Preparation method of antibacterial, antioxidant and hemostatic hydrogel dressing capable of slowly releasing phycocyanin

Abstract

The invention discloses a preparation method of an antibacterial, antioxidant and hemostatic hydrogel dressing capable of slowly releasing phycocyanin. The preparation method comprises the following steps: putting kaolin into an aqueous solution of lysozyme to fully adsorb the lysozyme, then putting the kaolin into a mixed aqueous solution of gamma-methacryloxypropyltrimethoxysilane and gamma-aminopropyltriethoxysilane, and enabling the two kinds of silane to generate polysiloxane on the surface of the kaolin. Mixing the kaolin which is subjected to silane treatment and adsorbs the lysozyme with an aqueous solution of spirulina and sodium alginate, and standing and defoaming to obtain a membrane casting solution; scraping the membrane casting solution into a membrane, and spraying an aqueous solution containing calcium chloride and lysozyme onto the surface of the membrane to cure and crosslink the membrane, thereby obtaining the antibacterial, antioxidant and hemostatic hydrogel dressing capable of slowly releasing phycocyanin. The lysozyme embedded in the hydrogel and the polysiloxane is slowly released near the spirulina to destroy the cell wall of the spirulina, so that the phycocyanin is slowly released. The lysozyme, the phycocyanin and the kaolin respectively endow the hydrogel dressing with antibacterial, antioxidant and hemostatic functions.

Description

Technical Field

[0001] The present invention relates to a preparation method of an antibacterial, antioxidant and hemostatic hydrogel dressing capable of sustained-release phycocyanin, belonging to the fields of biomaterials and medicine. Background Art

[0002] At present, the main hemostatic components of domestic and foreign military hemostatic materials are chitosan or clay particles (mainly kaolin and zeolite), and the carriers are traditional gauze or polymer dressings. Historically, various minerals have been used for traumatic hemostasis, such as zeolite and montmorillonite, which have gradually withdrawn due to their too large side effects. Zeolite can cause local scalding due to the exothermic reaction during hemostasis, while montmorillonite particles are prone to fall off, causing damage to the inner wall of blood vessels and organ embolism. Kaolin is mainly composed of tiny sheet-like, tubular, stacked sheet-like kaolinite cluster minerals smaller than 2 microns, belonging to aluminosilicate inert minerals, and has rich natural reserves in China. The chemical composition of kaolin contains a large amount of Al 2 O 3 、SiO 2 and a small amount of Fe 2 O 3 、TiO 2 as well as trace amounts of K 2 O, Na 2 O, CaO and MgO and other substances. When kaolin comes into contact with blood, it can directly activate coagulation factor XII, initiate the intrinsic coagulation pathway, and then activate coagulation factor XI, so that fibrinogen forms fibrin monomers, and the fibrin monomers combine into fibrin polymers to form water-insoluble blood fibers. In the case of the lack of coagulation factor XII, kaolin can directly activate coagulation factor XI to achieve the purpose of hemostasis. Another hemostatic mechanism of kaolin is physical hemostasis. When kaolin comes into contact with the blood flowing out of a damaged wound, it can quickly absorb the water molecules in the blood, concentrate the platelets and thrombin in the blood, and make the coagulation factors and platelets aggregate and deposit to achieve the purpose of hemostasis [Military Medical Sciences, 2017, 41(2): 141-145]. However, it is relatively difficult to clean the kaolin hemostatic material in powder form. Although these hemostatic materials have good hemostatic effects, their antibacterial properties are generally not strong, and some hemostatic gauzes even completely lack antibacterial functions and cannot meet the antibacterial requirements of acute infected wounds during wartime.

[0003] Wound dressings or artificial skin can prevent germs from invading the wound bed, effectively maintain the humidity of the wound, absorb wound exudate, and have good biocompatibility. Among commercial wound dressings, most are prepared from natural biopolymers. Gelatin or collagen crosslinked with glutaraldehyde can reduce immunogenicity and is suitable for the treatment of full-thickness skin wounds. Non-woven fabrics of chitin fibers are used for burn treatment and show good effects in clinical studies. Poly(L-leucine) sponges are also being clinically tested as dressings for full-thickness skin wounds. In order to improve the anti-infective property of this artificial skin and enhance its clinical application value, Matsuda et al. embedded microspheres of poly-L-lactic acid containing the antibiotic Tobramycin in the silicone layer of the upper layer of a double-layer artificial skin. As the microspheres degrade continuously, the antibiotic therein can be slowly released to prevent infection by external germs [Biomaterials, 1992, 13: 119]. Silver ions have been selected as antibacterial materials for a long time because of their strong antibacterial ability and low incidence of bacterial drug resistance. Silver ions have broad-spectrum antibacterial activity and have strong antibacterial effects on Gram-negative bacteria, Gram-positive bacteria, fungi, some viruses, etc. The research on antibacterial materials mostly endows the materials with antibacterial properties by loading antibiotics, silver ions, grafting quaternary ammonium groups or photothermal effects. These antibacterial methods all have certain defects: for example, overuse of antibiotics will lead to the emergence of drug-resistant bacteria, silver ions and quaternary ammonium groups have cytotoxicity, and the photothermal effect is complex to operate and difficult to reach deep tissues. Therefore, choosing an antibacterial method with good safety performance and convenient application is the research focus of wound antibacterial dressings.

[0004] Alginate is a natural polysaccharide isolated from brown seaweeds such as kelp and sargassum. It has a wide range of sources and low prices. As early as 1951, Blaine et al. explored the possibility of calcium alginate as a hemostatic material. The British scientist Winter found that the surface of the wound heals faster in a moist environment than in a dry environment. The moist environment accelerates the migration of epidermal cells from healthy skin to the wound, thus accelerating the wound healing rate. Under the guidance of the principle of "wet therapy", alginate-based medical dressings, gauzes, bandages, etc. have been widely used due to their superior moisture retention, easy removability as a whole and other characteristics [Knitting Industry, 2004, 32(5): 60 - 63]. In the early 1980s, the British company Courtaulds successfully made a medical gauze from alginate fiber and applied it to wounds with a lot of bleeding and pus. When the gauze comes into contact with pus and blood, the calcium alginate fiber undergoes an ion exchange with sodium ions in the human body, and the water-insoluble calcium alginate is slowly converted into water-soluble sodium alginate, so that a large amount of water enters the fiber interior to form a hydrogel, which endows the gauze with excellent properties such as extremely high hygroscopicity and easy removability. When Groves and Lawrence studied the application of alginate gauze on skin graft wounds, they found the good hemostatic effect of alginate gauze. Hemostasis of the wound surface can be achieved within 5 minutes after use [Ann R Coll Surg Engl., 1986, 68(1): 27 - 28]. Alginate dressings have functions such as oxygen permeability, promoting tissue growth and relieving pain. Qin Yimin et al. combined the antibacterial properties of silver and the hygroscopicity of alginate fiber to prepare an antibacterial medical dressing and confirmed its good antibacterial effect. Masahiro et al. found that alginate dressings do not adhere to tissues during wound dressing changes, which can reduce the pain of secondary trauma to patients. In addition, alginate dressings also have advantages such as wide sources, good biocompatibility, biodegradability, and no pollution to the environment.

[0005] Both kaolin and alginate lack antibacterial and bacteriostatic properties. The calcium alginate dressing loaded with nano-silver has good antibacterial properties, but too high silver ion concentration is likely to cause heavy metal ion toxicity, and silver ions are easily lost, resulting in a decline in antibacterial performance. Lysozyme, also known as muramidase, is a protein with no toxicity, harmlessness and high safety. It kills microorganisms by hydrolyzing the mucopolysaccharide of microorganisms, causing their cell walls to rupture and the contents to escape. It has no side effects on the human body, does not remain in the body, and has certain health care functions. The role of lysozyme in food preservation has attracted wide attention in society. It is now widely used in the anti-corrosion and preservation of aquatic products and meat products and can be an ideal antibacterial component for wound dressings.

[0006] Phycocyanin is a dark blue powder isolated from Spirulina. It is one of the rare pigment proteins in nature. Not only is it brightly colored, but it is also a protein rich in nutrients. Its amino acid composition is complete, and the content of essential amino acids is high. Phycocyanin has the effects of antioxidant, anti-infection, anti-allergy, anti-cancer, promoting blood cell regeneration, and promoting the synthesis of elastin in the human body. In countries such as Europe, America, and Japan, phycocyanin is widely used as a high-grade natural pigment in food and cosmetics and is made into biochemical drugs. Due to its good color characteristics and nutritional effects, as a natural blue pigment and nutritional and health care ingredient, it has been widely developed and applied in other fields such as food, cosmetics, diagnosis and treatment, rehabilitation health care products, and pharmaceuticals. Spirulina, as the source of phycocyanin, has also received increasing attention in recent years. Spirulina is a filamentous cyanobacterium rich in various proteins, vitamins, and trace elements. It is green, safe, easy to obtain, has no toxic and side effects, and has high nutritional value and disease prevention and health care value. It has also been found to have many pharmacological effects and can be used as a bioactive supplement, with physiological effects of promoting cell viability or survival, so it has attracted much attention in the biomedical industry. Spirulina is becoming increasingly important in the fields of health care products, food, and medicine. Moreover, there is currently no report on the toxic and side effects of spirulina products on humans. It can be used to antiviral, antioxidant, improve human immunity, and assist in the treatment of diseases. Choi et al. also used the electrospinning method and prepared an alginate / PCL nanofiber wound dressing containing spirulina extract through CaCl 2 crosslinking. The results showed that the dressing accelerated wound healing by improving the moisture retention, biosecurity, and effectiveness of the injured skin. The impregnation of alginate increased the skin adhesion and humidity, accelerated wound healing, and did not cause cytotoxicity [Biotechnology and Bioprocess Engineering, 2017, 22(6): 679-685]. However, the previous research of this invention showed that when spirulina or phycocyanin was directly added to the alginate hydrogel, the release of phycocyanin was too fast. Especially when the aqueous solution of spirulina and sodium alginate was formed into a film and then directly crosslinked with an aqueous calcium ion solution, water was lost during the crosslinking process. Therefore, a large amount of phycocyanin was lost into the calcium ion crosslinking aqueous solution.

[0007] In view of the problems such as the potential harm of heavy metal ion antibacterial agents in dressings, the generation of drug resistance in bacteria due to the abuse of antibiotics, the lack of antibacterial and antioxidant functions in traditional dressings, the easy loss and high price of directly added phycocyanin, etc., the present invention reports a preparation method of an antibacterial, antioxidant and hemostatic hydrogel dressing capable of slowly releasing phycocyanin. Kaolin is put into an aqueous solution of lysozyme to fully adsorb lysozyme, and then it is put into a mixed aqueous solution of γ-methacryloyloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane, and the two silanes generate polysiloxane on the surface of kaolin. The kaolin adsorbed with lysozyme after being treated with silane is mixed with an aqueous solution of spirulina and sodium alginate, and after standing and defoaming, a casting solution is obtained. After the casting solution is scraped into a film, it is sprayed with an aqueous solution containing calcium chloride and lysozyme on the film surface to cure and crosslink the film, and an antibacterial, antioxidant and hemostatic hydrogel dressing capable of slowly releasing phycocyanin is obtained. The lysozyme embedded in the hydrogel and polysiloxane is slowly released near the spirulina, destroying the cell wall of the spirulina, thereby slowly releasing phycocyanin. Lysozyme, phycocyanin and kaolin endow the hydrogel dressing of the present invention with antibacterial, antioxidant and hemostatic functions respectively. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the technical problem to be solved by the present invention is the problems such as the potential harm of heavy metal ion antibacterial agents in dressings, the generation of drug resistance in bacteria due to the abuse of antibiotics, the lack of antibacterial and antioxidant functions in traditional dressings, the easy loss and high price of directly added phycocyanin, etc. The technical solution of the present invention for the above-mentioned existing problems and deficiencies is to provide a preparation method of an antibacterial, antioxidant and hemostatic hydrogel dressing capable of slowly releasing phycocyanin.

[0009] The present invention provides a preparation method of an antibacterial, antioxidant and hemostatic hydrogel dressing capable of slowly releasing phycocyanin, which is characterized by including the following steps:

[0010] a) Prepare an aqueous solution of lysozyme with a mass percentage concentration of 0.1% - 5% with deionized water, disperse kaolin into the aqueous solution of lysozyme under stirring to obtain a kaolin mixed aqueous solution with a kaolin mass percentage concentration of 0.5% - 5%, improve the dispersion of kaolin by ultrasonic wave at low power, adjust the pH of the mixed aqueous solution to 4 - 5, and make lysozyme fully adsorb onto kaolin under stirring. The kaolin adsorbed with lysozyme is separated by filtration with filter paper, and then put into a mixture aqueous solution of γ-methacryloyloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane with a mass percentage concentration of 0.2% - 2% respectively. Ultrasonic wave at low power is used to uniformly disperse each component, and the mixture is allowed to stand and react for 12 - 36 hours. The precipitate is filtered, and the unreacted substances are washed away with deionized water to obtain kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane, which is reserved after freeze-drying;

[0011] b) The freeze-dried kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane obtained in step a) with a mass percentage concentration of 0.2% to 5% and spirulina with a mass percentage concentration of 0.5% to 2% are dispersed into deionized water under stirring, and then sodium alginate with a mass percentage concentration of 1.0% to 3% is added, and stirring is continued to fully dissolve the sodium alginate. After standing and defoaming, a casting solution is obtained for standby;

[0012] c) Prepare a mixed aqueous solution of lysozyme with a mass percentage concentration of 0.1% to 5% and a soluble calcium salt with a mass percentage concentration of 1.0% - 20% as a cross-linking agent aqueous solution, and pour the cross-linking agent aqueous solution into a spray bottle for standby;

[0013] d) Pour the casting solution obtained in step b) onto a clean and flat glass plate, and use a film scraping rod to scrape it into a liquid film with a uniform thickness. Then immediately spray the cross-linking agent aqueous solution in the spray bottle in step c) onto the surface of the liquid film. Calcium ions in the soluble calcium salt cross-link with sodium alginate to form a calcium alginate hydrogel film. After the calcium alginate hydrogel film is initially formed, it is taken down and immediately sprayed with the cross-linking agent aqueous solution to cross-link the other side of the film. After sufficient cross-linking, an antibacterial, antioxidant, and hemostatic hydrogel dressing capable of sustained release of phycocyanin is obtained; the hydrogel dressing is engraved into the required shape and size with a mold for standby;

[0014] e) Since lysozyme is adsorbed and embedded by polysiloxane, and lysozyme is easily adsorbed by alginate, the lysozyme inside the hydrogel dressing has a sustained release property. The released lysozyme destroys the cell wall of spirulina when it encounters spirulina, so as to achieve the purpose of sustained release of phycocyanin; the sustained release performance of phycocyanin in the hydrogel dressing obtained in step d) is tested in physiological saline. The hydrogel dressing of the present invention has a longer ability to release phycocyanin than the dressing directly added with phycocyanin and spirulina, and can continuously release phycocyanin for 24 to 60 hours;

[0015] f) The antibacterial rate of the hydrogel dressing of the present invention against Staphylococcus aureus reaches 65% to 100%;

[0016] g) The clearance rate of phycocyanin released by the hydrogel dressing of the present invention against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt reaches 60% to 96% after 7 hours.

[0017] The soluble calcium salt described in the present invention is any one or a mixture of two or more of calcium chloride, calcium dihydrogen phosphate, and calcium nitrate. The hemostatic time of the antibacterial, antioxidant, and hemostatic hydrogel dressing for the bleeding of the middle lobe of the rat liver after resection is 1 - 5 minutes. Detailed implementation manners

[0018] The following introduces specific embodiments of the present invention, but the present invention is not limited by the embodiments.

[0019] Example 1

[0020] a) Prepare an aqueous solution of lysozyme with a mass percentage concentration of 0.1% using deionized water. Disperse kaolin into the aqueous solution of lysozyme under stirring to obtain a kaolin mixed aqueous solution with a mass percentage concentration of 0.5% of kaolin. Ultrasonically improve the dispersibility of kaolin at low power, adjust the pH of the mixed aqueous solution to 4, and allow lysozyme to be fully adsorbed onto kaolin under stirring. Filter and separate the kaolin adsorbed with lysozyme using filter paper, and then place it into a mixed aqueous solution of γ-methacryloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane with a mass percentage concentration of 0.2% respectively. Ultrasonically disperse each component evenly at low power, allow it to stand and react for 12 hours, filter to obtain the precipitate, and wash away the unreacted substances with deionized water to obtain kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane. After freeze-drying, it is reserved for use;

[0021] b) Disperse the freeze-dried kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane obtained in step a) with a mass percentage concentration of 0.2% and spirulina with a mass percentage concentration of 0.5% into deionized water under stirring, and then add sodium alginate with a mass percentage concentration of 1.0%. Continue to stir until sodium alginate is fully dissolved. After standing and defoaming, a casting solution is obtained and reserved for use;

[0022] c) Prepare a mixed aqueous solution of lysozyme with a mass percentage concentration of 0.1% and calcium chloride with a mass percentage concentration of 1.0% as a cross-linking agent aqueous solution, and pour the cross-linking agent aqueous solution into a spray bottle and reserve it for use;

[0023] d) Pour the casting solution obtained in step b) onto a clean and flat glass plate, and use a film scraping rod to scrape it into a liquid film with a uniform thickness. Then immediately spray the cross-linking agent aqueous solution in the spray bottle in step c) onto the surface of the liquid film. Calcium ions in the soluble calcium salt cross-link with sodium alginate to form a calcium alginate hydrogel film. After the calcium alginate hydrogel film is initially formed, remove it immediately and spray the cross-linking agent aqueous solution on the other side of the film to cross-link. After sufficient cross-linking, an antibacterial, antioxidant, and hemostatic hydrogel dressing capable of sustained release of phycocyanin is obtained; Use a mold to cut the hydrogel dressing into the required shape and size and reserve it for use;

[0024] e) Since lysozyme is adsorbed and embedded by polysiloxane, and lysozyme is easily adsorbed by alginate, the lysozyme inside the hydrogel dressing has a sustained-release property. The sustained-release lysozyme destroys the cell wall of spirulina when it encounters spirulina, thereby achieving the purpose of sustained release of phycocyanin; Test the sustained-release property of phycocyanin in the hydrogel dressing obtained in step d) in physiological saline. The hydrogel dressing of the present invention has a longer ability to release phycocyanin than the dressing directly added with phycocyanin and spirulina, and can continuously release phycocyanin for up to 24 hours;

[0025] f) The antibacterial rate of the hydrogel dressing of the present invention against Staphylococcus aureus reaches 65%;

[0026] g) The scavenging rate of phycocyanin released from the hydrogel dressing of the present invention against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt reaches 60% after 7 hours, and the hemostasis time for the bleeding of the middle lobe resection of rat liver is 5 minutes.

[0027] Example 2.

[0028] a) Prepare an aqueous solution of lysozyme with a mass percentage concentration of 5% with deionized water. Disperse kaolin into the aqueous solution of lysozyme under stirring to obtain a mixed aqueous solution of kaolin with a mass percentage concentration of 5%. Ultrasonically improve the dispersibility of kaolin at low power, adjust the pH of the mixed aqueous solution to 5, and allow lysozyme to be fully adsorbed onto kaolin under stirring. Filter and separate the kaolin adsorbed with lysozyme with filter paper, and then put it into a mixed aqueous solution of 2% (by mass percentage) of γ-methacryloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane. Ultrasonically disperse each component evenly at low power, allow to stand and react for 36 hours, filter to obtain the precipitate, and wash away the unreacted substances with deionized water to obtain kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane. Freeze-dry and reserve for later use;

[0029] b) Disperse the freeze-dried kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane obtained in step a) with a mass percentage concentration of 5% and spirulina with a mass percentage concentration of 2% into deionized water under stirring, and then add sodium alginate with a mass percentage concentration of 3%. Continue stirring to fully dissolve sodium alginate, allow to stand and defoam to obtain a casting solution for later use;

[0030] c) Prepare a mixed aqueous solution of lysozyme with a mass percentage concentration of 5% and calcium nitrate with a mass percentage concentration of 20% as a cross-linking agent aqueous solution, and pour the cross-linking agent aqueous solution into a spray bottle for later use;

[0031] d) Pour the casting solution obtained in step b) onto a clean and flat glass plate, scrape it into a liquid film with uniform thickness with a film scraping rod, and then immediately spray the cross-linking agent aqueous solution in the spray bottle in step c) onto the surface of the liquid film. Calcium ions in the soluble calcium salt cross-link with sodium alginate to form a calcium alginate hydrogel film. After the calcium alginate hydrogel film is initially formed, take it down immediately and spray the cross-linking agent aqueous solution on the other side of the film. After sufficient cross-linking, obtain an antibacterial, antioxidant and hemostatic hydrogel dressing capable of sustained release of phycocyanin; Cut the hydrogel dressing into the required shape and size with a mold and reserve for later use;

[0032] e) Since lysozyme is adsorbed and encapsulated by polysiloxane, and lysozyme is easily adsorbed by alginate, the lysozyme inside the hydrogel dressing has a sustained-release property. The released lysozyme destroys the cell wall of Spirulina when it encounters Spirulina, thereby achieving the purpose of sustained release of phycocyanin. When testing the sustained-release property of phycocyanin in the hydrogel dressing obtained in step d) in physiological saline, the hydrogel dressing of the present invention has a longer ability to release phycocyanin than the dressing directly added with phycocyanin and Spirulina, and can continuously release phycocyanin for up to 60 hours.

[0033] f) The antibacterial rate of the hydrogel dressing of the present invention against Staphylococcus aureus reaches 100%.

[0034] g) The clearance rate of phycocyanin released by the hydrogel dressing of the present invention against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt reaches 96% after 7 hours, and the hemostasis time for the bleeding of the middle lobe of the rat liver after resection is 1 minute.

[0035] Example 3.

[0036] a) Prepare an aqueous solution of lysozyme with a mass percentage concentration of 0.5% using deionized water. Disperse kaolin into the aqueous solution of lysozyme under stirring to obtain a kaolin mixed aqueous solution with a mass percentage concentration of kaolin of 2.5%. Improve the dispersibility of kaolin by ultrasonic treatment at low power, adjust the pH of the mixed aqueous solution to 4.5, and allow lysozyme to be fully adsorbed onto kaolin under stirring. Filter and separate the kaolin adsorbed with lysozyme, and then place it into a mixed aqueous solution of γ-methacryloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane with a mass percentage concentration of 1.0% respectively. Ultrasonic at low power to uniformly disperse each component, let it stand and react for 24 hours, filter to obtain the precipitate, and wash away the unreacted substances with deionized water to obtain kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane, and reserve it after freeze-drying.

[0037] b) Disperse the freeze-dried kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane obtained in step a) with a mass percentage concentration of 3% and Spirulina with a mass percentage concentration of 1.5% into deionized water under stirring, and then add sodium alginate with a mass percentage concentration of 2.5%. Continue to stir to fully dissolve sodium alginate, and let it stand to defoam to obtain a casting solution for standby.

[0038] c) Prepare a mixed aqueous solution of lysozyme with a mass percentage concentration of 1.0% and calcium dihydrogen phosphate with a mass percentage concentration of 2.5% as the cross-linking agent aqueous solution, and pour the cross-linking agent aqueous solution into a spray bottle for standby.

[0039] d) Pour the casting solution obtained in step b) onto a clean and flat glass plate, and use a film scraping rod to scrape it into a liquid film with a uniform thickness. Then, immediately spray the crosslinking agent aqueous solution in the spray bottle in step c) onto the surface of the liquid film. The calcium ions in the soluble calcium salt crosslink with sodium alginate to form a calcium alginate hydrogel film. After the calcium alginate hydrogel film is initially formed, remove it and immediately spray the crosslinking agent aqueous solution on the other side of the film. After sufficient crosslinking, an antibacterial, antioxidant, and hemostatic hydrogel dressing capable of sustained release of phycocyanin is obtained. Cut the hydrogel dressing into the required shape and size with a mold for standby;

[0040] e) Since lysozyme is adsorbed and embedded by polysiloxane, and lysozyme is easily adsorbed by alginate, the lysozyme inside the hydrogel dressing has a sustained release property. The released lysozyme destroys the cell wall of Spirulina when it encounters Spirulina, so as to achieve the purpose of sustained release of phycocyanin. Test the sustained release property of phycocyanin in the hydrogel dressing obtained in step d) in physiological saline. The hydrogel dressing of the present invention has a longer ability to release phycocyanin than the dressing directly added with phycocyanin and Spirulina, and can continuously release phycocyanin for up to 48 hours;

[0041] f) The antibacterial rate of the hydrogel dressing of the present invention against Staphylococcus aureus reaches 90%;

[0042] g) The clearance rate of phycocyanin released by the hydrogel dressing of the present invention against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt reaches 90% after 7 hours, and the hemostasis time for the bleeding of the middle lobe of the rat liver after resection is 2 minutes.

[0043] Example 4.

[0044] a) Prepare an aqueous solution of lysozyme with a mass percentage concentration of 1.0% with deionized water. Disperse kaolin into the aqueous solution of lysozyme under stirring to obtain a kaolin mixed aqueous solution with a mass percentage concentration of kaolin of 2.0%. Ultrasonically improve the dispersion of kaolin at a low power, adjust the pH of the mixed aqueous solution to 4.2, and make lysozyme fully adsorbed onto kaolin under stirring. Filter and separate the kaolin adsorbed with lysozyme with filter paper, and then put it into a mixed aqueous solution of 2.0% by mass of γ-methacryloyloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane. Ultrasonically disperse each component evenly at a low power, stand and react for 24 hours, filter to obtain the precipitate, and wash away the unreacted substances with deionized water to obtain kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane. After freeze-drying, it is used for standby;

[0045] b) The kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane after freeze-drying obtained in step a) with a mass percentage concentration of 5% and spirulina with a mass percentage concentration of 1.5% are dispersed into deionized water under stirring, and then sodium alginate with a mass percentage concentration of 2.8% is added, and stirring is continued to fully dissolve the sodium alginate. After standing to defoam, a casting solution is obtained for standby;

[0046] c) Prepare an aqueous cross-linking agent solution of a mixed aqueous solution of lysozyme with a mass percentage concentration of 2.0% and calcium chloride with a mass percentage concentration of 5.0%, and pour the aqueous cross-linking agent solution into a spray bottle for standby;

[0047] d) Pour the casting solution obtained in step b) onto a clean and flat glass plate, and use a film scraping rod to scrape it into a liquid film with a uniform thickness. Then immediately spray the aqueous cross-linking agent solution in the spray bottle in step c) onto the surface of the liquid film. Calcium ions in the soluble calcium salt cross-link with sodium alginate to form a calcium alginate hydrogel film. After the calcium alginate hydrogel film is initially formed, it is taken down immediately and the other side of the film is sprayed with the aqueous cross-linking agent solution for cross-linking. After sufficient cross-linking, an antibacterial, antioxidant and hemostatic hydrogel dressing capable of sustained release of phycocyanin is obtained; the hydrogel dressing is engraved into the required shape and size with a mold for standby;

[0048] e) Since lysozyme is adsorbed and embedded by polysiloxane, and lysozyme is easily adsorbed by alginate, the lysozyme inside the hydrogel dressing has a sustained release property. The released lysozyme destroys the cell wall of spirulina when it encounters spirulina, so as to achieve the purpose of sustained release of phycocyanin; the sustained release performance of phycocyanin in the hydrogel dressing obtained in step d) is tested in physiological saline. The hydrogel dressing of the present invention has a longer ability to release phycocyanin than the dressing directly added with phycocyanin and spirulina, and can continuously release phycocyanin for up to 48 hours;

[0049] f) The antibacterial rate of the hydrogel dressing of the present invention against Staphylococcus aureus reaches 99.5%;

[0050] g) The clearance rate of phycocyanin released by the hydrogel dressing of the present invention against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt reaches 94% after 7 hours, and the hemostatic time for the bleeding of the middle lobe of the rat liver after resection is 1.5 minutes.

[0051] Example 5.

[0052] a) Prepare an aqueous solution of lysozyme with a mass percentage concentration of 1.0% using deionized water. Disperse kaolin into the aqueous solution of lysozyme under stirring to obtain a mixed aqueous solution of kaolin with a mass percentage concentration of 3%. Ultrasonically improve the dispersion of kaolin at low power, adjust the pH of the mixed aqueous solution to 4.8, and allow lysozyme to be fully adsorbed onto kaolin under stirring. Filter and separate the kaolin adsorbed with lysozyme using filter paper, and then place it into a mixed aqueous solution of γ-methacryloyloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane with a mass percentage concentration of 1.5% respectively. Ultrasonically disperse each component evenly at low power, allow it to stand and react for 32 hours, filter to obtain the precipitate, and wash away the unreacted substances with deionized water to obtain kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane. After freeze-drying, it is reserved for use;

[0053] b) Disperse the freeze-dried kaolin adsorbed with lysozyme wrapped with a layer of polysiloxane obtained in step a) with a mass percentage concentration of 4% and spirulina with a mass percentage concentration of 1% into deionized water under stirring, and then add sodium alginate with a mass percentage concentration of 2%. Continue to stir to fully dissolve sodium alginate, and after standing to defoam, obtain a casting solution for reserve;

[0054] c) Prepare a mixed aqueous solution of lysozyme with a mass percentage concentration of 0.5% and calcium chloride with a mass percentage concentration of 2% as the cross-linking agent aqueous solution, and pour this cross-linking agent aqueous solution into a spray bottle for reserve;

[0055] d) Pour the casting solution obtained in step b) onto a clean and flat glass plate, use a film scraping rod to scrape it into a liquid film with a uniform thickness, and then immediately spray the cross-linking agent aqueous solution in the spray bottle in step c) onto the surface of the liquid film. Calcium ions in the soluble calcium salt cross-link with sodium alginate to form a calcium alginate hydrogel film. After the calcium alginate hydrogel film is initially formed, remove it immediately and spray the cross-linking agent aqueous solution on the other side of the film to cross-link. After sufficient cross-linking, obtain an antibacterial, antioxidant, and hemostatic hydrogel dressing capable of sustained release of phycocyanin; Use a mold to cut the hydrogel dressing into the required shape and size for reserve;

[0056] e) Since lysozyme is adsorbed and embedded by polysiloxane, and lysozyme is easily adsorbed by alginate, the lysozyme inside the hydrogel dressing has a sustained release property. The released lysozyme destroys the cell wall of spirulina when it encounters spirulina, thereby achieving the purpose of sustained release of phycocyanin; Test the sustained release property of phycocyanin in the hydrogel dressing obtained in step d) in physiological saline. The hydrogel dressing of the present invention has a longer ability to release phycocyanin than the dressing directly adding phycocyanin and spirulina, and can continuously release phycocyanin for up to 52 hours;

[0057] f) The antibacterial rate of the hydrogel dressing of the present invention against Staphylococcus aureus reaches 92%;

[0058] The clearance rate of phycocyanin released from the hydrogel dressing of the present invention against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt reaches 86% after 7 hours, and the hemostasis time for the bleeding caused by partial hepatectomy of the middle lobe of the rat liver is 2.5 minutes.

Claims

1. A method for preparing an antibacterial, antioxidant and hemostatic hydrogel dressing capable of sustained-release phycocyanin, characterized in that The following steps are involved: a) preparing a lysozyme aqueous solution with a mass percentage concentration of 0.1% to 5% with deionized water, dispersing kaolin in the lysozyme aqueous solution under stirring to obtain a kaolin mixed aqueous solution with a mass percentage concentration of 0.5% to 5% of kaolin, Improving the dispersibility of kaolin by ultrasonic treatment at low power, adjusting the pH of the mixed aqueous solution to 4-5, fully adsorbing lysozyme onto the kaolin under stirring, filtering and separating the kaolin after adsorbing lysozyme with filter paper, and then putting it into a mixture aqueous solution of γ-methacryloxypropyltrimethoxysilane and γ-aminopropyltriethoxysilane with a mass percentage concentration of 0.2% to 2%, uniformly dispersing the components by ultrasonic treatment at low power, standing for reaction for 12-36 hours, filtering and collecting the precipitate, and washing the unreacted substances with deionized water to obtain a layer of polysiloxane-coated lysozyme-adsorbed kaolin, and freeze-drying it for later use; b) dispersing the lysozyme-adsorbing kaolin obtained in step a) and coated with a layer of polysiloxane after freeze-drying at a concentration of 0.2% to 5% by mass and spirulina at a concentration of 0.5% to 2% by mass into deionized water under stirring, and then adding sodium alginate at a concentration of 1.0% to 3% by mass, continuing to stir to fully dissolve the sodium alginate, and standing to defoam to obtain a casting solution for use; c) preparing a mixed aqueous solution of lysozyme with a mass percentage concentration of 0.1% to 5% and a soluble calcium salt with a mass percentage concentration of 1.0% to 20% as a cross-linking agent aqueous solution, and pouring the cross-linking agent aqueous solution into a spray bottle for later use; d) pouring the casting liquid obtained in step b) onto a clean and flat glass plate, scraping it into a liquid film with a film scraping rod, and then immediately spraying the crosslinking agent aqueous solution in the spray bottle in step c) onto the surface of the liquid film, so that the calcium ions in the soluble calcium salt cross-link with the sodium alginate to form a calcium alginate hydrogel film, and after the calcium alginate hydrogel film is initially formed, it is removed and immediately sprayed with the crosslinking agent aqueous solution to cross-link the other side of the film, and after sufficient cross-linking, an antibacterial, antioxidant and hemostatic hydrogel dressing capable of sustained-release phycocyanin is obtained; the hydrogel dressing is carved into a required shape and size with a mold for standby use; e) Since the lysozyme bacteria are adsorbed and embedded in the polysiloxane, and the lysozyme is easily adsorbed by alginate, the lysozyme inside the hydrogel dressing has a sustained release performance. The sustained-released lysozyme encounters spirulina and destroys the cell wall of the spirulina, thereby achieving the purpose of sustained release of phycocyanin; the sustained release performance of the phycocyanin in the hydrogel dressing obtained in step d) is tested in physiological saline. The hydrogel dressing of the present invention has a longer-lasting ability to release phycocyanin than the dressing directly adding phycocyanin and spirulina, and can sustainably release phycocyanin for 24 to 60 hours; f) The antibacterial rate of the hydrogel dressing of the present invention against Staphylococcus aureus reaches 65% to 100%; g) The phycocyanin released by the hydrogel dressing of the present invention has a clearance rate of 60% to 96% for 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt after 7 hours.

2. A method for preparing an antibacterial, antioxidant and hemostatic hydrogel dressing capable of sustained-release phycocyanin as claimed in claim 1, characterized in that The soluble calcium salt is any one of calcium chloride, calcium dihydrogen phosphate and calcium nitrate or a mixture of two or more thereof.

3. A method for preparing an antibacterial, antioxidant and hemostatic hydrogel dressing capable of sustained-release phycocyanin as claimed in claim 1, characterized in that The antibacterial and antioxidant hemostatic hydrogel dressing has a hemostatic time of 1 to 5 minutes for rat middle lobe resection bleeding.