is PAN, b is PAN-COOH, c is PAN-DIA, d is PAN-DIA-ITA, e is PAN-COOH-Th, and f is PAN-DIA-ITA-Th;

[0031] Figure 2 Area B shows the X-ray diffraction patterns of PAN, PAN-DIA, PAN-COOH, and PAN-DIA-ITA;

[0032] Figure 2 Area C is the H NMR spectrum of PAN-DIA-ITA. Figure 2 The D region is the structural formula of PAN-DIA-ITA. Figure 2 In regions C and D, a is a long carbon chain, b is -NH, c is -CH2-, d is -NH2, e is an unsaturated double bond, and f is -COOH.

[0033] Figure 3 This is the thermogravimetric analysis result of the surface modified PAN fiber membrane in performance test 3, where the horizontal axis of the left figure is temperature and the vertical axis represents weight percentage, and the horizontal axis of the right figure is temperature and the vertical axis represents heat flow.

[0034] Figure 4 This is a graph analyzing the test results of thrombin activity in performance test 4, wherein the abscissa represents time (min-hr) and the ordinate represents thrombin activity (HIN / unit).

[0035] Figure 5 The results of the performance test 5 adipose-derived stem cells (ADSC) and human skin fibroblasts (HSF) culture experiments are analyzed, among which, Figure 5 In area A, the horizontal axis is time (day), and the vertical axis is the cell activity (%) of adipose-derived stem cells (ADSC); Figure 5 In area B, the horizontal axis is time (day), and the vertical axis is the cell activity (%) of human skin fibroblasts (HSF); Figure 5 In area C, the horizontal axis represents time (days), and the vertical axis represents the cell density of adipose-derived stem cells (ADSCs), reflecting the cell proliferation; Figure 5 The horizontal axis of area D represents time (days), and the vertical axis represents the density of human skin fibroblasts (HSF), reflecting the cell proliferation.

[0036] Figure 6 SEM and Confocal images of adipose-derived stem cells (ADSC) and human skin fibroblasts (HSF) in the performance test 5 adipose-derived stem cells (ADSC) and human skin fibroblasts (HSF) culture experiment.

[0037] Figure 7 This is a result analysis chart of the hemostatic effect test of performance test 6, wherein the horizontal axis of the left figure is the sample type, and the vertical axis is the hemostasis time (min); the horizontal axis of the right figure is the sample type, and the vertical axis is the blood loss (%).

[0038] Figure 8 This is a result analysis diagram of the in vitro coagulation effect test of performance test 7, wherein the horizontal axis is time (sec) and the vertical axis is coagulation index (%).

[0039] Fig. 9 This is the result analysis diagram of observing the platelet adhesion in performance test 8, where a is the positive control group PC gauze, b is PAN, c is PAN-COOH, d is PAN-DIA, e is PAN-DIA-ITA, f is PAN-COOH-Th, and h is PAN-DIA-ITA-Th.

[0040] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0041] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0044] The present invention provides a method for preparing a thrombin-modified hemostatic membrane, and the method for preparing the thrombin-modified hemostatic membrane comprises the following steps:

[0045] S10, preparing PAN nanofiber membrane: dissolving PAN in an organic solvent to obtain a spinning solution, and then preparing a PAN fiber membrane by an electrospinning method, and drying it for later use.

[0046] It should be noted that the PAN is polyacrylonitrile.

[0047] The present invention does not limit the type of the organic solvent, but needs to achieve the purpose of dissolving PAN. Preferably, the organic solvent is at least one of N,N-dimethylformamide or dimethyl sulfoxide. Using at least one of the above organic solvents has less pollution and can effectively and quickly dissolve PAN.

[0048] The present invention does not limit the mass concentration of the spinning solution. Preferably, the mass concentration of the spinning solution is 8-10%. A relatively uniform PAN fiber membrane can be prepared within this mass concentration range. In addition, in some embodiments, PAN can be quickly and completely dissolved in the organic solvent by strong stirring for more than 24 hours.

[0049] The present invention does not limit the operating conditions of the electrospinning method for preparing PAN fiber membrane. In some embodiments, the electrospinning method for preparing the PAN fiber membrane includes the following steps: loading 2 ml of spinning solution into a 2.5 ml syringe, using a No. 21 needle at 23 KV and a sampling rate of 1 ml / h for spinning, and setting the speed of the cylindrical collector to 100 rpm, collecting samples at 12 cm away from the No. 21 needle to obtain a PAN fiber membrane, and placing the PAN fiber membrane in a vacuum oven at 40°C to dry for 24 hours for use.

[0050] S20, surface modification:

[0051] S21, carboxylation: soak the dried PAN fiber membrane in an alkaline solution, react at a constant temperature of 50-60°C, then soak and pickle in an acidic solution, and then rinse with deionized water until neutral, to obtain a surface carboxylated PAN fiber membrane.

[0052] The present invention does not limit the type of the alkaline solution. Preferably, the alkaline solution is at least one of a sodium hydroxide solution, a potassium hydroxide solution, a potassium hydroxide and ammonia solution mixture, and a sodium hydroxide and ammonia solution mixture, wherein the ammonia solution can play a role in adjusting the pH of the reaction system. The use of the alkaline solution as described above can effectively promote the reaction.

[0053] Further preferably, in some embodiments, the alkaline solution is a sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is 1 to 3 mol / L.

[0054] The carboxylation reaction in this example is carried out in a constant temperature shaker at 50-60° C. for 0.5-2 h, which can promote the reaction.

[0055] Similarly, the present invention does not limit the type of the acidic solution. The purpose of immersion pickling with an acidic solution is to remove the residual alkaline solution. Preferably, the acidic solution can be at least one of a hydrochloric acid solution, a nitric acid solution or a sulfuric acid solution. Further preferably, the acidic solution is a hydrochloric acid solution, and the molar concentration of the hydrochloric acid solution is 0.5 to 2 mol / L.

[0056] The present invention also does not limit the time of immersion pickling. Preferably, the immersion pickling time is 3 to 5 hours.

[0057] S22, amination: soaking the surface hydroxylated PAN fiber membrane in a mixed solution of ethylenediamine and ethanol, reacting at a constant temperature of 30-45°C, and then rinsing with deionized water until neutral, to obtain a surface aminated PAN fiber membrane.

[0058] Specifically, the volume ratio of ethylenediamine to ethanol is 4:5-8, and the reaction time is 8-12 hours.

[0059] S23, itaconic acid crosslinking and activation: soak the surface aminated PAN fiber membrane in an itaconic acid aqueous solution, react at a constant temperature of 50-65°C to obtain a cross-linked PAN fiber membrane, transfer the cross-linked PAN fiber membrane into an 80% ethanol mixture of EDC / NHS for immersion activation, and obtain a surface modified PAN fiber membrane.

[0060] It should be noted that the 80% ethanol mixture of EDC / NHS can be understood as: a mixture obtained by mixing EDC / NHS with an ethanol solution having a mass fraction of 80%. Specifically, the mass ratio of EDC to NHS is 5:1-3.

[0061] The surface-modified PAN fiber membrane can expose more active sites, which can bind to thrombin. After soaking and activation, the molecules on the surface-modified PAN fiber membrane become more active, providing excellent reaction conditions for subsequent loading of thrombin, and promoting the loading of thrombin.

[0062] In this embodiment, preferably, the mass fraction of the itaconic acid solution is 8% to 10%. If the mass fraction of the itaconic acid solution is high, excessive itaconic acid will remain after the cross-linking is completed. The residual itaconic acid will combine with thrombin, thereby affecting the loading amount of thrombin on the surface modified PAN fiber membrane. If the mass fraction of the itaconic acid solution is low, the cross-linking reaction is insufficient, the active sites on the surface modified PAN fiber membrane become less, and it is not conducive to the subsequent loading of thrombin.

[0063] The present invention does not limit the equipment for the cross-linking reaction of itaconic acid. The container has a constant temperature heating function. The temperature range of the constant temperature reaction is 50-65° C., which can maintain the activity of the raw materials for the cross-linking reaction. In some embodiments, a constant temperature box can be selected as the equipment for the cross-linking reaction of itaconic acid.

[0064] Specifically, the cross-linked PAN fiber membrane is moved into an 80% ethanol mixture of EDC / NHS. The present invention does not limit the number of immersion activations and the time of each immersion activation. Preferably, the number of immersion activations is 2 to 4 times, and each immersion activation is 6 to 8 hours. It should be noted that after each immersion activation, a new 80% ethanol mixture of EDC / NHS needs to be replaced before the next immersion activation. Through the above immersion activation conditions, the purpose of full activation can be achieved, making the molecules more active and providing more binding sites for loading thrombin.

[0065] In some embodiments, if the operation of loading thrombin is not performed immediately, the surface modified PAN fiber membrane can be placed under -20 ° C and freeze-dried for 12 hours, and then stored at -20 ° C. When the thrombin loading operation is required and the preparation of the hemostatic membrane is completed, it can be taken out for use. Because the surface modified PAN after cryopreservation is affected by the low temperature, the intermolecular movement is slow and not active enough. If the operation of loading thrombin is to be performed, the surface modified PAN fiber membrane stored by lyophilization needs to be immersed again for activation operation to promote the subsequent loading of thrombin. The re-immersion activation method is: immerse the surface modified PAN fiber membrane stored by lyophilization in an 80% ethanol mixture of EDC / NHS for 6 hours, wherein the mass ratio of EDC to NHS is 5:1 to 3. The surface modification operation of this embodiment and the step of loading thrombin may not be consistent, and a variety of production schemes can be provided for large-scale production, which can effectively control the production process.

[0066] S30, loading thrombin: transferring the surface modified PAN fiber membrane into a thrombin solution, incubating it at a constant temperature of 30-50° C., and then washing it with deionized water to obtain a thrombin-modified hemostatic membrane.

[0067] The present invention does not limit the concentration of the thrombin solution. Preferably, the concentration of the thrombin solution is 10 to 30 NIH units / ml. Within this concentration range, a hemostatic film with the surface evenly covered with thrombin can be obtained.

[0068] Likewise, the present invention does not impose any limitation on the culture time. Preferably, the culture time is 4 to 8 hours.

[0069] In some embodiments, the thrombin-modified hemostatic film is dried in a -20°C freeze dryer and stored frozen at -20°C, which can effectively maintain the activity of thrombin.

[0070] The thrombin-modified hemostatic membrane obtained by the present invention has a good skin feel, not only has a rapid hemostatic effect, but also can promote the growth and reproduction of adipose stem cells and human skin fibroblasts, accelerate wound healing, prevent secondary bleeding, and reduce blood loss.

[0071] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0072] Example 1

[0073] A method for preparing a thrombin-modified hemostatic membrane comprises the following steps:

[0074] S10, preparation of PAN nanofiber membrane: PAN was mixed with N,N-dimethylformamide solution, and stirred vigorously for 24 hours until PAN was completely dissolved to prepare a spinning solution with a mass concentration of 8%. Then 2 ml of the spinning solution was loaded into a 2.5 ml syringe, and a No. 21 needle was used to spin at a feed rate of 1 ml / h at 23 KV. The speed of the cylindrical collector was set to 100 rpm, and the sample was collected at 12 cm away from the No. 21 needle to obtain a PAN fiber membrane. The PAN fiber membrane was placed in a vacuum oven at 40°C and dried for 24 hours for use.

[0075] S20, surface modification:

[0076] S21, carboxylation: dissolve sodium hydroxide in deionized water to prepare a 2 mol / L sodium hydroxide solution, then soak the PAN fiber membrane dried in S10 in a culture dish containing the 2 mol / L sodium hydroxide solution, shake in a constant temperature shaker at 60°C for 1 hour, then soak and pickle with a 1 mol / L hydrochloric acid solution for 4 hours, and then rinse with deionized water until neutral to obtain a surface carboxylated PAN fiber membrane.

[0077] S22, amination: immersing the surface hydroxylated PAN fiber membrane in a mixed solution of ethylenediamine and ethanol in a volume ratio of 4:6, reacting in a 37°C thermostat for 12 h, and then rinsing with deionized water until neutral, to obtain a surface aminated PAN fiber membrane;

[0078] S23, itaconic acid crosslinking and activation: the surface aminated PAN fiber membrane is immersed in a 10% by mass itaconic acid aqueous solution, and placed in a 60°C constant temperature box for reaction for 6 hours to obtain a cross-linked PAN fiber membrane, and the cross-linked PAN fiber membrane is transferred into an EDC / NHS 80% ethanol mixture for immersion activation, the mass ratio of EDC / NHS is 5:2, after each immersion activation for 8 hours, replace with a new EDC / NHS 80% ethanol mixture for further immersion activation for 8 hours, repeat the above immersion activation 3 times to obtain a surface modified PAN fiber membrane.

[0079] S30, loading thrombin: transfer the activated surface-modified PAN fiber membrane to a culture dish containing a thrombin solution with a concentration of 20 NIH units / ml, place the culture dish in a constant temperature box at 40°C and culture it. Shake the culture dish every 1 hour to ensure that the thrombin and the PAN fiber membrane are fully and evenly contacted. After immersion and culture for 6 hours, take it out and rinse it with deionized water to obtain a thrombin-modified hemostatic membrane. Place the thrombin-modified clotting yarn in a -20°C freeze dryer to dry it and store it in a freezer at -20°C.

[0080] Example 2

[0081] A method for preparing a thrombin-modified hemostatic membrane comprises the following steps:

[0082] S10, preparation of PAN nanofiber membrane: PAN was mixed with N,N-dimethylformamide solution and stirred vigorously for 24 hours until PAN was completely dissolved to prepare a spinning solution with a mass concentration of 10%. Then 2 ml of the spinning solution was loaded into a 2.5 ml syringe and spun at 23 KV with a No. 21 needle at an injection rate of 1 ml / h. The speed of the cylindrical collector was set to 100 rpm, and the sample was collected at 12 cm away from the No. 21 needle to obtain a PAN fiber membrane. The PAN fiber membrane was placed in a vacuum oven at 40°C and dried for 24 hours for use.

[0083] S20, surface modification:

[0084] S21, carboxylation: sodium hydroxide is dissolved in deionized water to prepare a 3 mol / L sodium hydroxide solution, and then the PAN fiber membrane dried in S10 is immersed in a culture dish containing the 3 mol / L sodium hydroxide solution, shaken in a constant temperature shaker at 55°C for 0.5 h, then immersed in a 0.5 mol / L hydrochloric acid solution for acid washing for 5 h, and then rinsed with deionized water until neutral to obtain a surface carboxylated PAN fiber membrane.

[0085] S22, amination: the hydroxylated PAN fiber membrane was immersed in a mixed solution of ethylenediamine and ethanol in a volume ratio of 4:5, reacted in a constant temperature box at 30°C for 8 hours, and then rinsed with deionized water until neutral to obtain a surface aminated PAN fiber membrane.

[0086] S23, itaconic acid crosslinking and activation: soak the surface aminated PAN fiber membrane in an 8% by mass itaconic acid aqueous solution, and put it into a 60°C constant temperature box for reaction for 8 hours to obtain a cross-linked PAN fiber membrane, and move the cross-linked PAN fiber membrane into an 80% ethanol mixture of EDC / NHS for immersion activation, the mass ratio of EDC / NHS is 5:1, after 7 hours of immersion activation, replace with a new 80% ethanol mixture of EDC / NHS and continue immersion activation for 7 hours, repeat the above immersion activation twice to obtain a surface modified PAN fiber membrane.

[0087] S30, loading thrombin: transfer the activated surface-modified PAN fiber membrane to a culture dish containing a thrombin solution with a concentration of 10 NIH units / ml, place the culture dish in a constant temperature box at 50°C and culture it, shake the culture dish every 1 hour to allow the thrombin to fully and evenly contact with the PAN fiber membrane, take it out after immersion and culture for 4 hours, and rinse it with deionized water to obtain a thrombin-modified hemostatic membrane, place the thrombin-modified clotting yarn in a -20°C freeze dryer to dry, and store it in a freezer at -20°C.

[0088] Example 3

[0089] A method for preparing a thrombin-modified hemostatic membrane comprises the following steps:

[0090] S10, preparation of PAN nanofiber membrane: PAN was mixed with dimethyl sulfoxide solution, and stirred vigorously for 24 hours until PAN was completely dissolved to prepare a spinning solution with a mass concentration of 9%. Then 2 ml of the spinning solution was loaded into a 2.5 ml syringe, and a No. 21 needle was used to spin at a feed rate of 1 ml / h at 23 KV. The speed of the cylindrical collector was set to 100 rpm, and the sample was collected at 12 cm away from the No. 21 needle to obtain a PAN fiber membrane. The PAN fiber membrane was placed in a vacuum oven at 40°C and dried for 24 hours for use.

[0091] S20, surface modification:

[0092] S21, carboxylation: sodium hydroxide is dissolved in deionized water to prepare a 1 mol / L potassium hydroxide solution, and then the PAN fiber membrane dried in S10 is immersed in a culture dish containing the 1 mol / L sodium hydroxide solution, shaken in a constant temperature shaker at 55°C for 2 hours, then immersed in a 2 mol / L sulfuric acid solution for acid washing for 3 hours, and then rinsed with deionized water until neutral to obtain a surface carboxylated PAN fiber membrane.

[0093] S22, amination: immersing the surface hydroxylated PAN fiber membrane in a mixed solution of ethylenediamine and ethanol in a volume ratio of 4:8, reacting in a 45°C constant temperature box for 10 h, and then rinsing with deionized water until neutral, to obtain a surface aminated PAN fiber membrane;

[0094] S23, itaconic acid crosslinking and activation: the surface aminated PAN fiber membrane is immersed in an 8% by mass itaconic acid aqueous solution, and placed in a 65°C constant temperature box for reaction for 4 hours to obtain a cross-linked PAN fiber membrane, and the cross-linked PAN fiber membrane is transferred into an EDC / NHS 80% ethanol mixture for immersion activation, the mass ratio of EDC / NHS is 5:3, after immersion activation for 7 hours, replace with a new EDC / NHS 80% ethanol mixture for further immersion activation for 7 hours, repeat the above immersion activation 4 times to obtain a surface modified PAN fiber membrane.

[0095] S30, loading thrombin: transfer the activated surface-modified PAN fiber membrane to a culture dish containing a thrombin solution with a concentration of 30 NIH units / ml, place the culture dish in a constant temperature box at 30°C and culture it, shake the culture dish every 1 hour to allow the thrombin to fully and evenly contact with the PAN fiber membrane, take it out after immersion and culture for 4 hours, and rinse it with deionized water to obtain a thrombin-modified hemostatic membrane, place the thrombin-modified clotting yarn in a -20°C freeze dryer to dry, and store it in a freezer at -20°C.

[0096] Example 4

[0097] A method for preparing a thrombin-modified hemostatic membrane comprises the following steps:

[0098] S10, preparation of PAN nanofiber membrane: PAN was mixed with N,N-dimethylformamide solution, and stirred vigorously for 24 hours until PAN was completely dissolved to prepare a spinning solution with a mass concentration of 8%. 2 ml of the spinning solution was loaded into a 2.5 ml syringe, and a No. 21 needle was used to spin at a feed rate of 1 ml / h at 23 KV. The speed of the cylindrical collector was set to 100 rpm, and the sample was collected at 12 cm away from the No. 21 needle to obtain a PAN fiber membrane. The PAN fiber membrane was placed in a vacuum oven at 40°C and dried for 24 hours for use.

[0099] S20, surface modification:

[0100] S21, surface carboxylation: sodium hydroxide is dissolved in deionized water to prepare a 2 mol / L sodium hydroxide solution, the PAN fiber membrane dried in S10 is immersed in a culture dish filled with the sodium hydroxide solution, and the culture dish is placed in a constant temperature shaker at 60°C for 1.5 hours, taken out and immersed in a 1 mol / L hydrochloric acid solution for 4 hours, then taken out and repeatedly rinsed with deionized water until neutral, to obtain a PAN fiber membrane with surface carboxylation;

[0101] S22, amination: immersing the surface hydroxylated PAN fiber membrane in a mixed solution of ethylenediamine and ethanol in a volume ratio of 4:6, and placing the mixed solution in a 37° C. incubator for amination for 11 h, and then rinsing with deionized water until neutral, to obtain a surface aminated PAN fiber membrane;

[0102] S23, itaconic acid crosslinking and activation: soak the surface aminated PAN fiber membrane in a 10% itaconic acid aqueous solution, and put it into a 60°C thermostat for reaction for 6 hours to obtain a cross-linked PAN fiber membrane, move the cross-linked PAN fiber membrane into an 80% ethanol mixture of EDC / NHS for immersion activation, the mass ratio of EDC / NHS is 5:2, after 8 hours of immersion activation, replace with a new 80% ethanol mixture of EDC / NHS for continued immersion activation, repeat the above immersion activation 3 times to obtain a surface modified PAN fiber membrane, clean the surface modified PAN fiber membrane with deionized water, and then dry it in a freeze dryer at -20°C for 12 hours and store it in a freezer at -20°C.

[0103] S30, loading thrombin: The surface modified PAN fiber membrane stored in cryopreservation of S23 was immersed in a 80% ethanol mixed solution of EDC and NHS for activation for 6 hours, wherein the mass ratio of EDC to NHS was 5:2, and then immersed in a culture dish containing a thrombin solution with a concentration of 20 NIH units / ml, and the culture dish was placed in a constant temperature box at 40°C for culture, and the culture dish was shaken every 1 hour to ensure that the thrombin and the PAN fiber membrane were fully and evenly contacted, and after immersion and culture for 4 hours, it was taken out and rinsed with deionized water to obtain a thrombin-modified hemostatic membrane, and the thrombin-modified clotting yarn was placed in a -20°C freeze dryer for drying, and stored frozen at -20°C.

[0104] Comparative Example 1

[0105] Comparative Example 1: A PAN fiber membrane was prepared according to step S10 in Example 1.

[0106] Comparative Example 2

[0107] Comparative Example 2: A PAN fiber membrane was prepared according to step S10 in Example 1, and then a surface carboxylation PAN fiber membrane was prepared according to step S21 of carboxylation.

[0108] Comparative Example 3

[0109] Comparative Example 3: A PAN fiber membrane was prepared according to step S10 in Example 1, and then a surface aminated PAN fiber membrane was prepared according to step S21 of carboxylation and step S22 of amination.

[0110] Comparative Example 4

[0111] Comparative Example 4: A PAN fiber membrane was prepared according to step S10 in Example 1, and then a surface-modified PAN fiber membrane was prepared according to step S20.

[0112] Comparative Example 5

[0113] Comparative Example 5: A surface carboxylated PAN fiber membrane was prepared according to the operating methods of S10 and S21 in Example 1, and then thrombin was loaded on the surface carboxylated PAN fiber membrane according to the operating method of S30 in Example 1 to obtain a thrombin-modified surface carboxylated PAN fiber membrane.

[0114] Performance Testing

[0115] The product obtained in Example 1 is marked as PAN-DIA-ITA-Th, the product of Comparative Example 1 is marked as PAN, the product of Comparative Example 2 is marked as PAN-COOH, the product of Comparative Example 3 is marked as PAN-DIA, the product of Comparative Example 4 is marked as PAN-DIA-ITA, and the product of Comparative Example 5 is marked as PAN-COOH-Th.

[0116] 1. Morphology test

[0117] PAN-DIA-ITA-Th, PAN, PAN-COOH, PAN-DIA, PAN-DIA-ITA and PAN-COOH-Th were characterized by scanning electron microscopy. Figure 1 shown.

[0118] Depend on Figure 1 It can be seen that after a series of surface modification treatments such as carboxylation, amination, itaconic acid cross-linking and activation, the surface of PAN fiber membrane becomes relatively rough, which can provide more active sites and facilitate the subsequent loading of thrombin.

[0119] 2. Structural analysis

[0120] PAN-DIA-ITA-Th, PAN, PAN-COOH, PAN-DIA, PAN-DIA-ITA and PAN-COOH-Th were characterized by infrared spectroscopy. The characterization results are shown in Figure 2 As shown in the infrared spectrum of area A, a is PAN, b is PAN-COOH, c is PAN-DIA, d is PAN-DIA-ITA, e is PAN-COOH-Th, and f is PAN-DIA-ITA-Th.

[0121] Depend on Figure 2 From area A, we can see that PAN-DIA-ITA-Th obtained after surface modification and loading of thrombin still has the same characteristic peaks as PAN, indicating that the surface modification did not destroy the main structure of the PAN fiber membrane.

[0122] PAN, PAN-COOH, PAN-DIA and PAN-DIA-ITA were characterized by X-ray diffractometer. The characterization results are shown in Figure 2 The X-ray diffraction pattern of area B is shown.

[0123] Depend on Figure 2 From area B, it can be seen that PAN-DIA-ITA only retains the main peak similar to PAN, which means that the surface modification of PAN fiber membrane has been completed and the main structure has not been destroyed.

[0124] The molecular structure of PAN-DIA-ITA was analyzed. Figure 2 As shown in Area C and Area D. Among them, Figure 2 Area C is the H NMR spectrum of PAN-DIA-ITA. Figure 2 The D region is the molecular structure of PAN-DIA-ITA. Figure 2 In regions C and D, a is a long carbon chain, b is -NH, c is -CH2-, d is -NH2, e is an unsaturated double bond, and f is -COOH.

[0125] Depend on Figure 2 From area C and area D, we can see that: the characteristic peak of the long carbon chain on PAN-DIA-ITA is obvious and sharp, indicating that the PAN fiber membrane after surface modification still maintains a good PAN skeleton; the characteristic peaks of -NH2 and -CH2- obtained after amination are also relatively sharp, indicating that the amination modification is very successful; itaconic acid is cross-linked to the surface aminated PAN fiber membrane through -NH2 dehydrogenation, and -NH and the unsaturated double bonds and -COOH on itaconic acid all show stretching and bending vibrations, and the characteristic peaks are very sharp, indicating that itaconic acid has been successfully cross-linked.

[0126] In summary, the present invention can obtain a surface-modified PAN fiber membrane with stable structure through surface modification treatment, and the thrombin-modified hemostatic membrane obtained by loading thrombin retains the main structure of the PAN fiber membrane, that is, it has the better surface texture, excellent tensile strength and wear resistance brought by the PAN fiber membrane.

[0127] 3. Thermogravimetric analysis of surface modified PAN fiber membrane

[0128] Thermogravimetric analysis of PAN, PAN-COOH, PAN-DIA and PAN-DIA-ITA was performed. The results are shown in Figure 3 .

[0129] Depend on Figure 3It can be seen that PAN-DIA-ITA has a main decomposition stage between 200 and 400°C, with weight loss, but the weight loss is smaller than that of PAN, PAN-COOH and PAN-DIA, and it has good thermal stability. PAN without surface modification has a significant weight reduction at 300°C, a large heat flow, and the worst thermal stability, indicating that the surface-modified PAN fiber membrane that has been carboxylated, aminized, cross-linked and activated with itaconic acid has better thermal stability.

[0130] 4. Thrombin activity test

[0131] The activity of PAN-DIA-ITA-Th and PAN-COOH-Th thrombin was determined. The results are shown in Figure 4 .

[0132] Depend on Figure 4 It can be seen that PAN-COOH-Th is loaded with thrombin only after carboxylation without amination, itaconic acid cross-linking and activation, and the amount of thrombin attached is small and the activity is poor.

[0133] 5. Adipose-derived stem cells (ADSC) and human skin fibroblasts (HSF) culture experiments

[0134] Adipose-derived stem cell (ADSC) culture: PAN, PAN-COOH, PAN-DIA-ITA, PAN-COOH-Th and PAN-DIA-ITA-Th were cut into 1.0 × 1.0 cm squares and immersed in a solution containing 1 × 10 6 Adipose-derived stem cells (ADSCs) were cultured in a dish, and a blank control group TCP was designed. The blank control group TCP contained 1×10 6 The culture dishes of adipose-derived stem cells (ADSCs) were placed in a 37°C incubator for culture. On the first day, the cell activity of adipose-derived stem cells (ADSCs) in each group of culture dishes was measured. Figure 5 Area A.

[0135] Human skin fibroblast (HSF) culture: PAN, PAN-COOH, PAN-DIA-ITA, PAN-COOH-Th and PAN-DIA-ITA-Th were cut into squares with an area of ​​1.0 × 1.0 cm and infiltrated in a solution containing 1 × 10 6 A blank control group of TCP was designed, and the blank control group TCP contained 1×10 6 All culture dishes were placed in a 37°C incubator for culture. On the first day, the cell activity of human skin fibroblasts (HSF) in each group of culture dishes was measured. For details, see Figure 5 Area B.

[0136] The cell density of adipose-derived stem cells (ADSCs) and human skin fibroblasts (HSFs) in the culture dish on day 1, day 3, and day 7 was measured at OD 450 nm using a spectrophotometer: Figure 5 Area C is a comparison of the cell density of adipose-derived stem cells (ADSCs) in each group of culture medium on day 1, day 3, and day 7; Figure 5 Panel D is a comparison of the cell density of human skin fibroblasts (HSF) in each group of culture media on day 1, day 3, and day 7.

[0137] The morphology and distribution of adipose-derived stem cells (ADSCs) and human skin fibroblasts (HSFs) in the culture medium were observed by scanning electron microscopy and laser scanning confocal microscopy on days 0, 1, and 7, respectively. Figure 6 As shown, Figure 6 Area A shows the SEM and Confocal images of adipose-derived stem cells (ADSCs) and human skin fibroblasts (HSFs) on day 0; Figure 6 Panel B shows the SEM and Confocal images of adipose-derived stem cells (ADSCs) on day 1 and day 7; Figure 6 Panel C shows the SEM and Confocal images of human dermal fibroblasts (HSF) on the 1st and 7th days.

[0138] Depend on Figures 5 to 6 It can be seen that the thrombin-modified hemostatic membrane prepared by the technology of the present invention has a promoting effect on the proliferation and growth of adipose stem cells and human skin fibroblasts, and can then promote the rapid healing of the wound surface.

[0139] 6. Hemostasis effect test

[0140] Eight-week-old Balb / c female mice were randomly divided into groups. The tails of the mice were cut off and then wrapped with PC gauze of the blank control group, PAN, PAN-COOH, PAN-DIA, PAN-DIA-ITA, PAN-COOH-Th and PAN-DIA-ITA-Th. The time required for hemostasis of all samples and the blood loss of the corresponding mice were calculated. The results are shown in Figure 7 .

[0141] Depend on Figure 7 It can be seen that PAN-DIA-ITA-Th achieved the hemostatic effect within one minute and could control the blood from seeping out again, with minimal blood loss, thus reducing the blood loss of mice to the greatest extent.

[0142] 7. In vitro coagulation effect test

[0143] On the basis of test 6, the in vitro coagulation effects of PAN, PAN-COOH, PAN-DIA, PAN-DIA-ITA, PAN-COOH-Th and PAN-DIA-ITA-Th were determined, and a negative control group NC without any treatment was designed, and gauze was used as a positive control group PC. The test results are shown in Figure 8 .

[0144] Depend on Figure 8 It can be seen that with the progress of time, the coagulation indexes of PAN, PAN-COOH, PAN-DIA, PAN-DIA-ITA and PAN-COOH-Th all decreased, especially for PAN-DIA and PAN-COOH, whose coagulation index dropped sharply by about 20% within 30 to 120 s. Although the coagulation index of PAN-COOH-Th did not change much within 30 to 120 s, it dropped sharply by about 10% within 120 to 180 s. The coagulation index of PAN-DIA-ITA-Th remained basically unchanged within 30 to 180 s, indicating that the thrombin-modified hemostatic membrane of the present invention has excellent coagulation effect and can achieve long-term hemostasis.

[0145] 8. Observe platelet adhesion

[0146] After the in vitro coagulation effect test experiment of performance test 7 was completed, the gauze, PAN, PAN-COOH, PAN-DIA, PAN-DIA-ITA, PAN-COOH-Th and PAN-DIA-ITA-Th of the positive control group PC were characterized by scanning electron microscopy to observe the adhesion of platelets. The characterization results are shown in Fig. 9 Among them, a is the gauze of the positive control group PC, b is PAN, c is PAN-COOH, d is PAN-DIA, e is PAN-DIA-ITA, f is PAN-COOH-Th, and h is PAN-DIA-ITA-Th.

[0147] Depend on Fig. 9 It can be seen that PAN-DIA-ITA-Th, that is, the product of Example 1, has more platelets attached, indicating that the thrombin-modified hemostatic membrane prepared by the technology of the present invention has an excellent hemostatic effect.

[0148] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a thrombin-modified hemostatic membrane, characterized in that: It consists of the following steps: Preparation of PAN nanofiber membrane: dissolving PAN in an organic solvent to obtain a spinning solution, and then preparing a PAN fiber membrane by electrospinning, and drying for later use; Carboxylation: soak the dried PAN fiber membrane in an alkaline solution, react at a constant temperature of 50-60°C, then soak and pickle in an acidic solution, and then rinse with deionized water until neutral to obtain a surface carboxylated PAN fiber membrane; Amination: The surface hydroxylated PAN fiber membrane is immersed in a mixed solution of ethylenediamine and ethanol, reacted at a constant temperature of 30-45°C, and then rinsed with deionized water until neutral to obtain a surface aminated PAN fiber membrane; Itaconic acid crosslinking and activation: soaking the surface aminated PAN fiber membrane in an itaconic acid aqueous solution, reacting at a constant temperature of 50-65°C to obtain a crosslinked PAN fiber membrane, and then transferring the crosslinked PAN fiber membrane into an 80% ethanol mixture of EDC / NHS for immersion and activation to obtain a surface modified PAN fiber membrane; Loading thrombin: The surface-modified PAN fiber membrane is transferred into a thrombin solution, incubated at a constant temperature of 30-50°C, and then rinsed with deionized water to obtain a thrombin-modified hemostatic membrane.

2. The method for preparing the thrombin-modified hemostatic membrane according to claim 1, characterized in that: In the step of preparing the PAN nanofiber membrane, the organic solvent is at least one of N,N-dimethylformamide and dimethyl sulfoxide; and / or the mass concentration of the spinning solution is 8% to 10%.

3. The method for preparing the thrombin-modified hemostatic membrane according to claim 1, characterized in that: In the carboxylation step, the alkaline solution is at least one of a sodium hydroxide solution, a potassium hydroxide solution, a potassium hydroxide and ammonia solution mixture, and a sodium hydroxide and ammonia solution mixture; And / or, the acidic solution is at least one of a hydrochloric acid solution, a nitric acid solution or a sulfuric acid solution; And / or, the immersion pickling time is 3 to 5 hours.

4. The method for preparing the thrombin-modified hemostatic membrane according to claim 3, characterized in that: In the carboxylation step, the molar concentration of the sodium hydroxide is 1-3 mol / L; and / or the molar concentration of the hydrochloric acid solution is 0.5-2 mol / L.

5. The method for preparing the thrombin-modified hemostatic membrane according to claim 1, characterized in that: In the amination step, the volume ratio of ethylenediamine to ethanol is 4:5-8.

6. The method for preparing the thrombin-modified hemostatic membrane according to claim 1, characterized in that: In the itaconic acid cross-linking and activation step, the mass fraction of the itaconic acid aqueous solution is 8% to 10%.

7. The method for preparing the thrombin-modified hemostatic membrane according to claim 1, characterized in that: In the itaconic acid cross-linking and activation step, the activation is performed by immersion for 2 to 4 times, with each immersion for 6 to 8 hours.

8. The method for preparing the thrombin-modified hemostatic membrane according to claim 1, characterized in that: In the itaconic acid cross-linking and activation step, the mass ratio of EDC to NHS is 5:1-3.

9. The method for preparing the thrombin-modified hemostatic membrane according to claim 1, characterized in that: In the step of loading thrombin, the concentration of the thrombin solution is 10-30 NIH units / ml; and / or the culture time is 4-8 hours.

10. A thrombin-modified hemostatic membrane obtained by the preparation method according to any one of claims 1 to 9.

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