Preparation and application of visible light crosslinking composite gel type hemostatic dressing

Abstract

The present application provides a visible light crosslinking composite gel type hemostatic dressing, comprising 1-55% methacrylated natural polymer, 0.0001-0.05% visible light initiator, 2-15% thickening agent; the method for preparing the hemostatic dressing comprises the following steps: firstly, preparing a visible light initiator solution, and dissolving the methacrylated natural polymer with the visible light initiator solution to obtain solution A; dissolving the thickening agent with deionized water or a phosphate buffered saline solution to obtain solution B; uniformly mixing solution A and solution B to obtain the visible light crosslinking composite gel type hemostatic dressing. The present application (1) is suitable for first aid hemostasis; (2) is solidified under visible light irradiation and closely adheres to the wound tissue; (3) has good adhesion, flexibility and moisturizing property after being solidified under visible light irradiation, and has a certain healing promotion effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to the preparation and application of a visible light cross-linked composite gel-type hemostatic dressing. Background Technology

[0002] Uncontrollable bleeding following trauma and during surgery remains a leading cause of death worldwide. Currently, while the most effective method of hemostasis is still surgical sutures to close blood vessels, in outdoor environments and emergency situations lacking adequate medical resources, it is still necessary to first use methods such as packing and compression to temporarily stop the bleeding and close the wound before transferring the patient to a hospital emergency room for secondary disinfection and thorough hemostasis. With the rapid advancement of medical materials science, many experimental chemical agents have been used for rapid wound closure and hemostasis, such as fibrin glue, gelatin, polymers, and hydrogels. Hemostatic materials are available in various dosage forms, including dressings, powders, and expandable materials. Common hemostatic dressings include gelatin sponges and fibrin glue, which are convenient to use and have good hemostatic effects, making them the most commonly used in clinical practice. However, they are difficult to adapt to irregular wounds and deeper wounds to achieve both hemostasis and wound coverage. Common hemostatic powders include sodium alginate powder and chitosan powder. These materials are suitable for bleeding from superficial small blood vessels. If the wound is deep and the defect is large, additional filling and compression materials are needed to achieve hemostasis. Expandable hemostatic materials, such as polycaprolactone (PCL) nanofiber membranes, can be used for hemostasis in deep wounds. After rapid expansion, they provide temporary filling and compression functions. However, because the shape after expansion cannot be controlled, insufficient local adhesion or excessive local filling pressure can lead to complications such as necrosis of the wound edge tissue later. Therefore, current hemostatic materials all have certain limitations and cannot simultaneously achieve hemostasis and promote healing. Summary of the Invention

[0003] To overcome the shortcomings of existing materials, this invention proposes a visible light cross-linked composite gel-type hemostatic dressing, comprising a methacrylamide natural polymer, a visible light initiator, and a thickener;

[0004] The methacrylamide natural polymer is any one or a combination of methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide sodium alginate, and methacrylamide carboxymethyl chitosan.

[0005] The visible light initiator is a combination of eosin Y and N-phenylglycine in any proportion;

[0006] The thickener includes any one or a combination of polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, and glycerol.

[0007] On the other hand, this invention discloses a method for preparing a visible light crosslinked composite gel-type hemostatic dressing, the preparation method comprising:

[0008] Preparation of Solution A: Prepare a visible light initiator solution, and dissolve 0-30% methacrylamide gelatin, 0-10% methacrylamide hyaluronic acid, 1-10% methacrylamide sodium alginate and 0-5% methacrylamide carboxymethyl chitosan in the visible light initiator solution to obtain Solution A;

[0009] Preparation of solution B: Dissolve 0-2% polyvinyl alcohol, 0-5% polyvinylpyrrolidone, and 0-2% sodium carboxymethyl cellulose in deionized water or phosphate buffer solution, and mix in 0-3% glycerol to obtain solution B;

[0010] The steps for preparing a mixed solution of solution A and solution B are as follows: Solution A and solution B are mixed evenly to obtain a visible light cross-linked composite gel-type hemostatic dressing.

[0011] On the other hand, the present invention also discloses a method of use, the method comprising:

[0012] Inject or extrude visible light cross-linked composite gel-type hemostatic dressings onto the wound surface;

[0013] After the wound is completely covered, the visible light cross-linked composite gel hemostatic dressing is continuously irradiated with a visible light source for a predetermined time.

[0014] Depending on the condition of the wound, choose to use or not use sterile medical dressings for bandaging and fixation, including hydrocolloid dressings.

[0015] Compared with the prior art, the visible light cross-linked composite gel-type hemostatic dressing of the present invention has the following beneficial effects:

[0016] (1) Suitable for emergency hemostasis;

[0017] (2) It solidifies under visible light and adheres closely to the wound tissue;

[0018] (3) After curing under visible light, it has good adhesion, flexibility and moisture retention, and has a certain healing effect.

[0019] (4) The visible light cross-linked composite gel hemostatic dressing of the present invention is easy to prepare and use, and has good application prospects. Attached Figure Description

[0020] Figure 1 SEM image of the visible light crosslinked composite gel-type hemostatic dressing after freeze-drying in Example 2;

[0021] Figure 2 : Cytotoxicity test results of the photocrosslinkable composite hydrogel in Experiment Example 1;

[0022] Figure 3Blood compatibility test results of the photocrosslinkable composite hydrogel in Experiment Example 2;

[0023] Figure 4 : A bar chart showing the hemostasis time of the photocrosslinkable composite hydrogel in the mouse liver hemorrhage model in Experiment Example 3;

[0024] Figure 5 : A bar chart showing the bleeding volume of the photocrosslinkable composite hydrogel in the mouse liver hemorrhage model hemostasis experiment in Experiment Example 3;

[0025] Figure 6 : Statistical bar chart of coagulation time in in vitro coagulation experiment of photocrosslinkable composite hydrogel in Experiment Example 4. Detailed Implementation

[0026] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0027] Example 1

[0028] Dissolve 0.0001% of eosin Y and 0.001% of N-phenylglycine in deionized water to obtain a visible light initiator solution. Then, dissolve the photoinitiator solution and sodium methacrylamide in deionized water to prepare solution A, wherein sodium methacrylamide is 2%.

[0029] Under a 70℃ water bath, polyvinylpyrrolidone and sodium carboxymethyl cellulose were dissolved in deionized water for 60 minutes, and then glycerol was added to prepare solution B, which contained 4% polyvinylpyrrolidone, 2% sodium carboxymethyl cellulose, and 3% glycerol. Solution A and solution B were thoroughly mixed at a volume ratio of 1:1 and air bubbles were removed to prepare a visible light crosslinked composite gel-type hemostatic dressing.

[0030] Take 2 ml of solution A and apply it to the surface of a glass slide. Place it under white LED light (100 mW / cm2) for 2 minutes to cure.

[0031] Apply 2 ml of the composite gel to the surface of a glass slide, and cure it by irradiating it with a white LED light source (100 mW / cm2) for 2 minutes.

[0032] Example 2

[0033] At room temperature, eosin Y, N-phenylglycine, and sodium alginate with methacrylamide were dissolved in deionized water to prepare solution A', which contained 0.0002% eosin Y, 0.002% N-phenylglycine, and 4% sodium alginate with methacrylamide. Under a 70°C water bath, polyvinylpyrrolidone and sodium carboxymethyl cellulose were dissolved in deionized water for 60 minutes, and then glycerol was added to prepare solution B', which contained 4% polyvinylpyrrolidone, 2% sodium carboxymethyl cellulose, and 3% glycerol. Solutions A' and B' were thoroughly mixed at a volume ratio of 1:1 and air bubbles were removed to prepare a visible light crosslinked composite gel-type hemostatic dressing. 2 ml of the visible light crosslinked composite gel-type hemostatic dressing was applied to a glass slide and cured under a white LED (100 mW / cm²) for 2 minutes.

[0034] After solidification, the hydrogel was placed in deionized water until it swelled fully, then freeze-dried and surface-sprayed with gold. Its morphology and structure were characterized under a scanning electron microscope. The internal microstructure of the hydrogel is shown below. Figure 1 As shown.

[0035] Example 3

[0036] At room temperature, eosin Y, N-phenylglycine, sodium alginate with methacrylamide, and gelatin with methacrylamide were dissolved in deionized water to prepare solution A", which contained 0.0005% eosin Y, 0.005% N-phenylglycine, 4% sodium alginate with methacrylamide, and 8% gelatin with methacrylamide. Under a 70°C water bath, polyvinylpyrrolidone and sodium carboxymethyl cellulose were dissolved in deionized water for 60 minutes, and then glycerol was added to prepare solution B", which contained 4% polyvinylpyrrolidone, 2% sodium carboxymethyl cellulose, and 5% glycerol. Solutions A" and B" were thoroughly mixed at a volume ratio of 1:1 and air bubbles were removed to prepare a visible light crosslinked composite gel-type hemostatic dressing.

[0037] Apply 2 ml of visible light cross-linked composite gel-type hemostatic dressing to the surface of a glass slide and cure it under white LED light (100 mW / cm2) for 2 minutes.

[0038] Example 4

[0039] The visible light cross-linked composite gel hemostatic dressings prepared in Examples 1, 2, and 3 were injected or applied to the wound surface of mice.

[0040] After the visible light cross-linked composite gel-type hemostatic dressing completely covers the wound, it is cured by irradiation under white LED light (100mW / cm2) for 2 minutes.

[0041] The mouse wound was fixed with a hydrocolloid dressing.

[0042] Experimental Example 1: Cytotoxicity Test

[0043] According to standard GB / T 16886.5-2017 / ISO 10993-5:2009, the cytotoxicity of visible light crosslinked composite gel hemostatic dressings was evaluated using the CCK8 method via an extraction test. The specific experimental steps are as follows:

[0044] (1) Take samples of visible light cross-linked composite gel-type hemostatic dressings prepared in Examples 1, 2 and 3;

[0045] (2) Prepare an extract of the sample under sterile conditions at a ratio of 100 mg material / 1 mL complete culture medium. The extraction conditions are 37°C and 24 hours. Prepare a series of dilutions of the original extract.

[0046] (3) Mouse fibroblasts (3T3) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin antibiotics and placed in a cell culture incubator at 37°C with 5% CO2. After the cell density reached about 80%, the adherent cells were digested with trypsin-EDTA, centrifuged, resuspended in complete medium, and the cell density of the cell suspension was adjusted to 1×10⁵ cells / mL.

[0047] (4) Add 100 μL of cell suspension to each well of a 96-well plate and incubate in a cell culture incubator for 24 hours;

[0048] (5) Discard the culture medium and replace it with the original extract and a series of diluents. At the same time, set up positive control, negative control and blank control, and culture for 24 hours, 48 ​​hours and 72 hours according to the above culture conditions.

[0049] (6) At each observation time point, the cells were photographed under an optical microscope to record cell density and morphological changes. The original extract or culture medium was replaced with 100 μL of fresh complete culture medium per well and 10 μL of CCK8 reagent was added per well. The cells were incubated at 37°C for 3 hours.

[0050] (7) Place the 96-well plate in an ELISA reader to detect its absorbance at 450 nm, and calculate the relative cell viability of different samples under different concentrations of extract according to the formula.

[0051] Conclusion: According to the testing standards, the visible light cross-linked composite gel hemostatic dressing showed no cytotoxicity (relative cell viability > 90%) under the established experimental conditions. The experimental results obtained from steps 1-7 above are as follows: Figure 2 As shown.

[0052] Test Example 2: Blood Compatibility Test

[0053] The specific experimental steps are as follows:

[0054] (1) Take samples of visible light cross-linked composite gel-type hemostatic dressings prepared in Examples 1, 2 and 3;

[0055] (2) Weigh 1.25mg, 2.5mg, 5mg and 10mg samples respectively and place them in 1.5mL EP tubes. Add 1mL of physiological saline to each tube and incubate at 37℃ for 30 minutes. Set up positive control and negative control at the same time.

[0056] (3) C57BL / 6 mice were anesthetized by isoflurane inhalation. Whole blood was collected from the mice by enucleation. The mice were immediately euthanized by dislocation of the neck after blood collection. Fresh whole blood was mixed with EDTA-K2 anticoagulant (whole blood: anticoagulant = 9:1) to prepare anticoagulated blood. The blood was centrifuged at room temperature for 10 minutes at 100g to obtain layered blood. The supernatant was discarded and the red blood cells were washed three times with physiological saline.

[0057] (4) After adding 20 μL of red blood cell suspension to each sample tube, incubate at 37°C for 1 hour, centrifuge at 100g for 10 minutes, and then add the supernatant to a 96-well plate, 100 μL of liquid per well.

[0058] (5) Place the 96-well plate in an ELISA reader to detect its absorbance at 540 nm, and calculate the hemolysis rate of different samples at different concentrations according to the formula.

[0059] Conclusion: According to the testing standards, the visible light cross-linked composite gel hemostatic dressing exhibits good blood compatibility (hemolysis rate <5%) under the established experimental conditions. The experimental results obtained from the above steps are as follows: Figure 3 As shown.

[0060] Experimental Example 3: Verification of in vivo hemostasis function

[0061] The specific experimental steps are as follows:

[0062] (1) Take samples of visible light cross-linked composite gel-type hemostatic dressings prepared in Examples 1, 2 and 3;

[0063] (2) C57BL / 6 mice were anesthetized by pentobarbital intraperitoneal anesthesia. The mice were fixed in a supine position on a sterile operating table. After the skin of the anterior chest and upper abdomen of the mice was prepared and disinfected, a longitudinal incision of about 1 cm was made along the midline of the abdomen below the xiphoid process. The liver was cut layer by layer from the outside to the inside until it was exposed. The liver lobe was gently lifted out of the abdominal cavity and the body fluid around the liver was wiped away with sterile gauze. A sterile water-proof pad and a pre-weighed sterile gauze were placed under the liver.

[0064] (3) Make an incision at an angle of about 30° on the liver of a mouse using a syringe needle with an outer diameter of 1.6 mm, and wipe away any momentary bleeding with sterile gauze;

[0065] (4) The prepared photocrosslinkable composite gel was applied to the liver wound and irradiated under white LED (100mW / cm2) for a certain period of time. A control group was set up to conduct the experiment simultaneously.

[0066] (5) Remove the solidified hydrogel, observe the bleeding in the liver, weigh the sterile gauze again, and evaluate the hemostasis effect by the hemostasis time and the amount of bleeding.

[0067] Conclusion: Under the experimental conditions, the visible light cross-linked composite gel hemostatic dressing exhibited superior hemostatic effect (shorter hemostasis time and less bleeding compared to the control group). The experimental results obtained based on the above steps are as follows: Figure 4 , Figure 5 As shown.

[0068] Experiment Example 4: Verification of In Vitro Hemostasis Function

[0069] The specific experimental steps are as follows:

[0070] (1) Take samples of visible light cross-linked composite gel-type hemostatic dressings prepared in Examples 1, 2 and 3;

[0071] (2) C57BL / 6 mice were anesthetized by isoflurane inhalation. Whole blood was collected from the mice by enucleation. The mice were immediately euthanized by dislocation of the neck after blood collection. Fresh whole blood was mixed with sodium citrate anticoagulant (whole blood: anticoagulant = 9:1) to prepare anticoagulated blood.

[0072] (3) Weigh 10 mg of sample and place it in a 1.5 mL EP tube and preheat it at 37 °C for 30 minutes. Set up a control group and conduct the experiment simultaneously.

[0073] (4) Add 300 μL of anticoagulated blood to each tube, add 100 μL of 0.2 mol / L CaCl2 to the tube and start timing until the blood stops flowing completely. The in vitro hemostatic function of the material is measured by the clotting time.

[0074] Conclusion: Under the experimental conditions, the visible light cross-linked composite gel hemostatic dressing exhibited superior hemostatic effect (shorter in vitro coagulation time compared to the control group). The experimental results obtained based on the above steps are as follows: Figure 6 As shown.

[0075] Experimental Example 5: Validation of Wound Healing Promotion Function

[0076] The specific experimental steps are as follows:

[0077] (1) Take samples of visible light cross-linked composite gel-type hemostatic dressings prepared in Examples 1, 2 and 3;

[0078] (2) Anesthetize C57BL / 6 mice by isoflurane inhalation, fix the mice in a prone position on a sterile operating table, and after the back of the mice is prepared and disinfected, make a circular wound with a diameter of 1cm in the center of the back.

[0079] (3) The visible light cross-linked composite gel hemostatic dressing was covered on the wound and irradiated under white LED (100mW / cm2) for a certain period of time. A control group was set up to conduct the experiment simultaneously.

[0080] (4) After bandaging and fixing the dressing and wound, the experimental mice were fed and observed under suitable environmental conditions;

[0081] (5) Take photos to record the wound healing process at the set observation time points, collect materials and use pathological experimental techniques to evaluate the differences in wound healing among different experimental groups from the perspective of tissue repair and regeneration.

[0082] Conclusion: Under the experimental conditions, the visible light cross-linked composite gel hemostatic dressing showed a better effect in promoting wound repair (compared to the control group, the wound healing rate was faster, more new blood vessels were formed during the repair process, and the epithelial migration was faster).

[0083] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a visible light cross-linked composite gel-type hemostatic dressing, characterized in that, Includes the following steps: At room temperature, eosin Y, N-phenylglycine, and sodium methacrylamide were dissolved in deionized water to prepare solution A', which contained 0.0002% eosin Y, 0.002% N-phenylglycine, and 4% sodium methacrylamide. Under a 70°C water bath, polyvinylpyrrolidone and sodium carboxymethyl cellulose were dissolved in deionized water for 60 minutes, and glycerol was mixed in to prepare solution B', which contained 4% polyvinylpyrrolidone, 2% sodium carboxymethyl cellulose, and 3% glycerol. Solutions A' and B' were thoroughly mixed at a volume ratio of 1:1 and air bubbles were removed to obtain the visible light crosslinked composite gel-type hemostatic dressing.

2. A method for preparing a visible light cross-linked composite gel-type hemostatic dressing, characterized in that, Includes the following steps: At room temperature, eosin Y, N-phenylglycine, sodium alginate with methacrylamide, and gelatin with methacrylamide were dissolved in deionized water to prepare solution A", which contained 0.0005% eosin Y, 0.005% N-phenylglycine, 4% sodium alginate with methacrylamide, and 8% gelatin with methacrylamide. Under a water bath at 70°C, polyvinylpyrrolidone and sodium carboxymethyl cellulose were dissolved in deionized water for 60 minutes, and glycerol was mixed in to prepare solution B", which contained 4% polyvinylpyrrolidone, 2% sodium carboxymethyl cellulose, and 5% glycerol. Solution A" and solution B" were thoroughly mixed at a volume ratio of 1:1 and air bubbles were removed to obtain the visible light crosslinked composite gel-type hemostatic dressing.

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