Calcium-doped diatom shell-ulva pertusa polysaccharide composite hydrogel as well as preparation method and application thereof in wound repair

Abstract

The invention discloses a calcium-doped diatom shell-ulva pertusa polysaccharide composite hydrogel, which is characterized in that ulva pertusa polysaccharide and calcium-doped diatom shell are compounded to construct a unique three-dimensional cross-linked network structure. In the structure, calcium ions serve as an ion bridge to be connected with negative charge groups in polysaccharide, meanwhile, silicon hydroxyl on the surface of the diatom shell and Ca < 2 + > form a stable metal-diatom complex, and the nanopore structure of the diatom shell further enhances physical entanglement between polysaccharide chains. The composite hydrogel has excellent physical and chemical properties, mechanical toughness and conformability, and can be tightly attached to the surfaces of wounds in various complex shapes. Original wettability, biocompatibility and wound healing promoting activity of ulva pertusa polysaccharide are reserved, and the promoting effect on cell migration and tissue regeneration is further enhanced through a stable microenvironment and addition of calcium ions. Experiments prove that the hydrogel shows a remarkable wound healing promoting effect in a mouse full-thickness skin defect model.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharides, and particularly to a doped calcium diatom shell - ulva pertusa polysaccharide composite hydrogel, a preparation method thereof, and an application in wound repair. Background Art

[0002] The skin, as the largest organ of the human body, is the primary defense line against the external environment and plays an irreplaceable role in maintaining the internal environmental homeostasis and protecting human health. It can not only block the invasion of bacteria and the penetration of exogenous substances, but also effectively prevent the body from dehydration, being an important protective barrier of the human body. However, skin trauma has become a global medical challenge: 1. Diverse causes: including traffic accidents, sports injuries, daily accidents, engineering accidents, etc.; 2. Severe hazards: it may cause various complications and even endanger life in severe cases; 3. Social burden: According to the statistics of the World Health Organization, there are more than 40 million cases of severe skin trauma globally every year, and the related medical expenses are conservatively estimated to exceed 60 billion yuan. The physiological process of wound repair: The repair of skin trauma is a complex dynamic process, which successively experiences the following stages: 1. Hemostasis stage: The primary link after trauma; 2. Inflammatory reaction stage: Initiating the repair process; 3. Tissue formation stage: Growth of new tissue; 4. Structure and function remodeling stage: The final healing stage. Limitations of existing treatment options: Although there are various commercial dressings on the market, there are still significant deficiencies: 1. Functional limitations: Poor adaptability to complex wounds, limited ability to control inflammation and exudate, and difficulty in meeting multiple requirements such as moisturization and wound healing promotion simultaneously. 2. Physical property defects: Insufficient mechanical strength, poor flexibility, poor adhesion, easy to cause secondary trauma, and limited application in special parts such as joints. The latest progress in the field of biomaterials has brought new hope for solving these problems: Natural polymer materials and nanomaterials have shown excellent performance in wound healing, but the commercial application of existing products is still scarce. It is hoped that new materials can improve biocompatibility, enhance biodegradability, improve wound adaptability, and achieve multi - functional integration.

[0003] Diatoms are a type of single - cell algae widely distributed in marine, freshwater, and wetland environments, belonging to the Bacillariophyta in eukaryotes. As important primary producers globally, diatoms are of great significance to the marine ecosystem and the carbon cycle. Their most remarkable feature lies in the cell wall (i.e., diatom shell) structure, and this shell composed of silicon dioxide (siliceous) forms a complex micro - and nano - scale pattern structure. Due to its unique porous structure, large specific surface area, and excellent biocompatibility, the diatom shell has attracted much attention in the fields of materials science and biomedicine.

[0004] In the field of hydrogel preparation, diatom shells exhibit multiple advantages. As functional fillers or enhancers, they can significantly improve the mechanical properties, adsorption properties, and functionalization characteristics of hydrogels. The silicon hydroxyl groups (-OH) rich on the surface of diatom shells can interact with various functional groups (such as hydroxyl, carboxyl, or sulfate groups) through hydrogen bonding or electrostatic interactions. Especially under specific conditions (such as specific pH values or ionic environments), diatom shells can serve as physical crosslinking centers to promote the formation of a stable three-dimensional network structure.

[0005] What is more unique about the mechanism of action of diatom shells is that for macromolecular compounds containing negative charges (such as sulfuric acid or carboxylic acid), it can cooperate with divalent or multivalent metal ions (such as Ca 2+ 、Mg 2+ ) to form an ion crosslinking network, thereby enhancing the gelation ability of the hydrogel. During the crosslinking process, diatom shells not only play a bridging role as crosslinking centers, but their extremely high specific surface area and micro-nano structure can also provide structural support and more crosslinking sites, ultimately forming a more stable and functional hydrogel network. These characteristics make diatom shells show great potential in the application of hydrogels in fields such as drug delivery, biosensing, and tissue engineering. It is worth noting that there are currently no reports on the direct composite formation of diatom shells and ulvan to form hydrogels.

[0006] Ulvan is a polysaccharide extracted from the green alga Ulva pertusa, and its main components include rhamnose, galacturonic acid, glucuronic acid, galactose, and xylose. In addition, some of its functional groups also determine its potential in hydrogel applications. Functional groups such as sulfate groups and carboxyl groups can regulate inflammatory responses and reduce the release of inflammatory mediators. And ulvan can promote the proliferation and migration of fibroblasts, which is crucial for collagen synthesis and wound closure. Moreover, its good wettability, angiogenesis stimulation, antioxidant activity, biocompatibility, and gelation ability all indicate its potential in hydrogel preparation and application. Summary of the Invention

[0007] To solve the above technical problems, the inventors of the present invention used ulvan as a substrate and doped calcium diatom shells as a "bridge" to prepare a hydrogel that promotes wound healing. The present invention not only elaborates on the preparation method of the hydrogel but also deeply explores its molecular mechanism for promoting wound healing. Therefore, the first aspect of the present invention provides a doped calcium diatom shell-ulvan composite hydrogel, which is obtained by aqueous solution crosslinking of doped calcium diatom shell freeze-dried powder and ulvan freeze-dried powder;

[0008] The ulvan freeze-dried powder is obtained by water-extracting ulva seaweed powder, removing impurities with macroporous resin, and freeze-drying to obtain ulvan freeze-dried powder;

[0009] The freeze-dried calcium-doped diatom shells are obtained by mixing a diatom solution with an f / 2 medium containing Si and doped with calcium for culturing to obtain calcium-doped diatom cells, subjecting the calcium-doped diatom cells to shell burning treatment to obtain calcium-doped diatom shells, washing, and freeze-drying.

[0010] Furthermore, the total sugar content of the freeze-dried ulva pertusa polysaccharide is 39.86% ± 3.71, the uronic acid content is 25.11% ± 1.17, and the sulfate content is 14.06% ± 4.77.

[0011] Furthermore, the molecular weight of the freeze-dried ulva pertusa polysaccharide is mainly concentrated in the range of about several million, and the shape is spongy.

[0012] Furthermore, for the water-soluble crosslinking, 9 g of the freeze-dried ulva pertusa polysaccharide is dissolved in every 100 ml of water to prepare an ulva pertusa polysaccharide aqueous solution with a concentration of 9%, and then 0.5 - 2 g of the freeze-dried calcium-doped diatom shells is added, and frozen to obtain a calcium-doped diatom shell - ulva pertusa polysaccharide composite hydrogel.

[0013] Furthermore, the crosslinking temperature is 60 - 80 °C.

[0014] As Figure 13 shown, the preparation flow chart and the enlarged local structure diagram of the calcium-doped hydrogel in the present invention, where A is the preparation flow chart of the calcium-doped hydrogel in the present invention, and B is the enlarged local structure diagram. The diatom shells, as an ideal material for hydrogel preparation, significantly improve the hydrogel performance through their unique structure and surface chemical properties. The abundant silanol groups (-SiOH) on their surface can form hydrogen bonds or electrostatic interactions with various polar functional groups, acting as physical crosslinking centers under specific conditions to construct a stable three-dimensional network structure.

[0015] It is particularly noteworthy that the preparation method of the calcium-doped diatom shells in the present invention: by directly incorporating calcium ions during the diatom culture process, bio-mineralization doping of calcium elements on the surface of the diatom shells is achieved, forming a Si - O - Ca structure and a stable metal - diatom complex.

[0016] Meanwhile, the freeze-dried ulva pertusa polysaccharide used in the present invention is detected to contain uronic acid and sulfate, providing abundant carboxylate groups (-COO-) and sulfate groups (-OSO3 - ) and other negatively charged groups. These negatively charged groups undergo ion exchange or coordination reactions with the Ca on the surface of the calcium-doped diatom shells to form a crosslinked structure bridged by metal ions. Due to Ca 2+ 2+ ​It has a high charge density and can bind to multiple negatively charged groups simultaneously, effectively promoting the formation of a three-dimensional cross-linked network among polysaccharide chains. The unique nanoporous structure of diatom shells further enhances the physical entanglement between polysaccharide chains. The three-dimensional cross-linked network constructed by this dual mechanism endows the hydrogel with excellent mechanical properties, structural stability, and enhanced gelation ability, while completely retaining the inherent wettability, biocompatibility, and wound-healing-promoting bioactivity of ulva pertusa polysaccharide.

[0017] Further, the diatom solution is mixed and cultured with an f / 2 medium containing Si and doped with calcium, and the mixing volume ratio is 1:1.

[0018] Among them, the f / 2 medium containing Si and doped with calcium is as follows: add 0.075 g of NaNO3, 0.005 g of NaH2PO4·H2O, 0.030 g of Na2SiO3·9H2O, 1 ml of trace element stock solution, 1 ml of vitamin stock solution, and 1 ml of CaCl2 solution with a concentration of 0.5 mmol / L into 1 L of 25‰ seawater.

[0019] Among them, the trace element stock solution is prepared by dissolving chelates Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, and NaMoO4·2H2O in 25‰ seawater, and the concentrations of the chelates are 0.0044 g / L, 0.0032 g / L, 1.79×10 -4 g / L, 2.19×10 -5 g / L, 9.95×10 -6 g / L, 9.79×10 -6 g / L, and 6.18×10 -6 g / L respectively.

[0020] Among them, the vitamin stock solution is prepared by dissolving vitamin B1, vitamin H, and vitamin B 12 , in 25‰ seawater, and the concentrations of the vitamins are 1.00×10 -4 g / L, 5.00×10 -7 g / L, and 5.00×10 -7 g / L respectively.

[0021] The f / 2 medium used in the biomineralization doping process of calcium element on the surface of diatom shells in the present invention can be an existing f / 2 medium purchased on the market or a self-prepared f / 2 medium, which is the prior art in this field. It should be noted during the self-preparation process that since precipitation will occur when the components are directly mixed, they need to be prepared as stock solutions respectively and added during use. The preparation method is as follows:

[0022] (1) Prepare 25‰ seawater (dissolve 25 g of sea salt in every 1000 ml of water);

[0023] (2) Prepare nitrogen source and phosphorus source stock solutions. Dissolve NaNO3 and NaH2PO4·H2O in 25‰ seawater to obtain solutions with NaNO3 and NaH2PO4·H2O concentrations of 0.075 g / L and 0.005 g / L respectively;

[0024] (3) Prepare silicon source stock solution. Dissolve Na2SiO3·9H2O in 25‰ seawater to obtain a solution with a Na2SiO3·9H2O concentration of 0.030 g / L;

[0025] (4) Prepare trace element stock solution, including iron, zinc, manganese, cobalt, copper, and molybdenum, etc., which are usually provided in the form of chelates. Dissolve Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, and NaMoO4·2H2O in 25‰ seawater. The concentrations of the above chelates are 0.0044 g / L, 0.0032 g / L, 1.79×10 - 4 g / L, 2.19×10 -5 g / L, 9.95×10 -6 g / L, 9.79×10 -6 g / L, and 6.18×10 -6 g / L;

[0026] (5) Prepare vitamin stock solution, including vitamin B1, vitamin H, and vitamin B 12 . Vitamins are coenzymes required for the growth of diatoms or activators of metabolic reactions. Dissolve vitamin B1, vitamin H, and vitamin B 12 in 25‰ seawater. The concentrations of the above vitamins are 1.00×10 -4 g / L, 5.00×10 -7 g / L, and 5.00×10 -7 g / L;

[0027] (6) Prepare 0.5 mmol / L CaCl2 solution stock solution. Dissolve 55.49 mg of CaCl2 in every 1000 mL of 25‰ seawater.

[0028] (7) Preparation of f / 2 medium containing Si and doped with calcium: Take 1 L of 25‰ seawater, add 1 ml of nitrogen and phosphorus source storage solution, sterilize it at 121 °C for 15 min through an autoclave, cool it, and in a laminar flow hood, add 1 ml of membrane-filtered silicon source storage solution, 1 ml of membrane-filtered trace element storage solution, 1 ml of membrane-filtered vitamin storage solution, and 1 ml of membrane-filtered 0.5 mmol / L CaCl₂ solution storage solution to obtain the f / 2 medium containing Si and doped with calcium.

[0029] (8) Preparation of f / 2 medium containing Si without calcium doping: Take 1 L of 25‰ seawater, add 1 ml of nitrogen and phosphorus source storage solution, sterilize it at 121 °C for 15 min through an autoclave, cool it, and in a laminar flow hood, add 1 ml of membrane-filtered silicon source storage solution, 1 ml of membrane-filtered trace element storage solution, and 1 ml of membrane-filtered vitamin storage solution to obtain the f / 2 medium containing Si without calcium doping.

[0030] Further, the shell burning treatment of the calcium-doped diatom cells includes: mixing the calcium-doped diatom cells with the shell burning solution at a volume ratio of 1:1, and the shell burning solution is composed of 30% hydrogen peroxide and 2 mol / L hydrochloric acid at a volume ratio of 1:1 to obtain the calcium-doped diatom shells.

[0031] The shell burning treatment of the calcium-doped diatom cells in the present invention is also a commonly used technology in the art. In addition to the shell burning treatment technology using hydrogen peroxide and hydrochloric acid, low-temperature plasma technology, surfactant treatment technology, etc. can also be used.

[0032] Another aspect of the present invention also provides a preparation method of a calcium-doped diatom shell-Ulva pertusa polysaccharide composite hydrogel, and the preparation method includes the following steps:

[0033] (1) Mix the Ulva pertusa seaweed powder with water, extract it, remove impurities with macroporous resin, the eluent is deionized water, concentrate it, and freeze-dry it to obtain the freeze-dried powder of Ulva pertusa polysaccharide;

[0034] (2) Mix the diatom solution with the f / 2 medium containing Si and doped with calcium for culture to obtain calcium-doped diatom cells;

[0035] (3) Perform shell burning treatment on the calcium-doped diatom cells obtained in step (2), after the reaction, centrifuge, wash, and freeze-dry to obtain the freeze-dried powder of calcium-doped diatom shells;

[0036] (4) Perform water-soluble crosslinking reaction on the freeze-dried powder of calcium-doped diatom shells obtained in step (3) and the freeze-dried powder of Ulva pertusa polysaccharide obtained in step (1), and freeze to obtain the calcium-doped diatom shell-Ulva pertusa polysaccharide composite hydrogel.

[0037] Further, the macroporous resin model is NAK-9.

[0038] Further, the diatom solution described in step (2) is mixed and cultured with an f / 2 medium containing Si and doped with calcium, and the mixing volume ratio is 1:1;

[0039] Among them, the f / 2 medium containing Si and doped with calcium is: 0.075 g of NaNO3, 0.005 g of NaH2PO4·H2O, 0.030 g of Na2SiO3·9H2O, 1 ml of trace element stock solution, 1 ml of vitamin stock solution, and 1 ml of CaCl2 solution with a concentration of 0.5 mmol / L are added to every 1 L of 25‰ seawater;

[0040] Among them, the trace element stock solution is: Chelating agents Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, NaMoO4·2H2O are dissolved in 25‰ seawater for preparation, and the concentrations of the chelating agents are 0.0044 g / L, 0.0032 g / L, 1.79×10 -4 g / L, 2.19×10 -5 g / L, 9.95×10 -6 g / L, 9.79×10 -6 g / L, and 6.18×10 -6 g / L;

[0041] Among them, the vitamin stock solution is: Vitamins B1, H, and B 12 , are dissolved in 25‰ seawater for preparation, and the vitamin concentrations are 1.00×10 -4 g / L, 5.00×10 -7 g / L, and 5.00×10 -7 g / L.

[0042] Preferably, the temperature of the culture described in step (2) is 25 °C;

[0043] Preferably, the light-dark cycle of the culture described in step (2) is 12:12 h, and the total culture time is 14 days;

[0044] Preferably, the light intensity of the culture described in step (2) is 4000 Lux;

[0045] Preferably, the pH condition range of the culture described in step (2) is 7.5 - 8.5;

[0046] Further, the calcium-doped diatom cells described in step (2) are the cell walls of diatoms at the micron level, with nanopores and an inorganic silicon skeleton.

[0047] Further, the shell burning treatment of the calcium-doped diatom cells in step (3) includes: mixing the calcium-doped diatom cells with the shell burning solution at a volume ratio of 1:1, where the shell burning solution consists of 30% hydrogen peroxide and 2 mol / L hydrochloric acid at a volume ratio of 1:1, to obtain calcium-doped diatom shells.

[0048] Further, for the aqueous cross-linking in step (4), 9 g of the freeze-dried powder of Ulva pertusa polysaccharide is dissolved in every 100 ml of water to prepare an aqueous solution of Ulva pertusa polysaccharide with a concentration of 9%, and then 0.5 - 2 g of the freeze-dried powder of calcium-doped diatom shells is added;

[0049] Preferably, the cross-linking temperature in step (4) is 60 - 80 °C.

[0050] Further, the shell burning treatment of the calcium-doped diatom cells is carried out in a dark environment.

[0051] In this study, a comprehensive analysis method was used to comprehensively characterize Ulva pertusa polysaccharide, calcium-doped diatom shells, non-calcium-doped diatom shell-Ulva pertusa polysaccharide composite hydrogels, and a series of calcium-doped diatom shell-Ulva pertusa polysaccharide composite hydrogels with different concentrations (0%, 0.5%, 1%, 2%).

[0052] Through Fourier transform infrared spectroscopy (FTIR) analysis, the successful formation of Si - O - Si bonds (around 1100 cm-1) and Ca - O bonds (around 700 cm-1) was verified, confirming the molecular structural changes of calcium doping and polysaccharide cross-linking. X-ray diffraction (XRD) further confirmed the crystal structural changes of calcium-doped diatom shells, showing the displacement of characteristic diffraction peaks and the appearance of new peaks. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) indicated that the thermal stability of the hydrogel was significantly improved after calcium doping.

[0053] Scanning electron microscopy (SEM) observations showed that as the calcium doping concentration increased, the hydrogel network structure became denser and more uniform, and the porosity was most ideal at a concentration of 1%. The results of laser particle size analysis by Malvern Mastersizer 3000 showed that as the calcium doping concentration increased, the diatom shells in the hydrogel were more evenly dispersed and the aggregation phenomenon decreased. The Zeta potential analysis measured by Malvern Zatasizer showed that the surface potential of the hydrogel increased from -25.3 mV to -18.7 mV after calcium doping, confirming the effective binding of Ca 2+ with the negatively charged groups of the polysaccharide.

[0054] Rheological tests further confirmed that the 1% calcium-doped diatom shell-Ulva pertusa polysaccharide composite hydrogel exhibited the best mechanical properties, with both the storage modulus (G') and the loss modulus (G") higher than those of other concentration samples and remaining stable in a wide frequency range, indicating that it had a stronger internal network structure and anti-deformation ability. These comprehensive analysis results provided a scientific basis for optimizing the hydrogel formula and performance regulation.

[0055] The third aspect of the present invention provides an application of a calcium-doped diatom shell-ulva pertusa polysaccharide composite hydrogel as a wound healing dressing.

[0056] Furthermore, when the calcium-doped diatom shell-ulva pertusa polysaccharide composite hydrogel is used as a wound healing dressing and compared with the white control group PBS, the commercial dressing group 3M, and the ulva pertusa polysaccharide group, with a dosage of 200 μL each, the wound healing effect is more significant.

[0057] Furthermore, the wound in the full-thickness skin defect model of mice has a wound diameter of 10 mm.

[0058] Furthermore, the wounds include: burn wounds, chronic wounds, and daily skin injury wounds.

[0059] The calcium-doped diatom shell-ulva pertusa polysaccharide composite hydrogel developed by the present invention has good drug loading capacity and can be used as an ideal drug delivery system for wound treatment. With excellent biocompatibility and remarkable flexibility, the hydrogel can closely adhere to the surfaces of various complex-shaped wounds, ensuring that the therapeutic drugs fully contact the wound surface. Its unique structural design enables the hydrogel to maintain a stable form in the wound environment, while providing a suitable moist environment, effectively promoting cell migration, tissue regeneration, and the wound healing process, providing an efficient and safe advanced biomaterial for wound repair.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] (1) The present invention first successfully composited ulva pertusa polysaccharide with calcium-doped diatom shells to form a novel hydrogel, constructing a unique three-dimensional cross-linked network structure. In this structure, calcium ions act as "ionic bridges" to connect the negatively charged groups (sulfate groups, carboxyl groups) in the polysaccharide, and at the same time, the silanol groups on the surface of the diatom shell form a stable metal-diatom complex with Ca 2+ The nanoporous structure of the diatom shell further enhances the physical entanglement between the polysaccharide chains. This multiple cross-linking mechanism significantly improves the mechanical strength and structural stability of the hydrogel.

[0062] (2) The composite hydrogel not only exhibits excellent physicochemical properties, but also has excellent mechanical toughness and conformability, and can closely adhere to the surfaces of various complex-shaped wounds. At the same time, the hydrogel retains the original wettability, biocompatibility, and wound healing-promoting activity of ulva pertusa polysaccharide, and further enhances the promotion of cell migration and tissue regeneration through a stable microenvironment and the addition of calcium ions, providing ideal conditions for the wound healing process.

[0063] (3) Through the synergistic effect of this organic polysaccharide and inorganic diatom shells, the present invention overcomes the defect of insufficient mechanical strength of traditional polysaccharide hydrogels, provides a new biomaterial solution for the biomedical field, especially for wound repair, and has broad application prospects in the treatment of burn wounds, chronic wounds and various skin injuries. Description of the Drawings

[0064] Figure 1 It is the ultraviolet spectrum scanning diagram of the freeze-dried powder of Ulva pertusa polysaccharide;

[0065] Figure 2 It is the differential interference diagram of the non-doped calcium diatom cells in Example 2 of the present invention;

[0066] Figure 3 It is the SEM characterization diagram and electron energy spectrum diagram of the freeze-dried powder doped with calcium diatom shells and the freeze-dried powder not doped with calcium diatom shells in Example 3 of the present invention;

[0067] Figure 4 It is the SEM characterization diagram and electron energy spectrum diagram of the calcium-doped hydrogel fixed in the present invention;

[0068] Figure 5 It is the SEM characterization diagram and electron energy spectrum diagram of the non-doped calcium hydrogel fixed in the present invention;

[0069] Figure 6 It is the Fourier transform infrared spectrum diagram of the calcium-doped hydrogel and the non-doped calcium hydrogel in the present invention;

[0070] Figure 7 It is the differential scanning calorimetry diagram of the calcium-doped hydrogel and the non-doped calcium hydrogel in the present invention;

[0071] Figure 8 It is the thermogravimetric analysis diagram of the calcium-doped hydrogel and the non-doped calcium hydrogel in the present invention;

[0072] Figure 9 It is the X-ray diffraction diagram of the calcium-doped hydrogel and the non-doped calcium hydrogel in the present invention;

[0073] Figure 10 It is the particle size distribution analysis diagram of the calcium-doped hydrogel and the non-doped calcium hydrogel in the present invention;

[0074] Figure 11 It is the Zeta potential diagram of the calcium-doped hydrogel and the non-doped calcium hydrogel in the present invention;

[0075] Figure 12 It is the rheological property comparison diagram of the calcium-doped hydrogel and the non-doped calcium hydrogel in the present invention;

[0076] Figure 13 It is the preparation flow chart of the calcium-doped hydrogel in the present invention;

[0077] Figure 14 Figure showing the effect of Ulva pertusa polysaccharide on fibroblast migration;

[0078] Figure 15 Figure showing the effect of calcium-doped hydrogel on wound repair in mice. Specific embodiments

[0079] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0080] Example 1:

[0081] Extract Ulva pertusa polysaccharide from Ulva pertusa algal powder by hot water extraction, freeze-dry it and use a UV full-wavelength scanner to scan and identify the purity. The specific operation steps are as follows:

[0082] S1. Place the Ulva pertusa algal powder in hot water for extraction at a temperature of 85 °C, stir it every 30 minutes, and filter to obtain the extract after 2 hours; repeat the cycle twice to improve the leaching of hydrophilic products in the Ulva pertusa algal powder to obtain the extract;

[0083] S2. After cooling the extract obtained in S1 to room temperature, dilute the extract with deionized water at a ratio of 1:1, adsorb pigments and remove small molecules through macroporous resin. The macroporous resin model is NAK-9, and the eluent is deionized water to obtain the polysaccharide solution;

[0084] S3. Concentrate the polysaccharide solution obtained in S2 through a rotary evaporator, place it at -80 °C for at least 12 hours, and freeze-dry it through a freeze dryer to obtain the freeze-dried powder of Ulva pertusa polysaccharide;

[0085] S4. Take 1 mg of the freeze-dried powder of Ulva pertusa polysaccharide obtained in S3, dissolve it in 2 mL of distilled water and mix well to prepare a solution of 500 μg / mL. Using ultrapure water as the blank control group, perform a full-wavelength scan from 190 to 560 nm on a Thermo Fisher Evolution 300 UV full-wavelength scanner. The results are as Figure 1 shown. The Ulva pertusa polysaccharide contains almost no nucleic acid and has a low protein content, which can be almost ignored in the hydrogel reaction system. In addition, after detection, the total sugar content of the freeze-dried powder of Ulva pertusa polysaccharide is 39.86% ± 3.71, the uronic acid content is 25.11 ± 1.17, and the sulfate content is 14.06% ± 4.77.

[0086] Determination of sulfate content - barium sulfate turbidimetry:

[0087] The sulfate content of Ulva pertusa polysaccharide was determined by the BaCl2-gelatin solution method. After drying the K2SO4 standard product to constant weight, accurately weigh 0.181 g of the K2SO4 standard product, dissolve it in distilled water and make up the volume to 10 mL to prepare a 1.0 mg / mL SO4 2-Standard solution. Pipette 1 mL of the standard stock solution and dilute it to 0.05 mg / mL of SO4 2- Standard working solution. Pipette SO4 2- 0, 0.5, 1.0, 1.5, 2.0, 2.5 mL of the standard working solution into centrifuge tubes, perform three parallel replicates, and make up to 2.5 mL with distilled water to obtain SO4 2- The concentrations of the standard solutions are 0, 0.01, 0.02, 0.03, 0.04, 0.05 mg / mL respectively. Add 2.5 mL of BaCl2-gelatin solution to each tube, mix well, and measure the absorbance at 400 nm with an enzyme-labeled instrument. Using the concentration of the SO4 2- standard working solution as the abscissa and the absorbance as the ordinate, establish a standard curve and a linear regression equation.

[0088] Accurately weigh 6 mg of each Ulva pertusa polysaccharide sample, dissolve it in distilled water and make up to 2 mL. Add 1 mL of concentrated hydrochloric acid to the solution, mix well, and hydrolyze with acid at 100 °C for 6 h. After acid hydrolysis, let it stand to room temperature. Take 2.5 mL of the diluted acid hydrolysate, add 2.5 mL of BaCl2 gelatin solution, mix well, and measure the absorbance at 400 nm with an enzyme-labeled instrument. According to the detected absorbance value and the established regression equation, calculate the SO4 2- content.

[0089] Example 2: Use biotechnology to dope calcium onto the surface of the frustule of diatom cells. The specific operation steps are as follows:

[0090] S1. Prepare a stock solution of f / 2 medium containing Si to provide nutrients for diatoms, including nitrogen source, phosphorus source, trace elements, vitamins, and silicate.

[0091] Prepare 25‰ seawater (dissolve 25 g of sea salt in every 1000 ml of water);

[0092] Prepare stock solutions of nitrogen source and phosphorus source. Dissolve NaNO3 and NaH2PO4·H2O in 25‰ seawater to obtain solutions with concentrations of NaNO3 and NaH2PO4·H2O being 0.075 g / L and 0.005 g / L respectively;

[0093] Prepare a stock solution of silicon source. Dissolve Na2SiO3·9H2O in 25‰ seawater to obtain a solution with a concentration of Na2SiO3·9H2O being 0.030 g / L;

[0094] Prepare trace element stock solutions, including iron, zinc, manganese, cobalt, copper, molybdenum, etc., which are usually provided in the form of chelates. Dissolve Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, NaMoO4·2H2O in 25‰ seawater. The concentrations of the above chelates are 0.0044 g / L, 0.0032 g / L, 1.79×10 - 4 g / L, 2.19×10 -5 g / L, 9.95×10 -6 g / L, 9.79×10 -6 g / L and 6.18×10 -6 g / L;

[0095] Prepare vitamin stock solutions, including vitamin B1, vitamin H and vitamin B 12 . Vitamins are coenzymes required for the growth of diatoms or activators of metabolic reactions. Dissolve vitamin B1, vitamin H and vitamin B 12 in 25‰ seawater. The concentrations of the above vitamins are 1.00×10 -4 g / L, 5.00×10 -7 g / L and 5.00×10 -7 g / L;

[0096] Prepare a 0.5 mmol / L CaCl2 solution stock solution. Dissolve 55.49 mg of CaCl2 in 1000 mL of 25‰ seawater.

[0097] S2. Preparation of f / 2 medium containing Si and doped with calcium: Take 1 L of 25‰ seawater, add 1 ml of nitrogen source and phosphorus source stock solutions in S1, sterilize at 121 °C for 15 min in an autoclave, cool, and in a laminar flow hood, add 1 ml of membrane-filtered silicon source stock solution, 1 ml of membrane-filtered trace element stock solution, 1 ml of membrane-filtered vitamin stock solution, and 1 ml of membrane-filtered 0.5 mmol / L CaCl2 solution stock solution to obtain an f / 2 medium containing Si and doped with calcium.

[0098] S3. Preparation of f / 2 medium containing Si without doping with calcium: Take 1 L of 25‰ seawater, add 1 ml of nitrogen source and phosphorus source stock solutions in S1, sterilize at 121 °C for 15 min in an autoclave, cool, and in a laminar flow hood, add 1 ml of membrane-filtered silicon source stock solution, 1 ml of membrane-filtered trace element stock solution, and 1 ml of membrane-filtered vitamin stock solution to obtain an f / 2 medium containing Si without doping with calcium.

[0099] S4. Inoculate diatoms and culture: Inoculate 1 L of diatom solution into 1 L of f / 2 medium containing Si and doped with calcium prepared in step S2. Under the conditions of a temperature of 25°C, a light intensity of 4000 Lux, and a pH of 7.5 - 8.5, with a light-dark cycle of 12:12 hours, culture in an artificial light incubator for 14 days to dope calcium onto the surface of the frustules of diatoms, obtaining frustule cells doped with calcium.

[0100] S5. Inoculate diatoms and culture: Inoculate 1 L of diatom solution into 1 L of f / 2 medium containing Si without calcium doping prepared in step S3. Under the conditions of a temperature of 25°C, a light intensity of 4000 Lux, and a pH of 7.5 - 8.5, with a light-dark cycle of 12:12 hours, culture in an artificial light incubator for 14 days to obtain frustule cells without calcium doping.

[0101] S6. Take the frustule cells without calcium doping cultured for 14 days in step S5 and observe the morphology of the diatom cells through a differential interference microscope. The results are as Figure 2 shown.

[0102] Example 3: Use hydrogen peroxide and hydrochloric acid to perform shell burning treatment on frustule cells. The specific operation steps are as follows:

[0103] S1. Take 30% hydrogen peroxide and 2 mol / L hydrochloric acid and mix them in a volume ratio of 1:1 in an acid-resistant container to obtain a mixed solution;

[0104] S2. Add the frustule cells doped with calcium obtained in Example 2 to the mixed solution in S1 according to a liquid-solid ratio of 1:1 (mg / ml); add the frustule cells without calcium doping to the mixed solution in S1 according to a liquid-solid ratio of 1:1 (mg / ml); after reacting for 24 hours, centrifuge to mainly remove the organic matter and other pollutants on the surface of the biological sample through an oxidation reaction. Repeat this operation step 2 times to ensure that the organic matter and pollutants on the surface of the frustules are completely removed, obtaining clean frustules without calcium doping and clean frustules doped with calcium;

[0105] S3. Thoroughly wash the clean frustules without calcium doping and clean frustules doped with calcium after the reaction with deionized water respectively to remove the excess hydrochloric acid and hydrogen peroxide residues. Freeze-dry the clean frustules without calcium doping and clean frustules doped with calcium after the washing to obtain freeze-dried powder of frustules without calcium doping and freeze-dried powder of frustules doped with calcium, which are used as materials for preparing hydrogels in the following steps.

[0106] S4. Take the freeze-dried powder of frustules without calcium doping and the freeze-dried powder of frustules doped with calcium respectively for scanning electron microscopy observation. The results are as Figure 3 shown, where the upper part is the SEM characterization diagram and electron energy spectrum diagram of the freeze-dried powder of frustules doped with calcium, and the lower part is the SEM characterization diagram and electron energy spectrum diagram of the freeze-dried powder of frustules without calcium doping.

[0107] Example 4: Preparation of calcium-doped diatom shell hydrophilic polysaccharide hydrogel and non-calcium-doped diatom shell hydrophilic polysaccharide hydrogel. The specific operation steps are as follows:

[0108] S1. Prepare a 9% (w / v) Ulva pertusa polysaccharide solution: Add the Ulva pertusa polysaccharide freeze-dried powder obtained in Example 1 to deionized water (9 g of Ulva pertusa polysaccharide / 100 mL of water), heat and stir at 60 °C to ensure complete dissolution;

[0109] S2. Prepare non-calcium-doped hydrogel: Mix the non-calcium-doped diatom shell freeze-dried powder (DNP) in Example 3 at concentrations of 0%, 0.5%, 1%, and 2% (w / v) (i.e., add 0 g, 0.5 g, 1 g, and 2 g of DNP to each 100 mL of solution) with the 9% Ulva pertusa polysaccharide (UPP) solution in S1, mechanically stir at 60 °C for 20 - 30 minutes to mix evenly, pour the mixture into a mold and let it stand overnight to solidify, then freeze at -80 °C for at least 12 h, and freeze-dry to obtain non-calcium-doped hydrogels, named UPP, 0.5% DNP / UPP, 1% DNP / UPP, and 2% DNP / UPP respectively.

[0110] S3. Prepare calcium-doped hydrogel: Mix the calcium-doped diatom shell freeze-dried powder (Ca-DNP) in Example 3 at concentrations of 0%, 0.5%, 1%, and 2% (w / v) (i.e., add 0 g, 0.5 g, 1 g, and 2 g of Ca-DNP to each 100 mL of solution) with the 9% Ulva pertusa polysaccharide (UPP) solution in S1, mechanically stir at 60 °C for 20 - 30 minutes to mix evenly, pour the mixture into a mold and let it stand overnight to solidify, then freeze at -80 °C for at least 12 h, and freeze-dry to obtain calcium-doped hydrogels, named Ca-UPP, 0.5% Ca-DNP / UPP, 1% Ca-DNP / UPP, and 2% Ca-DNP / UPP respectively.

[0111] Example 5: Analyze the samples in Example 4 using a scanning electron microscope (SEM), including the scanning electron micrographs of the non-calcium-doped hydrogel series (0.5% DNP / UPP, 1% DNP / UPP, and 2% DNP / UPP) and the calcium-doped hydrogel series (0.5% Ca-DNP / UPP, 1% Ca-DNP / UPP, and 2% Ca-DNP / UPP). The results are as Figure 4 and Figure 5 shown.

[0112] Example 6: All samples in Example 4 were analyzed using a Fourier transform infrared spectrometer (FT-IR), including the non-doped calcium hydrogel series (UPP, 0.5% DNP / UPP, 1% DNP / UPP, and 2% DNP / UPP) and the doped calcium hydrogel series (Ca-UPP, 0.5% Ca-DNP / UPP, 1% Ca-DNP / UPP, and 2% Ca-DNP / UPP), as well as the non-doped calcium diatomite freeze-dried powder (DNP) and the doped calcium diatomite freeze-dried powder (Ca-DNP) for changes in chemical bonds.

[0113] Pretreatment for infrared spectrum testing: Weigh 2 mg of each sample powder precisely, mix it with potassium bromide, grind it, and press it into a transparent thin film. Use a pure potassium bromide tablet as a blank control, and use a Fourier transform infrared spectrometer (FT-IR) to scan and record the spectra of all samples.

[0114] Scanning parameters: 4000 - 400 cm -1 Range;

[0115] Experimental results: The infrared results are as Figure 6 shown, where A is the infrared spectrum of the doped calcium hydrogel and the doped calcium diatomite freeze-dried powder (DNP), and B is the infrared spectrum of the non-doped calcium hydrogel and the non-doped calcium diatomite freeze-dried powder (DNP). Analysis found that: in the range of 3600 - 3200 cm -1 is the stretching vibration absorption peak of O-H. The absorption peak in this region is a characteristic peak of sugars, and absorption peaks also appear in the hydrogel and diatom shell, but the absorption peak in this region of the prepared hydrogel is wider, indicating that more hydrogen bonds are formed in the hydrogel; the absorption peaks at 2936, 2932, and 2929 cm -1 are attributed to the stretching vibration of C-H; the absorption peaks in the range of 1079.84, 620.90, 590.24, and 467 cm -1 in this region are attributed to the stretching and bending vibrations of Si-O-Si, where the bending vibration of Ca-O also contributes to the absorption peaks at 620.90 and 590.24 cm -1 , and 1051.66 cm -1 in the polysaccharide of Ulva pertusa is attributed to the stretching vibration of C-O. In the hydrogel, the absorption peak broadens in the range of 1000 - 1200 cm -1 , and there is no peak at 467 cm -1 , suggesting that the diatom shell has reacted.

[0116] Example 7: The samples in Example 4 were analyzed by thermogravimetry-differential scanning calorimetry, including the calcium-free hydrogel series (0.5% DNP / UPP, 1% DNP / UPP, and 2% DNP / UPP) and the calcium-doped hydrogel series (0.5% Ca-DNP / UPP, 1% Ca-DNP / UPP, and 2% Ca-DNP / UPP), as well as the related transformation behaviors of the calcium-free diatomite shell freeze-dried powder (DNP) and the calcium-doped diatomite shell freeze-dried powder (Ca-DNP);

[0117] Thermal analysis experimental method: Weigh 10 mg of the sample precisely and place it in the sample crucible, ensuring full contact with the bottom of the crucible. Perform thermogravimetric analysis under nitrogen protection (flow rate 20 - 50 mL / min), with the temperature rising from 30 °C to 600 °C at a heating rate of 10 °C / min.

[0118] Thermal analysis results: Figure 7 (DTG) and Figure 8 (TG) showed that the decomposition rates of the prepared calcium-doped hydrogel series and the calcium-doped hydrogel series were faster than those of the raw material calcium-doped diatomite shell freeze-dried powder and the calcium-free diatomite shell freeze-dried powder. The mass loss of the sample mainly occurred in three stages: the first stage was water evaporation; the second stage had a larger mass loss, mainly due to the decomposition of Ulva pertusa polysaccharide; the third stage was the residual amount analysis. The results showed that the 2% Ca-DNP / UPP sample had the least residual amount and the best degradation ability.

[0119] Example 8: The samples in Example 4 were analyzed by X-ray diffraction, including the calcium-free hydrogel series (0.5% DNP / UPP, 1% DNP / UPP, and 2% DNP / UPP) and the calcium-doped hydrogel series (0.5% Ca-DNP / UPP, 1% Ca-DNP / UPP, and 2% Ca-DNP / UPP), as well as the structural changes of the calcium-free diatomite shell freeze-dried powder (DNP) and the calcium-doped diatomite shell freeze-dried powder (Ca-DNP);

[0120] X-ray diffraction analysis method: Weigh 50 mg of the sample precisely for wide-angle X-ray diffraction testing. The scanning angle range is 5° - 65° (2θ), the step size is 0.02°, and the scanning speed is 5° / min. Ensure that the sample surface is perpendicular to the X-ray beam to avoid signal deviation.

[0121] XRD analysis results: Figure 9 showed that DNP and Ca-DNP had relatively wide diffraction peaks near 2θ = 44°, indicating that their crystal sizes were small, which was consistent with the micron-scale structural characteristics. The peak intensity differences between the calcium-doped hydrogel and the calcium-free hydrogel near 2θ = 28° were obvious, indicating that the calcium-doped hydrogel and the calcium-free hydrogel had different molecular arrangement structures, reflecting that the addition of calcium ions changed the intermolecular interaction mode inside the hydrogel.

[0122] Example 9: Analyze the particle size distributions of the undoped calcium hydrogel 1% DNP / UPP and the calcium-doped hydrogel 1% Ca-DNP / UPP in Example 4 using a Malvern Mastersizer 3000 laser particle size analyzer;

[0123] Particle size analysis method: Accurately weigh 0.5 g of the sample. After ultrasonic dispersion pretreatment, add it to the dispersion unit using deionized water as the dispersion medium. Start stirring at an initial speed of 1000 rpm, and control the obscuration to be 10% - 20%. Repeat the test more than 3 times to ensure reliable results.

[0124] Particle size analysis results: Figure 10 It shows that A represents the particle size distribution diagram of the calcium-doped hydrogel 1% DNP / UPP, and B represents the particle size distribution diagram of the undoped calcium hydrogel 1% DNP / UPP. The bulk density analysis shows that the distribution of the calcium-doped hydrogel particles is more uniform and the particle size is smaller (Dv(50) = 44.6 μm). This may be due to the biomineralization of calcium ions on the surface of the diatom shell, converting Si-OH to Si-O-Ca structure, reducing the phenomenon of interparticle attraction and aggregation.

[0125] Example 10: Analyze the surface potential and surface properties of the samples in Example 4, including the undoped calcium hydrogel series (0.5% DNP / UPP, 1% DNP / UPP, and 2% DNP / UPP) and the calcium-doped hydrogel series (0.5% Ca-DNP / UPP, 1% Ca-DNP / UPP, and 2% Ca-DNP / UPP), using a Malvern Zetasizer Nano ZS 90 nanoparticle size and ZETA potential analyzer;

[0126] Zeta potential test method: Accurately weigh 0.5 g of the sample, and dilute it with deionized water to a diatom shell particle concentration of 0.01 - 0.1 wt%. Control the total volume to be 10 - 15 mL. Use a pipette to take 0.7 - 1 mL of the sample and inject it into the sample cell. Test at 25 °C, set the refractive index of the medium to 1.33 and the viscosity of the medium to 0.89 cP, and select an appropriate electric field strength according to the particle concentration and dispersion characteristics. Each group of samples is tested more than 3 times and the average value is taken.

[0127] Zeta potential analysis results: Figure 11 It shows that A represents the Zeta potential diagram of the calcium-doped hydrogel, and B represents the Zeta potential diagram of the undoped calcium hydrogel. Both the calcium-doped hydrogel and the undoped calcium hydrogel show negative potential values, indicating that the surface of the hydrogel particles is negatively charged. The absolute value of the potential of the calcium-doped hydrogel is higher, indicating better dispersion stability. This may be due to the mineralization of calcium ions during the growth of diatoms, reducing the chance of forming hydroxyl groups at the silicon terminus, thus affecting the surface charge distribution of the particles.

[0128] Example 11: The samples in Example 4 were tested by a modular intelligent advanced rheometer to study the rheological properties of undoped calcium hydrogel series (0.5% DNP / UPP, 1% DNP / UPP, and 2% DNP / UPP) and doped calcium hydrogel series (0.5% Ca-DNP / UPP, 1% Ca-DNP / UPP, and 2% Ca-DNP / UPP).

[0129] Rheological property test method: The rheological properties of the samples were measured using an Anton Paar MCR502 advanced rheometer. Samples with a diameter of 25 mm and a thickness of 1.5 mm were placed on a PP25 plate and tested in dynamic oscillation mode. The test conditions were a frequency of 10 rad / s, a temperature of 25 °C, and the oscillation strain was gradually increased from 0.01% to 1000%. Meanwhile, the relationships between the storage modulus (G') and the loss modulus (G") and the oscillation stress were recorded.

[0130] Results of rheological property analysis: Figure 12 The scanning results showed that A represents the strain scanning result diagram of the doped calcium hydrogel, B represents the strain scanning result diagram of the undoped calcium hydrogel, C represents the frequency scanning result diagram of the doped calcium hydrogel, and D represents the frequency scanning result diagram of the undoped calcium hydrogel. The storage modulus (G') of the hydrogel was greater than the loss modulus (G"), indicating that the system mainly exhibited elastic characteristics. The frequency scanning results showed that for the doped calcium hydrogel, both G' and G" gradually increased with increasing frequency, confirming that the three-dimensional doped calcium hydrogel network structure was effectively enhanced. In the high-frequency region, G" continued to increase, indicating that the doped calcium hydrogel exhibited more viscous behavior at high frequencies, suggesting that its structure tended to weaken under high-frequency conditions. This may be attributed to the formation of ionic bonds between polysaccharides and calcium ions on the surface of diatom shells and the interaction with silanol groups, reducing the interaction between the inorganic silicon skeleton and water, thereby enhancing the hydrophobicity.

[0131] Example 12: The effect of ulva pertusa polysaccharide on the migration of fibroblast L929 cells;

[0132] S1. Cultivation of fibroblast L929

[0133] Experimental content: 10% (v / v) fetal bovine serum and 1% (v / v) double antibody were added to the 1640 medium as the complete culture medium for fibroblast L929. After resuscitating the cryopreserved fibroblast L929, it was resuspended and mixed with the complete culture medium and placed in a sterile culture dish, and cultured in an incubator at 37 °C and 5% CO2. When the cells adhered and almost covered the bottom of the culture dish, they were passaged or plated for further experiments.

[0134] Experimental operation:

[0135] ① Recovery: Take out the fibroblast L929 in the cryopreservation tube from the liquid nitrogen tank, shake it in a 37°C water bath to quickly melt it, then transfer it to a centrifuge tube containing 5 mL of complete culture medium. After pipetting and mixing evenly, centrifuge at low speed (1000 rpm, 3 min), discard the supernatant, add 5 mL of fresh complete culture medium to resuspend the cells, transfer the cell suspension to a cell culture dish, and culture it in an incubator at 37°C and 5% CO2. Then replace the fresh complete cell culture medium and continue culturing. When the density of adherent cells reaches about 80%, subculture can be carried out.

[0136] ② Subculture: Aspirate and discard the old medium, wash the cells with PBS 1 - 2 times to remove residual serum. Add a small amount of 0.25% trypsin - EDTA solution to evenly cover the cell surface, digest for 1 minute, and observe the cells rounding up and detaching. Add an appropriate amount of medium to neutralize the trypsin, gently pipette to make a single - cell suspension. Inoculate the cell suspension into a new culture dish at a ratio of 1:3 or 1:4, and add an appropriate amount of fresh medium to continue culturing.

[0137] ③ Cryopreservation: Centrifuge the evenly pipetted cell suspension at low speed (1000 rpm, 3 min), discard the supernatant, add cell cryopreservation solution, pipette and mix evenly, transfer it to a sterile cryopreservation tube, and label the cell name and cryopreservation time. Keep it at - 80°C overnight, and finally store it in the liquid nitrogen tank.

[0138] S2. Effects of Ulva pertusa polysaccharide on the migration of fibroblast L929 cells

[0139] Experimental content: Use the scratch assay to simulate wounds and test the effects of different concentrations of Ulva pertusa polysaccharide on the migration of fibroblast L929, and then infer the repair ability of Ulva pertusa polysaccharide.

[0140] Experimental operation:

[0141] ① Prepare different concentration gradients of Ulva pertusa polysaccharide (12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL) for standby;

[0142] ② Inoculate fibroblast cells at an appropriate density (about 5×10 5 cells / well) into a 6 - well plate and culture until the cell confluence reaches 80% - 90%; use a sterile 10 μL pipette tip to draw a straight line vertically in the center of each well, try to ensure that the scratch width is consistent, then gently wash away the suspended cells with PBS, and add 2% serum medium containing Ulva pertusa polysaccharide; use a microscope to observe and take pictures of the cell migration in the scratched area at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h respectively; use the image analysis software ImageJ to measure the changes in the scratched area and calculate the cell migration rate.

[0143] Results of the scratch - healing experiment:Figure 14 Data showed that this in vitro experiment was used to evaluate cell migration and wound healing ability. Through comparative analysis of the control group, the groups treated with 100 μg / mL and 200 μg / mL Ulva pertusa polysaccharide, the group with 100 μg / mL Ulva pertusa polysaccharide showed the best cell migration and wound repair effects.

[0144] Example 13: Effect of calcium-doped hydrogel on wound repair in mice;

[0145] Experimental content: A full-thickness skin defect model of mice was established, and the calcium-doped hydrogel was applied to the created wounds. The wound healing rates of different groups were calculated by observing the wound healing speed of different groups.

[0146] Experimental operation:

[0147] C57BL / 6J male mice (about 20 g) were depilated one day before formal modeling. The next day, each mouse was anesthetized for surgery to create a circular full-thickness skin defect wound with a diameter of 10 mm. Then, different treatments were carried out for each group. The mice were grouped as follows:

[0148] Control: Blank control group,

[0149] 3M: 3M commercial dressing group,

[0150] UPP: Ulva pertusa polysaccharide group,

[0151] Ca-DNP / UPP: 1% calcium-doped hydrogel Ca-DNP / UPP group.

[0152] In vivo experimental results: Figure 15 It showed that the calcium-doped hydrogel showed a significant wound healing promoting effect in the mouse model, which was better than the control group.

[0153] The above are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above content. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Doped calcium diatom shell-enteromorpha polysaccharide composite hydrogel, characterized in that: The doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel is obtained by dissolving and cross-linking the freeze-dried powder of doped calcium diatom shell and the freeze-dried powder of Ulva pertusa polysaccharide in water. The freeze-dried powder of Ulva pertusa polysaccharide is obtained by extracting Ulva pertusa seaweed powder with water, removing impurities with macroporous resin, and freeze-drying. The freeze-dried powder of doped calcium diatom shell is obtained by mixing and culturing a diatom solution with an f / 2 medium containing Si and doped with calcium to obtain doped calcium diatom cells, burning the shells of the doped calcium diatom cells to obtain doped calcium diatom shells, washing, and freeze-drying.

2. The doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel according to claim 1, wherein: The total sugar content of the freeze-dried powder of Ulva pertusa polysaccharide is 39.86% ± 3.71, the uronic acid content is 25.11% ± 1.17, and the sulfate content is 14.06% ± 4.

77.

3. The doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel according to claim 1, wherein: The aqueous cross-linking is to dissolve 9 g of the freeze-dried powder of Ulva pertusa polysaccharide in every 100 ml of water to prepare an aqueous solution of Ulva pertusa polysaccharide with a concentration of 9%, and then add 0.5 - 2 g of the freeze-dried powder of doped calcium diatom shell, and freeze to obtain the doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel.

4. The doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel according to claim 3, characterized in that: The cross-linking temperature is 60 - 80 °C.

5. The doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel according to claim 1, characterized in that: The mixing and culturing of the diatom solution with the f / 2 medium containing Si and doped with calcium is carried out at a mixing volume ratio of 1:

1. Among them, the f / 2 medium containing Si and doped with calcium is: adding 0.075 g of NaNO3, 0.005 g of NaH2PO4·H2O, 0.030 g of Na2SiO3·9H2O, 1 ml of trace element stock solution, 1 ml of vitamin stock solution, and 1 ml of CaCl2 solution with a concentration of 0.5 mmol / L into every 1 L of 25‰ seawater. Among them, the trace element stock solution is prepared by dissolving the chelates Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, and NaMoO4·2H2O in 25‰ seawater, and the concentrations of the chelates are 0.0044 g / L, 0.0032 g / L, 1.79×10 -4 g / L, 2.19×10 -5 g / L, 9.95×10 -6 g / L, 9.79×10 -6 g / L, and 6.18×10 -6 g / L; Among them, the vitamin stock solution is prepared by dissolving vitamin B1, biotin and vitamin B 12 in 25‰ seawater, and the vitamin concentrations are 1.00×10 -4 g / L, 5.00×10 -7 g / L and 5.00×10 -7 g / L respectively.

6. The doped calcium diatom shell-porphyra polysaccharide composite hydrogel according to claim 1, characterized in that: The shell burning treatment of the doped calcium diatom cells includes: mixing the doped calcium diatom cells with the shell burning solution at a volume ratio of 1:1, and the shell burning solution is composed of 30% hydrogen peroxide and 2 mol / L hydrochloric acid at a volume ratio of 1:1 to obtain the doped calcium diatom shell.

7. A preparation method of the doped calcium diatom shell-ulva pertusa polysaccharide composite hydrogel according to any one of claims 1-6, characterized in that: The preparation method includes the following steps: (1) Mix Ulva pertusa seaweed powder with water, extract, remove impurities with macroporous resin, use deionized water as the eluent, concentrate, and freeze-dry to obtain the freeze-dried powder of Ulva pertusa polysaccharide. (2) Mix and culture the diatom solution with the f / 2 medium containing Si and doped with calcium to obtain doped calcium diatom cells. (3) Carry out shell burning treatment on the doped calcium diatom cells obtained in step (2), after the reaction, centrifuge, wash, and freeze-dry to obtain the freeze-dried powder of doped calcium diatom shell. (4) Carry out an aqueous cross-linking reaction on the freeze-dried powder of doped calcium diatom shell obtained in step (3) and the freeze-dried powder of Ulva pertusa polysaccharide obtained in step (1), and freeze to obtain the doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel.

8. The preparation method of the doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel according to claim 7, wherein: In step (2), the mixing and culturing of the diatom solution with the f / 2 medium containing Si and doped with calcium is carried out at a mixing volume ratio of 1:

1. Among them, the f / 2 medium containing Si and doped with calcium is: adding 0.075 g of NaNO3, 0.005 g of NaH2PO4·H2O, 0.030 g of Na2SiO3·9H2O, 1 ml of trace element stock solution, 1 ml of vitamin stock solution, and 1 ml of CaCl2 solution with a concentration of 0.5 mmol / L into every 1 L of 25‰ seawater. Among them, the trace element stock solution is prepared by dissolving the chelates Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, and NaMoO4·2H2O in 25‰ seawater, and the concentrations of the chelates are 0.0044 g / L, 0.0032 g / L, 1.79×10 -4 g / L, 2.19×10 -5 g / L, 9.95×10 -6 g / L, 9.79×10 -6 g / L, and 6.18×10 -6 g / L; Among them, the vitamin stock solution is prepared by dissolving vitamin B1, vitamin H and vitamin B 12 , in 25‰ seawater, and the vitamin concentrations are 1.00×10 -4 g / L, 5.00×10 -7 g / L and 5.00×10 -7 g / L respectively. Preferably, the temperature of the culture in step (2) is 25 °C. Preferably, the light-dark cycle of the cultivation in step (2) is 12:12 h, and the total cultivation time is 14 days; Preferably, the light intensity of the cultivation in step (2) is 4000 Lux; Preferably, the pH condition range of the cultivation in step (2) is 7.5 - 8.

5.

9. The doped calcium diatom shell-Ulva pertusa polysaccharide composite hydrogel according to claim 7, characterized in that: The calcined shell treatment of the calcium-doped diatom cells in step (3) includes: mixing the calcium-doped diatom cells with the shell calcining solution at a volume ratio of 1:1, and the shell calcining solution is composed of 30% hydrogen peroxide and 2 mol / L hydrochloric acid at a volume ratio of 1:1 to obtain the calcium-doped diatom shell; Preferably, for the aqueous solution crosslinking in step (4), 9 g of the freeze-dried powder of Ulva pertusa polysaccharide is dissolved in every 100 ml of water to prepare an aqueous solution of Ulva pertusa polysaccharide with a concentration of 9%, and then 0.5 - 2 g of the freeze-dried powder of the calcium-doped diatom shell is added; Preferably, the crosslinking temperature in step (4) is 60 - 80 °C, the reaction time is 30 min, and the preferred reaction temperature is 60 °C.

10. Use of the doped calcium diatom shell- Ulva pertusa polysaccharide composite hydrogel according to any one of claims 1-6, characterized in that: The calcium-doped diatom shell-Ulva pertusa polysaccharide composite hydrogel is used as a wound healing dressing.

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