A biological oxygen therapy and antioxidant double strategy soluble microneedle and a preparation method and application thereof

Abstract

The present application relates to a kind of biological oxygen therapy and antioxidant double strategy soluble microneedle and its preparation method and application, belong to medical technology field.The microneedle of the present application is integrated soluble microneedle, including the matrix formed by water-soluble polymer and the compound of microalgae and metal organic framework nanoenzyme uniformly dispersed in matrix, wherein the compound of microalgae and metal organic framework nanoenzyme is "core-shell" structure compound, and metal organic framework nanoenzyme as shell is wrapped in the surface of microalgae as core.The microneedle of the present application has the ability of in-situ rapid antioxidant and continuous deep layer oxygen delivery, is widely applied in the preparation of drug design or preparation for treating tumor, promoting wound healing, adjusting inflammatory microenvironment, skin burn and scald treatment and scar removal, and patient applicability is good, and its preparation process is green, simple and fast, mild condition is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to a soluble microneedle with a dual strategy of bio-oxygen therapy and anti-oxidation, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Oxidative stress arises from an imbalance between the generation of oxidants (free radicals and reactive oxygen species) and the elimination by antioxidants. Oxidative stress activates a series of intracellular proteolytic enzymes, which then break down the cellular structure, ultimately leading to apoptosis. These apoptotic cells or their products can also activate inflammatory cells. Oxidative stress and inflammation interact by regulating transcriptional levels, forming a vicious cycle. Oxidative stress is a concomitant phenomenon in the inflammatory process; it exacerbates the inflammatory response, while inflammation, through inflammatory mediators, further promotes oxidative stress. Immune cell infiltration, inflammatory cytokine retention, and increased reactive oxygen species levels can all cause hypoxia in the inflammatory microenvironment and mitochondrial damage, further aggravating oxidative damage and accelerating inflammation. Under continuous severe environmental stress, reactive oxygen species are generated and accumulate over a long period, causing serious damage to cell structure and function, and potentially inducing somatic mutations and tumorigenic transformation.

[0003] Gas therapy is a treatment modality based on the synergistic or direct regulation of the pathological microenvironment by specific gaseous signaling molecules (such as hydrogen sulfide, nitric oxide, carbon monoxide, hydrogen, and oxygen). It boasts advantages such as high efficiency, safety, and lack of toxic side effects. Decreased oxygen levels are a common pathological feature of the microenvironment at sites of inflammation, and oxygen therapy has been widely applied in various clinical conditions, including tumor treatment, wound healing, and skin disease recovery.

[0004] Chinese patent CN114557954A discloses soluble microneedles containing active microalgae, microneedle patches, preparation methods, and applications. It utilizes the in-situ continuous oxygen production of microalgae to continuously deliver oxygen and prepares soluble microneedle formulations for use in the preparation of drugs or reagents to improve tumor hypoxia, promote wound healing, treat skin burns, provide dental treatment, or improve the microenvironment of anemia. This invention utilizes gas therapy based on microalgae oxygen production, but does not involve the field of regulating oxidative stress and antioxidation, nor does it demonstrate its potential application in inflammation treatment. Chinese patent CN115444929A discloses a transdermal double-layer microneedle based on hydrogen-producing probiotics, its preparation method, and applications, utilizing the reducing hydrogen gas produced by probiotics to treat inflammatory skin diseases. This invention demonstrates the important role of hydrogen in regulating oxidative stress, but the mechanisms of hydrogen therapy and oxygen therapy differ. Chinese patent CN117122554A discloses the preparation of an oxidoreductase-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 to achieve an antioxidant effect, promote macrophage polarization towards the M2 subtype and promote the production of anti-inflammatory factors, while reducing the expression of pro-inflammatory factors such as iNOS and IL-1β. In an AIA mouse model, it has a significant anti-arthritis effect.

[0005] Currently, research on the combined application of bio-oxygen therapy and antioxidants remains limited, and traditional local dissolved oxygen delivery methods face challenges such as small delivery volume, difficulty in continuous delivery, and insufficient delivery depth. Therefore, the development of a delivery system that addresses both of these issues while ensuring delivery efficiency, therapeutic efficacy, and patient safety is still needed. Summary of the Invention

[0006] To address the technical deficiencies of existing technologies, this invention provides a soluble microneedle with a dual strategy of bio-oxygen therapy and anti-oxidation, along with its preparation method and applications. This invention involves loading a core-shell structured complex of an antioxidant metal-organic framework nanozyme and oxygen-producing microalgae onto a water-soluble polymer matrix, creating a soluble microneedle that integrates oxygen therapy and anti-oxidation treatment. This microneedle is then applied in therapeutic procedures. The bio-oxygen therapy and anti-oxidation dual-strategy microneedle designed in this invention exhibits significant therapeutic effects, is simple to prepare, has wide applications, and is suitable for large-scale production and application.

[0007] One objective of this invention is to provide a soluble microneedle with a dual strategy of bio-oxygen therapy and anti-oxidation. The microneedle is an integrated soluble microneedle comprising a matrix formed from a water-soluble polymer and a complex formed from microalgae and metal-organic framework nanozymes uniformly dispersed within the matrix.

[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, acting as the outer shell, is wrapped around the surface of the microalgae, which acts as the core.

[0009] The soluble microneedles of this invention are made from microalgae, which are a type of oxygen-producing microorganisms. Their function is to produce oxygen under light conditions, thereby achieving deep oxygen delivery and alleviating the hypoxia in the microenvironment caused by oxidative stress.

[0010] The soluble microneedles of the present invention contain metal-organic framework nanozymes with antioxidant activity. Their function is to first contact the microenvironment with highly reactive oxygen species and exert enzyme activity to scavenge reactive oxygen species.

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

[0012] The soluble microneedles of the present invention contain one or more of the following water-soluble polymers: hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, polylactic acid, carboxymethyl cellulose, amyl starch, chondroitin sulfate, sucrose, and fructose.

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

[0014] The soluble microneedles of the present invention are prepared by the following method: microalgae are centrifuged and dispersed in phosphate buffer solution to obtain microalgae suspension, metal ions are added to it, and finally organic ligands are added under stirring conditions to obtain a solution system containing microalgae-metal-organic framework nanoenzyme complex.

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

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

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

[0018] In the above technical solution, the microalgae are one or more of the following phyla: cyanobacteria, red algae, green algae, and golden algae.

[0019] Preferably, the microalgae is a type of green algae, and more preferably, it is Chlorella vulgaris.

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

[0021] Preferably, the metal ion is an iron ion.

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

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

[0024] In the above technical solution, the centrifugation conditions in the preparation process of the complex formed by microalgae and metal-organic framework nanozymes are centrifugation at 1000-5000 rpm for 1-30 min.

[0025] In the above technical solution, the stirring conditions during the preparation of the complex formed by the microalgae and the metal-organic framework nanozyme are 10-100 rpm for 10-60 min.

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

[0027] The soluble microneedles of this invention, wherein the outer "shell" structure of the complex formed by the microalgae and the metal-organic framework nanoenzyme first comes into contact with the microenvironment with highly reactive oxygen species, exerts enzyme activity to remove reactive oxygen species, and the complex undergoes the first step of degradation to expose the "core" structure of the microalgae. Under light conditions, the microalgae produce oxygen, achieving deep oxygen transport and alleviating the hypoxia in the oxidative stress microenvironment.

[0028] Another objective of this invention is to provide a method for preparing soluble microneedles with a dual strategy of bio-oxygen therapy and anti-oxidation, comprising the following steps: adding a water-soluble polymer to a solution system containing a microalgae-metal-organic framework nanoenzyme complex, stirring to dissolve, and then crosslinking in situ 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 soluble microneedles.

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

[0030] In the preparation method of soluble microneedles of the present invention, the centrifugation conditions are centrifugation at 1000-5000 rpm for 1-30 min.

[0031] In the preparation method of the soluble microneedles of the present invention, the drying conditions are 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 bio-oxygen therapy and antioxidant dual-strategy soluble microneedles in the design or formulation of drugs for tumor treatment, promoting wound healing, regulating the inflammatory microenvironment, treating skin burns and scalds, and removing scars.

[0033] Microalgae are tiny, single-celled algae that convert sunlight, water, and carbon dioxide into energy and oxygen through photosynthesis. The oxygen produced in this process often solves problems that are difficult to address in the treatment of many diseases. Microalgae can act as "natural oxygen generators," delivered to oxygen-deficient areas, and continuously produce oxygen under specific external conditions to alleviate environmental hypoxia. Microalgae have been proven to have nutritional supplementation, regulate gut microbiota metabolism, and possess anti-inflammatory and anti-aging properties, and are often used to promote wound healing in diabetic patients, treat inflammatory bowel disease, and protect against radiation damage. Iron is abundant in nature and has low toxicity; therefore, iron-based metal-organic frameworks possess diverse and high enzyme-like activities and good biocompatibility, making them very promising functional materials.

[0034] The beneficial effects of this invention are:

[0035] 1. The composite material formed by microalgae and metal-organic framework nanozymes prepared in this invention protects microalgal cells from damage caused by highly expressed reactive oxygen species in an oxidative stress microenvironment. Simultaneously, under light conditions, it promotes oxygen production and transport, helping to alleviate the hypoxic microenvironment caused by oxidative stress. This design, which encapsulates metal-organic framework nanozymes on the surface of microalgal cells, solves the problems of unstable storage and easy aggregation and inactivation of conventional nanoparticles. The composite design of this invention improves the stability of both microalgae and metal-organic framework nanozymes without affecting the performance of either monomer. The modification of microalgae by metal-organic framework nanozymes does not reduce the photosynthetic oxygen production capacity of microalgae, nor does it reduce the enzyme activity of the metal-organic framework nanozymes.

[0036] 2. This invention utilizes microalgae to generate oxygen to alleviate environmental hypoxia and utilizes the enzyme activity of metal-organic frameworks to regulate oxidative stress to achieve antioxidant effects. The combination of these two components into a complex can combine their advantages and synergistically enhance the therapeutic effects of oxygen generation and antioxidant regulation of oxidative stress.

[0037] 3. This invention combines oxygen-producing microalgae with antioxidant metal-organic framework nanozymes to form a complex with both bio-oxygen therapy and antioxidant functions. This complex is loaded into microneedles to simultaneously achieve antioxidant and oxygen therapy. The microneedle preparation process described in this invention is green, environmentally friendly, simple, rapid, and under mild conditions, making it suitable for large-scale production. The microneedle patch described in this invention has the ability to provide rapid in-situ antioxidant protection and continuous deep oxygen delivery. It has wide applications in the design or formulation of drugs for treating tumors, promoting wound healing, regulating the inflammatory microenvironment, treating burns, and removing scars, and has good patient suitability. The dual-strategy microneedles of bio-oxygen therapy and antioxidant design of this invention have significant therapeutic effects, are simple to prepare, have wide applications, and are suitable for large-scale production and application. Attached Figure Description

[0038] Figure 1 (A) is the preparation process of the pregel (Z-PBHA) of Chlorella-Prussian blue nanozyme complex and the microneedles of Chlorella-Prussian blue nanozyme complex obtained in Example 1. Figure 1 (B) is a scanning electron microscope image of Chlorella at a scale of 5 μm; Figure 1 (C) is a scanning electron microscope image of a single Prussian blue nanozyme at 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) shows the Fourier transform infrared spectra of the above substances.

[0039] Figure 2 (A) The dissolved oxygen content 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) To determine the dissolved oxygen content of 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 in a culture environment rich in hydroxyl radicals.

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

[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 microneedles obtained in Example 1, taken with an optical microscope; Figure 4(C) is a microneedle image obtained from Example 1, captured by scanning electron microscopy.

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

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

[0044] Figure 7 (A) is the experimental plan for in vivo treatment with the soluble microneedles obtained in Example 1; Figure 7 (B) is an image of the mouse's back 7 days after treatment; Figure 7 (C) shows the change in mouse body weight from day 1 to day 7; Figure 7 (D) Spleen index of mice in each group after 7 days of treatment; Figure 7 (E) PASI scores for each group of mice from day 1 to day 7; Figure 7 (F) represents the fraction of skin lesion area for each group of mice from day 1 to day 7; Figure 7 (G) represents the score of the degree of infiltration in each group of mice from day 1 to day 7; Figure 7 (H) represents the erythema score of each group of mice from day 1 to day 7; Figure 7 (I) Scale score for each group of mice from day 1 to day 7.

[0045] Figure 8 Immunohistochemical analysis of the dorsal skin of mice in each group during treatment with the soluble microneedles obtained in Example 1 was performed. Figure 8 (A) represents the amount of Ki-67 expressed in different experimental groups; Figure 8 (B) represents the expression level of HIF-α in different experimental groups; Figure 8 (C) represents the level of IL-17A expression in different experimental groups; Figure 8 (D) represents the amount of TNF-α expressed in different experimental groups.

[0046] Figure 9 This is a schematic diagram illustrating the use of the soluble microneedles obtained in this invention. Detailed Implementation

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

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

[0049] Example 1

[0050] The preparation of a soluble microneedle with a dual strategy of bio-oxygen therapy and antioxidation includes the following steps:

[0051] Chlorella was centrifuged (3000 rpm, 10 min) and redispersed in 10 mL of phosphate buffer (pH 7.4) to form a Chlorella suspension with an absorbance of 0.9 at 682 nm. 337.6 mg of potassium ferrocyanide (K4[Fe(CN)6]) was added to the algal suspension and stirred until dissolved. 216 mg of ferric chloride (FeCl3) was gradually added under stirring at 40 rpm for 60 min to prepare a solution containing the Chlorella-Prussian blue nanozyme complex (Z-PB). 0.8 g of hyaluronic acid was added to the system and stirred until dissolved. The mixture was then in-situ cross-linked at 37 °C for 2 h to obtain a pregel. The pregel was added to an 800 μm PDMS microneedle mold and centrifuged at 2000 rpm for 15 min to promote matrix filling into the needle tip. After centrifugation, the pregel was quantitatively replenished and dried at room temperature for 24 h. Finally, the microneedle formulation was obtained by demolding.

[0052] Example 2

[0053] The preparation of a soluble microneedle with a dual strategy of bio-oxygen therapy and antioxidation includes the following steps:

[0054] Chlorella was centrifuged (3000 rpm, 10 min) and redispersed in 10 mL of phosphate buffer (pH 7.4) to form a Chlorella suspension with an absorbance of 1.5 at 682 nm. 3376 mg of potassium ferrocyanide was added to the algal suspension and stirred until dissolved. 2160 mg of ferric chloride was gradually added under stirring at 40 rpm for 60 min to prepare a solution containing the Chlorella-Prussian blue nanozyme complex (Z-PB). 1.0 g of hyaluronic acid was added to the system and stirred until dissolved. The mixture was then in-situ cross-linked at 37 °C for 2 h to obtain a pregel. The pregel was added to an 800 μm PDMS microneedle mold and centrifuged at 2000 rpm for 15 min to promote matrix filling into the needle tip. After centrifugation, the pregel was quantitatively replenished and dried at room temperature for 24 h. Finally, the microneedle formulation was obtained by demolding.

[0055] Comparative Example 1

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

[0057] 216 mg of ferric chloride was added to 10 mL of phosphate buffer (pH 7.4), and 337.6 mg of potassium ferrocyanide was added while stirring at 40 rpm for 60 min to obtain a phosphate solution containing an iron-based metal-organic framework. Chlorella vulgaris was centrifuged (3000 rpm, 10 min) and redispersed in the phosphate solution, resulting in a Chlorella vulgaris suspension with an absorbance of 0.9 at 682 nm. 0.8 g of hyaluronic acid was added to the phosphate solution containing the iron-based metal-organic framework, and the mixture was stirred to dissolve and then in-situ crosslinked 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 matrix filling into the needle tip. After centrifugation, the pregel was quantitatively replenished and dried at room temperature for 24 h. Finally, the microneedle formulation was obtained by demolding.

[0058] Comparative Example 2

[0059] 2160 mg of ferric chloride was added to 10 mL of phosphate buffer (pH 7.4), and 3376 mg of potassium ferrocyanide was added while stirring at 40 rpm for 60 min to obtain a phosphate solution containing an iron-based metal-organic framework. Chlorella vulgaris was centrifuged (3000 rpm, 10 min) and redispersed in the phosphate solution, resulting in a Chlorella vulgaris suspension with an absorbance of 1.5 at 682 nm. 1.0 g of hyaluronic acid was added to the phosphate solution containing the iron-based metal-organic framework, stirred to dissolve, and then in-situ crosslinked 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 matrix filling into the needle tip. After centrifugation, the pregel was quantitatively replenished and dried at room temperature for 24 hours. Finally, the microneedle formulation was obtained by demolding.

[0060] The evaluation results of the Chlorella-Prussian blue nanozyme complex (Z-PB) obtained in Example 1 are as follows:

[0061] Scanning electron microscopy images show that *Chlorella protozoa* ( Figure 1 A) Individual Chlorella cells are approximately 3–5 μm in diameter, with a rough, network-like surface; Prussian blue nanozymes ( Figure 1 B) was obtained by directly mixing equimolar amounts (0.8 mmol) of FeCl3 and K4[Fe(CN)6] solution. The figure shows that the nanoparticles have a good cubic structure and a small size (approximately 100 nm); the Chlorella-Prussian blue nanozyme complex obtained in Example 1 (… Figure 1 C) It has characteristics different from the original Chlorella ( Figure 1The microscopic image in A) shows that the complex has a smooth surface, and the Prussian blue nanozyme is well modified on the surface of Chlorella. The diameter of Chlorella did not increase significantly, which proves the successful synthesis of the Chlorella-Prussian blue nanozyme complex.

[0062] The dissolved oxygen generation capabilities 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 are evaluated 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 each test sample and 10 mL of exposed Chlorella (CZ) suspension were cultured under light in an environment with the same concentration of hydroxyl radicals (the UV absorbance of both Chlorella and exposed Chlorella suspensions at 682 nm was 0.9; hydroxyl radical concentration: 10 mmol / L; light intensity: 3800 LUX). The changes in dissolved oxygen content in both groups of test samples over 6 hours were measured and recorded using a dissolved oxygen meter. The results are shown in Tables 1 and 2. Figure 2 It can be seen that the Chlorella-Prussian blue nanozyme complex obtained in Example 1 has an oxygen-generating capacity comparable to that of naked Chlorella under normal culture conditions. Figure 2 A); Under a culture environment rich in hydroxyl radicals, the Chlorella-Prussian blue nanozyme complex stably produces oxygen, while the oxygen production rate of the naked Chlorella and the "1+1" type mixture of Chlorella-Prussian blue nanozyme obtained in Comparative Example 1 gradually decreases. Figure 2 B).

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

[0065]

[0066] Table 2. Dissolved oxygen content of Chlorella-Prussian blue nanozyme complex, Chlorella-Prussian blue nanozyme "1+1" mixture, and naked Chlorella under conditions rich in hydroxyl radicals.

[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 increasing the concentration by 10 times is 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 samples. The stability of the two test samples was observed after standing at room temperature. The results are shown in […]. Figure 3 It can be seen that the Chlorella-Prussian blue nanozyme "1+1" type mixture (Z+PB) polymerizes and settles rapidly and settles completely within 20 minutes, while the Chlorella-Prussian blue nanozyme complex (Z-PB) can remain stable under the same conditions. This may be because the presence of Chlorella reduces the polymerization and sedimentation of Prussian blue nanozyme, thus increasing its stability.

[0070] The morphological evaluation results of the soluble microneedles with a dual strategy of bio-oxygen therapy and antioxidation obtained in Example 1 are as follows:

[0071] The structure of soluble microneedles loaded with Chlorella-Prussian blue nanozyme complex was analyzed using a mobile phone camera, optical microscope, and scanning electron microscope. (Image taken with a mobile phone camera...) Figure 4 A) It can be seen that the prepared microneedles have a square structure with neat and orderly tips; the backing layer is intact and without any bending. From the images under an optical microscope ( Figure 4 B) It can be seen that the tips of the microneedles are regular pyramidal in shape, evenly distributed, and morphologically complete, as observed from different angles. Further observation of the magnified microstructure of the prepared microneedles using scanning electron microscopy (SEM) images... Figure 4 C) It can be seen that the microneedles have a visible tetrahedral structure and the tips are intact and not bent.

[0072] The in vitro and in vivo solubility evaluation of the soluble microneedles with the dual strategy of bio-oxygen therapy and antioxidation obtained in Example 1 is as follows:

[0073] The soluble microneedles obtained in Example 1 were placed in 1.4% agarose gel, isolated porcine skin, and live mouse skin, respectively. After different maintenance times, the dissolution of the needle tips was observed under a microscope. The dissolution of soluble microneedles (MNs) in vitro and in vivo was observed, and the results are shown in the figure. Figure 5 .from Figure 5 As can be seen, the microneedle tip can completely dissolve in agarose gel within 30 seconds. Figure 5 A) After insertion into detached pigskin, the needle tip was observed to completely dissolve within 60 seconds. Figure 5 B) It can be observed that the needle tip can completely dissolve within 3 minutes in mouse skin.

[0074] The microneedle channel healing time of the soluble microneedles with the dual strategy of bio-oxygen therapy and antioxidant obtained in Example 1 is evaluated as follows:

[0075] The healing effect of the soluble microneedles obtained in Example 1 on the puncture site was evaluated through experiments on ex vivo pig skin and live mouse skin. Frozen pig skin was soaked in physiological saline. After thawing, the surface moisture of the pig skin was removed with filter paper. The pig skin was then placed on the filter paper with the stratum corneum facing outwards, and the microneedles were pressed vertically with a certain force for 30 seconds. The microneedles were then removed, and the puncture site was photographed and recorded at different time points. After Balb / c mice were normally fed for 7 days, most of the long hair of the mice was trimmed with scissors. Then, hair removal cream was evenly applied to the skin on the back of the mice for 3 minutes. The application time was appropriately extended or shortened as needed to avoid corroding the mouse skin. Finally, the hair removal cream and shed hair were wiped off with warm water, and the back of the mice was observed. When the skin condition of the mice was good, the microneedle patch was vertically inserted into the skin on the back of the mice with appropriate force. After 30 seconds, the patch was removed, and the skin healing process was recorded by taking pictures at different time points. The results are shown in […]. Figure 6 .

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

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

[0078] BALB / c mice were randomly divided into 5 groups: a blank control group (negative control for psoriasis model), a model group (positive control for psoriasis model), a drug treatment group (desonide cream treatment group), a microneedling treatment group (microneedling treatment only), and a microneedling + Light group (microneedling treatment plus 4 hours of light exposure). IMQ is an agonist used to establish a mouse psoriasis model. 62.5 mg of 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, and MNs + Light treatment were administered 6 hours after IMQ induction from day 1 to day 5 to minimize interference. In the drug treatment group, 50.0 mg of desonide cream was evenly applied to the back of the mice. In the two MNs groups, microneedling was applied directly to the shaved back skin; the difference was whether or not there was 4 hours of light exposure.

[0079] The severity of psoriasis in each group of mice was assessed using the Psoriasis Severity Index (PASI) score. Results are shown in [Figure number missing]. Figure 7 EI analysis shows that the PASI score in the blank group was 0 from day 1 to day 7. The model group had the highest scores in scaling, erythema, infiltration, and PASI. 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 shown in Figure E, compared with the model group, both the MNs group and the MNs+Light group exhibited lower PASI scores throughout the treatment period. This indicates that MNs treatment slowed the progression of psoriasis and alleviated its severity (lower scores for erythema, scaling, skin lesions, and skin infiltration). Meanwhile, mice in the MNs+Light group had fewer scores for erythema, scaling, skin lesions, and skin infiltration than mice in the MNs group, demonstrating that oxygen therapy can indeed alleviate the symptoms of psoriasis.

[0080] Immunohistochemistry was used to demonstrate the effective improvement in treatment efficiency and deep oxygen delivery performance. Ki-67 is a marker of cell proliferation; detecting the amount of Ki-67 can reflect the proliferation status of keratinocytes. From... Figure 8 A shows that Ki-67 expression was significantly increased in the model group, while MN treatment decreased Ki-67 expression, and the expression level 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 are significantly increased under hypoxic conditions; therefore, measuring HIF-α levels can reflect the degree of hypoxia in keratinocytes. Figure 8 B shows that the expression level 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, combined with light treatment, can effectively alleviate hypoxia at the pathological site. IL-17A is a key inflammatory cytokine in psoriasis; therefore, the expression of IL-17A in skin tissue was detected, and the results are shown in [Figure 1]. Figure 8 C shows that IL-17A expression was significantly increased in the model group, while it was significantly decreased in the MNs+Light group and the MNs group, indicating that MNs can inhibit IL-17A expression. In addition, the expression level of the pro-inflammatory cytokine TNF-α in mouse skin was observed, and the results are shown in [Figure C]. Figure 8 D shows 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 the expression level of TNF-α in the MNs+Light group was not significantly different from that in the blank group.

Claims

1. A soluble microneedle with a dual strategy of bio-oxygen therapy and antioxidation, characterized in that: The microneedles are integral soluble microneedles comprising a matrix formed of hyaluronic acid and a complex formed by Chlorella vulgaris and Prussian blue nanozymes uniformly dispersed in the matrix. The complex formed by Chlorella and Prussian blue nanozyme is a "core-shell" structure complex, with Prussian blue nanozyme, which acts as the outer shell, wrapped around the surface of Chlorella, which acts as the core. The preparation method of the complex formed by Chlorella and Prussian blue nanozyme is as follows: Chlorella is centrifuged and dispersed in phosphate buffer solution to obtain Chlorella suspension, then iron ions are added to it, and finally potassium ferrocyanide is added under stirring conditions to obtain a solution system containing Chlorella-Prussian blue nanozyme complex.

2. The soluble microneedles according to claim 1, characterized in that: The concentration of the Chlorella was 10. 5 ~10 9 The concentration of iron ions is 10-100 mg / mL; the concentration of potassium ferrocyanide is 100-1000 mg / mL.

3. The soluble microneedles according to claim 1, characterized in that: The soluble microneedles are made of PDMS microneedle mold, 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 of greater than 8×8.

4. The method for preparing soluble microneedles according to any one of claims 1 to 3, characterized in that: Hyaluronic acid was added to a solution containing Chlorella-Prussian blue nanozyme complex, and after stirring to dissolve, it was cross-linked in situ at 37°C for 2 h to obtain a pregel. The pregel was poured into a PDMS microneedle mold, centrifuged, dried, and then demolded to obtain soluble microneedles.

5. The method for preparing soluble microneedles according to claim 4, characterized in that: The hyaluronic acid has a mass percentage of 5% to 50%.

6. The method for preparing soluble microneedles according to claim 4, characterized in that: The centrifugation conditions are centrifugation at 1000~5000 rpm for 1~30 min; the drying conditions are drying at room temperature for 6~48 h.

7. The use of the soluble microneedles according to claim 1 in the preparation of a psoriasis treatment formulation.

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