Photo-thermal antibacterial and procoagulant wound dressing easy to peel as well as preparation method and application of photo-thermal antibacterial and procoagulant wound dressing

Abstract

The invention discloses a photo-thermal antibacterial and procoagulant wound dressing easy to peel and a preparation method and application thereof, and relates to the technical field of biological materials. The method comprises the following steps: dissolving sericin in water, then sequentially adding hemoglobin, carbodiimide and N-hydroxysuccinimide, and stirring for cross-linking reaction; obtaining a precursor of the compound protein dressing through a mechanochemical crosslinking strategy; pre-cooling and freeze-drying the precursor to obtain a sericin hemoglobin dressing; and dialyzing the sericin hemoglobin dressing in water for 2-3 days, and freeze-drying again to obtain the protein dressing only containing hemoglobin and sericin. After the prepared antibacterial dressing is in contact with blood, formation of a compact fibrous protein film can be promoted, and the antibacterial dressing can be easily stripped from a wound part while blood coagulation is promoted. The wound dressing is completely composed of protein, is good in biocompatibility and has a huge development prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a wound dressing with photothermal antibacterial and blood-promoting effects and easy peeling, and a preparation method and application thereof. Background Art

[0002] Uncontrolled massive bleeding from deep tissues in accidental trauma can lead to high mortality. In addition, since accidental trauma is usually exposed to a complex environment, the wound is exposed to bacterial infection, which leads to delayed wound healing. Therefore, the ideal wound dressing needs to have antibacterial activity and rapid hemostasis properties. Sponge hemostasis has high liquid absorption due to its porous structure. It shows excellent hemostatic effect in the hemostasis of uncontrolled massive bleeding in deep tissues and is considered to be a potential candidate hemostatic agent for pre-hospital treatment.

[0003] In recent years, hemostatic sponges made of multiple ingredients have been studied to achieve efficient hemostasis and antibacterial properties. Proteins from natural sources have good biocompatibility and degradation properties, and their degradation products are not only non-toxic but also usable by the human body. In particular, natural proteins with high abundance, availability and low cost are ideal raw materials for the preparation of biomaterials with practical application value.

[0004] Hemoglobin is a physiological oxygen transport metalloprotein present in red blood cells. Currently, the application of hemoglobin in biomedical materials is mainly concentrated in photothermal antibacterial and repair of chronic skin wounds. Its application in hemostasis has not been widely developed. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned background technology, the present invention provides a wound dressing with photothermal antibacterial, pro-coagulation and easy peeling, as well as a preparation method and application thereof. The protein wound dressing prepared by the method is composed only of hemoglobin and sericin, and has good biocompatibility. The composite protein dressing has hemostatic properties that a single component does not have. Rapid coagulation is achieved by promoting the aggregation of red blood cells and the formation of fibrin membranes. The fibrin membrane formed on the surface of the dressing during the hemostasis process can also isolate it from the injured tissue, so that the dressing is easy to peel off from the injured part to avoid secondary damage. In addition, it also has photothermal antibacterial activity.

[0006] The first object of the present invention is to provide a method for preparing a wound dressing having photothermal antibacterial and procoagulant effects and being easy to peel off, comprising the following steps: Dissolving sericin in water, then sequentially adding hemoglobin and carbodiimide and stirring evenly, then adding N-hydroxysuccinimide and stirring to perform a cross-linking reaction to obtain a cross-linking solution; Applying external force to the cross-linking solution causes the activated proteins in the cross-linking solution to aggregate and undergo a cross-linking reaction, and collecting the aggregates to obtain a hemoglobin sponge precursor; The hemoglobin sponge precursor is precooled and completely freeze-dried to obtain a sericin hemoglobin sponge; The sericin hemoglobin sponge was dialyzed in water for 2-3 days, pre-cooled again and freeze-dried to obtain the hemoglobin sericin dressing.

[0007] Preferably, the mass ratio of the sericin, carbodiimide and N-hydroxysuccinimide is 5:1-10:1-5; The mass ratio of the sericin to the hemoglobin is 10:1-5; The cross-linking reaction temperature is 0-50°C, and the reaction time is greater than 2 h.

[0008] Preferably, the method of applying external force includes centrifugation or pressurization.

[0009] Preferably, when an external force is applied to the cross-linking solution, centrifugation is used to aggregate the proteins in the cross-linking solution and cause a cross-linking reaction; during the centrifugation, the centrifugal speed is 1000-10000 rpm, and the centrifugal time is 1-20 min.

[0010] Preferably, the precooling temperature is -80 to -20°C, the precooling time is 12 to 24 hours, and the freeze-drying time is 10 to 24 hours.

[0011] Preferably, the molecular weights of the sericin and hemoglobin are both ≥8000 MW.

[0012] Preferably, during the dialyzing of the sericin hemoglobin sponge in water, the molecular weight cut-off of the dialysis bag used is 3500 MW.

[0013] Preferably, when the sericin is dissolved in water, it is heated in a boiling water bath until it is completely dissolved, and then cooled to room temperature.

[0014] The second object of the present invention is to provide a wound dressing which has photothermal antibacterial and blood-promoting effects and is easy to peel off.

[0015] The third object of the present invention is to provide a photothermal antibacterial and blood-promoting wound dressing and an easy-to-peel wound dressing for use in the preparation of an antibacterial or hemostatic dressing.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a wound dressing with photothermal antibacterial, blood coagulation-promoting and easy peeling properties, as well as a preparation method and application thereof. The sericin hemoglobin wound dressing prepared by the present invention has good biocompatibility, photothermal antibacterial activity, rapid blood coagulation and the characteristics of being easy to peel off from the wound site. Hemoglobin has a near-infrared photothermal effect, and can convert light energy into heat under the irradiation of 808 nm near-infrared light, thereby killing bacteria, so the dressing has photothermal antibacterial properties. In addition, the addition of hemoglobin reduces the pore size of the original sericin wound dressing, which is conducive to the aggregation of red blood cells and platelets during the coagulation process. Moreover, hemoglobin has the effect of promoting blood coagulation. When the composite protein dressing contacts blood, it can promote the formation of a dense fibrin network, thereby accelerating blood coagulation. At the same time, the dense fibrin network on the surface of the dressing can isolate the hemostatic dressing from the tissue, so that the wound dressing is easy to peel off from the injured site. The dressing prepared by the present invention has only sericin protein and hemoglobin as components, so it has good biocompatibility and has great development prospects.

[0017] The invention has high practical application value in terms of preparation method and performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 1 and 2 are scanning electron microscope images of the sericin hemoglobin dressings obtained in Examples 1 to 3 and the element scanning results of Example 2.

[0019] Figure 2 1 is the cyclic compression stress-strain curve of the sericin hemoglobin dressing obtained in Examples 1 to 3.

[0020] Figure 3 The antibacterial effect of the sericin hemoglobin dressing obtained in Examples 1 to 3 under 808 nm near-infrared light irradiation.

[0021] Figure 4 This is the scanning electron microscope morphology of the sericin hemoglobin dressing obtained in Examples 1 to 3 after interaction with red blood cells.

[0022] Figure 5 This is the scanning electron microscope morphology of the sericin hemoglobin dressing obtained in Examples 1 to 3 after interaction with plasma.

[0023] Figure 6 These are the statistical results of the coagulation index of the sericin hemoglobin dressings obtained in Examples 1 to 3. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0025] The purpose of the present invention is to provide a wound dressing with photothermal antibacterial, procoagulant and easy peeling properties, as well as a preparation method and application thereof. The antibacterial dressing prepared by the present invention can promote the formation of a dense fibrin network after contact with blood, thereby promoting coagulation. The dense fibrin network wrapped on the surface of the dressing makes it easy to peel the dressing from the wound site. In addition, by utilizing the photothermal effect of hemoglobin itself, the composite protein dressing can achieve photothermal antibacterial. The wound dressing is completely composed of protein, has good biocompatibility, and has great development prospects.

[0026] In order to achieve the above-mentioned object, the first aspect of the present invention provides a method for preparing a wound dressing having photothermal antibacterial and procoagulant effects and being easy to peel off, comprising the following steps: Dissolving sericin in water, then sequentially adding hemoglobin and carbodiimide and stirring evenly, then adding N-hydroxysuccinimide and stirring to perform a cross-linking reaction to obtain a cross-linking solution; Applying external force to the cross-linking solution causes the activated proteins in the cross-linking solution to aggregate and undergo a cross-linking reaction, and collecting the aggregates to obtain a hemoglobin sponge precursor; The hemoglobin sponge precursor is precooled and completely freeze-dried to obtain a sericin hemoglobin sponge; The sericin hemoglobin sponge was dialyzed in water for 2-3 days, pre-cooled again and freeze-dried to obtain the hemoglobin sericin dressing.

[0027] The preparation method provided by the present invention can construct a dressing with good porosity and has good photothermal conversion efficiency under 808 nm irradiation, thereby achieving an antibacterial effect.

[0028] The mass ratio of the sericin, carbodiimide and N-hydroxysuccinimide is 5:1-10:1-5, and the preferred mass ratio is 5:5:3; the mass ratio of the sericin to the hemoglobin is 10:1-5; The cross-linking reaction temperature is 0-50°C, and the reaction time is greater than 2 h.

[0029] The external force may be applied by centrifugation or pressurization. Specifically, when the external force is applied to the cross-linking solution, centrifugation is used to aggregate the proteins in the cross-linking solution and cause a cross-linking reaction; during the centrifugation, the centrifugal speed is 1000-10000 rpm and the centrifugal time is 1-20 min.

[0030] The precooling temperature is -80~-20°C, the precooling time is 12~24 hours, and the freeze-drying time is 10~24 hours.

[0031] The molecular weights of the sericin and hemoglobin are both ≥8000 MW.

[0032] During the dialysis of sericin hemoglobin sponge in water, the molecular weight cutoff of the dialysis bag used was 3500 MW.

[0033] When the sericin is dissolved in water, it is heated in a boiling water bath until it is completely dissolved, and then cooled to room temperature.

[0034] In one embodiment, a method for preparing a sericin hemoglobin wound dressing comprises the following steps: (1) Dissolve sericin in water and heat until completely dissolved. After cooling to room temperature, add hemoglobin and carbodiimide in sequence and stir for 15 min. Finally, add N-hydroxysuccinimide for cross-linking reaction to obtain a cross-linking reaction liquid of flocculent sericin and hemoglobin.

[0035] In the step (1), the molecular weights of sericin and hemoglobin are above 8000 MW.

[0036] In the step (1), the mass ratio of sericin: carbodiimide: N-hydroxysuccinimide is 5:1-10:1-5, and the mass ratio of sericin to hemoglobin is 10:1-5.

[0037] The cross-linking reaction time in step (1) is more than 2 h, and the cross-linking reaction temperature is 0-50°C.

[0038] The process is to cross-link sericin and hemoglobin using the carbodiimide protein coupling method.

[0039] (2) Centrifuging the cross-linking reaction solution obtained in step (1), removing the supernatant to obtain a sericin and hemoglobin sponge precursor. The purpose of centrifugation is to increase the probability of collision and cross-linking between the activated carboxyl groups and amino groups in sericin and hemoglobin.

[0040] (3) precooling and completely freeze-drying the sericin and hemoglobin sponge precursor obtained in step (2) to obtain a sericin hemoglobin sponge; (4) The sericin hemoglobin sponge obtained in step (3) is dialyzed in water for 2 to 3 days, and then pre-cooled and completely freeze-dried to obtain a wound dressing containing only sericin and hemoglobin.

[0041] The purpose of dialysis is to remove the residual carbodiimide and N-hydroxysuccinimide in the product of step (3), and finally obtain a wound dressing containing only sericin and hemoglobin.

[0042] The centrifugal speed in step (2) is above 1000 rpm, and the centrifugal time is above 1 min.

[0043] The precooling temperature during the preparation process is above 20°C and the precooling time is above 1 h.

[0044] In the step (4), the molecular weight cut-off of the dialysis bag used is 3500 MW.

[0045] The water used in the preparation process was deionized water.

[0046] The second aspect of the present invention provides a wound dressing which has photothermal antibacterial and blood-promoting effects and is easy to peel off.

[0047] A third aspect of the present invention provides a photothermal antibacterial and blood-promoting and easily peelable wound dressing for use in the preparation of antibacterial or hemostatic dressings.

[0048] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0049] The sericin (hereinafter referred to as sericin) and hemoglobin used in the following examples are both from the laboratory. The sericin is extracted by a conventional high-temperature water-soluble method. The hemoglobin is extracted by a conventional water-crushing extraction method.

[0050] Example 1 A wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling properties and a preparation method thereof, comprising the following steps: Dissolve 500 mg of sericin in 50 mL of deionized water and heat in a boiling water bath until completely dissolved, then cool to room temperature. Then add 50 mg of hemoglobin and 500 mg of carbodiimide in turn and stir for 15 min, and finally add 300 mg of N-hydroxysuccinimide and stir at room temperature for more than 2 h. Centrifuge the cross-linked solution (6000 rpm, 15 min), remove the supernatant and obtain a sericin hemoglobin sponge precursor. Precool the precursor at -80℃ for 12 h and freeze-dry for more than 10 h to obtain a sericin hemoglobin sponge with residual crosslinker. The sponge was dialyzed in deionized water with a 3500 MW dialysis bag for 2-3 days, precooled at -80℃ for 12 h again, and freeze-dried for more than 10 h to obtain a sericin hemoglobin sponge.

[0051] The obtained product has been experimentally determined to have antibacterial activity, high compression strength, red blood cell aggregation and rapid coagulation properties.

[0052] Example 2 A wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling properties and a preparation method thereof, comprising the following steps: Dissolve 500 mg of sericin in 50 mL of deionized water and heat until completely dissolved, then cool to room temperature. Then add 150 mg of hemoglobin and 500 mg of carbodiimide in turn and stir for 15 min, and finally add 300 mg of N-hydroxysuccinimide and stir at room temperature for 2 h. Centrifuge the cross-linked solution (6000 rpm, 15 min), remove the supernatant and obtain a sericin hemoglobin sponge precursor. Precool the precursor at -80℃ for 12 h and freeze-dry for more than 10 h to obtain a sericin hemoglobin sponge with residual crosslinker. The sponge was dialyzed in deionized water with a 3500 MW dialysis bag for 2-3 days, and then precooled at -80℃ for 12 h and freeze-dried for more than 10 h to obtain a sericin hemoglobin sponge.

[0053] The obtained product has been experimentally determined to have antibacterial activity, high compression strength, red blood cell aggregation and rapid coagulation properties.

[0054] Example 3 A wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling properties and a preparation method thereof, comprising the following steps: Dissolve 500 mg of sericin in 50 mL of deionized water and heat until completely dissolved, then cool to room temperature. Then add 250 mg of hemoglobin and 500 mg of carbodiimide in turn and stir for 15 min, and finally add 300 mg of N-hydroxysuccinimide and stir at room temperature for 2 h. Centrifuge the cross-linked solution (6000 rpm, 15 min), remove the supernatant to obtain a sericin hemoglobin sponge precursor. Precool the precursor at -80℃ for 12 h and freeze-dry for more than 10 h to obtain a sericin hemoglobin sponge with residual crosslinker. The sponge was dialyzed in deionized water with a 3500 MW dialysis bag for 2-3 days, precooled at -80℃ for 12 h again, and freeze-dried for more than 10 h to obtain a sericin hemoglobin sponge.

[0055] The obtained product has been experimentally determined to have antibacterial activity, high compression strength, red blood cell aggregation and rapid coagulation properties.

[0056] Comparative Example 1 A wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling properties and a preparation method thereof, comprising the following steps: Dissolve 500 mg of sericin in 50 mL of deionized water and heat until completely dissolved, then cool to room temperature. Then add 500 mg of carbodiimide and stir for 15 min, and finally add 300 mg of N-hydroxysuccinimide and stir to react at room temperature for 2 h. Centrifuge the cross-linked solution (6000 rpm, 15 min), remove the supernatant to obtain a sericin sponge precursor. Precool the precursor at -80°C for 12 h and freeze-dry for more than 10 h to obtain a sericin sponge with residual crosslinker. The sponge was dialyzed in deionized water with a 3500 MW dialysis bag for 2-3 days, precooled again at -80°C for 12 h, and freeze-dried for more than 10 h to obtain a sericin sponge.

[0057] The experimental results show that the obtained product has weak antibacterial activity, high compression strength, red blood cell aggregation and weak coagulation performance.

[0058] In order to illustrate the relevant properties of the photothermal antibacterial, pro-coagulation and easily peelable wound dressing provided by the present invention, it is described in conjunction with the accompanying drawings.

[0059] Figure 1 The following are scanning electron microscope images of the sericin hemoglobin dressings obtained in Examples 1 to 3 and the element scanning results of Example 2. Taking Example 2 as an example, the element scanning results show that the sericin hemoglobin dressing contains iron, proving the presence of hemoglobin. The scanning results show that the dressings with added hemoglobin are similar to the comparative examples, both of which have an interconnected porous structure, which is conducive to absorbing blood, forming a coagulation barrier at the bleeding site, and accelerating the coagulation process in hemostasis of heavy bleeding.

[0060] Figure 2 : The cyclic compression stress-strain curves of the sericin hemoglobin dressings obtained in Examples 1 to 3 after wetting. The sericin dressing showed a compressive stress of 12.39 kPa after the first compression, and the compressive stress after 10 cycles of compression was 10.73 kPa. Example 1 showed a compressive stress of 16.08 kPa after the first compression, and the compressive stress after 10 cycles of compression was 14.95 kPa. Example 2 showed a compressive stress of 18.01 kPa after the first compression, and the compressive stress after 10 cycles of compression was 16.35 kPa. Example 3 showed a compressive stress of 18.37 kPa after the first compression, and the compressive stress after 10 cycles of compression was 16.47 kPa. Compared with the comparative example, the hemoglobin dressing has a higher compressive stress. After 10 cycles of compression, Examples 1 to 3 can still maintain 93.03%, 90.78%, and 89.65% of the compressive stress, respectively. The control group can only maintain 86.60% of the compression stress after 10 cycles of compression. This proves that the hemoglobin sericin dressing has excellent mechanical properties and can play an effective role as a coagulation barrier during hemostasis.

[0061] Figure 3 The antibacterial effect of the sericin hemoglobin dressings obtained in Examples 1 to 3 under 808 nm near-infrared light irradiation. As can be seen from the figure, Example 2 showed 100% bactericidal effect after 3 minutes of near-infrared light irradiation, and the photothermal bactericidal effect of Example 3 was even better, showing 100% bactericidal activity after 2 minutes of irradiation.

[0062] Figure 4 The scanning electron microscopic morphology of the hemoglobin sericin dressing obtained in Examples 1 to 3 after interaction with red blood cells. The ability of wound dressings to aggregate red blood cells is one of the criteria for evaluating the hemostatic performance of wound dressings. From the scanning electron microscopic results, it can be observed that the hemoglobin sericin dressings have excellent aggregation effects on red blood cells, indicating that they have the potential for efficient hemostasis.

[0063] Figure 5 The scanning electron microscope morphology of the hemoglobin sericin dressing obtained in Examples 1 to 3 after interaction with plasma. It can be seen from the figure that after the hemoglobin sericin dressing interacted with plasma, a dense fibrin film was formed on the surface. In contrast, after the sericin dressing in the comparative example interacted with plasma, a sparse fibrin network was formed. In comparison, the dense fibrin film has a stronger effect of aggregating red blood cells and platelets. At the same time, the dense film with a smooth surface is also conducive to the dressing being peeled off from the wound tissue.

[0064] Figure 6 The coagulation index of the sericin hemoglobin dressings obtained in Examples 1 to 3 is statistically analyzed. The coagulation index is one of the important indicators for evaluating the hemostatic ability of wound dressings. The results show that the coagulation index of the hemoglobin sericin dressing and the sericin dressing are significantly lower than that of the blank group and the commercial gelatin group. Among them, the coagulation index of Examples 2 and 3 is significantly lower than that of the other groups. It proves that hemoglobin has a procoagulant effect. Dressings with high hemoglobin content have better in vitro coagulation effects.

[0065] The present invention describes preferred embodiments and their effects. However, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0066] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a wound dressing having photothermal antibacterial and procoagulant properties and easy to peel off, characterized in that: The following steps are involved: Dissolving sericin in water, then sequentially adding hemoglobin and carbodiimide and stirring evenly, then adding N-hydroxysuccinimide and stirring to perform a cross-linking reaction to obtain a cross-linking solution; Applying external force to the cross-linking solution causes the activated proteins in the cross-linking solution to aggregate and undergo a cross-linking reaction, and collecting the aggregates to obtain a hemoglobin sponge precursor; The hemoglobin sponge precursor is precooled and completely freeze-dried to obtain a sericin hemoglobin sponge; The sericin hemoglobin sponge was dialyzed in water for 2-3 days, pre-cooled again and freeze-dried to obtain the hemoglobin sericin dressing.

2. The method for preparing the wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling according to claim 1, characterized in that: The mass ratio of the sericin, carbodiimide and N-hydroxysuccinimide is 5:1-10:1-5; The mass ratio of the sericin to the hemoglobin is 10:1-5; The cross-linking reaction temperature is 0-50°C, and the reaction time is greater than 2 h.

3. The method for preparing the wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling according to claim 1, characterized in that: The external force can be applied by centrifugation or pressure.

4. The method for preparing the wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling according to claim 1, characterized in that: When an external force is applied to the cross-linking solution, centrifugation is used to aggregate the proteins in the cross-linking solution and cause a cross-linking reaction; during the centrifugation, the centrifugal speed is 1000 ~10000 rpm, and the centrifugal time is 1~20 min.

5. The method for preparing the wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling according to claim 1, characterized in that: The precooling temperature is -80~-20°C, the precooling time is 12~24 hours, and the freeze-drying time is 10~24 hours.

6. The method for preparing the photothermal antibacterial and blood-promoting and easily peelable wound dressing according to claim 1, characterized in that: The molecular weights of the sericin and hemoglobin are both ≥8000 MW.

7. The method for preparing the wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling according to claim 1, characterized in that: During the dialysis of sericin hemoglobin sponge in water, the molecular weight cutoff of the dialysis bag used was 3500 MW.

8. The method for preparing the wound dressing with photothermal antibacterial and blood-promoting properties and easy peeling according to claim 1, characterized in that: When the sericin is dissolved in water, it is heated in a boiling water bath until it is completely dissolved, and then cooled to room temperature.

9. A wound dressing having photothermal antibacterial and blood-promoting properties and being easy to peel off, prepared by the method according to any one of claims 1 to 8.

10. Use of the photothermal antibacterial and procoagulant wound dressing as claimed in claim 9 in the preparation of antibacterial or hemostatic dressings.

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