Micro-ecological hydrogel loaded with photosynthetic bacteria and chlorella as well as preparation method and application of micro-ecological hydrogel

Abstract

The invention belongs to the technical field of biomedical materials, and particularly discloses micro-ecological hydrogel loaded with photosynthetic bacteria and chlorella as well as a preparation method and application of the micro-ecological hydrogel. According to the invention, chlorella and photosynthetic bacteria are jointly loaded in methylacryloylated gelatin, so that the micro-ecological tandem hydrogel with metabolic circulation mutually promoted among internal microorganisms is developed. The micro-ecological hydrogel loaded with the photosynthetic bacteria and the chlorella not only has high biocompatibility, but also has the effects of reducing inflammatory response, stimulating proliferation and migration of fibroblasts, inducing angiogenesis, promoting skin reepithelization and collagen deposition and the like, and can effectively accelerate wound healing. The method can be applied to preparation of wound healing dressings, and is particularly suitable for diabetic hard-to-heal wounds.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and relates to a microecological hydrogel loaded with photosynthetic bacteria and chlorella, and a preparation method and application thereof. Background Art

[0002] Skin wounds are generally divided into chronic wounds and acute wounds. Chronic wounds are generally difficult to heal due to internal reasons, such as burn wounds or diabetic wounds. Since the high blood sugar level of diabetic patients can cause specific damage to blood vessels, nervous system and immune system, once a wound is formed, it is easy to develop into chronic inflammation and difficult to heal.

[0003] In the occurrence, development and prognosis of chronic wounds in diabetes, hypoxia, resistant bacterial infection and fragile angiogenesis are three key issues. Insufficient blood oxygen transport caused by vascular damage can lead to insufficient oxidative energy supply, low metabolism, and dysfunction of inflammatory cells and repair cells. In addition, as a subsequent symptom of hypoxia, due to neurological malnutrition and reduced body resistance, multiple pathogen infections may occur, leading to tissue apoptosis and persistent inflammation. At present, traditional dressings in clinical wound treatment are limited to preventing bacterial infection of wounds and cannot solve the problems of wound hypoxia and fragile angiogenesis. In view of the porous structure and suitable swelling ratio of hydrogel matrix, a variety of multifunctional hydrogels for wound repair have been reported. However, the current multifunctional hydrogels generally have shortcomings such as cumbersome preparation steps and low synergistic efficiency. Summary of the invention

[0004] In view of the shortcomings of the multifunctional hydrogel in the prior art, the present invention provides a microecological hydrogel loaded with photosynthetic bacteria and Chlorella, which has a relatively simple preparation process, can effectively improve the hypoxic microenvironment at the wound, and the loaded photosynthetic bacteria and Chlorella can biologically synergistically promote wound healing. Furthermore, the prepared microecological hydrogel is used to prepare wound healing dressings.

[0005] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution: In a first aspect, the present invention provides a microecological hydrogel loaded with photosynthetic bacteria and Chlorella, comprising a base hydrogel layer, and photosynthetic bacteria and Chlorella loaded thereon; Wherein, the raw material for preparing the base hydrogel layer includes methacrylated gelatin solution.

[0006] The present invention comprises active chlorella ( Chlorella vulgaris , referred to as Cv) and photosynthetic bacteria ( Rhodopseudomonas palustrisIn the present study, a series of active microecological hydrogels were developed by co-encapsulating photosynthetic bacteria and Chlorella in gelatin (GelMA). The microecological hydrogel (PSB-Cv@GelMA) loaded with photosynthetic bacteria and Chlorella produces dissolved oxygen through photosynthesis of Chlorella during the day, actively consumes glucose and consumes reactive oxygen using inherent antioxidants (such as carotenoids, lutein, vitamin C, etc.). At night, under specific conditions, the photothermal properties of photosynthetic bacteria are stimulated, which can not only photothermally sterilize, but also change the fluidity of Chlorella cell membrane by high temperature to break part of Chlorella and release nutrients including tryptophan. On the one hand, the tryptophan released by Chlorella is the synthetic substrate for photosynthetic bacteria to synthesize indoleacetic acid, and the synthesized indoleacetic acid can promote the growth of Chlorella; on the other hand, other nutrients help to supplement nutrition in situ in the wound and reduce inflammation; on the third hand, the photothermal properties of photosynthetic bacteria can also terminate the oxygen consumption of Chlorella in dark respiration. Photosynthetic bacteria and Chlorella form a mutually reinforcing metabolic cycle, synergistically promoting the healing of diabetic chronic wounds. The microecological hydrogel loaded with photosynthetic bacteria and Chlorella not only has high biocompatibility, but also can reduce inflammatory response, stimulate the proliferation and migration of hypoxic tissues, induce angiogenesis, promote skin re-epithelialization and collagen deposition, and can effectively accelerate wound healing.

[0007] Preferably, the final concentration of the photosynthetic bacteria added is 1×10 7 CFU / mL~1×10 9 CFU / mL; The final concentration of the added Chlorella is 1×10 7 CFU / mL~1×10 9 CFU / mL.

[0008] Preferably, the ratio of the number of viable bacteria of the photosynthetic bacteria to the number of cells of Chlorella is 100:1 to 1:10.

[0009] Preferably, the photosynthetic bacteria is Rhodopseudomonas palustris ( Rhodopseudomonas palustris ), in the present invention, Rhodopseudomonas palustris numbered NBRC16661 is taken as an example for illustration.

[0010] According to zoological verification, Rhodopseudomonas palustris ( Rhodopseudomonas palustris ) has no toxic or pathogenic effect on the test animals, has no adverse effect on fertility and growth, and is not potentially harmful. It is rich in nutrients such as protein, various vitamins, amino acids, etc., and has been approved by the Ministry of Agriculture and Rural Affairs of my country for use as a feed additive, which can significantly improve the survival rate of animals and enhance disease resistance. Based on the good biological safety of photosynthetic bacteria and the ability to produce photothermal effects, the present invention selects this bacterium as the research object.

[0011] The Chlorella ( Chlorella vulgaris), in the present invention, the Chlorella vulgaris numbered FACHB-482 purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences is used as an example for explanation.

[0012] Chlorella has the advantages of rapid proliferation and strong metabolic activity, and can be used as a tool for glucose consumption, thus making full use of the high glucose microenvironment of diabetic wound tissue.

[0013] In a second aspect, the present invention also provides a method for preparing the above-mentioned microecological hydrogel loaded with photosynthetic bacteria and Chlorella, the preparation method comprising the following steps: mixing photosynthetic bacteria, Chlorella and methacrylated gelatin solution, and performing photocuring to obtain the microecological hydrogel loaded with photosynthetic bacteria and Chlorella.

[0014] The preparation method of the microecological hydrogel loaded with photosynthetic bacteria and chlorella provided by the present invention has the advantages of simple operation steps, short process flow and low synthesis cost. The prepared microecological hydrogel loaded with photosynthetic bacteria and chlorella not only has good biocompatibility and high stability, but also can achieve antibacterial and anti-inflammatory effects, stimulate the proliferation and migration of cells in hypoxic tissues, induce angiogenesis, promote skin re-epithelialization, promote collagen deposition and granulation tissue formation, etc. by improving the hypoxic microenvironment, photothermal sterilization, and wound in situ nutrition supplementation, thereby effectively promoting wound healing. Therefore, the microecological hydrogel loaded with photosynthetic bacteria and chlorella has a wide range of application prospects.

[0015] In the present invention, during the photocuring, the methacrylylated gelatin solution containing photosynthetic bacteria and Chlorella is irradiated with a 405nm ultraviolet lamp for 30s to solidify it and form a hydrogel filled with photosynthetic bacteria and Chlorella.

[0016] Preferably, the method for preparing the methacrylated gelatin solution comprises: dissolving the methacrylated gelatin in a photoinitiator solution at 45° C. to 55° C., and mixing the mixture evenly to obtain the methacrylated gelatin solution.

[0017] Preferably, the photoinitiator comprises phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite; The concentration of the photoinitiator solution is 0.23% (w / v) to 0.27% (w / v); The mass concentration of methacryloyl gelatin in the methacryloyl gelatin solution is 9% to 11%.

[0018] In a third aspect, the present invention also provides the use of the above-mentioned microecological hydrogel loaded with photosynthetic bacteria and Chlorella in the preparation of wound healing dressings.

[0019] In view of the fact that the microecological hydrogel loaded with photosynthetic bacteria and Chlorella provided by the present invention has the effects of antibacterial and anti-inflammatory, improving the hypoxic microenvironment, promoting cell proliferation and migration, inducing angiogenesis, promoting skin re-epithelialization, collagen deposition and granulation tissue formation, etc., it is suitable for use as a skin wound healing dressing, and is particularly suitable for use as a diabetic wound healing dressing.

[0020] In a fourth aspect, the present invention further provides a method for using the microecological hydrogel loaded with photosynthetic bacteria and chlorella, the method comprising: applying the microecological hydrogel loaded with photosynthetic bacteria and chlorella to a wound, and irradiating the microecological hydrogel loaded with photosynthetic bacteria and chlorella with near-infrared light having a wavelength of 800nm ​​to 810nm for 7min to 10min at night.

[0021] Preferably, by irradiating the microecological hydrogel loaded with photosynthetic bacteria and Chlorella with near-infrared light of a specific wavelength at night, the photothermal performance of the photosynthetic bacteria is stimulated, not only the purpose of photothermal sterilization is achieved, but also the high temperature can change the fluidity of the Chlorella cell membrane, thereby promoting the release of nutrients, supplementing nutrients in situ in the wound, reducing inflammation, and stopping the oxygen consumption of Chlorella in dark respiration. The photosynthetic bacteria and Chlorella alternate day and night to achieve the purpose of synergistically promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is the photothermal capacity test result of Rhodopseudomonas palustris in the present invention, wherein Figure 1 a) Different concentrations of Rhodopseudomonas palustris at 808nm, 1W / cm 2 The results of the photothermal ability test under near-infrared light, Figure 1 b) is the photothermal capacity test result of Rhodopseudomonas palustris under four different powers of near-infrared light. Figure 1 c) is the test result of cyclic photothermal capacity of Rhodopseudomonas palustris; Figure 2 The oxygen production capacity test results of Chlorella with different concentrations in the present invention are shown; Figure 3 The test results of the cyclic oxygen production capacity of Chlorella in the present invention are as follows; Figure 4 This is a SEM image of the effect of photothermal treatment of Rhodopseudomonas palustris on Chlorella vulgaris in the present invention, wherein Figure 4 a) Normal Chlorella; Figure 4b) The chlorella has been broken; Figure 5 The results are the test results of the effects of different concentrations of Chlorella on the photothermal capacity of Rhodopseudomonas palustris in the present invention; Figure 6 The test results of the effects of different concentrations of Rhodopseudomonas palustris on the oxygen production performance of Chlorella vulgaris in the present invention are as follows; Figure 7 This is a macroscopic image of the microecological hydrogel loaded with photosynthetic bacteria and Chlorella in Example 1 of the present invention; Figure 8 This is a SEM image of the microecological hydrogel loaded with photosynthetic bacteria and Chlorella in Example 1 of the present invention; Fig. 9 The results of the test on the ability of different groups in the present invention to promote the cell proliferation of NIH-3T3 cells in vitro; Fig.10 The results of in vitro antibacterial ability test of different hydrogels in the present invention; Fig.11 The wound healing conditions of diabetic mice in different groups during the treatment period of the present invention; Fig.12 The wound areas of the wounds of diabetic mice treated in different groups during the treatment period of the present invention; Fig.13 The wound tissue sections of the wounds of diabetic mice treated in different groups in the present invention are shown. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The raw materials and reagents used in the present invention, unless otherwise specified, are conventional commercial products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0025] The photosynthetic bacteria selected in the present invention are Rhodopseudomonas palustris ( Rhodopseudomonas palustris ), strain number NBRC16661.

[0026] The chlorella selected in the present invention ( Chlorella vulgaris ) was purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences with the serial number FACHB-482.

[0027] In order to better explain the present invention, the following four aspects are described here.

[0028] 1. In order to evaluate the photothermal capacity of Rhodopseudomonas palustris, the following experiments were conducted. Figure 1 shown.

[0029] Different concentrations (10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL and 10 9 CFU / mL) of Rhodopseudomonas palustris was washed three times with PBS and resuspended in PBS. 2 The samples were irradiated with near infrared light (NIR) for 8 minutes and the temperature was recorded with a thermal imager. The test results are as follows: Figure 1 a).

[0030] Take another 4 portions with a concentration of 10 8 1 mL of each bacterial solution of Rhodopseudomonas palustris was washed three times with PBS and resuspended in PBS to obtain 10 CFU / mL. 8 CFU / mL bacterial suspension, respectively, with 0.5W / cm 2 , 1W / cm 2 , 1.5W / cm 2 and 2W / cm 2 The bacterial suspension was irradiated with 808nm NIR at four power densities for 8 minutes. The temperature change was recorded by a thermal imager. The test results are as follows: Figure 1 b) as shown.

[0031] In addition, in order to verify whether Rhodopseudomonas palustris can produce heat in a cycle, the above concentration of 10 8 1 mL of a suspension of Rhodopseudomonas palustris with a CFU / mL was exposed to 808 nm and 1 W / cm 2 After cooling to room temperature, the NIR wavelength was continuously increased to 808 nm and 1 W / cm 2 The near-infrared light was irradiated for 8 minutes for 4 cycles, and the temperature change was recorded with a thermal imager. The test results are as follows Figure 1 c) as shown.

[0032] Depend on Figure 1 It can be seen that when the power density of the NIR laser with a wavelength of 808nm is kept constant, the temperature of Rhodopseudomonas palustris increases with the increase of bacterial concentration. When the concentration of Rhodopseudomonas palustris is kept constant, the photothermal effect increases with the increase of laser power. In summary, under the irradiation of 808nm NIR, Rhodopseudomonas palustris can convert the absorbed light into heat.

[0033] 2. The oxygen production capacity of Chlorella was measured. The specific contents are as follows: Different concentrations of Chlorella were collected, washed three times with PBS and resuspended in PBS to obtain concentrations of 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL and 10 9 CFU / mL of Chlorella suspension. Using PBS as the measurement medium, the wavelength was 635nm and the light dose was 50mW / cm 2 Under laser irradiation, the oxygen content was measured using a dissolved oxygen meter. Each group was measured three times in parallel. The results are shown in Figure 2 As shown; The cyclic oxygen production performance of Chlorella was measured at 25°C. The results are as follows: Figure 3 shown.

[0034] Depend on Figure 2~3 It can be seen that under the irradiation of sunlight, Chlorella can produce a large amount of oxygen through photosynthesis, and the oxygen production is positively correlated with the concentration of Chlorella. Chlorella has good cyclic oxygen production performance.

[0035] 3. In order to investigate the effects of light and heat on Chlorella, the following experiments were carried out: Photothermal 808nm, 1W / cm 2 The Chlorella was treated under NIR for 8 minutes, gold was sprayed onto the surface of the Chlorella, and the structure of the Chlorella was observed using a cold field emission scanning electron microscope (SEM). The SEM image is shown in the figure. Figure 4 As shown, Figure 4 a) Normal Chlorella; Figure 4 b) Broken Chlorella.

[0036] Depend on Figure 4 It can be seen that the light and heat of Rhodopseudomonas palustris can change the fluidity of the cell membrane of Chlorella, thereby breaking the Chlorella. Furthermore, after the Chlorella is broken, tryptophan and other nutrients can be released.

[0037] 4. In order to investigate whether Chlorella has an effect on the photothermal capacity of Rhodopseudomonas palustris and whether Rhodopseudomonas palustris has an effect on the oxygen production capacity of Chlorella, the following experiments were conducted: (1) Take the concentration as 10 8 CFU / mL of Rhodopseudomonas palustris suspension, respectively, with a concentration of 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL of Chlorella suspension was mixed to determine the effect of different concentrations of Chlorella suspension on the photothermal capacity of Rhodopseudomonas palustris. The test results are as follows Figure 5 shown.

[0038] Depend on Figure 5 It can be seen that 1×10 9 CFU / mL of Chlorella reduced the photothermal temperature of Rhodopseudomonas palustris. The reason may be that the high concentration of Chlorella affected the absorption of light by Rhodopseudomonas palustris, thus affecting the photothermal effect. 6 CFU / mL, 1×10 7 CFU / mL and 1×10 8 CFU / mL of Chlorella had little effect on the photothermal effect of Rhodopseudomonas palustris.

[0039] (2) Select PBS resuspended at a concentration of 10 8 CFU / mL of Chlorella suspension, respectively, with a concentration of 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL of the palustris suspension was mixed to determine the effect of different concentrations of palustris on the oxygen production capacity of Chlorella. The test results are as follows Figure 6 shown.

[0040] Depend on Figure 6 It can be seen that 1×10 9 CFU / mL of Rhodopseudomonas palustris reduced the oxygen release of Chlorella. The possible reason is that the respiratory oxygen consumption of high concentrations of Rhodopseudomonas palustris affected the total oxygen release. 6 CFU / mL, 1×10 7 CFU / mL and 1×10 8 The respiratory oxygen consumption of Rhodopseudomonas palustris CFU / mL has little effect on the total oxygen release.

[0041] Example 1 This embodiment provides a method for preparing a microecological hydrogel loaded with photosynthetic bacteria and Chlorella, the preparation method comprising the following steps: (1) Cultivation of photosynthetic bacteria In the clean bench, take an appropriate amount of activated Rhodopseudomonas palustris numbered NBRC16661 ( Rhodopseudomonas palustris ) bacterial solution, inoculated into sterilized culture medium, and added sterilized liquid paraffin as a sealing layer into the bacterial culture medium. Placed in a constant temperature bacterial culture box at 30°C, cultured under incandescent light for one week, and the cultured Rhodopseudomonas palustris was obtained.

[0042] (2) Cultivation of Chlorella In the clean bench, take the Chlorella vulgaris numbered FACHB-482 ( Chlorella vulgaris) was inoculated into 200 mL of sterilized BG-11 medium and cultured statically in a light incubator at 25°C and 1500 Lux, with 12 h of light and 12 h of dark treatment per day. After culturing for 8 days, the cultured Chlorella was obtained.

[0043] (3) Preparation of microecological hydrogel loaded with photosynthetic bacteria and Chlorella At 50°C, freeze-dried methacryloyl gelatin was dissolved in 0.25% (w / v) photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP) solution and mixed well to obtain a 10% (w / v) GelMA solution. The final concentration of Rhodopseudomonas palustris and Chlorella vulgaris was 1×10 8 The cultured Rhodopseudomonas palustris and the cultured Chlorella were evenly mixed with the GelMA solution, and the solution was irradiated with a 405nm ultraviolet lamp for 30s to solidify it, thereby obtaining a microecological hydrogel loaded with photosynthetic bacteria and Chlorella. In the present invention, the prepared microecological hydrogel loaded with photosynthetic bacteria and Chlorella is recorded as PSB-Cv@GelMA.

[0044] The macroscopic image of the microecological hydrogel loaded with photosynthetic bacteria and Chlorella is shown in Figure 7 As shown, the SEM images of the microecological hydrogel loaded with photosynthetic bacteria and Chlorella are shown in Figure 8 shown.

[0045] Depend on Figure 6 It can be seen that the color of the microecological hydrogel loaded with photosynthetic bacteria and Chlorella prepared in this embodiment is greener than that of conventional GelMA. In addition, the microecological hydrogel has a certain degree of adhesion and can be adhered to the back skin of mice.

[0046] Depend on Figure 7 It can be seen that the microecological hydrogel loaded with photosynthetic bacteria and Chlorella prepared in this embodiment successfully loads the rod-shaped Rhodopseudomonas palustris and the spherical Chlorella into the three-dimensional network space structure of the hydrogel.

[0047] Example 2 This embodiment provides a method for preparing a microecological hydrogel loaded with photosynthetic bacteria and Chlorella, the preparation method comprising the following steps: (1) Cultivation of photosynthetic bacteria The cultivation method of photosynthetic bacteria in this embodiment is the same as that in Example 1.

[0048] (2) Cultivation of Chlorella In the clean bench, take the Chlorella vulgaris numbered FACHB-482 ( Chlorella vulgaris) was inoculated into 200 mL of sterilized BG-11 medium and cultured statically in a light incubator at 23°C and 1000 Lux, with 12 h of light and 12 h of dark treatment every day. After culturing for 10 days, the cultured Chlorella was obtained.

[0049] (3) Preparation of microecological hydrogel loaded with photosynthetic bacteria and Chlorella At 55°C, freeze-dried methacryloyl gelatin was dissolved in 0.27% (w / v) photoinitiator LAP solution and mixed well to obtain 9% (w / v) GelMA solution. Rhodopseudomonas palustris was added to a final concentration of 1×10 7 CFU / mL, the final concentration of Chlorella was 1×10 8 The cultured Rhodopseudomonas palustris and the cultured Chlorella are evenly mixed with the GelMA solution, and the solution is irradiated with a 405 nm ultraviolet lamp for 30 seconds to solidify it, thereby obtaining the microecological hydrogel loaded with photosynthetic bacteria and Chlorella.

[0050] Example 3 This embodiment provides a method for preparing a microecological hydrogel loaded with photosynthetic bacteria and Chlorella, the preparation method comprising the following steps: (1) Cultivation of photosynthetic bacteria The cultivation method of photosynthetic bacteria in this embodiment is the same as that in Example 1.

[0051] (2) Cultivation of Chlorella In the clean bench, take the Chlorella vulgaris numbered FACHB-482 ( Chlorella vulgaris ) was inoculated into 200 mL of sterilized BG-11 medium and cultured statically in a light incubator at 23°C and 1000 Lux, with 12 h of light and 12 h of dark treatment every day. After culturing for 10 days, the cultured Chlorella was obtained.

[0052] (3) Preparation of microecological hydrogel loaded with photosynthetic bacteria and Chlorella At 45°C, freeze-dried methacryloyl gelatin was dissolved in 0.23% (w / v) photoinitiator LAP solution and mixed well to obtain 11% (w / v) GelMA solution. Rhodopseudomonas palustris was added to a final concentration of 1×10 8 CFU / mL, the final concentration of Chlorella was 1×10 9 The cultured Rhodopseudomonas palustris and the cultured Chlorella are evenly mixed with the GelMA solution, and the solution is irradiated with a 405 nm ultraviolet lamp for 30 seconds to solidify it, thereby obtaining the microecological hydrogel loaded with photosynthetic bacteria and Chlorella.

[0053] Example 4 This embodiment provides a method for preparing a microecological hydrogel loaded with photosynthetic bacteria and Chlorella, the preparation method comprising the following steps: (1) Cultivation of photosynthetic bacteria The cultivation method of photosynthetic bacteria in this embodiment is the same as that in Example 1.

[0054] (2) Cultivation of Chlorella In the clean bench, take the Chlorella vulgaris numbered FACHB-482 ( Chlorella vulgaris ) was inoculated into 200 mL of sterilized BG-11 medium and cultured statically in a light incubator at 23°C and 1000 Lux, with 12 h of light and 12 h of dark treatment every day. After culturing for 10 days, the cultured Chlorella was obtained.

[0055] (3) Preparation of microecological hydrogel loaded with photosynthetic bacteria and Chlorella At 55°C, freeze-dried methacryloyl gelatin was dissolved in 0.27% (w / v) photoinitiator LAP solution and mixed well to obtain 10% (w / v) GelMA solution; Rhodopseudomonas palustris was added to a final concentration of 1×10 9 CFU / mL, the final concentration of Chlorella was 1×10 7 The cultured Rhodopseudomonas palustris and the cultured Chlorella are evenly mixed with the GelMA solution, and the solution is irradiated with a 405 nm ultraviolet lamp for 30 seconds to solidify it, thereby obtaining the microecological hydrogel loaded with photosynthetic bacteria and Chlorella.

[0056] Comparative Example 1 This comparative example provides a method for preparing a hydrogel, and the preparation method comprises the following steps: At 50°C, freeze-dried methacryloyl gelatin was dissolved in 0.25% (w / v) photoinitiator LAP solution and mixed well to obtain a 10% (w / v) GelMA solution. The GelMA solution was irradiated with a 405 nm ultraviolet lamp for 30 seconds to solidify it, thereby obtaining the hydrogel, which was recorded as GelMA.

[0057] Comparative Example 2 This comparative example provides a method for preparing a photosynthetic bacteria-loaded microecological hydrogel. The preparation method is basically the same as that in Example 1, except that: (1) Chlorella is not cultured; (2) Chlorella is not loaded in GelMA. The other conditions and parameters are the same as those in Example 1. The prepared photosynthetic bacteria-loaded microecological hydrogel is recorded as PSB@GelMA.

[0058] Comparative Example 3 This comparative example provides a method for preparing a microecological hydrogel loaded with Chlorella. The preparation method is basically the same as that in Example 1, except that: (1) Rhodopseudomonas palustris is not cultured; (2) Rhodopseudomonas palustris is not loaded in GelMA. The other condition parameters are the same as those in Example 1. The prepared microecological hydrogel loaded with photosynthetic bacteria is recorded as Cv@GelMA.

[0059] Effect example The present invention evaluates the effects of different hydrogels in promoting cell proliferation, antibacterial ability, and wound healing. Among them, when investigating the effects on cell proliferation and wound healing, PBS is set as a blank control. In this effect example, the microecological hydrogel loaded with photosynthetic bacteria and Chlorella prepared in Example 1 is used as an example for illustration. The microecological hydrogel loaded with photosynthetic bacteria and Chlorella prepared in Examples 2-4 can also achieve the same effect as the microecological hydrogel prepared in Example 1. The following PSB-Cv@GelMA also represents the microecological hydrogel prepared in Example 1. The specific contents are as follows: (1) Evaluation of the ability to promote NIH-3T3 cell proliferation in vitro Select NIH-3T3 cells with a confluence of 80%, and use a 200µL sterile pipette tip to draw 3 straight lines horizontally and vertically in a 6-well plate. Wash with PBS 3 times to ensure that there are no cells and foreign proteins remaining in the scratches, and the parallel width of the scratches is uniform. Divide into five groups: Con (i.e. PBS), GelMA, PSB@GelMA, Cv@GelMA and PSB-Cv@GelMA, and irradiate the PSB@GelMA and PSB-Cv@GelMA groups with 808nm laser for 8 minutes at night. Three parallel experiments were made for each group. Observe and take pictures under a microscope, record the photos of each group at 0h, and then put the cells in a 37℃ incubator for incubation. The PSB@GelMA group and PSB-Cv@GelMA group were only taken out during irradiation, and the other conditions were the same as other groups. After the five groups of cells were incubated in the incubator for 24h, the floating cells and bacteria were washed off, and the photos were recorded under a microscope. The evaluation results of the ability of different groups to promote NIH-3T3 cell proliferation in vitro are shown as follows Fig. 9 shown.

[0060] Depend on Fig. 9It can be seen that compared with the Con group and the GelMA group, the PSB@GelMA group, the Cv@GelMA group and the PSB-Cv@GelMA group can enhance the migration ability of NIH-3T3 cells. However, compared with the PSB@GelMA group and the Cv@GelMA group, the PSB-Cv@GelMA group has a more significant effect in promoting the migration ability of NIH-3T3 cells. Promoting the directional migration of fibroblasts can make the cells move to the scratch area, which can make the wound heal faster.

[0061] (2) Evaluation of in vitro antibacterial ability By detecting methicillin-resistant Staphylococcus aureus ( Methicillin-Resistant Staphylococcus Aureus , referred to as MRSA) and Escherichia coli ( Escherichia coli , referred to as E. coli ) to evaluate the antibacterial effect of different groups of hydrogels. The sterilized hydrogels of different groups were placed in 48-well plates. MRSA bacterial liquid (10 6 CFU / mL) and E. coli Bacterial liquid (10 6 CFU / mL) were centrifuged and washed with PBS, and 500 µL of bacterial solution was added to the surface of the hydrogels of the above different groups. Subsequently, 1 mL of sterile PBS was introduced into each well, and the remaining bacteria were resuspended and inoculated on the agar plate. The plates were cultured in a 37°C constant temperature incubator for 24 h. During the culture process, all groups of hydrogels were irradiated with 808 nm NIR (1.5 W / cm 2 ) irradiated for 8 min. After 24 h of culture, the in vitro antibacterial ability of different hydrogels was as follows Fig.10 Colony forming units were counted on agar plates as shown.

[0062] Depend on Fig.10 It can be seen that compared with the GelMA group and the Cv@GelMA group, the PSB@GelMA group and the PSB-Cv@GelMA group can effectively kill pathogens and inhibit the growth of bacteria, and are effective against both Gram-negative and Gram-positive bacteria.

[0063] (3) Evaluation of wound healing promotion ability in diabetic mice The wound diameter was 0.8 ± 0.05 cm. 2The diabetic mouse wound model was successfully established and grouped for treatment 24 hours after the model was successfully established. Among them, the Con group treated the wound with 1mL PBS buffer, and the GelMA, PSB@GelMA, Cv@GelMA and PSB-Cv@GelMA groups applied equal amounts of hydrogel to the wound. At the same time, the PSB@GelMA and PSB-Cv@GelMA groups were irradiated with 808nm laser for 8min at night. The wound was treated every four days, and the corresponding hydrogels were replaced every four days in different hydrogel groups for 12 days. The wound area of ​​the mouse was measured and recorded once a day, and the wound tissue of the mouse was photographed every four days.

[0064] During the treatment period, the wound healing of diabetic mice in different groups was as follows Fig.11 As shown in Figure 2, the wound areas of diabetic mice treated with different groups are shown in Figure 2. Fig.12 shown.

[0065] Depend on Fig.11 It can be seen that on the 4th day, the wound area of ​​each group showed a trend of shrinking. Compared with the GelMA group, the wound area of ​​the PSB@GelMA group and the Cv@GelMA group decreased by about 40%, and the wound area of ​​the PSB-Cv@GelMA group decreased by 50%. On the 8th and 12th days, the PSB-Cv@GelMA group showed a significant acceleration of the wound healing process, the epidermal tissue became smooth, and the healing effect was the best. On the 12th day, the wound was almost completely healed. Compared with other groups, the PSB-Cv@GelMA group had the best recovery effect.

[0066] Depend on Fig.12 It can be seen that the wound area of ​​the Con group was the largest during the entire wound healing process; the wound area of ​​the GelMA group was slightly smaller than that of the Con group, because GelMA can absorb the exudate in the wound site and keep the wound site dry. The wound areas of the PSB@GelMA group and the Cv@GelMA group were smaller than those of the GelMA group, but not as good as the healing effect of the PSB-Cv@GelMA group, while the wound area of ​​the PSB-Cv@GelMA group was the smallest, which was more conducive to wound healing.

[0067] (4) Analysis of wound tissue sections after treatment In order to evaluate the epidermal regeneration and inflammation in the wound area, the skin specimens collected on the 4th, 8th and 12th days were fixed and dehydrated in 10% formaldehyde solution, and 5 µm thick paraffin sections were made, followed by H&E staining and analysis. Fig.13 shown.

[0068] Depend on Fig.13The process of skin re-epithelialization, collagen deposition, and granulation tissue formation can be seen. H&E staining showed that after 4 days of treatment, all five groups showed varying degrees of inflammatory cell infiltration and granulation tissue formation. However, more fibroblasts migrated and proliferated in the PSB-Cv@GelMA group. On day 8, compared with the other groups, the PSB-Cv@GelMA group had complete dermal tissue and more capillaries. On day 12, the dermal tissue in the PSB-Cv@GelMA group was almost completely regenerated, showing more hair follicles and almost no inflammation. Compared with the other groups, on day 12, the PSB-Cv@GelMA group had the highest values ​​of epithelial gap and granulation tissue thickness, a more complete epithelial structure on the skin wound, the highest collagen content, and the best healing effect.

[0069] In summary, the microecological hydrogel loaded with photosynthetic bacteria and Chlorella provided by the present invention can not only provide enough dissolved oxygen for the diabetic wound through photosynthesis, but also improve the hypoxic microenvironment of the wound; under the corresponding laser irradiation, it also has a photothermal effect, achieving photothermal sterilization and promoting the fluidity of the Chlorella cell membrane, breaking part of the Chlorella, thereby releasing a series of nutrients including tryptophan, wherein tryptophan can be used by Rhodopseudomonas palustris to synthesize indoleacetic acid. Indoleacetic acid is not only a natural bacterial biofilm inhibitor, thereby effectively inhibiting bacterial growth at the wound, but also promoting the growth of Chlorella; and other released nutrients can supplement nutrition for the wound in situ and promote wound healing. Rhodopseudomonas palustris and Chlorella form a mutually promoting metabolic cycle to form a series of microecology. In addition, the microecological hydrogel loaded with Rhodopseudomonas palustris and Chlorella provided by the present invention also has the effects of antibacterial and anti-inflammatory, promoting cell migration and proliferation, and inducing angiogenesis.

[0070] Compared with other groups, the microecological hydrogel group loaded with photosynthetic bacteria and Chlorella provided by the present invention has more fibroblasts migrating and proliferating in the early stage of treatment, more complete dermal tissue in the middle and late stages, and more capillaries generated, thus having the best therapeutic effect on diabetic wound healing.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A microecological hydrogel loaded with photosynthetic bacteria and Chlorella, characterized in that: The microecological hydrogel loaded with photosynthetic bacteria and Chlorella comprises a base hydrogel layer, and photosynthetic bacteria and Chlorella loaded thereon; Wherein, the raw material of the base hydrogel layer includes methacrylated gelatin solution.

2. The microecological hydrogel loaded with photosynthetic bacteria and Chlorella as claimed in claim 1, characterized in that: The final concentration of the photosynthetic bacteria added was 1×10 7 CFU / mL~1×10 9 CFU / mL; The final concentration of the added Chlorella is 1×10 7 CFU / mL~1×10 9 CFU / mL.

3. The microecological hydrogel loaded with photosynthetic bacteria and Chlorella as claimed in claim 1, characterized in that: The ratio of the number of viable bacteria of the photosynthetic bacteria to the number of cells of Chlorella is 100:1 to 1:

10.

4. The microecological hydrogel loaded with photosynthetic bacteria and Chlorella as claimed in claim 1, characterized in that: The photosynthetic bacteria is Rhodopseudomonas palustris ( Rhodopseudomonas palustris ).

5. The method for preparing the microecological hydrogel loaded with photosynthetic bacteria and Chlorella according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: mixing photosynthetic bacteria, chlorella and methacrylated gelatin solution, and performing photocuring to obtain a microecological hydrogel loaded with photosynthetic bacteria and chlorella.

6. The method for preparing the microecological hydrogel loaded with photosynthetic bacteria and Chlorella as claimed in claim 5, characterized in that: The preparation method of the methacrylic gelatin solution comprises: dissolving the methacrylic gelatin in a photoinitiator solution at 45° C. to 55° C., and mixing the mixture evenly to obtain the methacrylic gelatin solution.

7. The method for preparing the microecological hydrogel loaded with photosynthetic bacteria and Chlorella as claimed in claim 4, characterized in that: The photoinitiator comprises phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite; and / or The concentration of the photoinitiator solution is 0.23% (w / v) to 0.27% (w / v); and / or The mass concentration of methacryloyl gelatin in the methacryloyl gelatin solution is 9% to 11%.

8. Use of the microecological hydrogel loaded with photosynthetic bacteria and Chlorella as claimed in any one of claims 1 to 4 in the preparation of wound healing dressings.

9. The use according to claim 8, characterized in that: The wound comprises a diabetic wound.

10. A method for using the microecological hydrogel loaded with photosynthetic bacteria and Chlorella as claimed in any one of claims 1 to 4, characterized in that: The microecological hydrogel loaded with photosynthetic bacteria and Chlorella is attached to the wound, and near-infrared light with a wavelength of 800nm-810nm is used to irradiate the microecological hydrogel loaded with photosynthetic bacteria and Chlorella for 7min-10min at night.

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