Gel dressing based on algae particles and capable of promoting wound healing as well as preparation method and application of gel dressing

Abstract

The invention discloses a gel dressing based on algae particles and capable of promoting wound healing as well as a preparation method and application of the gel dressing, and belongs to the technical field of biological medicines. The gel dressing is prepared from Pluronic F127, dopamine-loaded algae and platinum nanoparticles, and is characterized in that the dopamine-loaded algae and the platinum nanoparticles are added into the Pluronic F127; the platinum nanoparticles are prepared in situ through biomineralization by utilizing the advantages of natural active microbial algae. The synthesis method has the beneficial effects that the synthesis method is simple and green, raw materials are rich in source, and the synthesis method has the advantages of low cost, low toxicity and high efficiency. The gel dressing provided by the invention is simple to prepare, can effectively promote healing of diabetic wounds, has relatively high biological safety, and has a wide application prospect in the aspect of wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a gel dressing based on algae particles that can promote wound healing, and a preparation method and application thereof. Background Art

[0002] Diabetes affects nearly 425 million people, of which about 19% to 34% of diabetic patients develop diabetic chronic wounds due to high blood sugar, which makes wounds difficult to heal, and may further lead to delayed wound healing, severe infection, and even lower limb amputation. Currently, up to 25% of diabetic patients are at risk of chronic wounds that will not heal for life. In diabetic wounds, high blood sugar can lead to excessive glycosylation and expression of proinflammatory cytokines, induce angiogenesis disorders, change the extracellular matrix, and hinder re-epithelialization. In addition, since oxygen (O2) is essential for the proliferation of fibroblasts, keratinocytes, and endothelial cells, and the diabetic wound microenvironment is highly hypoxic, this will further hinder tissue regeneration. Therefore, the persistence of chronic inflammation and the hypoxic state of local tissues are key factors affecting wound healing. Solving the potential problems of chronic inflammation and hypoxia provides an effective way to treat diabetic wounds.

[0003] However, existing oxygen therapy strategies rely on gaseous oxygen as an oxygen source, which makes it difficult to provide the wound site with sufficient and continuous oxygen supply. In clinical practice, hyperbaric oxygen therapy (HBO) and local gaseous oxygen therapy (TGO) have been shown to be effective in promoting some cases of delayed wound healing, but the efficacy is limited and cannot significantly improve multiple types of wounds. For example, HBO therapy may produce risks such as damage and oxygen poisoning caused by changes in air pressure; TGO therapy is easily hindered by the limited penetration of external gases into tissues. In addition, during TGO / HBO treatment, oxygen levels can only be temporarily increased for 1-2 hours, and once the patient is removed from the chamber, they will return to baseline levels within a few minutes. In clinical practice, oxygen therapy usually takes a long time (>3 hours) to take effect. At present, studies have been conducted to continuously generate oxygen through dynamic Schiff base and boronate cross-linked glycopeptide hydrogels, but the preparation method is relatively cumbersome. Therefore, it is very necessary to develop an oxygen therapy strategy with simple preparation and sustainable supply of dissolved oxygen to alleviate wound hypoxia.

[0004] Chlorella is one of the most widely cultivated microalgae due to its rapid reproduction. It can continuously produce oxygen through photosynthesis, achieving sustainable and controllable oxygen production to improve the hypoxic microenvironment. In addition, algae can solidify glucose, which can become a tool for consuming glucose, and can further improve the microenvironment of diabetic wounds. In recent years, hydrogels have attracted much attention in the repair of diabetic chronic wounds due to their unique three-dimensional porous network structure and high hydrophilicity. However, there are major problems in the design of multifunctional hydrogels, such as complex separation, cumbersome preparation, and low synergistic efficiency. In addition, during the inflammatory phase of the wound, macrophages and neutrophils produce a large amount of reactive oxygen species in response to high blood sugar. This reduces angiogenesis and worsens wound tissue. Platinum nanoparticles (PtNPs) are widely used as effective antioxidants because of their powerful antioxidant, anti-inflammatory and antibacterial properties. They can prevent oxidative damage and promote cell proliferation by inhibiting the production of excessive reactive oxygen species (ROS). However, the traditional preparation method of platinum nanoparticles is relatively complicated, and most of them use chemical reduction methods, which makes it difficult to ensure the economy, environmental protection and non-toxicity of nanoparticles.

[0005] The Chinese patent application document with publication number CN113559314A discloses an extracellular matrix-mimicking hydrogel dressing for diabetic foot ulcers and a preparation method thereof, comprising: dispersing polydopamine nanoparticles loaded with deferoxamine mesylate in a modified human-like collagen solution, then mixing with a hyaluronic acid solution grafted with epigallocatechin gallate dimer, and adding a tyrosinase solution to obtain an extracellular matrix-mimicking hydrogel dressing. The patent discloses an extracellular matrix-mimicking hydrogel dressing prepared by dissolving polydopamine nanoparticles loaded with deferoxamine mesylate in a modified human-like collagen solution, mixing with a hyaluronic acid solution grafted with epigallocatechin gallate dimer, and then quickly adding a tyrosinase solution, which specifically solves the problems of impaired angiogenesis and long-term chronic inflammation of diabetic foot ulcer wounds, can effectively promote angiogenesis and anti-inflammation, and has a high wound healing rate. However, the oxygen production and antibacterial properties of the auxiliary material are still poor, so it needs to be further improved. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to solve the problem that the existing wound healing auxiliary materials have poor oxygen production capacity and anti-inflammatory properties.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The first aspect of the present invention provides a gel dressing based on algae particles that can promote wound healing, comprising a dressing matrix, algae particles and platinum nanoparticles, wherein the algae particles and platinum nanoparticles are loaded on the dressing matrix, and the components of the dressing matrix include Pluronic F127 and polydopamine (PDA).

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned gel dressing, comprising the following steps:

[0010] S1: Mixing the Chlorella (CH) solution with the chloroplatinic acid (H2PtCl6) solution, culturing in a constant temperature and light shaking incubator, centrifuging, washing, and obtaining the Chlorella (CHPT) after mineralization of platinum nanoparticles;

[0011] S2: PDA, Pluronic F127 and water are mixed to obtain the dressing matrix;

[0012] S3: Add the CHPT obtained in S1 to the dressing matrix of S2, mix well, and obtain a gel dressing.

[0013] Preferably, in S1, the volume ratio of the Chlorella (CH) solution to the chloroplatinic acid (H2PtCl6) solution is (9-19):(0.5-3), and more preferably 9:1.

[0014] Preferably, in S1, the concentration of Chlorella is (0.5-2)×10 7 cell / mL; the concentration of chloroplatinic acid (H2PtCl6) solution is 0.5-3mM; more preferably 1×10 7 cell / mL, 1mM.

[0015] Preferably, in S1, the illumination time is 10 to 14 h, the illumination intensity is 2000-3000 Lux, the temperature is 24 to 26° C., and the shaking speed is 40 to 60 rpm; more preferably, 12 h, 2500 Lux, 25° C., and 50 rpm.

[0016] Preferably, in S1, the centrifugal conditions are: 4000-6000 rpm, 2-5 min; more preferably, 5000 rpm, 3 min.

[0017] Preferably, in S2, the usage ratio of polydopamine (PDA), Pluronic F127 and water is (80-120) mg: (1-5) g: (5-15) mL, and more preferably: 100 mg: 2.2 g: 10 mL.

[0018] Preferably, in S2, the mass concentration of polydopamine (PDA) in the obtained dressing matrix is ​​0.5-2%, and the mass concentration of Pluronic F127 is 17-20%; more preferably, 1% and 18%.

[0019] Preferably, in S3, the dosage ratio of CHPT to dressing matrix is: 5×10 6 ~5×10 7 cell / mL: 1mL.

[0020] Preferably, the gel dressing is a thermosensitive hydrogel, which is in a liquid state at 4°C and forms a gel when the temperature is at 37°C, that is, the liquid component will be converted into a gel dressing when it contacts the skin.

[0021] The third aspect of the present invention provides the use of the above-mentioned gel dressing or the gel dressing prepared by the above-mentioned preparation method in promoting wound healing.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention develops a new method for preparing platinum nanoparticles based on in situ mineralization of organisms. The advantages of natural active microorganisms and algae are utilized to realize biomineralization and preparation of platinum nanoparticles. The preparation conditions and process are simple, convenient and green, and the source of raw materials is abundant, avoiding the use of chemical reagents, and only simple illumination is required, which has the advantages of low cost, low toxicity and high efficiency. It has been verified that the algae platinum gel dressing prepared by the present invention, based on the algae light oxygen production and the antioxidant properties of platinum nanoparticles, can improve hypoxia in the wound site, reduce oxidative stress, and promote angiogenesis to promote diabetic wound healing.

[0024] 2. Develop a greener method to prepare platinum nanoparticles with simple composition, hydrogel dressings that can reduce ROS and have a continuous oxygen production therapy strategy.

[0025] 3. Pluronic F127 and polydopamine (PDA) are used as hydrogel matrix to form a thermosensitive hydrogel. At the same time, algae are loaded inside the hydrogel pores. Algae not only have photosynthesis that can achieve continuous oxygen production, but also have various reducing cytochromes, lipids, sugars and other substances, which can serve as host cells for intracellular biomineralized metal ions. While performing photosynthesis, electrons are generated, allowing electrons to be transferred in the photosynthetic chain [H2O→ 1 / 2O2+2H + +2e - (PSII)], achieving Pt 4+ Conversion to platinum nanoparticles.

[0026] 4. The gel dressing loaded with algae and platinum nanoparticles prepared by the present invention has multifunctionality. The present invention combines algae and platinum nanoparticles to achieve continuous oxygen production to promote cell respiration; and effectively removes reactive oxygen to reduce oxidative stress; at the same time, the hypoxic microenvironment is improved, promoting angiogenesis and accelerating diabetic wound healing. In addition, algae can reduce glucose and is more suitable for treating diabetic wounds. Pluronic F127 and PDA as gel carriers have high biocompatibility, and the addition of PDA enhances the adhesion performance of the gel dressing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the process of in-situ mineralization of platinum nanoparticles by Chlorella in Example 1 of the present invention;

[0028] Figure 2 The ultraviolet spectra of Chlorella (CH), chloroplatinic acid (H2PtCl6), a mixture of Chlorella and chloroplatinic acid (H2PtCl6), and Chlorella (CHPT) after mineralization with platinum nanoparticles in Example 1 of the present invention;

[0029] Figure 3 is the potential change of CH and CHPT in Example 1 of the present invention;

[0030] Figure 4 is the particle size distribution of CH and CHPT in Example 1 of the present invention;

[0031] Figure 5 is the infrared spectrum of CH and CHPT in Example 1 of the present invention;

[0032] Figure 6 TEM images of CH and CHPT in Example 1 of the present invention, with a scale bar of 1 μm;

[0033] Figure 7 TEM element mapping diagram of CHPT in Example 1 of the present invention, with a scale of 500 nm;

[0034] Figure 8 The oxygen production of CH and CHPT in Example 1 of the present invention;

[0035] Fig. 9 The scanning electron microscope images of Gel and CHPT gel in Example 1 of the present invention are shown;

[0036] Fig.10 The modulus change diagram of Gel and CHPT gel;

[0037] Fig.11 The in vitro glucose consumption capacity of the gel dressing of Application Example 1 of the present invention;

[0038] Fig.12 The ability of the gel dressing of Application Example 1 of the present invention to decompose hydrogen peroxide in vitro;

[0039] Fig.13 This is the in vitro cell compatibility of the gel dressing of Application Example 2 of the present invention;

[0040] Fig.14 The in vitro blood compatibility of CHPT in Application Example 2 of the present invention;

[0041] Fig.15The in vitro active oxygen scavenging ability of the gel dressing of Application Example 3 of the present invention, the scale bar is 200 μm;

[0042] Fig.16 This is the wound healing condition of diabetic mice after treatment with the gel dressing in Application Example 4 of the present invention;

[0043] Fig.17 This is the hematoxylin and eosin staining (H&E) and Masson's trichrome staining of the wound healing tissue in vivo of diabetic mice treated for 12 days in Application Example 4 of the present invention, the scale bar is 100 μm;

[0044] Fig.18 This is the immunofluorescence staining of CD31 and HIF-1α in diabetic mice treated for 12 days in Application Example 4 of the present invention. The scale bar is 100 μm.

[0045] In the figure, ** represents P < 0.01, *** represents P < 0.001, and ns represents no significant difference. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0048] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0049] The chlorella described below is Chlorella, which was purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences.

[0050] Embodiment 1:

[0051] A method for preparing a gel dressing based on algae particles that can promote wound healing, comprising the following steps:

[0052] (1) Synthesis and characterization of CHPT: 9 mL of Chlorella vulgaris containing kanamycin (1×10 7cell / mL) and 1mL chloroplatinic acid (H2PtCl6, 1mM) were mixed and placed in a conical flask, and then the conical flask was placed in a constant temperature light shaking incubator at 25°C and 50rpm for 12h. After that, the Chlorella solution after mineralization of platinum nanoparticles was taken into a 10mL centrifuge tube, centrifuged at 5000rpm for 5min, washed with deionized water, and repeated 3 times. Chlorella after mineralization of platinum nanoparticles (CHPT) was obtained.

[0053] Schematic diagram of the process of in situ mineralization of platinum nanoparticles by Chlorella Figure 1 As shown;

[0054] UV spectra of Chlorella (CH), chloroplatinic acid (H2PtCl6), a mixture of Chlorella and chloroplatinic acid, and Chlorella after mineralization with platinum nanoparticles (CHPT) Figure 2 As shown in Figure 2, the absorption peak of Chlorella at 261nm after mineralization of platinum nanoparticles disappears, proving that the existence form of platinum ions has changed; the potential diagram of Chlorella and Chlorella after mineralization of platinum nanoparticles is shown in Figure 2. Figure 3 As shown, the potential of Chlorella decreased after mineralization, which was due to the negative charge of Pt nanoparticles;

[0055] The particle size diagram of CH and CHPT is shown in Figure 4 As shown, the particle size of Chlorella becomes larger after mineralization;

[0056] The infrared spectra of CH and CHPT are shown in Figure 5 As shown, the infrared spectra of CH and CHPT showed high similarity, which indicated that the carbohydrates, lipids, and proteins of Chlorella were not damaged;

[0057] Transmission electron microscopy images of Chlorella before and after mineralization Figure 6 As shown, it can be seen that compared with Chlorella alone, the Chlorella after mineralization with platinum nanoparticles has black particles.

[0058] Transmission electron microscopy element mapping diagram Figure 7 As shown, the mapping shows the presence of platinum nanoparticles. The above shows that Chlorella successfully mineralized platinum nanoparticles in situ.

[0059] The oxygen production capacity of Chlorella before and after mineralization Figure 8 As shown, it can be seen that the oxygen production performance of Chlorella is not affected after mineralization with platinum nanoparticles.

[0060] (2) Preparation and characterization of gel dressing: 100 mg of PDA powder was added to a 15 mL centrifuge tube, 9.9 mL of deionized water was added, and then 2.195 g of Pluronic F127 was added to the 15 mL centrifuge tube. The mixture was stirred and placed in a 4°C refrigerator to dissolve, thereby obtaining an 18% Pluronic F127 and 1% PDA solution.

[0061] (3) Add the CHPT obtained in step (1) to the above 18% Pluronic F127 and 1% PDA solution, mix well, and obtain a gel dressing (CHPT gel). Store in a refrigerator at 4°C and place on the skin to form a gel when used.

[0062] Scanning electron microscopy images of blank gel (Gel) and gel dressing (CHPT gel) are shown in Fig. 9 As shown in the figure, it can be seen that Gel and CHPT gel present a reticular porous structure, and small round particles can be seen in the CHPT gel group, which shows that the gel dressing was successfully prepared.

[0063] Comparative Example 1:

[0064] A method for preparing a wound dressing comprises the following steps: (the difference from Example 1 is that CHPT is not contained)

[0065] Preparation and characterization of gel dressing: Take 100 mg of PDA powder and add it to a 15 mL centrifuge tube, add 9.9 mL of deionized water, then add 2.195 g of Pluronic F127 to the above 15 mL centrifuge tube, stir to mix, and place in a 4°C refrigerator to dissolve to obtain 18% Pluronic F127 and 1% PDA solution, store in a 4°C refrigerator, and place on the skin to form a gel when used.

[0066] The modulus changes of Gel and CHPT gel are shown in the figure Fig.10 As shown, compared with Gel, the storage modulus and loss modulus of CHPT gel are basically the same, that is, the addition of mineralized platinum nanoparticles to Chlorella has little effect on the gel properties.

[0067] Embodiment 2:

[0068] A method for preparing a wound dressing comprises the following steps:

[0069] (1) Synthesis of CHPT: 9 mL of Chlorella vulgaris containing kanamycin (1×10 7 cell / mL) and 1mL chloroplatinic acid (H2PtCl6, 1mM) were mixed and placed in a conical flask, and then the conical flask was placed in a constant temperature light shaking incubator at 25°C and 50rpm for 12h. After that, the Chlorella solution after mineralization of platinum nanoparticles was taken into a 10mL centrifuge tube, centrifuged at 5000rpm for 5min, washed with deionized water, and repeated 3 times. Chlorella after mineralization of platinum nanoparticles (CHPT) was obtained.

[0070] (2) Preparation and characterization of gel dressing: 100 mg of PDA powder was added to a 15 mL centrifuge tube, 9.9 mL of deionized water was added, and then 2.048 g of Pluronic F127 was added to the above 15 mL centrifuge tube, stirred and mixed, and placed in a 4°C refrigerator to dissolve, to obtain a 17% Pluronic F127 and 1% PDA solution. The CHPT obtained in step (1) was added to the above 17% Pluronic F127 and 1% PDA solution, mixed evenly, to obtain a gel dressing (CHPT gel). It was stored in a 4°C refrigerator and placed on the skin to form a gel when used.

[0071] Embodiment 3:

[0072] A method for preparing a wound dressing comprises the following steps:

[0073] (1) Synthesis of CHPT: 9 mL of Chlorella vulgaris containing kanamycin (1×10 7 cell / mL) and 1mL chloroplatinic acid (H2PtCl6, 1mM) were mixed and placed in a conical flask, and then the conical flask was placed in a constant temperature light shaking incubator at 25°C and 50rpm for 12h. After that, the Chlorella solution after mineralization of platinum nanoparticles was taken into a 10mL centrifuge tube, centrifuged at 5000rpm for 5min, washed with deionized water, and repeated 3 times. Chlorella after mineralization of platinum nanoparticles (CHPT) was obtained.

[0074] (2) Preparation and characterization of gel dressing: 100 mg of PDA powder was added to a 15 mL centrifuge tube, 9.9 mL of deionized water was added, and then 2.346 g of Pluronic F127 was added to the above 15 mL centrifuge tube, stirred and mixed, and placed in a 4°C refrigerator to dissolve, to obtain a 9% Pluronic F127 and 1% PDA solution. The CHPT obtained in step (1) was added to the above 19% Pluronic F127 and 1% PDA solution, mixed evenly, to obtain a gel dressing (CHPT gel). It was stored in a 4°C refrigerator and placed on the skin to form a gel when used.

[0075] Embodiment 4:

[0076] A method for preparing a wound dressing comprises the following steps:

[0077] (1) Synthesis of CHPT: 9 mL of Chlorella vulgaris containing kanamycin (1×10 7cell / mL) and 1mL chloroplatinic acid (H2PtCl6, 1mM) were mixed and placed in a conical flask, and then the conical flask was placed in a constant temperature light shaking incubator at 25°C and 50rpm for 12h. After that, the Chlorella solution after mineralization of platinum nanoparticles was taken into a 10mL centrifuge tube, centrifuged at 5000rpm for 5min, washed with deionized water, and repeated 3 times. Chlorella after mineralization of platinum nanoparticles (CHPT) was obtained.

[0078] (2) Preparation and characterization of gel dressing: 100 mg of PDA powder was added to a 15 mL centrifuge tube, and 9.9 mL of deionized water was added. Then 2.500 g of Pluronic F127 was added to the above 15 mL centrifuge tube, stirred and mixed, and placed in a 4°C refrigerator to dissolve, to obtain a 20% Pluronic F127 and 1% PDA solution. The CHPT obtained in step (1) was added to the above 20% Pluronic F127 and 1% PDA solution, and mixed evenly to obtain a gel dressing (CHPT gel). The dressing was stored in a 4°C refrigerator and placed on the skin to form a gel when used.

[0079] Application Example 1:

[0080] This application example tests the ability of the gel dressing prepared in Example 1 and Comparative Example 1 to reduce glucose and decompose hydrogen peroxide according to the following steps.

[0081] Divide into three groups: PBS, Gel, CHPT gel, each group has three parallels, a total of 9 samples. The gel dressings prepared in Example 1 and Comparative Example 1 were co-cultured with BG11 medium containing 33mM glucose under light conditions for 12h. Then, 5μL of the supernatant was taken and the glucose content was detected using a glucose detection kit.

[0082] Divide into three groups: PBS, Gel, CHPT gel, each group has three parallels, a total of 9 samples. The gel dressings prepared in Example 1 and Comparative Example 1 were co-cultured with BG11 culture medium containing 16.3mM H2O2 under light conditions for 1 hour. Then, 25μL of the supernatant was taken and the hydrogen peroxide content was detected using a hydrogen peroxide content detection kit.

[0083] The results of the in vitro glucose-lowering ability test of the gel dressing are as follows Fig.11 As shown in the figure, the results of the in vitro test on the ability of the gel dressing to reduce hydrogen peroxide are as follows Fig.12 As shown in the figure, compared with the PBS and Gel groups, the glucose content and hydrogen peroxide content of CHPT gel after co-culture were significantly reduced, indicating that CHPT gel has the ability to metabolize glucose and has good anti-inflammatory properties.

[0084] Application Example 2:

[0085] In this application example, the biocompatibility of the gel dressings prepared in Example 1 and Comparative Example 1 was tested according to the following steps.

[0086] MTT test for cell compatibility: The gel dressing prepared in Example 1 and Comparative Example 1 was incubated with 1640 medium (10% serum + 1% double antibody) at 37°C for 24 hours, and the supernatant was taken for use. HUVEC cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. The relative cell viability of the gel dressing was determined by the MTT method. Briefly, 100 μL (5×10 4 / mL) HUVEC cell suspension was inoculated into a 96-well plate and cultured for 24 hours until it adhered to the bottom of the well. It was divided into two groups: Gel and CHPT gel. The two groups were divided into five groups according to the weight of the gel, namely 100 mg / mL, 50 mg / mL, 10 mg / mL, 2 mg / mL and 0 mg / mL, with four replicate wells in each group. After 24 hours, the supernatant of the HUVEC cell suspension was discarded, and the above dressing co-incubation solution was co-cultured with the HUVEC cells that had adhered to the well. Only 1640 culture medium was added to the PBS group as a blank control. The culture was continued for 24 hours, and MTT detection solution (culture medium: MTT = 10:1) was added, and incubated in a 37°C incubator in the dark for 4 hours. After 4 hours, the supernatant was discarded and 150 μL DMSO was added. After tapping to dissolve, the OD value was measured at 570 nm under an enzyme reader to analyze the cytotoxicity. The calculation formula is as follows: Relative cell viability (%) = (OD 样本 -OD 空白 ) / (OD 对照 -OD 空白 )×100%.

[0087] Hemolysis experiment to detect blood compatibility: Take eye blood from healthy Balb / c mice, put it in an anticoagulant tube, centrifuge it at 5000rpm for 5 minutes, wash it 5 times with 0.9% NaCl solution to obtain red blood cells (RBCs), and disperse the red blood cells in 0.9% NaCl solution. Red blood cells incubated with deionized water and 0.9% NaCl were used as positive and negative controls, respectively. The red blood cells were incubated with CHPT prepared in Example 1 (1) at 37°C for 30 minutes and shaken at 200rpm. After incubation, centrifuge it at 5000rpm for 5 minutes, aspirate the supernatant and place it in a 96-well plate. Use an enzyme reader to measure the absorbance at 540nm. The hemolysis rate was calculated according to the following formula: Hemolysis rate (%) = (OD 样本 -OD 0.9％NaCl ) / (OD ddH2O -OD 0.9％NaCl )×100%.

[0088] The results of in vitro cell compatibility test of gel dressing are as follows Fig.13 As shown, compared with the untreated group (0 mg / mL), the cell activity of the Gel and CHPT gel groups can reach 90-100%, with no significant difference, indicating a higher cell compatibility.

[0089] The results of the in vitro blood compatibility test of CHPT are as follows Fig.14 As shown, the hemolysis rate of CHPT is about 1%, which is less than 5%, indicating that the material has high blood compatibility.

[0090] Application Example 3:

[0091] This application example tests the antioxidant properties of the gel dressings prepared in Example 1 and Comparative Example 1 according to the following steps.

[0092] The reactive oxygen detection kit (DCFH-DA) detects the level of intracellular reactive oxygen. The gel dressing prepared in the above Example 1 and Comparative Example 1 was co-incubated with 1640 culture medium (10% serum + 1% double antibody) at 37°C for 24 hours, and the supernatant was taken for standby use. HUVEC was inoculated into a 24-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere to the wall. The supernatant was discarded, and 1640 culture medium containing 100 μM H2O2 was added to the cells and cultured for 12 hours to induce high expression of intracellular reactive oxygen. H2O2 was not added to the PBS group. After 24 hours, the supernatant was discarded, and the dressing co-incubation solution was added, and the 1640 culture medium was used as a negative control. After 6 hours of co-culture, the supernatant was discarded, and DCFH-DA was diluted with serum-free culture medium at a volume ratio of 1:1000 to a final concentration of 10 μmol / mL. Add 500 μL of diluted DCFH-DA to each group and incubate in a 37°C cell culture incubator in the dark for 20 min. Wash the cells three times with PBS to fully remove the DCFH-DA that has not entered the cells. Observe the images of the stained cells under an inverted fluorescence microscope.

[0093] The results of the in vitro active oxygen scavenging experiment of the gel dressing are as follows Fig.15 As shown in the figure, compared with the positive control H2O2 group, after adding CHPT gel dressing, the fluorescence intensity of reactive oxygen species was significantly reduced, which seemed to be equivalent to that of the PBS group. This was due to the role of platinum nanoparticles and algae in resisting oxidative stress. This showed that CHPT gel dressing had a higher ability to reduce oxidative stress.

[0094] Application example 4:

[0095] This application example tests the therapeutic effects of the gel dressings prepared in Example 1 and Comparative Example 1 in a diabetic wound model according to the following steps.

[0096] Balb / c mice (6-8 weeks) were randomly divided into three groups: diabetic mice group (PBS), blank gel dressing group (Gel), drug-loaded gel dressing group (CHPT gel), with 6 mice in each group. The diabetic mouse modeling method is as follows: after all mice were continuously fed with high-sugar and high-fat feed and 12h normal water / 12h 10% sugar water for one week, they were intraperitoneally injected with 60mg / Kg of streptozotocin (STZ) for five consecutive days. They were fasted before each injection, and were kept fed with high-sugar and high-fat feed plus sugar water during the period. After one week of STZ injection, blood sugar was basically stable. Mice with fasting blood sugar higher than 11.1mmol / L were defined as diabetic mice for further experiments. Subsequently, the back of the mice was depilated, and after the mice were anesthetized by isoflurane inhalation, a circular wound with a diameter of 10mm was made on their backs, and the modeling was successful.

[0097] Diabetic mouse group: only wounds were constructed without any treatment.

[0098] Dressing group: The gel dressings prepared in Example 1 and Comparative Example 1 were applied to the wounds on the back of mice, respectively. The dressings were changed once a day for treatment. The wound status was recorded and observed every other day. On day 12, circular skin tissue was taken, fixed with 4% paraformaldehyde, embedded in paraffin blocks, and sliced ​​with a paraffin slicer. The tissue was stained with hematoxylin and eosin (H&E), and the stained slices were observed with a Leica microscope; Masson trichrome staining was used to evaluate the collagen deposition activity during wound healing. Endothelial cell adhesion molecule-1 (CD31) was used to detect neovascularization in wound tissue; hypoxia-inducible factor-1α (HIF-1α) was used to detect the improvement of hypoxia in wound tissue.

[0099] Wound healing evaluation results of diabetic mice 0 to 12 days after gel dressing treatment Fig.16 As shown. It can be observed that the healing speed of the drug-loaded gel dressing group is the fastest compared with the other two groups, and the healing speed of the PBS group is the slowest. This shows that the gel dressing prepared by loading Chlorella and platinum nanoparticles has the ability to promote diabetic wound healing.

[0100] H&E staining and Masson staining of wound healing tissue in diabetic mice after 12 days of treatment Fig.17 As shown in the figure, compared with the other two groups, the H&E staining shows that the drug-loaded gel dressing group has more new blood vessels and fewer inflammatory cells; the Masson staining shows that the drug-loaded gel dressing group has more collagen deposition. The above shows that the wound healing effect after treatment with the drug-loaded gel dressing group is better, indicating that the gel dressing has the ability to promote angiogenesis and collagen deposition.

[0101] Immunofluorescence images of CD31 and HIF-1α in diabetic mice after 12 days of treatment Fig.18As shown. The analysis results showed that compared with the untreated diabetic mice (PBS) group and the blank gel (Gel) group, the drug-loaded gel dressing group significantly promoted the positive expression of CD31. The more CD31 expression, the stronger the ability to promote angiogenesis. At the same time, the expression of HIF-1α was downregulated. The lower the HIF-1α, the better the hypoxic environment. These data indicate that CHPT gel can promote diabetic wound healing by reducing inflammation, accelerating collagen deposition, promoting angiogenesis, and improving the hypoxic microenvironment.

[0102] Embodiment 5:

[0103] A method for preparing a wound dressing comprises the following steps:

[0104] (1) Synthesis of CHPT: 12 mL of Chlorella vulgaris containing kanamycin (0.5 × 10 7 cell / mL) was mixed with 2mL chloroplatinic acid (H2PtCl6, 0.5mM) and placed in a conical flask, and then the conical flask was placed in a constant temperature light shaking incubator at 24°C and 60rmp for 10h. After that, the Chlorella solution after mineralization of platinum nanoparticles was taken into a 10mL centrifuge tube, centrifuged at 6000rmp for 2min, washed with deionized water, and repeated 3 times. Chlorella after mineralization of platinum nanoparticles (CHPT) was obtained.

[0105] (2) Preparation and characterization of gel dressing: 80 mg of PDA powder was added to a 15 mL centrifuge tube, 5 mL of deionized water was added, and then 1 g of Pluronic F127 was added to the above 15 mL centrifuge tube, stirred and mixed, and placed in a 4°C refrigerator to dissolve, thereby obtaining a Pluronic F127 and PDA solution. The CHPT obtained in step (1) was added to the above Pluronic F127 and PDA solution, and mixed evenly to obtain a gel dressing (CHPT gel). The dressing was stored in a 4°C refrigerator and placed on the skin to form a gel when used.

[0106] Embodiment 6:

[0107] A method for preparing a wound dressing comprises the following steps:

[0108] (1) Synthesis of CHPT: 19 mL of Chlorella vulgaris containing kanamycin (2 × 10 7 cell / mL) was mixed with 3mL chloroplatinic acid (H2PtCl6, 3mM) and placed in a conical flask, and then the conical flask was placed in a constant temperature light shaking incubator at 26°C and 40rmp for 14h. After that, the Chlorella solution after mineralization of platinum nanoparticles was taken into a 10mL centrifuge tube, centrifuged at 4000rmp for 5min, washed with deionized water, and repeated 3 times. Chlorella after mineralization of platinum nanoparticles (CHPT) was obtained.

[0109] (2) Preparation and characterization of gel dressing: 120 mg of PDA powder was added to a centrifuge tube, 15 mL of deionized water was added, and then 5 g of Pluronic F127 was added to the above centrifuge tube, stirred and mixed, and placed in a 4°C refrigerator to dissolve, thereby obtaining a Pluronic F127 and PDA solution. The CHPT obtained in step (1) was added to the above Pluronic F127 and PDA solution, and mixed evenly to obtain a gel dressing (CHPT gel). The dressing was stored in a 4°C refrigerator and placed on the skin to form a gel when used.

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gel dressing based on algae particles that can promote wound healing, characterized in that: The dressing matrix comprises a dressing matrix, algae particles and platinum nanoparticles, wherein the algae particles and the platinum nanoparticles are loaded on the dressing matrix, and the components of the dressing matrix include Pluronic F127 and PDA.

2. The method for preparing the gel dressing according to claim 1, characterized in that: The following steps are involved: S1: mixing the Chlorella (CH) solution with the chloroplatinic acid solution, culturing in a constant temperature and light shaking incubator, centrifuging, washing, and obtaining the Chlorella (CHPT) with mineralized platinum nanoparticles; S2: PDA, Pluronic F127 and water are mixed to obtain the dressing matrix; S3: Add the CHPT obtained in S1 to the dressing matrix in S2, mix well, and obtain a gel dressing.

3. The preparation method according to claim 2, characterized in that: In S1, the volume ratio of the Chlorella (CH) solution to the chloroplatinic acid (H2PtCl6) solution is (9-19): (0.5-3).

4. The preparation method according to claim 2, characterized in that: In S1, the concentration of Chlorella is (0.5-2)×10 7 cell / mL; the concentration of chloroplatinic acid solution is 0.5~3mM.

5. The preparation method according to claim 2, characterized in that: In S1, the illumination time is 10-14 h, the illumination intensity is 2000-3000 Lux, the temperature is 24-26° C., and the shaking speed is 40-60 rpm.

6. The preparation method according to claim 2, characterized in that: In S1, the centrifugal conditions are: 4000-6000 rpm, 2-5 min.

7. The preparation method according to claim 2, characterized in that: In S2, the usage ratio of PDA, Pluronic F127 and water is (80-120) mg: (1-5) g: (5-15) mL.

8. The preparation method according to claim 2, characterized in that: In S2, the mass concentration of PDA in the obtained dressing matrix is ​​0.5-2%, and the mass concentration of Pluronic F127 is 17-20%.

9. The preparation method according to claim 2, characterized in that: The gel dressing needs to gel at 37°C.

10. Use of the gel dressing according to claim 1 or the gel dressing prepared by the preparation method according to any one of claims 2 to 9 in promoting wound healing.

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