A time-release oxygen / antibacterial / osteointegration-promoting layered hydrogel dressing and a preparation method and application thereof

Abstract

The application belongs to the technical field of medical materials, and discloses a time sequence oxygen-releasing / antibacterial / osteointegration-promoting layered hydrogel dressing as well as a preparation method and application thereof. The layered hydrogel dressing comprises, from bottom to top, a lower layer, an interface layer and an upper layer in sequence. The lower layer is composed of sodium alginate, calcium peroxide nanoparticles and carrageenan. The upper layer is composed of carrageenan, chlorella and sodium alginate. The interface layer is a calcium ion-mediated carboxyl-sulfate cross-linked isolation zone. The layered hydrogel dressing has the following advantages: 1. spatiotemporal precise regulation: the lower layer responds quickly to control early infection, and the upper layer continuously acts to support long-term healing; 2. deep repair penetration: the released oxygen and calcium ions diffuse to the bone defect area to promote prosthesis osteointegration; 3. infection blocking mechanism: the surface antibacterial effect blocks the invasion of bacteria to deep bone tissue.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and specifically relates to a time-released oxygen / antibacterial / osteointegration-promoting layered hydrogel dressing, its preparation method and application. Background Technology

[0002] Postoperative infection after joint replacement surgery is a major complication leading to surgical failure, significantly increasing patients' physical suffering and financial burden, and posing a significant challenge to clinical treatment. Currently, prevention and treatment technologies for this problem suffer from three key limitations, hindering further improvements in efficacy. First, existing antibiotic delivery systems mostly rely on single-drug sustained-release mechanisms. Long-term local application of low-dose antibiotics can easily induce bacterial resistance and may even lead to dysbiosis, further exacerbating the difficulty of infection control. Second, traditional dressings have limited functionality, only providing basic wound coverage and exudate absorption, failing to meet the dynamic needs of different postoperative stages: in the early postoperative period, the wound faces a high risk of infection, requiring potent antibacterial and anti-inflammatory effects; while in the later repair stage, a microenvironment conducive to osseointegration needs to be created, and traditional materials struggle to simultaneously address these two differentiated and crucial treatment goals. Furthermore, current treatment strategies generally lack the ability to actively regulate the repair of deep bone defect areas. After infection is controlled, effectively promoting bone ingrowth into the prosthesis interface and achieving robust biological fixation remains an unsolved problem.

[0003] In existing technologies, once a bacterial biofilm forms on the surface of the prosthesis, it generates a strong immune escape and drug resistance, causing the infection to become chronic. The accompanying persistent inflammation will aggravate local tissue hypoxia, forming a hypoxic microenvironment that is not conducive to tissue regeneration. The deterioration of the microenvironment and the physical barrier effect of the biofilm together hinder the ingrowth and integration of host bone cells on the surface of the prosthesis, ultimately leading to prosthesis loosening and surgical failure.

[0004] Therefore, there is an urgent need to develop a novel multifunctional dressing to comprehensively address the challenges of coordinating infection control, wound repair, and prosthesis-osseointegration after joint replacement surgery. This dressing needs to provide rapid antibacterial and sustained oxygenation in the early postoperative period, and ensure that its active ingredients can effectively penetrate the bone-implant interface, thereby significantly simplifying postoperative care, reducing the risk of infection, and accelerating the osseointegration process. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a time-released oxygen / antibacterial / osteointegration-promoting layered hydrogel dressing, its preparation method, and its application.

[0006] The layered hydrogel dressing of the present invention has the following advantages: 1. Precise spatiotemporal control: the lower layer responds quickly to control early infection, while the upper layer provides continuous support for long-term healing; 2. Deep repair and penetration: the released oxygen and calcium ions diffuse to the bone defect area, promoting prosthesis-bone integration; 3. Infection barrier mechanism: the surface antibacterial effect blocks bacteria from invading deep bone tissue.

[0007] A first aspect of the present invention provides a layered hydrogel dressing.

[0008] A layered hydrogel dressing, from bottom to top, includes a lower layer, an interface layer and an upper layer; The lower layer consists of sodium alginate, calcium peroxide nanoparticles, and carrageenan. The upper layer includes carrageenan, chlorella, and sodium alginate; The interface layer is a calcium ion-mediated carboxyl-sulfate cross-linked isolation band.

[0009] The lower layer releases hydrogen peroxide (H2O2) upon contact with tissue fluid, achieving rapid antibacterial action, while simultaneously providing a burst of oxygen to alleviate acute hypoxia. The upper layer continuously releases oxygen under light, promoting wound repair. The interface layer prevents H2O2 from damaging Chlorella cells and achieves synergistic dual-mode oxygen supply.

[0010] Preferably, the upper layer further comprises EDTA (ethylenediaminetetraacetic acid) and gluconolactone.

[0011] Preferably, the lower layer further comprises EDTA (ethylenediaminetetraacetic acid) and gluconolactone.

[0012] Preferably, the carrageenan is k-carrageenan.

[0013] Preferably, in the composition of the lower layer, the mass ratio of sodium alginate to calcium peroxide nanoparticles is 5:(300-500), more preferably 5:(400-500). For example, 5:450.

[0014] Preferably, the mass ratio of EDTA (ethylenediaminetetraacetic acid) to gluconolactone in the upper and lower layers is 1:(0.5-1.5). For example, 1:1.

[0015] A second aspect of the present invention provides a method for preparing a layered hydrogel dressing.

[0016] A method for preparing a layered hydrogel dressing includes the following steps: (1) Preparation of the lower layer: Sodium alginate, calcium peroxide nanoparticles, and carrageenan were mixed and cross-linked to form the lower layer; (2) Preparation of the upper layer: Chlorella suspension is mixed with carrageenan solution and sodium alginate, and then coated on the surface of the lower layer. Then, ionic cross-linking is performed to obtain the layered hydrogel dressing.

[0017] Preferably, the calcium peroxide nanoparticles are synthesized by precipitation of calcium chloride and hydrogen peroxide in an ethanol system.

[0018] Preferably, the preparation of the calcium peroxide nanoparticles includes the following steps: (a) Dissolve anhydrous calcium chloride and polyvinylpyrrolidone in ethanol; (b) Add ammonia water while stirring, then add hydrogen peroxide solution, and continue the reaction to obtain a mixture; (c) The mixture was collected by centrifugation and washed with ethanol to obtain calcium peroxide nanoparticles.

[0019] Preferably, the preparation of the calcium peroxide nanoparticles includes the following steps: (a) Dissolve 300 mg of anhydrous calcium chloride and 60 mg of polyvinylpyrrolidone in 30 mL of ethanol; (b) Add 0.5 mL of 10 M ammonia water while stirring, and add 0.4 mL of 10 M hydrogen peroxide solution at a rate of 0.04 mL / min. Continue the reaction for 1 h to obtain a mixture. (c) The mixture was centrifuged at 11,000 rpm for 10 min to collect the product, washed three times with ethanol to obtain calcium peroxide nanoparticles, which were then dispersed in 5 mL of ethanol for later use.

[0020] Preferably, the Chlorella suspension is prepared by the following steps: (a) Take a clean and dry culture dish, prepare Chlorella culture medium, purify the original Chlorella solution by streak plate method, pick up the purified Chlorella seeds with a clean pipette tip, place them in a Chlorella culture dish, and culture at room temperature, providing light and dark environments every day. (b) Take Chlorella cells in the exponential growth phase, remove excess culture medium by centrifugation, add buffer solution for centrifugation and washing to obtain Chlorella cell precipitate, add deionized water for resuspending to obtain Chlorella suspension.

[0021] Preferably, the Chlorella suspension is prepared by the following steps: (a) Take a clean and dry culture dish, prepare a special culture medium for Chlorella, purify the original Chlorella solution by streak plate method, pick up the purified Chlorella seeds with a clean pipette tip, place them in a Chlorella culture dish, and culture at room temperature, providing 12 hours of light and 12 hours of darkness per day. (b) Take 15 mL of microalgae (Chlorella) cells in the exponential growth phase (OD)750 = 0.6), centrifuge at 6000 rpm for 5 min to remove excess culture medium, and add PBS to centrifuge and wash once to obtain Chlorella cell precipitate. Add deionized water to resuspend and obtain Chlorella suspension.

[0022] Preferably, sodium alginate, EDTA (ethylenediaminetetraacetic acid), and gluconolactone are also added during the preparation of the upper and lower layers.

[0023] A third aspect of the present invention provides an application of a layered hydrogel dressing.

[0024] The above-mentioned layered hydrogel dressings are used in the preparation of medical products.

[0025] The aforementioned layered hydrogel dressing can be applied to the prosthesis-bone interface during surgery; postoperative light exposure activates the upper layer's photosynthetic oxygen supply, releasing active ingredients that penetrate into the bone defect area to promote healing.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: The layered hydrogel dressing of the present invention has the following advantages: 1. Precise spatiotemporal control: the lower layer responds quickly to control early infection, while the upper layer provides continuous support for long-term healing; 2. Deep repair and penetration: the released oxygen and calcium ions diffuse to the bone defect area, promoting prosthesis-bone integration; 3. Infection barrier mechanism: the surface antibacterial effect blocks bacteria from invading deep bone tissue.

[0027] Specifically, the layered hydrogel dressing of this invention achieves comprehensive therapeutic effects through a three-stage cascade reaction: At the wound level: H2O2 disrupts the bacterial biofilm structure, while Chlorella photosynthesis maintains the epithelial regeneration microenvironment; Prosthesis interface: Continuous oxygen supply promotes osteoblast differentiation, and calcium ions enhance hydroxyapatite deposition; In bone defect areas: the antibacterial barrier blocks the downward spread of infection, and oxygen diffusion activates vascular ingrowth and bone remodeling. Attached Figure Description

[0028] Figure 1 The samples are: original Chlorella solution, Chlorella cultured for 0 days, and Chlorella cultured for 7 days. Figure 2 SEM image of calcium peroxide nanoparticles in a solution; Figure 3 The infrared spectrum of calcium peroxide nanoparticles in a solution; Figure 4 SEM image of the layered hydrogel dressing prepared in Example 1; Figure 5 An optical microscope image of the layered hydrogel dressing prepared in Example 1; Figure 6 Infrared spectra of the products prepared in Example 1 and Comparative Examples 1-3; Figure 7 The images show actual photos of the layered hydrogel dressings prepared in Examples 1, 2, and 4. Figure 8 The oxygen release performance test results are for the layered hydrogel dressing prepared in Example 1; Figure 9 The results of comparative tests on the oxygen release performance of the layered hydrogel dressings prepared in Example 1 and Comparative Example 5 are shown. Figure 10 The antibacterial zones of the gel products prepared in Example 1 and Comparative Examples 1-3; Figure 11 The diameter of the inhibition zone of the gel products prepared in Example 1 and Comparative Examples 1-3; Figure 12 The image shows the live / dead staining of the gel product prepared in Example 1 in the NIH3T3 cell compatibility test. Figure 13 The fluorescence image of the gel product prepared in Example 1 in the NIH3T3 cell compatibility test; Figure 14 This is a wound image from an in vitro wound healing experiment in mice. Figure 15 This is an overlay image of the wound surface during an in vitro wound healing experiment in mice. Figure 16 This is a diagram of wound healing during an in vitro wound healing experiment in mice. Figure 17 The images show the physical images of the layered hydrogel dressings prepared in Example 1 and Comparative Example 6. Figure 18 The results show the oxygen release performance test results of the layered hydrogel dressings prepared in Example 1 and Comparative Example 6. Detailed Implementation

[0029] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0030] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0031] The Chlorella suspension and calcium peroxide nanoparticle solution used in the following examples were prepared by the following methods.

[0032] Preparation of Chlorella suspension: (a) Take a clean and dry culture dish, prepare a special culture medium for Chlorella, purify the original Chlorella solution by streak plate method, pick up the purified Chlorella seeds with a clean pipette tip, place them in a Chlorella culture dish, and culture at room temperature, providing 12 hours of light and 12 hours of darkness per day. (b) Take 15 mL of microalgae (Chlorella) cells in the exponential growth phase (OD) 750 = 0.6), centrifuge at 6000 rpm for 5 min to remove excess culture medium, and add PBS to centrifuge and wash once to obtain Chlorella cell precipitate. Add deionized water to resuspend and obtain Chlorella suspension.

[0033] Figure 1 The original Chlorella solution, Chlorella cultured for 0 days, and Chlorella cultured for 7 days; from Figure 1 ( Figure 1 As can be seen from Figure (a) which corresponds to the original Chlorella solution, Figure (b) which corresponds to Chlorella cultured for 0 days, and Figure (c) which corresponds to Chlorella cultured for 7 days, the Chlorella is growing stably, has a normal morphology, and is free of contaminants.

[0034] Preparation of calcium peroxide nanoparticle solution: (a) Dissolve 300 mg of anhydrous calcium chloride and 60 mg of polyvinylpyrrolidone in 30 mL of ethanol; (b) Add 0.5 mL of 10 M ammonia water while stirring, and add 0.4 mL of 10 M hydrogen peroxide solution at a rate of 0.04 mL / min. Continue the reaction for 1 h to obtain a mixture. (c) The mixture was centrifuged at 11,000 rpm for 10 min to collect the product, washed three times with ethanol, and then dispersed in 5 mL of ethanol to obtain a calcium peroxide nanoparticle solution.

[0035] Figure 2 SEM image of calcium peroxide nanoparticles in a solution; Figure 3 The image shows the infrared spectrum of calcium peroxide nanoparticles in a solution.

[0036] from Figure 2 As can be seen, calcium peroxide particles are mainly spherical or nearly spherical in shape, but there is agglomeration. The particle surface looks relatively smooth without obvious cracks or defects. This smooth surface may help calcium peroxide to exhibit activity in chemical reactions.

[0037] from Figure 3 ( Figure 3 In this context, "Transmittance" refers to transmittance, and "Wavenumber" refers to wavenumber (3500-3200 cm⁻¹). -1Within the range and 1500-1700cm -1 There are peak values ​​within the range, which are consistent with the characteristics of CaO2 infrared peaks.

[0038] Example 1 A method for preparing a layered hydrogel dressing includes the following steps: (a) Preparation of M / Alg / Cgn: (1) Under continuous stirring conditions, 100 μL of the above Chlorella suspension was added to 15 mL of 30 mg / mL sodium alginate solution and stirred until well mixed (dark green) to obtain mixture a, denoted as M / Alg; (3) Add 1g of carrageenan (CAS 9000-07-1) to the above mixture and stir until well mixed (at this time the viscosity of the solution begins to increase), to obtain mixture b; (4) Add 0.18g EDTA and 0.18g gluconolactone to the above mixture b, stir and mix well, and the resulting product is denoted as M / Alg / Cgn.

[0039] (b) Preparation of CaO2 / Alg / Cgn: (1) Under continuous stirring conditions, 100 μL of 50 mg / mL calcium peroxide nanoparticle solution was added to 15 mL of 30 mg / mL sodium alginate solution and stirred until homogeneous. The resulting mixture c was denoted as CaO2 / Alg.

[0040] (2) Add 1g of carrageenan (CAS 9000-07-1) to the above mixture and stir until well mixed (at this time the viscosity of the solution begins to increase), and the resulting mixture d; (3) Add 0.18g EDTA and 0.18g gluconolactone to mixture d, stir and mix well, and the resulting product is denoted as CaO2 / Alg / Cgn.

[0041] (c) Preparation of layered hydrogel dressings: (1) Using a 24-well plate as a mold, take 0.5 mL of CaO2 / Alg / Cgn and 0.5 mL of M / Alg / Cgn respectively, and add them to the well plate in the following order (i.e., add CaO2 / Alg / Cgn first, spread it out and then add M / Alg / Cgn), and shake it slightly. (2) Place at room temperature to allow ionic cross-linking for 15 minutes, carefully remove the material and wash with deionized water; (3) The layered hydrogel dressing was obtained and kept at 4°C.

[0042] Figure 4 SEM image of the layered hydrogel dressing prepared in Example 1; Figure 5An optical microscope image of the layered hydrogel dressing prepared in Example 1. From Figure 4 As shown in Figure (a), the layered hydrogel dressing has a porous structure, with each pore cross-linked to form a three-dimensional network. Figure 4 Figure (b) shows that the layered hydrogel dressing contains spherical particles. Figure 5 It can be seen that the surface of the layered hydrogel dressing contains a large number of dark green Chlorella cells, and there are a large number of surviving Chlorella cells on both the surface and the inside of the layered hydrogel dressing.

[0043] Example 2 Compared with Example 1, the only difference in Example 2 is that the amount of calcium peroxide nanoparticles used in Example 2 is increased by 10 times.

[0044] Comparative Example 1 Compared with Example 1, the difference in Comparative Example 1 is that Chlorella and calcium peroxide nanoparticles were not used in the preparation of the gel dressing, and the resulting product was designated as GEL-1.

[0045] Comparative Example 2 Compared with Example 1, the difference in Comparative Example 2 is that calcium peroxide nanoparticles are not used in the preparation of the gel dressing, and the resulting product is designated as GEL-2.

[0046] Comparative Example 3 Compared with Example 1, the difference in Comparative Example 3 is that Chlorella was not used in the preparation of the gel dressing, and the resulting product is denoted as GEL-3.

[0047] Figure 6 Infrared spectra of the products prepared in Example 1 and Comparative Examples 1-3; from Figure 6 It can be seen that the stretching vibration of the hydroxyl group is typically between 3200 and 3500 cm⁻¹. -1 Within a certain range, it reflects the vibrations of water molecules and hydroxyl groups; carboxyl bending vibrations: typically between 1500-1700 cm⁻¹. -1 Within the range; Carrageenan CO vibration: typically 1500-1700 cm -1 Within the specified range, this demonstrates that the gel dressing prepared in Example 1 conforms to the experimental design concept.

[0048] Comparative Example 4 Compared with Example 1, the only difference in Comparative Example 4 is that the carrageenan used was replaced with an equal amount of chitosan.

[0049] Figure 7 The images show actual photos of the layered hydrogel dressings prepared in Examples 1, 2, and 4. Figure 7 (a) in the text corresponds to the layered hydrogel dressing of Example 2. Figure 7 (b) corresponds to the layered hydrogel dressing in proportion 4. Figure 7 (c) in the figure corresponds to the layered hydrogel dressing of Example 1. Due to the excessive amount of calcium peroxide nanoparticles in Example 2, the bottom gelation was uneven. The layered hydrogel dressing of Comparative Example 4 was slightly cloudy, the gel was brittle, the cross-linking was weak, and the adhesion was also smaller than that of Example 1.

[0050] Comparative Example 5 Compared with Example 1, the difference in Comparative Example 5 is that a calcium chloride solution of equal concentration and volume is used instead of the calcium peroxide nanoparticle solution in Example 1.

[0051] Comparative Example 6 Compared with Example 1, Comparative Example 6 differs in that an equal amount of diatoms (small crescent-shaped rhomboid algae) is used instead of Chlorella in Example 1.

[0052] Product effectiveness test 1. Oxygen production performance test In an environment of 25°C, the layered hydrogel dressings prepared in Example 1 and Comparative Example 5 were placed in 10 mL of PBS (pH 7.4), respectively, and the results were as follows. Figure 9 As shown.

[0053] Figure 9 The results of comparative tests on the oxygen release performance of the layered hydrogel dressings prepared in Example 1 and Comparative Example 5 are shown. Figure 9 Figure (a) in the figure corresponds to scale 5. Figure 9 Figure (b) corresponds to Example 1. Observation shows that no bubbles were generated in Comparative Example 5 within a short time, while bubbles were generated in Example 1, verifying the oxygen production performance of calcium peroxide nanoparticles in the early stage.

[0054] In an environment of 25 °C, the layered hydrogel dressing prepared in Example 1 was placed in 10 mL of PBS (pH 7.4), and the changes in oxygen concentration at different time points (0 min, 10 min, 30 min, 60 min, 2 h, 6 h, 12 h, 24 h) were monitored using a dissolved oxygen meter under both normal and non-normal light conditions (8000 lux LED light). The results are as follows. Figure 8 As shown.

[0055] Figure 8 The oxygen release performance test results are for the layered hydrogel dressing prepared in Example 1; from Figure 8(The text indicates that the layered hydrogel dressing initially released oxygen rapidly, presumably due to the release of oxygen primarily from CaO2. After 5 hours, the curve slope gradually decreased, with the oxygen release from the light-exposed layered hydrogel dressing being faster and greater than that from the dark-exposed layered hydrogel dressing. This suggests that the oxygen release was mainly from Chlorella algae within the layered hydrogel dressing. Subsequently, the oxygen release curve of the dark-exposed layered hydrogel dressing gradually flattened, while the oxygen content of the light-exposed layered hydrogel dressing continued to increase, fully demonstrating the oxygen production performance of Chlorella algae within the layered hydrogel dressing.)

[0056] 2. In vitro antibacterial performance test Take 4 clean and dry petri dishes, prepare LB agar medium, and make 4 solid culture media. Use a pipette to take 50 μL of diluted Staphylococcus aureus inoculum (S). aureus The concentration is 10 6 The gel (CFU / mL) was evenly spread into four solid culture media. Then, the gel products prepared in Comparative Example 1 (without calcium peroxide and Chlorella), Comparative Example 3 (without Chlorella), Comparative Example 2 (without calcium peroxide), and the layered hydrogel dressing from Example 1 were added sequentially to each media. After sealing in a clean bench, the media were incubated at 37°C for 24 hours. Afterward, the media were removed, photographed, and the presence of inhibition zones was observed. If inhibition zones were present, their diameter was measured and photographed. The results are as follows: Figure 10 and Figure 11 As shown.

[0057] Figure 10 The antibacterial zones of the gel products prepared in Example 1 and Comparative Examples 1-3; Figure 10 (a) corresponds to response ratio 1, (b) corresponds to response ratio 3, (c) corresponds to response ratio 2, and (d) corresponds to Example 1.

[0058] Figure 11 The diameter of the inhibition zone is the diameter of the gel products prepared in Example 1 and Comparative Examples 1-3 (where "Inhibition zone diameter" refers to the diameter of the inhibition zone).

[0059] Figure 10 and Figure 11 The experimental results showed that Comparative Example 1 did not have a significant antibacterial effect, while Example 1 showed a very obvious inhibition zone with D=12.5mm, indicating that the gel product prepared in Example 1 had a good antibacterial effect against Staphylococcus aureus, which is in line with the product design concept.

[0060] 3. NIH 3T3 cell compatibility test NIH3T3 cells were cultured in DMEM medium (Gibco) supplemented with 10% (v / v) inactivated FBS (Gibco, i.e., fetal bovine serum) and 1% (v / v) antibiotic / antifungal solution (AB / AM, Gibco). A DMEM mixture containing antibiotic / antifungal agents (v / v = 1:89) was prepared, and the gel product prepared in Example 1 was immersed in serum-free medium containing both antibiotics (v / v = 1:10) and incubated at 37°C for 24 h (a control medium was set up). The extract was filtered using a 0.22 μm filter, and a complete extract was prepared according to 10% (v / v) FBS. A 10% extract was prepared according to a material extract: control medium ratio of 1:9 (v / v). NIH3T3 cells were seeded in 96-well plates at a density of 2000 cells / well and pre-cultured for 12 hours to allow the cells to adhere completely. The original culture medium was then removed, and 200 μL of complete extract solution was added for further culture.

[0061] 2 μL of calcein solution (5 mg / mL) and 3 μL of propidium iodide (PI) solution (5 mg / mL) were added to 1 mL of PBS to prepare the live / dead staining solution. When cells were cultured for 1, 3, and 5 days, the original extract was removed, and 100 μL of the live / dead staining solution was added to each well under dark conditions. The cells were incubated at 37°C in a 5% CO2 incubator for 20 min. Fluorescence images were captured using a fluorescence microscope. The results are shown below. Figure 12 and Figure 13 As shown.

[0062] Figure 12 Live / dead staining images of the gel product prepared in Example 1 in the NIH3T3 cell compatibility test (where "Live" indicates live, "Dead" indicates dead, "Merge" indicates fusion, "Day 1" indicates day 1, "Day 3" indicates day 3, and "Day 5" indicates day 5). Figure 13 ("Time (day)" indicates time (day), "Live / Dead Fluorescence Intensity" indicates the fluorescence intensity of live / dead staining) is a fluorescence image of the gel product prepared in Example 1 in the NIH3T3 cell compatibility test.

[0063] from Figure 12 and Figure 13 It can be seen that as the culture period increases, the number of cells continues to increase, and the cells maintain normal proliferative and metabolic activity.

[0064] 4. In vitro wound healing experiment in Balb / c mice 4.1 Experimental Grouping: Ten 6-week-old male Balb / c mice of similar size and weight were used in the experiment. Identical wound models were created on both sides of the spine of each mouse using the "skin lesion + smear" method. The mice were then randomly divided into five groups of two. The first group served as the control group, receiving only saline solution without dressing treatment. The second group received a layered hydrogel dressing prepared in Example 1. The third group received a gel dressing prepared in Comparative Example 3 containing only calcium peroxide. The fourth group received a gel dressing prepared in Comparative Example 2 containing only Chlorella. The fifth group received a gel dressing prepared in Comparative Example 1 without calcium peroxide and Chlorella. The wound healing process was observed and recorded in all five groups.

[0065] 4.2 Experimental Procedure: (1) The mice were anesthetized. During the operation, the skin was prepared on both sides of the spine on the back of the mice, and a circular mark with a diameter of 1 cm was made on each side. The full thickness of the skin was cut and the fascia layer was reached. After the wound was fully hemostatic, Staphylococcus aureus was inoculated.

[0066] (2) If the wound shows purulent discharge, redness and swelling of the skin edge, and increased skin temperature three days after the operation, the model can be considered successful.

[0067] (3) Mice were randomly divided into five groups: Group 1 was a blank control, receiving only saline; Group 2 had the layered hydrogel dressing prepared in Example 1 applied to the wounds; Group 3 had the gel dressing prepared in Comparative Example 3 containing only calcium peroxide applied to the wounds; Group 4 had the gel dressing prepared in Comparative Example 2 containing only Chlorella applied to the wounds; and Group 5 had the gel dressing prepared in Comparative Example 1 applied to the wounds. The wound healing status of the five groups of mice was continuously recorded at different time points, and the mice's vital signs were monitored to continuously compare the wound healing status of the five groups.

[0068] (4) About two weeks after the operation, the mice in the five groups were euthanized.

[0069] The results are as follows Figure 14 , Figure 15 and Figure 16 As shown.

[0070] Figure 14 This is a wound image from an in vitro wound healing experiment in mice. Figure 15 This is an overlay image of the wound surface during an in vitro wound healing experiment in mice. Figure 16 This is a diagram of wound healing during an in vitro wound healing experiment in mice (where “Woundarea” represents the wound area and “Time (day)” represents the time (days)).

[0071] From the above Figure 14 , Figure 15 and Figure 16 As can be seen, the layered hydrogel dressing prepared in Example 1 has a better effect on wound healing in mice. This dressing not only combines the excellent biocompatibility and moisturizing properties of hydrogel, but also achieves continuous oxygen release and uniform distribution of antibacterial drugs through an innovative layered cross-linking preparation method. Through the continuous release of oxygen by CaO2 and Chlorella, an oxygen-rich environment is created for the wound, promoting wound healing. At the same time, the antibacterial components of Chlorella and CaO2 inside it effectively inhibit the growth of pathogens, reduce the risk of infection, and enable the wounds of mice to heal better.

[0072] 5. Comparison of morphology and oxygen release effect between diatomaceous earth gel and chlorella gel Figure 17 Images of the layered hydrogel dressings prepared in Example 1 and Comparative Example 6; where (a) corresponds to Comparative Example 6 and (b) corresponds to Example 1. Figure 17 It can be seen that *Nyctaginus simulans* contains yellowish-brown plate-like chloroplasts, appearing yellowish-brown or brown in water, while *Chlorella vulgaris* from Example 1 contains chlorophyll, appearing green or dark green in water.

[0073] In an environment of 25 °C, the gel products prepared in Comparative Example 6 and Example 1 were placed in 10 mL of PBS (pH 7.4), respectively. Under normal lighting conditions (8000 lux LED lamp), the changes in oxygen concentration were monitored and measured using a dissolved oxygen meter at 12 h and 24 h. The results are as follows. Figure 18 As shown.

[0074] Figure 18 The results show the oxygen release performance test results of the layered hydrogel dressings prepared in Example 1 and Comparative Example 6.

[0075] from Figure 18 It can be seen that, whether at 12h or 24h, the oxygen content released by the gel product of Example 1 is much greater than that released by the gel product of Comparative Example 1, which fully demonstrates that the oxygen production performance of Chlorella in Example 1 is better than that of Rhomboidella simonii (diatom) in Comparative Example 6.

Claims

1. A layered hydrogel dressing, characterized in that, From bottom to top, it includes a lower layer, an interface layer, and an upper layer; the lower layer consists of sodium alginate, calcium peroxide nanoparticles, and carrageenan. The upper layer includes carrageenan, chlorella, and sodium alginate; The interface layer is a calcium ion-mediated carboxyl-sulfate cross-linked isolation band; The carrageenan is κ-carrageenan; In the composition of the lower layer, the mass ratio of sodium alginate to calcium peroxide nanoparticles is 300-500:5; The upper layer also includes ethylenediaminetetraacetic acid and gluconolactone; The lower layer also includes ethylenediaminetetraacetic acid and gluconolactone; The mass ratio of ethylenediaminetetraacetic acid to gluconolactone in the upper or lower layer is 1:(0.5-1.5).

2. The method for preparing the layered hydrogel dressing according to claim 1, characterized in that, Includes the following steps: (1) Preparation of the lower layer: Sodium alginate was mixed with calcium peroxide nanoparticles, carrageenan, ethylenediaminetetraacetic acid and gluconolactone and cross-linked to form the lower layer; (2) Preparation of the upper layer: Chlorella suspension is mixed with carrageenan solution, sodium alginate, ethylenediaminetetraacetic acid and gluconolactone, and then coated on the surface of the lower layer. Then, ionic cross-linking is performed to obtain the layered hydrogel dressing.

3. The preparation method according to claim 2, characterized in that, The calcium peroxide nanoparticles are synthesized by precipitation of calcium chloride and hydrogen peroxide in an ethanol system.

4. The preparation method according to claim 2, characterized in that, The suspension of Chlorella is prepared by the following steps: (a) Take a clean and dry culture dish, prepare Chlorella culture medium, purify the original Chlorella solution by streak plate method, pick up the purified Chlorella seeds with a clean pipette tip, place them in a Chlorella culture dish, and culture at room temperature, providing light and dark environments daily. (b) Take Chlorella cells in the exponential growth phase, remove excess culture medium by centrifugation, add buffer solution for centrifugation and washing to obtain Chlorella cell precipitate, add deionized water for resuspending to obtain Chlorella suspension.

5. The application of the layered hydrogel dressing according to claim 1 in the preparation of medical products.

Images