Chlorella-loaded silk protein microneedle patch, preparation and application of chlorella-loaded silk protein microneedle patch in promoting healing of diabetic wounds

Abstract

The invention discloses a silk protein microneedle patch loaded with chlorella, preparation and application of the silk protein microneedle patch in promoting healing of diabetic wounds, and belongs to the field of medicines. The microneedle patch is a microneedle array formed by a plurality of microneedles arranged on one surface of the base; the base and the microneedles are made of silk protein materials; active chlorella is loaded on the tip part of the silk protein microneedle. The preparation method comprises the following steps: mixing chlorella with a fibroin solution, injecting the mixture into the microneedle patch, carrying out vacuum suction filtration, injecting the fibroin solution to fill the base, carrying out suction filtration, and drying. The oxygen generated by the efficient photosynthesis of the chlorella is used for continuously conveying local dissolved oxygen to the wound of the diabetic patient, and the oxygen can promote tissue regeneration and vascular repair. Meanwhile, the silk fibroin is used as a carrier for embedding the chlorella, so that the activity of the chlorella is protected in the environment, the oxygen production capacity of the chlorella is ensured, and the storage time of the active chlorella in the microneedle is prolonged. Therefore, the problems that diabetic chronic wounds are difficult to heal and active materials are difficult to store are solved.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to an oxygen-producing silk fibroin microneedle patch coated with active Chlorella vulgaris, its preparation and its application in promoting the healing of diabetic wounds. Technical Background

[0002] Diabetes is one of the most common diseases that induce chronic wounds, and up to 25% of diabetic patients are at risk of lifelong chronic wound non-healing or even non-healing. Diabetic patients are in a state of hyperglycemia for a long time, and hyperglycemia-induced hypoxia can damage angiogenesis, which is the main cause of chronic wounds in diabetic patients. High blood sugar levels interfere with the function of hypoxia-inducible factor-1α (HIF-1α) by damaging post-translational rapid hydroxylation and transactivation. As a result, it is unable to upregulate vascular endothelial growth factor, and the process of generating new blood vessels at the wound site is relatively slow, and the new blood vessels are damaged. Therefore, alleviating the hypoxic environment of chronic wounds is an important strategy for treating diabetic wound healing. At present, oxygen delivery and in-situ oxygen generation are common strategies to provide sufficient oxygen to relieve hypoxia. However, traditional oxygen delivery methods are not easy to maintain or deliver sufficient oxygen to the wound. For example, hyperbaric oxygen delivery (HBO2) does not allow self-renewing oxygen generation, cannot generate oxygen for specific sites, and there is a risk of immune rejection with calcium peroxide oxygen-generating particles. When using local gaseous oxygen therapy, the penetration ability of external gas to tissues is limited, and its effect in penetrating the skin is poor compared with local dissolved oxygen, which limits its application in wound healing.

[0003] In recent years, there has been an increasing interest in using photosynthesis as an alternative source of oxygen for tissue regeneration. This therapeutic strategy involves harnessing the natural oxygen-producing ability of live algae to increase oxygen levels and balance the oxygen gradient within larger wound structures. In the present invention, Chlorella vulgaris is a single-celled alga of the family Chlorellaceae, which is a microorganism existing in fresh water, salt water, and soil. Chlorella vulgaris contains a large amount of chlorophyll, can absorb light in a relatively wide wavelength range, and conducts photosynthesis within a certain wavelength range, producing a large amount of dissolved oxygen to alleviate the severe hypoxia problem of chronic wounds. In addition to its photosynthetic ability, Chlorella vulgaris also has the ability to solidify glucose at the wound site, which can serve as a nutrient for ischemic tissues and exhibits anti-inflammatory properties. However, the therapeutic effects of local administration of live algae are usually limited by some challenges. The lack of a protective carrier for live algae will cause the loss of the activity of live algae during storage and use, thus affecting oxygen production. Therefore, a material with relatively high biocompatibility is needed as a carrier to protect Chlorella vulgaris. Silk is a high-quality natural fiber obtained from silkworm cocoons, mainly composed of silk fibroin (SF, or silk protein) and sericin. SF can self-assemble into larger fiber structures through recombinant weak hydrogen bonds. This self-assembly of intramolecular fibrin leads to strong physical interactions, thus endowing SF with good mechanical properties. In addition to its superior mechanical properties, SF has been proven to have a high degree of biocompatibility both in vitro and in vivo. We used silk fibroin to encapsulate Chlorella vulgaris to prepare micro-needles in the form of micro-needles. The Chlorella vulgaris micro-needles have good solubility and biocompatibility. Most importantly, the protective effect of silk protein on Chlorella vulgaris can increase the storage time of Chlorella vulgaris in the micro-needles, maintain the activity of Chlorella vulgaris, and improve the application value of Chlorella vulgaris. At the same time, when the Chlorella vulgaris micro-needles are administered at the wound site, the micro-needles quickly dissolve under the action of body fluids to release Chlorella vulgaris. Under light, Chlorella vulgaris conducts photosynthesis to release oxygen, improving the hypoxic environment at the wound site, thereby promoting wound healing.

[0004] Before the present invention, a Chinese invention patent with the application number CN202210654296.3 in the prior art disclosed a separable GelMA micro-needle loaded with active microalgae for treating diabetic wound healing. However, the oxygen-producing ability of the microalgae in the micro-needle patch decreased significantly within 7 days, and the activity of the microalgae could not be guaranteed for a long time. The storage period of the microalgae in the GelMA micro-needles was short, lacking practical medical application value, which was also a key factor restricting its application. Secondly, after the tip of the micro-needle inhaled the Chlorella vulgaris solution, it needed to be cured by ultraviolet lamp irradiation first, and then PVA solution was added as the base of the micro-needle and dried. This process was cumbersome and not conducive to large-scale production.

[0005] For another example, a Chinese invention patent with the application number CN201711299062.7 discloses a preparation method of silk fibroin microneedles. In this invention, glutaraldehyde, vanillin and other aldehyde substances are introduced as chemical cross-linking agents, and the microneedles are prepared by high-temperature treatment with water vapor. This microneedle does not consider the problem of loading active organisms. On the one hand, the introduction of chemical reagents is toxic to loading active organisms. On the other hand, high-temperature treatment with water vapor will cause the inactivation of active organisms, resulting in the inability of this preparation method to load active substances.

[0006] Therefore, there is an urgent need for a way to solve the key problem of long-term preservation of active microalgae. Summary of the Invention

[0007] The purpose of the present invention is to provide an oxygen-producing silk fibroin microneedle patch coated with Chlorella vulgaris, which uses the efficient photosynthesis of Chlorella vulgaris to produce oxygen and continuously deliver locally dissolved oxygen to the wounds of diabetic patients. Oxygen can promote tissue regeneration and blood vessel repair. At the same time, using silk fibroin as a carrier for embedding Chlorella vulgaris can protect the activity of Chlorella vulgaris in the environment, increase the storage time of Chlorella vulgaris, and ensure the oxygen-producing ability of Chlorella vulgaris. Thus, the problems of difficult healing of diabetic chronic wounds and difficult storage of active materials are solved, and its practical application value is indeed improved.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] A silk fibroin microneedle patch loaded with Chlorella vulgaris, characterized in that the microneedle patch is a microneedle array composed of a plurality of microneedles provided on one side of a base; both the base and the microneedles are made of pure silk fibroin material; active Chlorella vulgaris is loaded at the tip of the silk fibroin microneedle.

[0010] The area of the microneedle patch is 1 cm 2 -4 cm 2 , and it is composed of an array of 100 - 400 microneedles (such as a 10×10 - 20×20 array), preferably a 10×10 array. The height of each microneedle is 1000 - 1200 μm, preferably 1200 μm, the needle spacing is 800 - 1000 μm, preferably 1000 μm. The connection between the needle body and the back lining is a square base, and the height of the active Chlorella vulgaris loaded at the tip of the microneedle is 700 - 900 μm, preferably 900 μm.

[0011] The active Chlorella vulgaris is mixed with a silk fibroin solution and then embedded in the tip of the silk fibroin microneedle.

[0012] The preparation method of the silk fibroin microneedle patch loaded with Chlorella vulgaris is characterized by including the following steps:

[0013] Step 1) Cut the silkworm cocoons, remove the silkworm pupae, add the silkworm cocoons to the boiling sodium carbonate solution, tear the silkworm cocoons into flocs, steam and degum them, then wash them, and air-dry the cleaned silk at room temperature for later use;

[0014] Step 2) Add the silk to the preheated lithium bromide solution and heat to dissolve it, then transfer it to a dialysis bag for dialysis. After dialysis is completed, centrifuge to remove impurities to obtain a silk protein solution;

[0015] Step 3) Centrifuge and wash Chlorella vulgaris with PBS. After washing the Chlorella vulgaris clean, mix it with the silk protein solution to obtain a Chlorella vulgaris and silk protein mixed solution. Load the mixed solution into the microneedle patch mold in batches and perform vacuum filtration to make the silk protein solution wrapping the Chlorella vulgaris fill the corresponding needle tip part of the mold;

[0016] Step 4) Drop the silk protein solution into the microneedle mold obtained in Step 3) to make the silk protein solution fill the entire mold and further remove the solvent, and dry it at a certain temperature to obtain microneedles;

[0017] Step 5) Demold the microneedles obtained in Step 4) and store them in the refrigerator.

[0018] According to the above preparation method, the specific parameter settings in the preparation are as follows:

[0019] In the above Step 1), the steaming time of the silkworm cocoons is 30 min.

[0020] In the above Step 2), a 9.3 M lithium bromide solution is preferably used, and constant temperature heating is carried out at 60 °C, such as constant temperature heating in an oven at 60 °C;

[0021] In the above Step 2), it is preferably that 20 mL of 9.3 M lithium bromide solution is used for every 5 g of silk, dissolved at 60 °C, and the silk is stirred every 1 h for a total dissolution time of 4 h;

[0022] In the above Step 2), the molecular weight cut-off of the dialysis bag is 3000 Da, the dialysis time is 55 h - 60 h, and the centrifugation conditions after dialysis are a rotation speed of 8000 rpm, a centrifugation temperature of 8 °C, and a centrifugation time of 10 min.

[0023] In the above Step 3), the centrifugation conditions for washing Chlorella vulgaris are a rotation speed of 4500 rpm and a centrifugation temperature of 10 - 20 °C;

[0024] In the above Step 3), the concentration of Chlorella vulgaris in the Chlorella vulgaris and silk protein mixed solution is 5×10 7 -5×10 8 cell / mL, preferably 5×10 8 cell / mL, and the mass percentage concentration of the used silk protein solution is 6%;

[0025] In step 3), the Chlorella vulgaris and silk fibroin mixed solution are co-loaded into the microneedle mold in multiple times, and then suction filtration is carried out to obtain the corresponding needle tip part containing Chlorella vulgaris. For example, 100 μL of the mixed solution loaded into the microneedle mold is loaded in five times, 20 μL each time, and the total suction filtration time is 100 min - 120 min.

[0026] In step 4) above, the mass percentage concentration of the silk fibroin solution used is 6%, the solvent is water, the drying temperature of the obtained microneedles is 15 - 20 °C, and the drying time is 12 - 18 h.

[0027] In step 5) above, the storage temperature of the microneedle patch in the refrigerator is 4 °C.

[0028] The technical solution proposed by the present invention has the following advantages:

[0029] (1) A silk fibroin microneedle patch loaded with Chlorella vulgaris provided by the present invention is prepared by the vacuum suction filtration method. The preparation method is simple, and Chlorella vulgaris highly aggregates at the tip of the microneedles, making the treatment process efficient.

[0030] (2) A silk fibroin microneedle patch loaded with Chlorella vulgaris provided by the present invention. Chlorella vulgaris has rich sources, is cheap and easily available, has good biocompatibility and degradability, and is an ideal oxygen producer.

[0031] (3) A silk fibroin microneedle patch loaded with Chlorella vulgaris provided by the present invention. Silk fibroin, as a carrier for encapsulating Chlorella vulgaris, can protect the activity of microalgae in the environment, enhance the storage capacity of active microalgae, and solve the problem of difficult storage of active materials.

[0032] (4) A silk fibroin microneedle patch loaded with Chlorella vulgaris provided by the present invention has excellent dissolution properties and mechanical properties, can easily pierce the skin of mice, can effectively deliver Chlorella vulgaris to the deep skin and release it, and continuously deliver dissolved oxygen through light irradiation, effectively improving the hypoxic environment of the wound. Description of the Drawings

[0033] Figure 1 It is a physical picture of free Chlorella vulgaris in BG-11 medium.

[0034] Figure 2 It is a growth curve of free Chlorella vulgaris.

[0035] Figure 3 It is a preparation flow chart of Chlorella vulgaris microneedles.

[0036] Figure 4 It is a physical picture of Chlorella vulgaris microneedles.

[0037] Figure 5 It is a scanning electron microscope picture of Chlorella vulgaris microneedles.

[0038] Figure 6 It is a 3D fluorescence image of the tip part of the Chlorella vulgaris microneedle.

[0039] Figure 7 It is a graph of the mechanical property data of the pure silk fibroin microneedle and the Chlorella vulgaris microneedle.

[0040] Figure 8 It is a graph of the needle hole recovery after the Chlorella vulgaris microneedle punctures the mouse skin.

[0041] Figure 9 It is a dissolution visualization graph of the pure silk fibroin microneedle and the Chlorella vulgaris microneedle.

[0042] Figure 10 It is a graph of the experimental results of the in vitro dissolution and release of the Chlorella vulgaris microneedle.

[0043] Figure 11 It is a graph of the oxygen production data of the Chlorella vulgaris microneedle at different concentrations.

[0044] Figure 12 It is a graph of the oxygen production data of the Chlorella vulgaris microneedle at different glucose concentrations.

[0045] Figure 13 It is a graph of the oxygen production data of the Chlorella vulgaris microneedle and free Chlorella vulgaris at different storage times.

[0046] Figure 14 It is a graph of the viability analysis of the Chlorella vulgaris microneedle and free Chlorella vulgaris at different storage times.

[0047] Figure 15 It is a graph of the wound healing and wound area histogram of different groups in the mouse diabetes model.

[0048] Figure 16 It is a graph of the H&E and Masson staining of the skin at the wound of different groups in the mouse diabetes model.

[0049] Figure 17 It is a graph of the analysis of the epidermal thickness of the skin of different groups in the mouse diabetes model.

[0050] Figure 18 It is a graph of the analysis of the collagen deposition of the skin of different groups in the mouse diabetes model.

[0051] Figure 19 It is a graph of the immunofluorescence staining of the skin of different groups in the mouse diabetes model. Specific implementation mode

[0052] The following will be described in detail through specific implementation cases and the accompanying drawings of the specification, but the present invention is not limited to the following embodiments.

[0053] Preparation of silk fibroin solution

[0054] (1) Degumming of silk. Remove the silkworm pupae from the cocoons for later use. Heat 4 L of water to boiling, dissolve 8.48 g of anhydrous sodium carbonate, and wait for the anhydrous sodium carbonate to dissolve completely. Then add 10 g of processed cocoons, and tear the cocoons into flocs with tweezers. Boil for 30 min. After cooking, rinse the silk with running water repeatedly to make the sericin on the silk surface fall off completely. Soak the degummed silk in deionized water and change the water every 2 h. After 4 h, wring out the water from the washed degummed silk and let it air-dry naturally at room temperature.

[0055] (2) Dissolving silk in lithium bromide. Slowly add 155 g of lithium bromide to 128 mL of water, stir and release heat to prepare a 9.3 mol / L lithium bromide solution. Measure 20 mL of the 9.3 mol / L lithium bromide solution into a beaker, preheat it to 60 °C in a constant temperature drying oven, add 5 g of degummed silk, stir well to dissolve, seal it with tin foil, and heat and dissolve at 60 °C for 4 h, stirring once every 1 h.

[0056] (3) Dialysis. Pour the fully dissolved silk protein solution into a 3000 Da dialysis bag, clamp both ends in a cross shape with a sealing clip and fix it with a rubber band. Place the dialysis bag in a beaker containing 4000 mL of deionized water and carry out dialysis on a magnetic stirrer. Change the water every 2 h for the first 6 h, and then change the water every 12 h. Dialyze for a total of 60 h.

[0057] (4) Centrifugation to remove impurities. Transfer the dialyzed silk protein solution to a 50 mL centrifuge tube and centrifuge at 8000 rpm and 8 °C for 10 min. After centrifugation, take the supernatant to remove impurities and repeat 2 - 3 times.

[0058] (5) Determining the concentration of the silk protein solution. Take 3 1.5 mL centrifuge tubes, weigh the mass of the empty tube X1 with an analytical balance. Add 100 μL of the impurity-removed silk protein solution to the bottom of the centrifuge tube and weigh its mass X2. Place the centrifuge tube in a constant temperature mixer and dry it (100 °C, 300 rpm, 90 min), cool to room temperature and then weigh the mass X3. Calculate the concentration of the silk protein solution according to the following formula.

[0059] SF%=(X3 - X1) / (X2 - X1)×100%

[0060] Cultivation of Chlorella and determination of its growth curve:

[0061] Chlorella FACHB - 482 (10 6 cells / mL) was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences. As Figure 1As shown, Chlorella vulgaris was cultured in BG-11 medium. Weigh 1.7 g of BG-11 and dissolve it in 1000 mL of deionized water. Pour 150 mL into each conical flask, seal it with a sealing film, and sterilize it at 121 °C for 15 min. Inoculate Chlorella vulgaris into the BG-11 medium at an inoculation amount of 20%. The inoculated Chlorella vulgaris medium was cultured in a light incubator under light conditions of an intensity of 2000 Lux, a light-dark ratio of 12 h / 12 h, and 25 °C. Every month, Chlorella vulgaris was subcultured in the BG-11 medium at the same inoculation amount (20%).

[0062] Take 4 mL of Chlorella vulgaris and perform serial dilutions by a 2-fold gradient to prepare 6 different concentration gradients. Use a UV spectrophotometer to measure the absorbance value of each gradient sample at a wavelength of 680 nm. At the same time, use a hemocytometer to measure the algal cell density of each gradient sample under a microscope. Plot the relationship curve between the Chlorella vulgaris density and the absorbance, with the absorbance value as the abscissa and the algal cell density as the ordinate. The results are shown Figure 2 . In the following experiment, this curve will be used to calculate the specific density of Chlorella vulgaris.

[0063] The preparation of silk fibroin microneedles loaded with Chlorella vulgaris is as Figure 3 shown:[[]]END]]

[0064] (1) The Chlorella vulgaris solution was first centrifuged to obtain a Chlorella vulgaris precipitate, resuspended and washed with PBS buffer solution, and then centrifuged again to obtain a precipitate. This washing process was repeated three times in total. The centrifugation conditions were 4500 rpm for 5 min. The Chlorella vulgaris was resuspended with a 6% silk fibroin solution to determine the concentration, and the concentrations of Chlorella vulgaris were 5×10 7 , 10 8 , 5×10 8 cells / mL.

[0065] (2) Cut off the tip part of the syringe needle and bend it to serve as a drug spreader. Turn on the vacuum filtration pump, drop 20 μL of the Chlorella vulgaris and silk fibroin mixed solution onto the microneedle mold, and use the drug spreader to evenly spread the solution over the mold needle holes. After the solution is completely filtered to the tip of the needle, drop 20 μL of the mixed solution onto the center of the mold again, and repeat the above steps until a total of 100 μL is dropped. Subsequently, slowly drop 500 μL of 6% silk fibroin solution from the periphery to the center of the mold, use a pipette gun to remove the air bubbles in the corners of the mold, and turn on the filtration pump to continue operating for 10 - 15 min.

[0066] (3) Then, dry the mold with the microneedles overnight (temperature maintained at 15 - 20 °C). After drying, peel the Chlorella vulgaris silk fibroin microneedles from the mold. The prepared Chlorella vulgaris microneedles are as Figure 4As shown, the microneedle consists of a sharp tip and a stable substrate part. Due to the loaded Chlorella, the tip part of the microneedle is dark green. The microneedles are in a 10×10 array, with a height of 1200 μm, a needle pitch of 1000 μm, and the connection between the needle body and the backing is a square base with a side length of 600 μm.

[0067] The morphology of the microneedles was characterized by scanning electron microscopy (SEM) and laser confocal microscopy. The complete Chlorella microneedles were fixed on the double-sided tape on the sample stage, sputter-coated with gold, and then subjected to SEM testing. The results are as Figure 5 shown. It was observed that the tips of 4 complete microneedles presented a standard quadrangular pyramid morphology, which enables the microneedles to better penetrate the skin and achieve the purpose of non-invasive drug delivery. The Chlorella microneedles were vertically fixed on a glass slide, and the 3D imaging function of the laser confocal microscope was used to observe the distribution of Chlorella in the microneedles. The excitation wavelength was set at 436 nm and the emission wavelength was set at 680 nm. The results are as Figure 6 shown. Chlorella was evenly distributed in the tip part of the microneedles at 0 - 800 μm from the tip, indicating that in the treatment, Chlorella can penetrate deep into the skin first for treatment.

[0068] The puncture performance of the microneedles was characterized by mechanical property measurement. The Chlorella filamentous protein microneedles and pure silk protein microneedles at three concentrations of 5×10 7 , 10 8 , 5×10 8 cells / mL were cut into a complete 3×3 array and flatly adhered to the sample stage of the universal testing machine to ensure that each microneedle could receive uniform test conditions. The compression mode of the universal testing machine was used to test the breaking force of the Chlorella microneedles, and the compression speed was 1 mm / min. The results are as Figure 7 shown. The breaking force of the pure silk protein microneedles at 300 μm was 2.25 N / needle, and the breaking forces of the Chlorella microneedles at 5×10 7 , 10 8 , 5×10 8 cells / mL concentrations at 300 μm were 1.82, 2.25, and 1.37 N / needle respectively. The breaking forces of the Chlorella microneedles were all greater than the reported minimum breaking force (0.058 N), indicating that the Chlorella microneedles not only have sufficient mechanical strength to easily penetrate the skin but are also more likely to maintain the integrity and stability of the structure in practical applications. In addition, a puncture experiment was carried out using mouse skin to simulate human skin. As Figure 8 shown, the needle holes left by the Chlorella microneedles on the back skin of the mice were evenly distributed, indicating the stability of the puncture performance of the Chlorella microneedles. The needle holes on the skin disappeared within 20 min, demonstrating the superiority of non-invasive drug delivery of the Chlorella microneedles.

[0069] To evaluate the ability of Chlorella vulgaris micro - needles to dissolve and release Chlorella vulgaris during actual application, 3% agarose gel was used to simulate human skin. The Chlorella vulgaris micro - needles were pricked on the agarose gel, and the changes in the tips of the Chlorella vulgaris micro - needles and the release of Chlorella vulgaris on the agarose gel were observed. As Figure 9 shown, the silk fibroin micro - needles can be completely dissolved in 30 s, and the Chlorella vulgaris micro - needles can be completely dissolved in 120 s. This indicates that the silk fibroin micro - needles have excellent dissolution and release properties. After loading Chlorella vulgaris, the micro - needles have a sustained - release ability, which can increase the retention time of Chlorella vulgaris in the skin. This characteristic makes the Chlorella vulgaris micro - needles have broad application prospects in the biomedical field and is expected to provide a more effective and convenient method for disease treatment.

[0070] To explore the solubility of the Chlorella vulgaris micro - needles, one Chlorella vulgaris micro - needle was placed in 1 mL of PBS solution. Every once in a while, 10 μL of the solution was taken, and the absorbance of the sample at 280 nm was measured using a NanoDrop micro - spectrophotometer. The results are as Figure 10 shown. From 0 - 11 min, the concentration of silk fibroin in the solution increased sharply; at 11 min, the concentration of silk fibroin in the solution was 61.5% of the total concentration, indicating that the Chlorella vulgaris micro - needles have good solubility.

[0071] The oxygen - producing ability of the micro - needles was characterized by a dissolved oxygen meter. The Chlorella vulgaris micro - needles have photosynthetic oxygen - producing performance. Measuring the dissolved oxygen content in the solution is an important means to characterize the oxygen - producing ability of CV - MN. 80 μL of 0.05 g / mL anhydrous sodium sulfite was added to 10 mL of PBS solution to prepare anaerobic water. 5×10 7 、10 8 、5×10 8 cells / mL of Chlorella vulgaris micro - needles were respectively dissolved in 10 mL of anaerobic water, placed in a light incubator with a light intensity of 2000 Lux and a temperature of 25 °C. The dissolved oxygen content in the solution was measured every 1 h using a dissolved oxygen meter, and each group was measured in parallel three times. The results are as Figure 11 shown. During the 5 - h detection, the oxygen content in the solution increased significantly. For the Chlorella vulgaris micro - needles with 5×10 8 cells / mL, the oxygen content increased slowly within 1 h and then at a higher rate after 1 h. This result shows that the Chlorella vulgaris micro - needles with a high concentration not only have a high oxygen - producing ability but also have a high adaptability to the environment, and can quickly recover the oxygen - producing ability and maintain a high oxygen - producing rate after release.

[0072] To explore the influence of the high - glucose environment at the wound on the oxygen - producing ability of Chlorella vulgaris, the oxygen - producing abilities of 5×10 8 cells / mL of Chlorella vulgaris micro - needles were measured at glucose concentrations of 0, 25, 250, and 500 μM respectively. The results are as Figure 12 shown. In the high - glucose environment, for 5×10 8The oxygen production of Chlorella micro needles at cells / mL was significantly increased, and the higher the glucose concentration, the higher the oxygen production. This result indicates that the high sugar environment at the wound promotes the oxygen production ability of Chlorella. While using glucose to enhance its oxygen production ability, Chlorella also reduces the glucose concentration at the wound, thus reducing the inhibitory effect of the high sugar environment on wound healing.

[0073] To explore the change in the oxygen production ability of Chlorella micro needles after storage for a period of time, the Chlorella micro needles were stored for three months, and the oxygen production ability of the Chlorella micro needles was measured once a month. Similarly, the oxygen production ability of an equal amount of free Chlorella was measured under the same conditions. The results are as Figure 13 shown. The oxygen production of free Chlorella decreased sharply in the first month, and the downward trend was more obvious. This indicates that silk fibroin ensures the stability of the activity of Chlorella in the Chlorella micro needles and also ensures its oxygen production ability.

[0074] The key to the function of Chlorella micro needles is to ensure the activity of Chlorella. Therefore, the change in the activity of Chlorella in the Chlorella micro needles was mainly detected. Chlorella micro needles with a concentration of 5×10 8 cells / mL were prepared and stored at 4°C for three months. During the storage process, one micro needle was taken out every month, dissolved in 1 mL of PBS solution, centrifuged at 8000 rpm for 5 min, and the supernatant was removed. 1.75 mL of 90% methanol was added, and the mixture was heated in a water bath at 60°C in the dark for 2 h. After cooling to room temperature, it was centrifuged at 8000 rpm for 5 min, and the supernatant contained all the chlorophyll of Chlorella. The absorbance of the supernatant at 435 nm was measured, and three parallels were set for each group. The absorbance value of the Chlorella micro needles in the 0th month was recorded as 100% of the activity of Chlorella in the Chlorella micro needles, and the activities of the remaining Chlorella micro needles were converted according to this value. The results are as Figure 14 shown. The activity of Chlorella in the micro needles was basically stable in the first two months (completely reaching 90%, or even higher than 90%). When free Chlorella was stored under the same conditions, its activity showed an obvious downward trend, indicating that silk fibroin prepared in the form of micro needles has a protective effect on the activity of Chlorella.

[0075] To verify the therapeutic ability of Chlorella microneedles on wounds in a diabetes model, type I diabetes was induced by intraperitoneal injection of STZ at a dose of 150 mg / kg. Four hours after injection, the mice were supplemented with food and glucose water, and the mouse feed and deionized water were changed every other day. After two weeks, the blood glucose levels of the mice were measured, and those exceeding 16.7 mM were considered successful in inducing type I diabetes, and subsequent experiments were carried out. After the diabetes model was completed, the mice were randomly divided into four groups: the PBS group, the MN group, the MA group, and the MAL group. The PBS group was added with PBS at the wound site, the MN group used blank silk fibroin microneedles to treat the wound, the MA group used Chlorella microneedles to treat the wound, and the MAL group irradiated the wound with 660 nm light for 2 h after inserting the Chlorella microneedles into the wound. Treatment was performed once every 3 days, and the wound healing situation was recorded. The results are as Figure 15 shown. On the 12th day, the wounds in the MAL group had almost completely healed, the wound surface was covered by regenerated skin tissue, and the wound area was only 4.4%. In contrast, the wound areas of the other groups were still larger, and the open wounds were more obvious.

[0076] The therapeutic effect of Chlorella microneedles on wounds in the diabetes model was evaluated by H&E staining, Masson staining, and immunofluorescence staining. After the wounds in the MAL group had basically healed on the 12th day, the mice were anesthetized and sacrificed with isoflurane. The skin at the wound site was removed, embedded, sectioned, and subjected to H&E staining, Masson staining, HIF-1α immunofluorescence staining, and CD31 immunofluorescence staining. The results are as Figure 16 、 17 、18, and 19 shown. The MAL group had fewer inflammatory factors, significantly increased collagen fibers, and relatively regular arrangements, indicating that tissue repair and reconstruction were relatively successful. The expression of HIF-1α in the MAL group was the lowest, indicating that the MAL group might significantly improve the hypoxic state and contribute to rapid wound healing. The CD31 signal in the MAL group was the strongest, showing the highest level of angiogenesis. It was shown that MAL treatment had the best effect on angiogenesis and contributed to accelerated healing. In summary, the MAL group had the best effect on promoting the healing of wounds in the diabetes model.

Claims

1. A fibroin microneedle patch loaded with Chlorella vulgaris, characterized in that, The microneedle patch has a microneedle array composed of multiple microneedles on one side of the base; both the base and the microneedles are made of silk fibroin; active chlorella is loaded at the tip of the silk fibroin microneedles.

2. The silk fibroin microneedle patch according to claim 1, wherein The active chlorella is mixed with the silk fibroin solution and then embedded in the tips of the silk fibroin microneedles.

3. The silk fibroin microneedle patch according to claim 1 or 2, characterized in that, The area of the described microneedle patch is 1 cm 2 -4 cm 2 , which consists of an array of 100 - 400 microneedles (such as a 10×10 - 20×20 array), preferably a 10×10 array. The height of each microneedle is 1000 - 1200 μm, preferably 1200 μm, the needle pitch is 800 - 1000 μm, preferably 1000 μm. The connection between the needle body and the backing is a square base, and the height of the active Chlorella vulgaris loaded at the tip of the microneedle is 700 - 900 μm, preferably 900 μm.

4. The preparation method of the silk fibroin microneedle patch according to any one of claims 1-3, characterized in that, It includes the following steps: Step 1) Cut the silkworm cocoon, remove the silkworm pupa, add the silkworm cocoon to the boiling sodium carbonate solution, tear the silkworm cocoon into flocs, wash it after degumming by steaming, and air-dry the cleaned silk at room temperature for later use; Step 2) Add the silk to the preheated lithium bromide solution and heat to dissolve it, then transfer it to a dialysis bag for dialysis. After dialysis is completed, centrifuge to remove impurities to obtain a silk fibroin solution; Step 3) Centrifuge and wash the chlorella with PBS. The chlorella-silk fibroin mixed solution obtained by mixing the cleaned chlorella with the silk fibroin solution is loaded into the microneedle patch mold in batches, and vacuum filtration is carried out to make the silk fibroin solution containing chlorella fill the corresponding needle tip part of the mold; Step 4) Drop the silk fibroin solution into the microneedle mold obtained in Step 3) to make the silk fibroin solution fill the entire mold and further remove the solvent, and dry it at a certain temperature to obtain microneedles; Step 5) Demold the microneedles obtained in Step 4) and store them in the refrigerator.

5. The method according to claim 4, characterized in that, In the above Step 1), the steaming time of the silkworm cocoon is 30 min.

6. The method according to claim 4, characterized in that, In the above Step 2), it is preferably that 20 mL of 9.3 M lithium bromide solution is used for every 5 g of silk, dissolved at 60 °C, and the silk is stirred every 1 h for a total dissolution time of 4 h; In the above Step 2), the molecular weight cut-off of the dialysis bag is 3000 Da, the dialysis time is 55 h - 60 h, and the centrifugation conditions after dialysis are a rotation speed of 8000 rpm, a centrifugation temperature of 8 °C, and a centrifugation time of 10 min.

7. The method according to claim 4, characterized in that In the above Step 3), the centrifugation conditions for cleaning the chlorella are a rotation speed of 4500 rpm and a centrifugation temperature of 10 - 20 °C; In the above step 3), the concentration of Chlorella in the Chlorella and silk fibroin mixed solution is 5×10 7 -5×10 8 cell / mL, preferably 5×10 8 cell / mL, and the mass percentage concentration of the silk fibroin solution used is 6%; In Step 3), the chlorella-silk fibroin mixed solution is loaded into the microneedle mold in multiple times, and then vacuum filtration is carried out to obtain the corresponding needle tip part containing chlorella. For example, 100 μL of the mixed solution loaded into the microneedle mold is loaded in five times, 20 μL each time, and the total vacuum filtration time is 100 min - 120 min.

8. The method according to claim 4, characterized in that, In the above Step 4), the mass percentage concentration of the silk fibroin solution used is 6%, the solvent is water, the drying temperature of the obtained microneedles is 15 - 20 °C, and the drying time is 12 - 18 h.

9. The method according to claim 4, characterized in that, In the above Step 5), the storage temperature of the microneedle patch in the refrigerator is 4 °C, and the activity can remain basically unchanged for up to 2 months.

10. The application of the silk fibroin microneedle patch according to any one of claims 1 - 3, as an external patch for promoting the healing of diabetic wounds.

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