Bio-oxygen therapy and anti-oxidation dual-strategy soluble microneedle as well as preparation method and application thereof

Abstract

The invention relates to a biological oxygen therapy and antioxidation dual-strategy soluble microneedle as well as a preparation method and application thereof, and belongs to the technical field of medicines. The microneedle is an integrated soluble microneedle and comprises a matrix formed by a water-soluble polymer and a compound which is uniformly dispersed in the matrix and formed by microalgae and metal organic framework nano-enzyme, the compound formed by the microalgae and the metal organic framework nano-enzyme is a compound of a'core-shell 'structure, the metal organic framework nano-enzyme serving as the shell is wrapped on the surface of the microalgae serving as the core. The microneedle disclosed by the invention has the capability of rapidly resisting oxidation in situ and continuously and deeply conveying oxygen, is widely applied to preparation of medicine design or preparations for treating tumors, promoting wound healing, regulating an inflammation microenvironment, treating skin burns and scalds and removing scars, and is good in patient applicability; and the preparation process is green, environment-friendly, simple, rapid, mild in condition and suitable for large-scale production.

Description

Technical Field

[0001] The invention relates to a biological oxygen therapy and anti-oxidation dual-strategy soluble microneedle and a preparation method and application thereof, belonging to the technical field of medicine. Background Art

[0002] Oxidative stress is caused by an imbalance between the generation of oxidants (free radicals and reactive oxygen species) and the elimination of antioxidants. Oxidative stress activates a series of proteolytic enzymes in cells, which in turn decompose the cell structure, ultimately leading to cell apoptosis, and these apoptotic cells or their products can also activate inflammatory cells. Oxidative stress and inflammation interact by regulating transcription levels, forming a vicious cycle. Oxidative stress is a concomitant phenomenon in the inflammatory process. Oxidative stress aggravates the inflammatory response, and inflammation promotes further oxidative stress through inflammatory mediators. Immune cell infiltration, inflammatory factor retention, and increased reactive oxygen content can cause microenvironmental hypoxia and mitochondrial damage at the site of inflammation, further aggravating oxidative damage and further accelerating the development of inflammation. Under continuous malignant environmental stress, reactive oxygen species will be generated and accumulated for a long time, causing serious damage to cell structure and function, and may induce somatic cell mutations and tumorigenic transformation.

[0003] Gas therapy is a treatment mode based on special gas signal molecules (such as hydrogen sulfide, nitric oxide, carbon monoxide, hydrogen and oxygen, etc.) through synergistic enhancement or direct regulation of the pathological microenvironment. It has the advantages of high efficiency, safety and no toxic side effects. The reduction of oxygen content is a common pathological feature of the microenvironment at the site of inflammation. Oxygen therapy has been widely used in a variety of clinical diseases such as tumor treatment, wound healing and skin disease recovery.

[0004] Chinese patent CN114557954A discloses soluble microneedles, microneedle patches, preparation methods and applications containing active microalgae, which utilize the characteristics of continuous oxygen production in situ by microalgae to continuously transport oxygen, and are prepared into soluble microneedle preparations for use in the preparation of drugs or reagents for improving tumor hypoxia, promoting wound healing, skin burn recovery, dental treatment or improving anemic microenvironment. This invention utilizes gas therapy to produce oxygen from microalgae, does not involve the field of regulating oxidative stress and anti-oxidation, and does not reflect the potential of the invention for application in the treatment of inflammation. Chinese patent CN115444929A discloses a transdermal double-layer microneedle based on hydrogen-producing probiotics, its preparation method and application, which utilizes the characteristics of probiotics to produce reducing hydrogen to treat inflammatory skin diseases. This invention demonstrates the important role of hydrogen in regulating oxidative stress, but the treatment mechanism of hydrogen therapy is different from that of oxygen therapy. Chinese patent CN117122554A discloses the preparation of a redox enzyme active drug carrier and its application in arthritis. The carrier itself has catalase / peroxidase activity, which can decompose hydrogen peroxide into oxygen and hydroxyl free radicals, achieve antioxidant effects, promote macrophage polarization to M2 type and promote the production of anti-inflammatory factors, while reducing the expression of pro-inflammatory factors such as iNOS and IL-1β, and has significant anti-arthritis effects in the AIA mouse model.

[0005] At present, the research on the combined application of biological oxygen therapy and antioxidants is still relatively limited, and the traditional local delivery of dissolved oxygen faces problems such as small delivery volume, difficulty in continuous delivery, and insufficient delivery depth. Therefore, a delivery system that can simultaneously ensure delivery efficiency, therapeutic effect, and patient safety for the above two problems still needs to be developed. Summary of the invention

[0006] In order to solve the technical defects of the prior art, the present invention provides a biological oxygen therapy and antioxidant dual strategy soluble microneedle and its preparation method and application. The present invention loads a complex with a "core-shell" structure formed by a metal organic framework nanozyme with antioxidant effect and a microorganism microalgae that can produce oxygen into a water-soluble polymer matrix to prepare a soluble microneedle that integrates oxygen therapy and antioxidant therapy, and applies it to the treatment process. The biological oxygen therapy and antioxidant dual strategy microneedle designed by the present invention has significant efficacy, simple preparation, wide application, and is suitable for large-scale production and application.

[0007] One of the purposes of the present invention is to provide a soluble microneedle with a dual strategy of biological oxygen therapy and antioxidant, wherein the microneedle is an integrated soluble microneedle, comprising a matrix formed by a water-soluble polymer and a complex formed by microalgae and metal organic framework nanozymes uniformly dispersed in the matrix, wherein:

[0008] The complex formed by the microalgae and the metal organic framework nanozyme is a "core-shell" structure complex, in which the metal organic framework nanozyme as the outer shell is wrapped on the surface of the microalgae as the inner core.

[0009] The soluble microneedles of the present invention, wherein the microalgae is a type of oxygen-producing microorganism, functions to produce oxygen under light conditions, thereby achieving the purpose of deep oxygen transport and alleviating hypoxia in the oxidative stress microenvironment.

[0010] The soluble microneedle described in the present invention, the metal organic framework nanozyme is a metal organic framework nanozyme with antioxidant activity, which first contacts the microenvironment with high reactive oxygen expression and exerts enzyme activity to remove reactive oxygen.

[0011] The soluble microneedles of the present invention integrate oxygen therapy and antioxidant therapy.

[0012] In the soluble microneedle of the present invention, the water-soluble polymer is one or more of hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, polylactic acid, carboxymethyl cellulose, amylose, chondroitin sulfate, sucrose and fructose.

[0013] Preferably, the water-soluble polymer is hyaluronic acid.

[0014] The soluble microneedles of the present invention, the preparation method of the complex formed by the microalgae and the metal organic framework nanozyme is: the microalgae are centrifuged and dispersed in a phosphate buffer solution to obtain a microalgae suspension, and then metal ions are added thereto, and finally an organic ligand is added under stirring conditions to obtain a solution system containing the microalgae-metal organic framework nanozyme complex.

[0015] In the above technical solution, the concentration of the microalgae is 10 5 ~10 9 Pieces / mL.

[0016] In the above technical solution, the concentration of the metal ions is 1 to 100 mg / mL.

[0017] In the above technical solution, the concentration of the organic ligand is 10 to 1000 mg / mL.

[0018] In the above technical solution, the microalgae are one or more of Cyanobacteria, Rhodophyta, Chlorophyta and Chrysophyta.

[0019] Preferably, the microalgae is a species within the Chlorophyta, more preferably Chlorella vulgaris within the Chlorophyta.

[0020] In the above technical solution, the metal ions are one or more of zinc, copper, iron, cobalt and nickel ions.

[0021] Preferably, the metal ions are iron ions.

[0022] Preferably, the organic ligand is potassium ferrocyanide.

[0023] In the above technical solution, the pH of the phosphate buffer solution during the preparation of the complex formed by the microalgae and the metal organic framework nanozyme is 7.4.

[0024] In the above technical solution, the centrifugation condition during the preparation process of the complex formed by the microalgae and the metal organic framework nanozyme is centrifugation at 1000-5000 rpm for 1-30 minutes.

[0025] In the above technical solution, the stirring condition during the preparation process of the complex formed by the microalgae and the metal organic framework nanozyme is stirring at 10 to 100 rpm for 10 to 60 minutes.

[0026] The soluble microneedle of the present invention is made of a microneedle mold PDMS, has a needle height of 400-1500 μm, a needle body diameter of 300-500 μm, a base diameter of 5-30 mm, a needle tip diameter of less than 10 μm, and an array number greater than 8×8.

[0027] The soluble microneedles described in the present invention, the outer "shell" structure of the complex formed by the microalgae and the metal organic framework nanozyme first contacts the microenvironment with high reactive oxygen expression, exerts enzyme activity to remove reactive oxygen, and the first step of degradation of the complex exposes the "core" structure of the microalgae. Under light conditions, the microalgae produces oxygen, achieving deep oxygen transport to alleviate hypoxia in the oxidative stress microenvironment.

[0028] Another object of the present invention is to provide a method for preparing soluble microneedles with a dual strategy of biological oxygen therapy and antioxidant, comprising the following steps: adding a water-soluble polymer to a solution system containing a microalgae-metal organic framework nanoenzyme complex, stirring and dissolving, and then in situ cross-linking at 37°C for 2 hours to obtain a pregel; pouring the pregel into a PDMS microneedle mold, centrifuging, drying, and then demolding to obtain a soluble microneedle.

[0029] In the method for preparing the soluble microneedle of the present invention, the mass percentage of the water-soluble polymer is 5% to 50%.

[0030] In the method for preparing the soluble microneedle of the present invention, the centrifugation condition is centrifugation at 1000-5000 rpm for 1-30 min.

[0031] In the method for preparing the soluble microneedles of the present invention, the drying condition is drying at room temperature for 6 to 48 hours.

[0032] Another object of the present invention is to provide the application of the above-mentioned biological oxygen therapy and antioxidant dual strategy soluble microneedles in the preparation of drug design or preparation for tumor treatment, promoting wound healing, regulating inflammatory microenvironment, treating skin burns and scalds, and removing scars.

[0033] Microalgae are a type of tiny single-cell algae that convert sunlight, water and carbon dioxide into energy and oxygen through photosynthesis. In this process, the oxygen produced by microalgae can often solve difficult problems in the treatment of many diseases. Microalgae can be delivered to hypoxic areas as a "natural oxygen generator" and continuously produce oxygen through external specific conditions to alleviate environmental hypoxia. Microalgae have been proven to have nutritional supplements, regulate bacterial metabolism, and have anti-inflammatory and anti-aging effects. They are often used to promote diabetic wound healing, treat inflammatory bowel disease, and resist radiation damage. Iron is abundant in nature and has low toxicity. Therefore, iron-based metal organic frameworks have diverse and high enzyme-like activities and good biosafety, making them functional materials with great application prospects.

[0034] Beneficial effects of the present invention:

[0035] 1. The complex formed by the microalgae and metal organic framework nanozymes prepared by the present invention protects the microalgae cells from the damage of highly expressed reactive oxygen species in the oxidative stress microenvironment, and can promote the generation and delivery of oxygen under light conditions, which helps to alleviate the hypoxic microenvironment of oxidative stress. The design of the present invention that wraps the metal organic framework nanozyme on the surface of the microalgae cells can solve the problem of unstable storage and easy aggregation and inactivation of conventional nanoparticles. The design of the complex of the present invention can improve the stability of microalgae and metal organic framework nanozymes without affecting the performance of the two monomers. The modification of the microalgae by the metal organic framework nanozyme does not reduce the photosynthetic oxygen production capacity of the microalgae, and the enzyme activity of the metal organic framework nanozyme is not reduced.

[0036] 2. The present invention utilizes microalgae to produce oxygen to alleviate environmental hypoxia, and utilizes the enzyme activity of metal organic frameworks to regulate oxidative stress to achieve the purpose of anti-oxidation. The two are made into a complex to combine the advantages of the two, and synergistically enhance the effect to achieve the therapeutic purpose of oxygen production and anti-oxidation regulation of oxidative stress.

[0037] 3. The present invention combines oxygen-producing microorganisms and microalgae with metal organic framework nanozymes with antioxidant properties to form a complex with both biological oxygen therapy and antioxidant functions, which is loaded in microneedles to achieve antioxidant therapy and oxygen therapy at the same time. The microneedle preparation process described in the present invention is green and environmentally friendly, simple and fast, and has mild conditions and is suitable for large-scale production. The microneedle patch described in the present invention has the ability of rapid in-situ antioxidant and continuous deep oxygen delivery, and is widely used in the preparation of drug design or preparations for treating tumors, promoting wound healing, regulating inflammatory microenvironment, treating skin burns and scar removal, and has good patient applicability. The dual-strategy microneedles designed by the present invention for biological oxygen therapy and antioxidant therapy have significant efficacy, simple preparation, wide application, and are suitable for large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 (A) is the preparation process of the pregel (Z-PBHA) loaded with the Chlorella-Prussian blue nanozyme complex and the microneedles loaded with the Chlorella-Prussian blue nanozyme complex obtained in Example 1; Figure 1 (B) is a scanning electron microscope image of Chlorella, with a scale of 5 μm; Figure 1 (C) is a scanning electron microscope image of a single Prussian blue nanozyme, with a scale of 200 nm; Figure 1 (D) is a scanning electron microscope image of the Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 1, with a scale bar of 5 μm; Figure 1 (E) is the Fourier transform infrared spectrum of the above substances.

[0039] Figure 2 (A) is the content of dissolved oxygen produced by naked Chlorella (CZ) and the Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 1 under normal culture conditions; Figure 2 (B) is the dissolved oxygen content produced by the Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 1, the Chlorella-Prussian blue nanozyme "1+1" mixture (Z+PB) obtained in Comparative Example 1, and naked Chlorella under a culture environment rich in hydroxyl radicals.

[0040] Figure 3 This is a color change diagram of the polymerization sedimentation stability test of the Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 2 and the Chlorella-Prussian blue nanozyme "1+1" mixture (Z+PB) obtained in Comparative Example 2 under the same high concentration conditions.

[0041] Figure 4 (A) is a mobile phone image of the soluble microneedles obtained in Example 1; Figure 4 (B) is an image of the microneedle obtained in Example 1 taken using an optical microscope; Figure 4(C) is an image of the microneedles obtained in Example 1 taken by scanning electron microscopy.

[0042] Figure 5 The dissolution ability of the soluble microneedles obtained in Example 1 in agarose gel (A), ex vivo pig skin (B), and in vivo mouse skin (C).

[0043] Figure 6 The healing time of the soluble microneedle channels obtained in Example 1 on in vitro pig skin (A) and in vivo mouse skin (B).

[0044] Figure 7 (A) is the experimental plan for in vivo treatment of the soluble microneedles obtained in Example 1; Figure 7 (B) is an image of the back of mice after 7 days of treatment; Figure 7 (C) is the weight change of mice from day 1 to day 7; Figure 7 (D) is the spleen index of mice in each group after 7 days of treatment; Figure 7 (E) PASI scores of each group of mice from day 1 to day 7; Figure 7 (F) is the skin lesion area score of each group of mice from day 1 to day 7; Figure 7 (G) The scores of the infiltration degree of mice in each group from day 1 to day 7; Figure 7 (H) Erythema scores of each group of mice from day 1 to day 7; Figure 7 (I) Scaling scores of each group of mice from day 1 to day 7.

[0045] Figure 8 This is the immunohistochemical analysis of the back skin of each group of mice during treatment with the soluble microneedles obtained in Example 1, wherein: Figure 8 (A) is the expression amount of Ki-67 in different experimental groups; Figure 8 (B) The expression amount of HIF-α in different experimental groups; Figure 8 (C) is the expression amount of IL-17A in different experimental groups; Figure 8 (D) is the expression amount of TNF-α in different experimental groups.

[0046] Fig. 9 Schematic diagram of the use of the soluble microneedles obtained in the present invention. DETAILED DESCRIPTION

[0047] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0048] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0049] Example 1

[0050] A preparation method of a biological oxygen therapy and antioxidant dual strategy soluble microneedle comprises the following steps:

[0051] After the Chlorella was centrifuged (3000rpm, 10min), it was redispersed with 10mL of pH=7.4 phosphate buffer to form a Chlorella suspension with an ultraviolet absorbance of 0.9 at 682nm. 337.6mg of potassium ferrocyanide (K4[Fe(CN)6]) was added to the algae solution and stirred to dissolve. 216mg of ferric chloride (FeCl3) was gradually added under stirring conditions of 40rpm and stirred for 60min to prepare a solution system containing Chlorella-Prussian blue nanoenzyme complex (Z-PB). 0.8g of hyaluronic acid was added to the system and stirred and dissolved, and then in situ cross-linked at 37℃ for 2h to obtain a pre-gel. The pre-gel was added to a PDMS microneedle mold with a needle length of 800μm, centrifuged at 2000rpm for 15min to promote the filling of the matrix into the needle tip. After the centrifugation was completed, the pre-gel was quantitatively supplemented and dried at room temperature for 24 hours. Finally, the microneedle preparation was demolded.

[0052] Example 2

[0053] A preparation method of a biological oxygen therapy and antioxidant dual strategy soluble microneedle comprises the following steps:

[0054] After the Chlorella was centrifuged (3000rpm, 10min), it was redispersed with 10mL of pH=7.4 phosphate buffer to form a Chlorella suspension with an ultraviolet absorbance of 1.5 at 682nm. 3376mg of potassium ferrocyanide was added to the algae solution and stirred to dissolve. 2160mg of ferric chloride was gradually added and stirred for 60min under stirring conditions of 40rpm to prepare a solution system containing Chlorella-Prussian blue nanoenzyme complex (Z-PB). 1.0g of hyaluronic acid was added to the system and stirred and dissolved, and then in situ cross-linked for 2h at 37°C to obtain a pre-gel. The pre-gel was added to a PDMS microneedle mold with a needle length of 800μm, centrifuged at 2000rpm for 15min to promote the filling of the matrix into the needle tip. After the centrifugation was completed, the pre-gel was quantitatively supplemented and dried at room temperature for 24 hours. Finally, the microneedle preparation was demolded.

[0055] Comparative Example 1

[0056] A preparation method of a microneedle loaded with a "1+1" mixture (Z+PB) of Chlorella-Prussian blue nanozyme comprises the following steps:

[0057] 216 mg of ferric chloride was added to 10 mL of phosphate buffer with a pH of 7.4, and 337.6 mg of potassium ferrocyanide was added under stirring at 40 rpm, and stirred for 60 min to obtain a phosphate solution containing an iron-based metal organic framework. Chlorella was centrifuged (3000 rpm, 10 min) and then redispersed in the phosphate solution containing an iron-based metal organic framework to form a Chlorella suspension with an ultraviolet absorbance of 0.9 at 682 nm. 0.8 g of hyaluronic acid was added to the above-mentioned phosphate solution containing an iron-based metal organic framework, and stirred and dissolved, and then in situ cross-linked at 37 ° C for 2 h to obtain a pregel. The pregel was added to a PDMS microneedle mold with a needle length of 800 μm, and centrifuged at 2000 rpm for 15 min to promote the matrix filling into the needle tip. After the centrifugation was completed, the pregel was quantitatively supplemented and dried at room temperature for 24 hours. Finally, the microneedle preparation was demolded.

[0058] Comparative Example 2

[0059] 2160 mg of ferric chloride was added to 10 mL of phosphate buffer with a pH of 7.4, and 3376 mg of potassium ferrocyanide was added under stirring at 40 rpm, and stirred for 60 min to obtain a phosphate solution containing an iron-based metal organic framework. The chlorella was centrifuged (3000 rpm, 10 min) and then redispersed in the phosphate solution containing an iron-based metal organic framework to form a chlorella suspension with an ultraviolet absorbance of 1.5 at 682 nm. 1.0 g of hyaluronic acid was added to the above phosphate solution containing an iron-based metal organic framework, and stirred and dissolved, and then in situ cross-linked at 37 ° C for 2 h to obtain a pregel. The pregel was added to a PDMS microneedle mold with a needle length of 800 μm, and centrifuged at 2000 rpm for 15 min to promote the matrix filling into the needle tip. After the centrifugation was completed, the pregel was quantitatively supplemented and dried at room temperature for 24 hours. Finally, the microneedle preparation was demolded.

[0060] The evaluation results of the Chlorella-Prussian Blue Nanozyme Complex (Z-PB) obtained in Example 1 are as follows:

[0061] Scanning electron microscope images show that Chlorella vulgaris ( Figure 1 A) The diameter of a single Chlorella is about 3 to 5 μm, and the surface is rough and mesh-like; Prussian blue nanozyme ( Figure 1 B) obtained by directly mixing equimolar amounts (0.8 mmol) of FeCl3 and K4[Fe(CN)6] solution. It can be seen from the figure that the nanoparticles have a good cubic structure and a small size (about 100 nm); the Chlorella-Prussian blue nanozyme complex obtained in Example 1 ( Figure 1 C) has different characteristics from Chlorella protothecoides ( Figure 1A) shows that the complex has a smooth surface, the Prussian blue nanozyme is well modified on the surface of Chlorella, and the diameter of Chlorella does not increase significantly, proving the successful synthesis of the Chlorella-Prussian blue nanozyme complex.

[0062] The evaluation of the dissolved oxygen generation capacity of the Chlorella-Prussian Blue nanozyme complex (Z-PB) obtained in Example 1 and the Chlorella-Prussian Blue nanozyme "1+1" mixture (Z+PB) obtained in Comparative Example 1 is as follows:

[0063] The Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 1 and the Chlorella-Prussian blue nanozyme "1+1" mixture (Z+PB) obtained in Comparative Example 1 were used as test samples. 10 mL of the test sample and 10 mL of naked Chlorella (CZ) suspension were respectively taken and cultured under light under the same concentration of hydroxyl radicals (the ultraviolet absorbance of the Chlorella and naked Chlorella suspensions in the test sample at 682 nm was 0.9; hydroxyl radical concentration: 10 mmol / L; light intensity: 3800 LUX). The changes in the dissolved oxygen content of the two groups of test samples within 6 hours were measured and recorded using a dissolved oxygen meter. The results are shown in Tables 1, 2 and Figure 2 It can be seen that the ability of the Chlorella-Prussian blue nanozyme complex obtained in Example 1 to produce oxygen under normal culture conditions is comparable to that of naked Chlorella ( Figure 2 A); In a culture environment rich in hydroxyl radicals, the Chlorella-Prussian blue nanozyme complex stably produces oxygen, while the oxygen production rate of the Chlorella-Prussian blue nanozyme "1+1" mixture obtained in naked Chlorella and Comparative Example 1 gradually decreases ( Figure 2 B).

[0064] Table 1 Dissolved oxygen content produced by Chlorella-Prussian blue nanozyme complex and naked Chlorella under normal culture conditions

[0065]

[0066] Table 2 Dissolved oxygen content produced by Chlorella-Prussian blue nanozyme complex, Chlorella-Prussian blue nanozyme "1+1" mixture and naked Chlorella under hydroxyl radical-rich conditions

[0067]

[0068] The polymerization sedimentation stability of the Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 2 and the Chlorella-Prussian blue nanozyme "1+1" mixture (Z+PB) obtained in Comparative Example 2 after the concentration was increased to 10 times was evaluated as follows:

[0069] The Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 2 and the Chlorella-Prussian blue nanozyme "1+1" mixture (Z+PB) obtained in Comparative Example 2 were used as test products. The stability of the two test products was observed at room temperature. The results are shown in Table 2. Figure 3 It can be seen that the Chlorella-Prussian blue nanozyme "1+1" mixture (Z+PB) aggregates and settles quickly and completely in 20 minutes, while the Chlorella-Prussian blue nanozyme complex (Z-PB) can exist stably under the same conditions. This may be because the presence of Chlorella reduces the aggregation and sedimentation phenomenon of Prussian blue nanozyme, thereby increasing its stability.

[0070] The morphological evaluation results of the biological oxygen therapy and antioxidant dual strategy soluble microneedles obtained in Example 1 are as follows:

[0071] The structure of the soluble microneedles loaded with the Chlorella-Prussian blue nanozyme complex was analyzed using a mobile phone camera, optical microscopy, and scanning electron microscopy. Photo taken from a mobile phone camera ( Figure 4 A) It can be seen that the prepared microneedles have a square structure, the needle tips are neat and orderly, and the backing layer is intact without any bending. From the image under an optical microscope ( Figure 4 B) It can be seen that the tip of the microneedle is a regular pyramid shape, evenly distributed, and complete in shape when observed from different angles. The magnified microstructure of the prepared microneedle is further observed by scanning electron microscope images ( Figure 4 C), it can be seen that the microneedle has a visible quadrangular pyramid structure, and the tip is intact without bending.

[0072] The solubility of the biological oxygen therapy and antioxidant dual strategy soluble microneedles obtained in Example 1 in vivo and in vitro was evaluated as follows:

[0073] The soluble microneedles obtained in Example 1 were placed in 1.4% agarose gel, in vitro pig skin and in vivo mouse skin, respectively. After different time periods, the dissolution of the needle tip was observed under a microscope. The dissolution of the soluble microneedles (MNs) in vitro and in vivo was observed. Figure 5 .from Figure 5 It can be seen that the microneedle tip can be completely dissolved in agarose gel within 30 seconds ( Figure 5 A), after being inserted into the ex vivo pig skin, it was observed that the needle tip completely dissolved within 60 seconds ( Figure 5 B), The needle tip was observed to completely dissolve in mouse skin within 3 minutes.

[0074] The microneedle pore healing time of the biological oxygen therapy and antioxidant dual strategy soluble microneedles obtained in Example 1 was evaluated as follows:

[0075] Through in vitro pig skin and live mouse skin experiments, the skin healing of the punctured site after the soluble microneedle obtained in Example 1 was evaluated. Take frozen pig skin and soak it in physiological saline. After the pig skin is thawed, remove the surface moisture with filter paper, then spread the pig skin on the filter paper with the stratum corneum facing outward, press the microneedle vertically for 30 seconds with a certain force, take out the microneedle, and take pictures and record the punctured site at different time points. After the Balb / c mice were raised normally for 7 days, most of the long hair of the mice was trimmed with scissors, and then the depilatory cream was evenly applied to the back skin of the mice for 3 minutes. The application time can be appropriately extended or shortened according to the situation to avoid corrosion of the mouse skin. Finally, wipe with warm water to remove the depilatory cream and shed hair, observe the back of the mouse, and when the mouse skin is in good condition, insert the microneedle patch vertically into the back skin of the mouse with appropriate force, remove the patch after 30 seconds, take pictures with a camera at different time points, and record the skin healing process. The results are shown in Figure 6 .

[0076] from Figure 6 It can be seen that the recovery time of the soluble microneedle on the ex vivo pig skin is 50 minutes ( Figure 6 A); The pinholes induced on the skin of mice gradually recovered and disappeared within 40 minutes ( Figure 6 B), and no side effects of redness and inflammation appeared on the skin surface of the mice, and the skin showed a satisfactory healing response, indicating that the living skin has better self-healing properties.

[0077] Application of a dual-strategy soluble microneedle with biological oxygen therapy and antioxidant in the treatment of psoriasis mouse model:

[0078] BALB / c mice were randomly divided into 5 groups: blank group (negative control group of psoriasis model), model group (positive control group of psoriasis model), drug treatment group (desonide cream treatment group), MNs treatment group (microneedle treatment only) and MNs+Light group (microneedle treatment plus 4 hours of light exposure group). IMQ is an agonist used to establish a mouse psoriasis model. 62.5 mg IMQ cream (Mingxin Pharmaceutical Co., Ltd, China) was applied to the backs of mice in the model group, drug treatment group, MNs treatment group and MNs+Light group for six consecutive days. Drug treatment, MNs treatment group and MNs+Light treatment group were performed 6 hours after IMQ induction modeling from day 1 to day 5 to minimize interference. Among them, the drug treatment group was to evenly apply 50.0 mg of the therapeutic drug desonide cream on the back of the mouse, and the two MNs groups were to apply microneedles directly on the shaved back skin, the difference being whether there was 4 hours of light exposure.

[0079] The severity of psoriasis in each group of mice was evaluated using the Psoriasis Severity Index (PASI) score. Figure 7 EI, it can be seen that the PASI score of the blank group was 0 from day 1 to day 7, the scaling score, erythema score, infiltration score, and PASI score of the model group were the highest, and there was no statistically significant difference between the drug treatment group and the model group, indicating that the therapeutic effect of conventional drugs is limited. Figure 7 As can be seen in E, compared with the model group, the MNs group and the MNs+Light group showed lower PASI scores throughout the treatment cycle, that is, the treatment with MNs slowed down the progression of psoriasis and alleviated the disease progression (lower scores for erythema, scaling, lesions, and skin infiltration); at the same time, the mice in the MNs+Light group had lower erythema, scaling, lesions, and skin infiltration scores than the mice in the MNs group, proving that oxygen therapy can indeed alleviate the symptoms of psoriasis.

[0080] Immunohistochemistry was used to demonstrate the effective improvement of treatment efficiency and deep oxygen delivery performance. Ki-67 is a marker for cell proliferation. The amount of Ki-67 detected can reflect the proliferation of stratum corneum cells. Figure 8 A shows that the expression of Ki-67 in the model group increased significantly, MNs treatment decreased the expression of Ki-67, and the expression in the MNs+Light group was significantly different from that in the model group. HIF-α is located in the cytoplasm, and its stability and transcriptional activity increase significantly under hypoxic conditions. Therefore, measuring the content of HIF-α can reflect the degree of hypoxia in stratum corneum cells. Figure 8 B shows that the expression of HIF-α in the MNs+Light group was not significantly different from that in the blank group, proving that the soluble microneedles (MNs) prepared in Example 1 in combination with light treatment can effectively alleviate hypoxia in the pathological site. IL-17A is a key inflammatory cytokine in psoriasis, so the expression of IL-17A in skin tissue was detected, and the results are shown in Figure 8 C, it can be seen that the expression of IL-17A in the model group increased significantly, while the expression of IL-17A in the MNs+Light group and the MNs group decreased significantly, indicating that MNs can inhibit the expression of IL-17A. In addition, the expression level of the proinflammatory cytokine TNF-α in the mouse skin was also observed. Figure 8 D. It can be seen that compared with the blank group, the expression of TNF-α in the model group was significantly increased, while the expression of TNF-α in the drug group, MNs group, and MNs+Light group was significantly decreased after treatment, and there was no significant difference in the expression of TNF-α between the MNs+Light group and the blank group.

Claims

1. A biological oxygen therapy and anti-oxidation dual strategy soluble microneedle, characterized by: The microneedle is an integrated soluble microneedle, comprising a matrix formed by a water-soluble polymer and a complex formed by microalgae and metal organic framework nanozymes uniformly dispersed in the matrix, wherein the complex formed by the microalgae and the metal organic framework nanozymes is a "core-shell" structure complex, and the metal organic framework nanozyme as the outer shell is wrapped on the surface of the microalgae as the inner core.

2. The soluble microneedle according to claim 1, characterized in that: The preparation method of the complex formed by microalgae and metal organic framework nanozyme is: after centrifuging the microalgae, disperse it in a phosphate buffer solution to obtain a microalgae suspension, then add metal ions thereto, and finally add an organic ligand under stirring conditions to obtain a solution system containing the microalgae-metal organic framework nanozyme complex.

3. The method for preparing the complex formed by microalgae and metal organic framework nanozyme according to claim 2, characterized in that: The concentration of the microalgae is 10 5 ~10 9 The concentration of the metal ions is 10 to 100 mg / mL; the concentration of the organic ligand is 100 to 1000 mg / mL.

4. The method for preparing the complex formed by microalgae and metal organic framework nanozyme according to claim 2, characterized in that: The metal ions are one or more of zinc, copper, iron, cobalt and nickel ions; the organic ligands are one or more of benzoic acid derivatives, ferrocyanide, dimethylimidazole and its derivatives; the microalgae are one or more of cyanobacteria, red algae, green algae and golden algae.

5. The soluble microneedle according to claim 1, characterized in that: The water-soluble polymer is one or more of hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, polylactic acid, carboxymethyl cellulose, amylose, chondroitin sulfate, sucrose and fructose.

6. The soluble microneedle according to claim 1, characterized in that: The soluble microneedle is made of a microneedle mold PDMS, with a needle height of 400-1500 μm, a needle body diameter of 300-500 μm, a base diameter of 5-30 mm, a needle tip diameter of less than 10 μm, and an array number greater than 8×8.

7. The method for preparing the soluble microneedle according to any one of claims 1 to 6, characterized in that: A water-soluble polymer was added to the solution system containing the microalgae-metal organic framework nanozyme complex, and after being stirred and dissolved, the solution was in situ cross-linked at 37°C for 2 hours to obtain a pre-gel; The pre-gel is poured into a PDMS microneedle mold, and after centrifugation and drying, the mold is demoulded to obtain a soluble microneedle.

8. The method for preparing soluble microneedles according to claim 7, characterized in that: The mass percentage of the water-soluble polymer is 5% to 50%.

9. The method for preparing soluble microneedles according to claim 7, characterized in that: The centrifugation condition is centrifugation at 1000-5000 rpm for 1-30 min; the drying condition is drying at room temperature for 6-48 h.

10. Use of the soluble microneedle according to claim 1 in the preparation of drugs or preparations for treating tumors, promoting wound healing, regulating inflammatory microenvironment, treating skin burns and scalds, and removing scars.

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