Asymmetric layered porous antibacterial hemostatic sponge dressing as well as preparation method and application thereof

Abstract

The invention discloses an asymmetric layered porous antibacterial hemostatic sponge dressing as well as a preparation method and application thereof. The method comprises the following steps: pre-crosslinking chitosan and halloysite nanotubes, further crosslinking with keratin, carrying out directional freeze drying on the crosslinked mixed solution to obtain a sponge material, and coating one surface of sponge with a PDMS solution to obtain the asymmetric layered porous antibacterial hemostatic sponge dressing. The layered hemostatic sponge dressing with rapid and effective hemostatic performance, antibacterial activity and biocompatibility is prepared by combining a freeze drying technology and asymmetric design and utilizing feather keratin, halloysite nanotubes (HNTs) and chitosan (CS), and the layered hemostatic sponge dressing has a wide application prospect in the field of wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of medical products, and in particular relates to an asymmetric layered porous antibacterial hemostatic sponge dressing and a preparation method and application thereof. Background Art

[0002] On the battlefield, in traffic accidents and in daily life, massive bleeding and wound infection are the main causes of death. Therefore, rapid intervention in patient bleeding is crucial to improving survival rates. There are currently many types of hemostatics, including polysaccharides (such as chitosan (CS), oxidized cellulose, starch), proteins (such as thrombin, gelatin, collagen) and clay minerals. Among these hemostatics, clays are particularly noteworthy, including montmorillonite, zeolite, kaolin, halloysite and lithophane, because of their many advantages in practical applications. Clay has a strong ability to absorb blood due to its huge specific surface area and unique crystal structure. Its hemostatic mechanism mainly stimulates coagulation factors through negative charges, quickly absorbs the outflowing fluid and concentrates the coagulation factors.

[0003] In addition to the hemostatic component, porosity is an essential feature of hemostatic dressings. Sponges with high porosity have a larger surface area, which is conducive to the adsorption and enrichment of red blood cells. In particular, the interconnected structure of the pores can provide oxygen and nutrients, provide a good physiological environment for cells, and promote wound healing and cell proliferation. The ability to keep the wound moist has a significant advantage in this process. Summary of the invention

[0004] We used directional and conventional freeze-drying methods to prepare layered sponges with highly regular longitudinal channels, transverse layers and porous structures. By adjusting the use of HNTs, we also developed a new layered sponge dressing with through holes. The resulting sponge was modified to form an asymmetric structure, which is more conducive to preventing reverse osmosis of blood.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first object of the present invention is to provide a method for preparing an asymmetric layered porous antibacterial hemostatic sponge dressing, which comprises the following steps: firstly pre-crosslinking chitosan and halloysite nanotubes, then further crosslinking with keratin, directionally freeze-drying the cross-linked mixed solution to obtain a sponge material, and coating a PDMS solution on one side of the sponge to obtain an asymmetric layered porous antibacterial hemostatic sponge dressing.

[0007] Preferably, the mass ratio of chitosan, keratin and halloysite nanotubes is 1:(0.1-1):(0.5-1)).

[0008] Preferably, the mass ratio of chitosan, keratin and halloysite nanotubes is 1:1:(0.5-1).

[0009] Preferably, the viscosity of the chitosan is 100-200 mPa.s.

[0010] Preferably, the PDMS solution comprises a PDMS prepolymer, a curing agent and THF.

[0011] Preferably, the coating PDMS solution is: weigh 3.0g PDMS prepolymer and 0.3g curing agent, add them to 25mL THF organic solvent, stir magnetically for 1 hour to dissolve, use a spray pen to spray the mixed PDMS solution on the sponge surface at a constant speed under an air pressure of 0.4MPa, and after spraying, place the sponge in a vacuum drying oven at 60°C for 2h.

[0012] Preferably, the specific steps are:

[0013] 1) dissolving chitosan in an acetic acid aqueous solution and filtering for later use;

[0014] 2) mixing the ultrasonically treated halloysite nanotubes with the CS aqueous solution of step 1) and stirring for 1 hour;

[0015] 3) adding KP with a molecular weight less than 1KD to the mixed solution of step 2) and stirring for 3 hours;

[0016] 4) placing the mixed solution in step 3) in a mold for freezing, and then performing directional freeze drying to obtain a layered porous sponge dressing;

[0017] 5) Weigh 3.0 g of PDMS prepolymer and 0.3 g of curing agent, add them to 25 mL of THF organic solvent, stir magnetically for 1 hour to dissolve the two components, use a spray pen with a nozzle diameter of 0.5 mm to spray the mixed PDMS solution on the sponge surface at a constant speed under an air pressure of 0.4 MPa. After spraying, put the sponge into a vacuum drying oven at 60° C. for 2 hours. After curing, an asymmetric layered porous antibacterial hemostatic sponge dressing is obtained.

[0018] The second object of the present invention is to provide an asymmetric layered porous antibacterial hemostatic sponge dressing prepared by the above method.

[0019] The third object of the present invention is to provide the use of the above-mentioned sponge dressing in antibacterial hemostasis.

[0020] Preferably, the antibacterial hemostasis is antibacterial hemostasis of wounds.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) All sponge components are derived from nature, and each component is both a structural unit and a functional unit;

[0023] (2) The synthesis method is simple and mild, requiring only a self-crosslinking reaction at room temperature to ensure the biosafety and bioactivity of the material.

[0024] (3) The introduction of KP not only makes each sheet full of pores, which helps to quickly absorb blood in wounds with heavy bleeding, but also improves the antibacterial effect of the sponge.

[0025] (4) Directed freezing technology gives the sponge axially ordered and regular pore channels, enabling it to have a more efficient liquid transfer rate.

[0026] (5) The introduction of HNTs not only improves the hemostatic rate of sponge dressings, but also regulates the pore structure of the sponge, greatly improving the porosity of the layered sponge.

[0027] (6) By coating one side of the sponge with polydimethylsiloxane (PDMS), an asymmetric effect is achieved. The PDMS coating can prevent reverse osmosis of blood, block foreign substances from entering the wound through the pores, reduce the risk of wound infection, and maintain the air permeability of the hemostatic dressing. The air circulation on the wound surface can also promote the aggregation of coagulation factors, platelets, and fibrin, thereby achieving coagulation.

[0028] (7) The present invention uses directional freeze-drying technology to successfully prepare an axially ordered layered porous sponge, thereby accelerating the hemostasis process. The addition of HNTs not only improves the hemostasis speed of the sponge dressing, but also can adjust the pore structure of the sponge and increase the porosity of the material. The synthetic sponge exhibits an antibacterial effect. One side of the sponge is coated with polydimethylsiloxane (PDMS) to make it hydrophobic, forming an asymmetric structure that can effectively prevent blood reverse osmosis. This specially designed sponge can not only prevent the reverse osmosis of blood and prevent foreign substances from entering the wound through the pores, thereby reducing the risk of wound infection, but also maintain the air permeability of the hemostatic dressing. Compared with pure CS / HNTs sponge material, the introduction of keratin not only makes the material exhibit the characteristics of a porous lamellar structure, improves the liquid transmission rate, but also enhances the antibacterial effect of the sponge dressing. The sponge dressing of the present invention has broad application prospects in the process of wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a physical picture of the layered porous keratin sponge material of Examples 3 to 4.

[0030] Figure 2 These are SEM images of the layered sponge materials of Examples 1 to 2 before and after hemostasis.

[0031] Figure 3 These are SEM images of the layered sponge materials of Examples 3 to 4 before and after hemostasis.

[0032] Figure 4The antibacterial properties of the sponge materials of Control and Examples 1 to 4 against S. aureus and E. coil.

[0033] Figure 5 The antibacterial rates of the sponge materials of Control and Examples 1 to 4 against S. aureus and E. coil.

[0034] Figure 6 This is the water contact angle of the hydrophobic surface of the asymmetric sponge material in Example 4.

[0035] Figure 7 This is the barrier effect of the composite sponge with or without PDMS coating on blood in Example 4.

[0036] Figure 8 The hemostatic properties of gauze and sponge materials of Examples 1 to 4 on femoral artery injury in rats (n=6). DETAILED DESCRIPTION

[0037] The content of the present invention is further described in detail below through specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial sources or can be obtained by prior art methods unless otherwise specified. Unless otherwise specified, the experiments or test methods are conventional methods in the art. The feather keratin of the present invention was purchased from Shaanxi Zhongyi Kangjian Biotechnology Co., Ltd.

[0038] Embodiment 1:

[0039] (1) Weigh 2 g of CS at a mass volume ratio of 2% and dissolve it in 100 mL of acetic acid aqueous solution (the volume ratio of acetic acid to water is 2:100), filter and set aside. The viscosity of CS is 100-200 mPa.s.

[0040] (2) The CS solution was placed in a mold with a copper block at the bottom for directional freezing. The ice crystals grown along the temperature gradient were treated as a reverse template by a directional freeze-drying method, so that the material had a porous structure in the lateral direction and formed highly regular tunnels in the longitudinal direction, and the CS layered sponge dressing (CS-SM) was obtained by directional freeze-drying.

[0041] (3) The antibacterial activity of the sponge was tested using S. aureus and E. coil as the test strains. The bacteria were reactivated twice for the experiment. The bacterial stock solution was added to the sterilized LB liquid culture medium and cultured for 12 h at 37 °C and 150 rpm / min. Before the bacterial culture, the sponge sample was exposed to ultraviolet light for 24 h. The initial concentration of the bacterial suspension was adjusted to 10 7CFU / mL, cut 10 mg of sponge into pieces and mix it with 25 mL of bacterial solution and put it into a test tube (without sponge as the control), and place it on a constant temperature incubator shaker at 37°C and 120 r / min for 1 h. Then, take 100 μL of the suspension and inoculate it on a sterilized LB agar plate. After incubating it in a 37°C incubator for 24 h, count the number of bacteria and calculate the inhibition rate.

[0042] (4) 3.0 g of PDMS prepolymer and 0.3 g of curing agent were weighed at a mass ratio of 10:1 and added to 25 mL of THF organic solvent. The two components were dissolved by magnetic stirring for 1 h. The mixed PDMS solution was sprayed on the sponge surface at a constant speed using a spray pen (nozzle diameter of 0.5 mm) under an air pressure of 0.4 MPa. After spraying, the sponge was placed in a vacuum drying oven at 60 °C for 2 h. After curing, an asymmetric CS sponge with a PDMS coating was obtained.

[0043] (5) To test the barrier effect of the PDMS hydrophobic layer on blood, an equal amount of blood was dropped onto a sponge sprayed or not sprayed with PDMS, and the blood loss was recorded.

[0044] (6) The ethical requirements for all animal experiments in this work strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals. Male SD rats weighing between 400 and 500 g were selected as the femoral artery injury model. Due to their large amount of bleeding, they were anesthetized with sodium pentobarbital and fixed on the operating board before surgery. First, the hair in the groin of the rat's thigh was shaved to expose the skin, which was then disinfected with 75% ethanol solution, and then an incision was made to expose the femoral artery. Subsequently, the femoral blood vessels were cut, resulting in heavy bleeding. Immediately, a pre-sterilized asymmetric hemostatic sponge (diameter: 3 cm) was applied to the wound, ensuring that the hydrophobic side of the sponge faced outward. The coagulation was observed every 10 seconds until the bleeding stopped completely. The coagulation time was recorded, and the blood loss was calculated after weighing the hemostatic material. The wound was photographically recorded. Three rats were used in each group.

[0045] Embodiment 2:

[0046] (1) Same as Example 1-(1).

[0047] (2) The HNTs after ultrasound (40 KHz, 20 min) were mixed with the above CS aqueous solution (the mass ratio of HNTs to CS was 1:1, both were 400 mg) at 600 rpm and stirred for 1 hour.

[0048] (3) The same as Example 1-(2), the obtained layered sponge was named (CS-1HCNs-SM).

[0049] (4) Same as Example 1-(3).

[0050] (5) Same as Example 1-(4).

[0051] (6) Same as Example 1-(5).

[0052] (7) Same as Example 1-(6).

[0053] Embodiment 3:

[0054] (1) Same as Example 1-(1).

[0055] (2) The HNTs after ultrasound (40 KHz, 20 min) were mixed with the above CS aqueous solution (the mass ratio of HNTs to CS was 0.5:1, 200 mg and 400 mg, respectively) at 600 rpm and stirred for 1 hour.

[0056] (3) Add 400 mg of KP (feather keratin) with a molecular weight less than 1 KD into the mixture in step 2 and continue stirring for 3 hours.

[0057] (4) The same as Example 1-(2) to obtain a layered sponge (CSKP-0.5HCNs-SM).

[0058] (5) Same as Example 1-(3).

[0059] (6) Same as Example 1-(4).

[0060] (7) Same as Example 1-(5).

[0061] (8) Same as Example 1-(6).

[0062] Embodiment 4:

[0063] (1) Same as Example 1-(1).

[0064] (2) The HNTs after ultrasound (40 KHz, 20 min) were mixed with the above CS aqueous solution (the mass ratio of HNTs to CS was 1:1, both were 400 mg) at 600 rpm and stirred for 1 hour.

[0065] (3) Add 400 mg of KP with a molecular weight less than 1 KD to the mixture in step 2 and continue stirring for 3 hours.

[0066] (4) The same as Example 1-(2) to obtain a layered sponge (CSKP-1HCNs-SM).

[0067] (5) Same as Example 1-(3).

[0068] (6) Same as Example 1-(4). The water contact angle of the hydrophobic surface of the asymmetric sponge material is as follows: Figure 6As shown, the water contact angle of the hydrophobic surface of the PDMS coating is 135.8°.

[0069] (7) Same as Example 1-(5). The barrier effect of composite sponge with or without PDMS coating on blood is shown in Figure 7 As shown, after spraying PDMS, the blood loss of the layered sponge CSKP-1HCNs-SM was 0.

[0070] (8) Same as Example 1-(6).

[0071] Test example:

[0072] 1. Scanning electron microscope (SEM) observation

[0073] (1) Methods:

[0074] Freeze-dry the sponge sample first, and cut a cube-sized sample of about 5 mm from the sponge material. Fix the sponge sample on the SEM sample stage. Use conductive tape to ensure good electrical contact between the sample and the sample stage. Then coat the sample with a conductive film (gold). Turn on the SEM equipment, place the sample in the SEM sample chamber, and use the microscope eyepiece or monitor to perform preliminary alignment and positioning of the sample. Make sure the sample is in the field of view and adjust the focus to obtain a clear image.

[0075] (2) Results:

[0076] The actual pictures of the layered porous keratin sponge materials of Examples 3 to 4 are as follows Figure 1 As shown, the SEM images of the layered sponge materials of Examples 1 to 2 before and after hemostasis are as follows Figure 2 As shown, the SEM images of the layered sponge materials of Examples 3 to 4 before and after hemostasis are as follows Figure 3 As shown. Figure 1-3 It can be seen that compared with pure CS / HNTs sponge materials, CSKP-0.5HCNs-SM and CSKP-0.5HCNs-SM exhibit the characteristics of lamellar structure with holes, with highly regular longitudinal channels, lateral layers and porous structures.

[0077] 2. Calculation of bacterial inhibition rate

[0078] (1) Calculation formula:

[0079] Inhibition rate (%) = (bacterial number of blank group - bacterial number of sample group) / bacterial number of sample group.

[0080] (2) Results:

[0081] The antibacterial properties of the sponge materials of Examples 1-4 against S. aureus and E. coil are shown in Table 1. Figure 4 As shown, the antibacterial rate is Figure 5 shown.

[0082] 3. Hemostasis performance test

[0083] Examples 1 to 4 Hemostatic properties of sponge materials on femoral artery injury in rats (n=6) Figure 8 shown.

[0084] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing an asymmetric layered porous antibacterial hemostatic sponge dressing, characterized in that: The method comprises the following steps: pre-crosslinking chitosan and halloysite nanotubes, further crosslinking with keratin, directionally freeze-drying the crosslinked mixed solution to obtain a sponge material, and coating a PDMS solution on one side of the sponge to obtain an asymmetric layered porous antibacterial hemostatic sponge dressing.

2. The method according to claim 1, characterized in that: The mass ratio of chitosan, keratin and halloysite nanotubes is 1:(0.1-1):(0.5-1).

3. The method according to claim 2, characterized in that The mass ratio of chitosan, keratin and halloysite nanotubes is 1:1:(0.5-1).

4. The method according to claim 1, characterized in that The viscosity of the chitosan is 100-200 mPa.s.

5. The method according to claim 1, characterized in that The PDMS solution includes a PDMS prepolymer, a curing agent and THF.

6. The method according to claim 1, characterized in that The PDMS solution coating is as follows: 3.0 g of PDMS prepolymer and 0.3 g of curing agent are weighed, added to 25 mL of THF organic solvent, magnetically stirred for 1 hour to dissolve, and the mixed PDMS solution is sprayed on the sponge surface at a constant speed using a spray pen under an air pressure of 0.4 MPa. After spraying, the sponge is placed in a vacuum drying oven at 60° C. for 2 hours.

7. The method according to claim 1, characterized in that The specific steps are: 1) dissolving chitosan in an acetic acid aqueous solution, filtering and setting aside; 2) mixing the ultrasonically treated halloysite nanotubes with the CS aqueous solution of step 1) and stirring for 1 hour; 3) adding KP with a molecular weight less than 1KD to the mixed solution of step 2) and stirring for 3 hours; 4) placing the mixed solution in step 3) in a mold for freezing, and then performing directional freeze drying to obtain a layered porous sponge dressing; 5) Weigh 3.0 g of PDMS prepolymer and 0.3 g of curing agent, add them to 25 mL of THF organic solvent, stir magnetically for 1 hour to dissolve the two components, use a spray pen with a nozzle diameter of 0.5 mm to spray the mixed PDMS solution on the sponge surface at a constant speed under an air pressure of 0.4 MPa. After spraying, put the sponge into a vacuum drying oven at 60° C. for 2 hours. After curing, an asymmetric layered porous antibacterial hemostatic sponge dressing is obtained.

8. An asymmetric layered porous antibacterial hemostatic sponge dressing prepared by the method according to any one of claims 1 to 7.

9. Use of the asymmetric layered porous antibacterial hemostatic sponge dressing according to claim 8 in antibacterial hemostasis.

10. The use according to claim 9, characterized in that: The antibacterial hemostasis is the antibacterial hemostasis of wounds.

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