Microneedle patch containing purslane exosome as well as preparation method and application of microneedle patch
By preparing microneedle patches containing purslane exosomes and antioxidant carrier materials, the problems of unsatisfactory efficacy and difficulty in transdermal absorption in the treatment of atopic dermatitis were solved, and effective skin treatment effects were achieved.
Patent Information
- Application Number
- CN202510505188.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-07-11
AI Technical Summary
The treatment methods for atopic dermatitis in the prior art have problems such as unsatisfactory efficacy, many long-term side effects, and difficult to absorb topical preparations through the skin. Traditional Chinese medicine treatment methods lack effective external skin forms.
Ulslane exosomes were prepared by ultracentrifugation and size exclusion methods using microneedle patches containing purslane exosomes and antioxidant carrier material 4-amino-TEMPO modified hyaluronic acid, and were loaded into microneedles to achieve transdermal delivery.
It significantly regulates the immune response, reduces skin scabs and mast cell infiltration, reduces Th2 cytokine levels, improves the therapeutic effect of atopic dermatitis, and solves the problem of transdermal absorption of topical preparations.
Smart Images
Figure CN120284840A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of drugs, and particularly to a microneedle patch containing purslane exosomes. Background Art
[0002] Atopic dermatitis is a type of chronic inflammatory skin disease with diverse clinical manifestations, including erythema, papules, lichenoid changes, severe pruritus, etc. It mostly occurs in infants and young children, and the incidence rate in children under 3 years old can reach 44.0%. At present, the pathogenesis of atopic dermatitis has not been clarified, and there is no cure plan in Western medicine clinical practice. Clinically, it is mainly treated by taking glucocorticoids, local immunomodulators, antihistamine drugs, etc. orally or topically. Although the relevant symptoms can be relieved temporarily, the condition is prone to recurrence, the long-term curative effect is not ideal, there are many adverse reactions, and the epidermis of atopic dermatitis patients is thickened, making it difficult for topical preparations to penetrate the skin and be absorbed.
[0003] In ancient Chinese medicine, there is no clear corresponding disease name for atopic dermatitis, but according to its characteristics, symptoms, etc., it can be classified into "Siwan Feng", "Nai Xian", "Tai Chuang", etc. Traditional Chinese medicine has a long history of treating it, with flexible and diverse methods. It can implement a treatment that takes both the root and the branch on the basis of combining the four diagnostic methods and differentiating syndromes and types. According to the condition, traditional Chinese medicine is taken orally and combined with external treatment of traditional Chinese medicine, acupuncture, etc., gradually forming a diversified, unique and highly effective traditional Chinese medicine treatment system.
[0004] Purslane, also known as purslane herb, five-element vegetable, longevity vegetable, etc., is a common medicinal and edible plant on the roadside of fields. "Chinese Pharmacopoeia" records that purslane is "cold in nature and sour in taste" and has the effects of "clearing heat and detoxifying, stopping bleeding and cooling blood, and relieving dysentery", and is used to treat "heat-toxic bloody dysentery, carbuncles and sores, eczema, erysipelas, snake and insect bites, hematochezia, hemorrhoids, metrorrhagia and metrostaxis". Purslane is rich in various active ingredients, such as alkaloids, polysaccharides, flavonoids, organic acids, etc. Research shows that purslane has various effects, such as antioxidant, hypoglycemic, hypolipidemic, antihypertensive, antibacterial, liver-protecting, nerve-protecting, etc.
[0005] Exosomes secreted by traditional Chinese medicine have been taken seriously in recent years. These exosomes are secreted by the plant itself and contain various components, including proteins, lipids, nucleic acids and secondary metabolites, and the lipid bilayer of exosomes can protect the internal components. Plant exosomes are mostly used for oral administration in the treatment of gastrointestinal diseases, and there is currently no relevant report on applying them in the form of microneedles to the treatment of skin diseases. Summary of the Invention
[0006] To solve the above technical problems, the present invention includes the following aspects:
[0007] The first aspect of the present invention provides a microneedle patch for treating atopic dermatitis. The microneedle patch is composed of soluble needles and a backing layer, and the soluble needles contain purslane exosomes and an antioxidant carrier material.
[0008] Preferably, each microneedle patch contains 10 - 100 μg of protein - containing purslane exosomes. More preferably, each microneedle patch contains 30 - 80 μg of protein - containing purslane exosomes. Even more preferably, each microneedle patch contains 50 μg of protein - containing purslane exosomes.
[0009] Preferably, the antioxidant carrier material is hyaluronic acid modified with 4 - amino - TEMPO.
[0010] Preferably, each microneedle patch contains 0.5 - 2 mg of the antioxidant carrier material. More preferably, each microneedle patch contains 1 - 1.5 mg of the antioxidant carrier material. Even more preferably, each microneedle patch contains 1.2 mg of the antioxidant carrier material.
[0011] Preferably, the weight ratio of the protein amount of purslane exosomes to the antioxidant carrier material in the microneedle patch is 1:(10 - 50). More preferably, the weight ratio of the protein amount of purslane exosomes to the antioxidant carrier material in the microneedle patch is 1:(20 - 30). Even more preferably, the weight ratio of the protein amount of purslane exosomes to the antioxidant carrier material in the microneedle patch is 1:(22 - 26). Even more preferably, the weight ratio of the protein amount of purslane exosomes to the antioxidant carrier material in the microneedle patch is 1:24.
[0012] Preferably, the particle size of the purslane exosomes is 100 - 300 nm. More preferably, the particle size of the purslane exosomes is 150 - 250 nm. Even more preferably, the particle size of the purslane exosomes is 200 - 250 nm.
[0013] Preferably, the preparation method of the purslane exosomes is as follows: The crude purslane exosomes are separated from purslane by ultra - centrifugation, and then purified by size - exclusion chromatography or density - gradient centrifugation to obtain the purslane exosomes.
[0014] Preferably, the size - exclusion chromatography uses an Exo - spin TM midi purification column for purification.
[0015] Preferably, the preparation method of the purslane exosomes is as follows:
[0016] (1) Soak purslane in buffer solution and then juice it, and filter to remove residues;
[0017] (2) Centrifuge the filtrate obtained in step (1) multiple times, and take the supernatant after each centrifugation;
[0018] (3) Centrifuge the filtrate obtained in step (2) at low temperature and high speed, and remove the supernatant after centrifugation;
[0019] (4) Resuspend the precipitate obtained in step (3) with a buffer to obtain a crude exosome product;
[0020] (5) Purify the crude exosome product in step (4) using size exclusion or density gradient centrifugation to obtain the purified purslane exosomes.
[0021] Preferably, the buffer in step (1) is MES buffer.
[0022] Preferably, in step (1), purslane and MES buffer are mixed at a weight-to-volume ratio (g / ml) of 1:1.
[0023] Preferably, in step (2), first centrifuge at 500 g - 2000 g for 5 - 15 min, take the supernatant; then centrifuge the supernatant at 2000 g - 5000 g for 15 - 45 min, take the supernatant; then centrifuge the supernatant at 5000 g - 20000 g for 0.5 - 2 h, take the supernatant. More preferably, in step (2), first centrifuge at 1000 g for 10 min, take the supernatant; then centrifuge the supernatant at 3000 g for 30 min, take the supernatant; then centrifuge the supernatant at 10000 g for 1 h, take the supernatant.
[0024] Preferably, in step (3), centrifuge at 50000 g - 200000 g for 0.5 - 2 h at 2 - 8°C. More preferably, in step (3), centrifuge at 100000 g for 1 h at 4°C.
[0025] Preferably, the buffer in step (4) is PBS buffer.
[0026] Preferably, the size exclusion method in step (5) uses an Exo-spin TM midi purification column for purification.
[0027] Preferably, the operating method of the density gradient centrifugation method in step (5) is as follows:
[0028] (i) Prepare sucrose into 4 concentrations of 60%, 40%, 20%, and 10% with PBS; add each concentration of sucrose solution into an ultracentrifugation tube;
[0029] (ii) Add the crude exosome product solution in step (4) into the ultracentrifugation tube and centrifuge at low temperature and high speed;
[0030] (iii) Discard the upper part of the solution and collect the solution in the middle part;
[0031] (iv) Centrifuge the solution in step (iii) at low temperature and high speed, remove the supernatant, and resuspend with a buffer to obtain the product.
[0032] Preferably, 4-7 mL of sucrose solution at each concentration is added in step (i). More preferably, 5-6 mL of sucrose solution at each concentration is added in step (i). Even more preferably, 5.5 mL of sucrose solution at each concentration is added in step (i).
[0033] Preferably, 0.4-0.8 mL of crude exosome solution is in the ultracentrifugation tube in step (ii). More preferably, 0.5-0.7 mL of crude exosome solution is in the ultracentrifugation tube in step (ii). Even more preferably, 0.6 mL of crude exosome solution is in the ultracentrifugation tube in step (ii).
[0034] Preferably, in steps (ii) and (iv), centrifugation is carried out at 1-10 °C at 20000 g - 200000 g for 0.2-2 h. More preferably, in step (ii), centrifugation is carried out at 2-6 °C at 50000 g - 150000 g for 0.5-1.5 h. Even more preferably, in step (ii), centrifugation is carried out at 4 °C at 100000 g for 1 h.
[0035] Preferably, in step (iii), the top 3-7 mL of the solution is discarded, and the middle 4-7 mL of the solution is collected. More preferably, in step (iii), the top 4-6 mL of the solution is discarded, and the middle 5-6 mL of the solution is collected. Even more preferably, in step (iii), the top 5 mL of the solution is discarded, and the middle 5.5 mL of the solution is collected.
[0036] Preferably, the antioxidant carrier material is prepared from hyaluronic acid and 4-amino-TEMPO in the presence of an acid-binding agent and a hydroxyl activating reagent.
[0037] Preferably, the molecular weight of the hyaluronic acid is 5000-50000. More preferably, the molecular weight of the hyaluronic acid is 8000-20000. Even more preferably, the molecular weight of the hyaluronic acid is 10000.
[0038] Preferably, the acid-binding agent is sodium acetate.
[0039] Preferably, the hydroxyl activating reagent is selected from one or more of benzyl chloroformate, isopropyl chloroformate, and ethyl chloroformate. More preferably, the hydroxyl activating reagent is benzyl chloroformate.
[0040] Preferably, the weight ratio of hyaluronic acid to 4-amino-TEMPO is (5-50):1. More preferably, the weight ratio of hyaluronic acid to 4-amino-TEMPO is (10-30):1. Even more preferably, the weight ratio of hyaluronic acid to 4-amino-TEMPO is (20-25):1. Even more preferably, the weight ratio of hyaluronic acid to 4-amino-TEMPO is 22:1.
[0041] Preferably, the weight ratio of the acid binding agent to the hydroxyl activating agent is 1:(1-5). More preferably, the weight ratio of the acid binding agent to the hydroxyl activating agent is 1:(1-3). Further preferably, the weight ratio of the acid binding agent to the hydroxyl activating agent is 1:2.
[0042] Preferably, the preparation method of the antioxidant carrier material is as follows:
[0043] (1) dissolving hyaluronic acid in water, adding an acid binding agent and a hydroxyl activating agent in sequence, and stirring the mixture to react;
[0044] (2) adding 4-amino-TEMPO to the reaction solution obtained in step (1), adjusting the pH value of the solution to 7-9, and stirring the reaction sufficiently;
[0045] (3) placing the reaction solution obtained in step (2) in a dialysis bag for dialysis, and then freeze-drying the reaction solution in the dialysis bag to obtain the product.
[0046] Preferably, the weight ratio of the hyaluronic acid in step (1) to the 4-amino-TEMPO in step (2) is (5-50):1. More preferably, the weight ratio of the hyaluronic acid in step (1) to the 4-amino-TEMPO in step (2) is (10-30):1. Further preferably, the weight ratio of the hyaluronic acid in step (1) to the 4-amino-TEMPO in step (2) is (20-25):1. Further preferably, the weight ratio of the hyaluronic acid in step (1) to the TEMPO in step (2) is 22:1.
[0047] Preferably, the weight ratio of the acid binding agent to the hydroxyl activating agent in step (1) is 1:(1-5). More preferably, the weight ratio of the acid binding agent to the hydroxyl activating agent in step (1) is 1:(1-3). Further preferably, the weight ratio of the acid binding agent to the hydroxyl activating agent in step (1) is 1:2.
[0048] Preferably, the molecular weight of the hyaluronic acid in step (1) is 5000-50000. More preferably, the molecular weight of the hyaluronic acid is 8000-20000. Further preferably, the molecular weight of the hyaluronic acid is 10000.
[0049] Preferably, the acid binding agent in step (1) is sodium acetate.
[0050] Preferably, the hydroxyl activating agent in step (1) is selected from one or more of benzyl chloroformate, isopropyl chloroformate, and ethyl chloroformate. More preferably, the hydroxyl activating agent is benzyl chloroformate.
[0051] Preferably, in step (1), an acid-binding agent is added first and stirred for 1-3 hours, then a hydroxyl activating reagent is added and the stirring reaction is continued for 12-36 hours. More preferably, in step (1), an acid-binding agent is added first and stirred for 2 hours, then a hydroxyl activating reagent is added and the stirring reaction is continued for 24 hours.
[0052] Preferably, in step (2), the pH value of the solution is adjusted to 7.5-8.8. More preferably, in step (2), the pH value of the solution is adjusted to 7.8-8.5.
[0053] Preferably, the stirring reaction time in step (2) is 12-36 hours. More preferably, the stirring reaction time in step (2) is 24 hours.
[0054] Preferably, the cut-off molecular weight of the dialysis bag in step (3) is 500-5000. More preferably, the cut-off molecular weight of the dialysis bag in step (3) is 1000-3000. Further preferably, the cut-off molecular weight of the dialysis bag in step (3) is 2000.
[0055] Preferably, the dialysis time in step (3) is 1-3 days. More preferably, the dialysis time in step (3) is 2 days.
[0056] Preferably, the backing layer is made of one or more selected from polylactic acid, polyvinylpyrrolidone, polyethylene glycol, and hydroxypropyl methylcellulose. More preferably, the backing layer is made of polyvinylpyrrolidone. Further preferably, the backing layer is made of PVP-K90.
[0057] Preferably, the length and width of the microneedle patch are both 0.5-2 cm, and the height of the backing is 0.5-1.5 mm. More preferably, the length and width of the microneedle patch are both 0.5-1 cm, and the height of the backing is 1-1.5 mm. Further preferably, the length and width of the microneedle patch are both 1 cm, and the height of the backing is 1 mm.
[0058] Preferably, the height of each needle body in the microneedle patch is 400-800 μm, and the tip diameter is 200-400 μm. More preferably, the height of each needle body in the microneedle patch is 500-700 μm, and the tip diameter is 250-350 μm. Further preferably, the height of each needle body in the microneedle patch is 600 μm, and the tip diameter is 300 μm.
[0059] Preferably, the tip array in the microneedle patch is 12×12, and there are 144 needles in each patch in total.
[0060] The second aspect of the present invention provides a preparation method of the above microneedle patch, and the preparation method comprises the following steps:
[0061] (1) Preparation of soluble microneedle polymer solution: Take purslane exosomes, discard the supernatant after centrifugation, dissolve with water, mix the antioxidant carrier material with the purslane exosome solution to obtain a mixed solution, and centrifuge to remove air bubbles;
[0062] (2) Preparation of soluble microneedles: Inject the mixed solution obtained in step (1) into a microneedle mold, centrifuge to cover the mold, remove the excess solution, and dry the microneedle mold containing the soluble microneedles;
[0063] (3) Preparation of backing layer polymer solution: Add the backing layer polymer material to water for dissolution, and centrifuge to remove air bubbles;
[0064] (4) Preparation of microneedle patch: Aspirate the backing layer polymer solution prepared in step (3), inject it into the microneedle mold containing the soluble microneedles in step (2), centrifuge to cover the mold, and after drying, peel it off from the microneedle mold to obtain the microneedle patch.
[0065] The third aspect of the present invention provides the application of the above-mentioned microneedle patch in the preparation of a drug for treating inflammation.
[0066] Preferably, the inflammation is atopic dermatitis.
[0067] Technical effects produced by the present invention:
[0068] 1. The present invention unexpectedly discovers that purslane exosomes have significant immunomodulatory and anti-inflammatory effects, and creatively combines the ultracentrifugation method with the size exclusion method to efficiently prepare purslane exosomes. The yield of exosomes quantified by BCA protein is as high as 3470 μg / 100 g purslane.
[0069] 2. In addition to immune disorders and inflammation, the pathogenesis of atopic dermatitis also has a large accumulation of ROS to generate oxidative stress. The present invention uses an antioxidant carrier material to load purslane exosomes, and the antioxidant matrix therein is low molecular weight hyaluronic acid (HA) modified with 4-amino-2,2,6,6-tetramethylpiperidine oxide (4-amino-TEMPO), while purslane exosomes play a role in regulating immunity and anti-inflammation, effectively avoiding the defect of limited effects of single traditional Chinese medicine. The test results show that the antioxidant carrier material loaded with purslane exosomes of the present invention can treat atopic dermatitis mice, reduce the skin scab area, reduce the epidermal thickness and mast cell infiltration, and reduce Th2 cytokines.
[0070] 3. The microneedle patch preparation of the present invention can transdermally deliver the drug to the dermis layer, playing a role in treating atopic dermatitis, and effectively solving the problem that it is difficult for topical preparations to transdermally absorb through the thickened epidermis of atopic dermatitis patients. Description of the Drawings
[0071] Figure 1 These are the morphological characterization results of purslane exosomes, where Figure 1 A is the transmission electron microscope image of purslane exosomes, Figure 1 B is the particle size diagram of purslane exosomes;
[0072] Figure 2 These are the characterization results of the antioxidant carrier material HA-TEMPO, where Figure 2 A is the synthesis chemical formula of HA-TEMPO, Figure 2 B is the NMR spectrum of HA-TEMPO (the arrow indicates the characteristic peak of TEMPO), Figure 2 C is the electron microscope image of HA-TEMPO, Figure 2 D is the anti-ROS result of HA-TEMPO in vitro in HaCaT cells;
[0073] Figure 3 These are the characterization results of purslane exosome-loaded micro-needle patches with antioxidant carrier materials, where Figure 3 A is the bright field image and electron microscope image of the micro-needle patch, Figure 3 B is the schematic diagram after the micro-needles penetrate the rat skin (the blue small holes represent the positions where the micro-needles penetrate the skin), Figure 3 C is the distribution map of purslane exosomes in the needle body (the red fluorescence is the purslane exosomes stained with DiD), Figure 3 D is the release rate result of purslane exosomes in the micro-needle patch;
[0074] Figure 4 These are the pharmacodynamic test results of the micro-needle patch of the present invention in atopic dermatitis mice, where Figure 4 A is the scabbing condition and area ratio of the back skin of mice in each test group; Figure 4 B is the levels of IFN-γ, IL-13, IgE and IL-4 in the skin of mice in each test group; Figure 4 C is the immunofluorescence staining images of oxidative damage markers 4-HNE and 8-OHDG in the skin of mice in each test group; Figure 4 D is the level of 4-HNE in the skin of mice in each test group; Figure 4 E is the level of 8-OHDG in the skin of mice in each test group. Detailed implementation mode
[0075] The present invention will be further described below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.
[0076] Preparation Example 1. Extraction of purslane exosomes (ultracentrifugation combined with size exclusion method)
[0077] (1) Weigh 100 g of purslane, soak it in MES overnight according to the ratio of purslane:MES buffer (g / ml) = 1:1 after cleaning;
[0078] (2) Use a juicer to juice the purslane and MES, and filter it through gauze to remove plant residues;
[0079] (3) Centrifuge the filtrate at 1000 g for 10 min and take the supernatant;
[0080] (4) Centrifuge the supernatant in step (3) at 3000 g for 30 min and take the supernatant;
[0081] (5) Centrifuge the supernatant in step (4) at 10000 g for 1 h and take the supernatant;
[0082] (6) Centrifuge the supernatant in step (5) at 100000 g for 1 h at 4 °C, and then remove the supernatant;
[0083] (7) Add 1 ml of PBS to resuspend the precipitate in step (6) to obtain a crude exosome product, and perform exosome purification according to the instructions of the Exo-spin TM midi purification column;
[0084] (8) Remove the screw cap of the Exo-spin TM midi purification column, pour out and retain the anti-corrosion buffer in the column top, and remove the plug at the bottom outlet of the column;
[0085] (9) Balance the purification column by continuously adding 2 times 10 ml of PBS to the column top, and let the PBS drain naturally under gravity;
[0086] (10) After the purification column is balanced, install the purification column on the clamp seat and place a 1.5 ml centrifuge tube at the bottom outlet of the column;
[0087] (11) Carefully add half of the volume, that is, 500 μl of the exosome sample in step (7) from the column top, collect the flowing liquid by gravity, and label this centrifuge tube as fraction 1;
[0088] (12) Place a 1.5 ml centrifuge tube at the bottom outlet of the column, add the remaining 500 μl of the exosome sample in step (7) from the column top, collect the flowing liquid by gravity, and label this centrifuge tube as fraction 2;
[0089] (13) Place a 1.5 ml centrifuge tube at the bottom outlet of the column, add 500 μl of PBS from the column top, collect the flowing liquid by gravity, and label this centrifuge tube as fraction 3;
[0090] (14) Repeat step (13) 21 times and number them continuously until 24 fractions of 500 μl are collected;
[0091] (15) Collect fractions 7 - 12, and combine them to obtain about 3 ml of highly pure purslane exosomes;
[0092] (16) Fill the column with PBS from the top of the column and wait for it to drain to wash the column. Then close the outlet plug at the bottom of the column, and add the anti - corrosion buffer solution collected in step (8) to the top of the column to preserve the purification column.
[0093] Using the size - exclusion method of this preparation example to purify exosomes, the yield of exosomes quantified by BCA protein is 3470 ± 502.7 μg / 100 g purslane.
[0094] Preparation Example 2. Extract purslane exosomes (ultra - centrifugation method combined with density gradient centrifugation method)
[0095] (1) Weigh 100 g of purslane, soak it in MES overnight according to the ratio of purslane:MES buffer (g / ml) = 1:1 after washing;
[0096] (2) Use a juicer to juice purslane with MES, and filter it through gauze to remove plant residues;
[0097] (3) Centrifuge the filtrate at 1000 g for 10 min, and take the supernatant;
[0098] (4) Centrifuge the supernatant in step (3) at 3000 g for 30 min, and take the supernatant;
[0099] (5) Centrifuge the supernatant in step (4) at 10000 g for 1 h, and take the supernatant;
[0100] (6) Centrifuge the supernatant in step (5) at 100000 g for 1 h at 4 °C, then remove the supernatant, and resuspend the precipitate in this step with 0.6 ml PBS;
[0101] (7) Prepare 4 concentrations of sucrose with PBS: 60%, 40%, 20%, 10%;
[0102] (8) Use a syringe to add the prepared 60%, 40%, 20%, 10% sucrose solutions to the ultra - centrifugation tube along the tube wall in sequence, and add 5.5 ml of each concentration of sucrose solution;
[0103] (9) Then add the 0.6 mL exosome solution in step (6) on the top, for a total of 22.6 mL;
[0104] (10) Centrifuge the sample solution in step (9) at 100000 g for 1 h at 4 °C;
[0105] (11) Discard the top 5 mL of the solution and collect the middle 5.5 mL of the solution;
[0106] (12) Centrifuge the solution in step (11) at 100,000 g for 1 h at 4 °C, then remove the supernatant and resuspend the precipitate in this step with 1 mL of PBS.
[0107] The density gradient centrifugation method of this preparation example was used to purify exosomes, and the yield of exosomes quantified by BCA protein was 413.3 ± 124.2 μg / 100 g of purslane. Thus, it can be seen that the exosome yield of this method is significantly lower than that of the size exclusion method of Preparation Example 1.
[0108] Preparation Example 3: Synthesis of antioxidant carrier material HA-TEMPO
[0109] (1) Dissolve 3.79 g of hyaluronic acid (HA) with a molecular weight of 10,000 in 15 ml of ultrapure water and place it on a magnetic stirrer until dissolved;
[0110] (2) Add 84 mg of sodium acetate to step (1) and stir continuously for 2 h;
[0111] (3) Under vigorous stirring of the magnetic stirrer, add 172 mg of benzyl chloroformate to step (2) to activate the hydroxyl group; stir and react at room temperature for 24 h;
[0112] (4) Add 172 mg of 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidine oxide) to step (3), adjust the pH value to 7.8 - 8.5 with 1 M NaOH, and stir and react at room temperature for 24 h;
[0113] (5) Add the solution in step (4) to a dialysis bag with a molecular weight cut-off of 2000, place it in a large beaker filled with ultrapure water, and dialyze in the dark for 2 days;
[0114] (6) Lyophilize the reaction solution in step (5) to obtain the HA-TEMPO product.
[0115] Preparation Example 4: Preparation of antioxidant carrier material-loaded purslane exosome soluble microneedle patch
[0116] (1) Prepare a soluble needle body polymer solution
[0117] Take purslane exosomes with a protein content of 5 mg, centrifuge at 110,000 g for 70 min, discard the supernatant, and dissolve with 200 μl of ultrapure water. Weigh 120 mg of HA-TEMPO and add it to the above exosome solution for mixing to obtain a mixed solution.
[0118] (2) Prepare soluble needles
[0119] Place a syringe containing 50 μl of the mixed solution from step (1) on one side of the microneedle mold, centrifuge at 4000 rpm for 3 min to cover the mold, scrape off the excess polymer solution. Each microneedle patch contains approximately 2 μl of the mixed solution from step (1) in the needle body, and place it in a silica gel desiccator to dry for 12 h.
[0120] (3) Prepare the backing polymer solution
[0121] Weigh 8 g of PVP-K90, add 20 ml of ultrapure water and stir to dissolve, centrifuge at 4000 rpm for 3 min to remove air bubbles.
[0122] (4) Prepare the microneedle patch
[0123] Draw 100 μl of the solution prepared in step (3) and inject it into the microneedle mold containing the soluble needle body in step (2), centrifuge at 4000 rpm for 3 min to cover the mold, place it in a silica gel desiccator to dry for 24 h, and after drying, peel off the microneedle mold to obtain a soluble microneedle patch.
[0124] Experimental Example 1. Morphological characterization of purslane exosomes
[0125] 1. Test method
[0126] Draw 10 μl of the purslane exosome solution extracted by the method of Preparation Example 1, drop it on a special copper mesh for transmission electron microscopy, after staying for 1 min, suck it off with filter paper. Carefully drop 20 μl of uranyl acetate on the copper mesh for staining, after staying for 1 min, suck it off with filter paper. Take pictures of the exosome morphology through the transmission electron microscope.
[0127] Draw 50 μl of the purslane exosome solution extracted by the method of Preparation Example 1, add it to the cuvette in the particle size analyzer to measure the particle size.
[0128] 2. Test results
[0129] As Figure 1 shown, purslane exosomes have a cup-shaped structure, conforming to the structure of exosomes, and their average particle size is 205.6 nm.
[0130] Experimental Example 2. Characterization of the antioxidant carrier material HA-TEMPO
[0131] 1. Test method
[0132] Use nuclear magnetic resonance hydrogen spectrum to identify the HA-TEMPO synthesized in Preparation Example 3. Weigh 6 g of the freeze-dried powder of HA-TEMPO, add 10 ml of ultrapure water, place it at room temperature for 2 h, and observe its gelation situation. Place the freeze-dried powder of HA-TEMPO on a metal bracket, spray gold and then observe the material structure under a scanning electron microscope.
[0133] HaCaT cells were incubated in a 12-well plate at a density of 60,000 cells per well. After 24 h, HA-TEMPO was dissolved in a hydrogen peroxide medium with a concentration of 1 mM to make its concentrations 0.125, 0.25, and 0.5 mg / mL. 1 mL of the drug solution was added to each well, and the ROS level was measured by flow cytometry after 4 h.
[0134] 2. Test results
[0135] As Figure 2 shown in B, the characteristic peaks of TEMPO in HA-TEMPO were identified by 1H NMR, indicating successful synthesis. As Figure 2 shown in C, HA-TEMPO exhibited a lamellar structure. Figure 2 The results in D showed that HA-TEMPO had the effect of scavenging ROS.
[0136] Experimental Example 3. Characterization of soluble microneedle patches loaded with antioxidant carrier materials and purslane exosomes
[0137] 1. Test method
[0138] The soluble microneedle patch was prepared by referring to the method of Preparation Example 4, and its morphology was observed under an upright microscope. Then, the microneedle patch was adhered to a metal bracket, sputter-coated with gold, and its morphology was observed under a scanning electron microscope.
[0139] Approximately 5 mg of purslane exosome solution was co-incubated with 10 μM cell membrane probe DiD for exosome staining. Then, the DiD-stained microneedle patch was prepared by referring to the method of Preparation Example 4, and the distribution of DiD-labeled purslane exosomes in the microneedles was observed under a laser confocal microscope.
[0140] The above microneedle patch was placed in 2 mL of PBS and stirred continuously. 100 μL of the leaching solution was taken out at 5, 30, 60, 120, 180, and 300 s, and the same volume of PBS was immediately replenished. The protein concentration in the leaching solution was measured using BCA.
[0141] 2. Test results
[0142] As Figure 3 shown in A, the base of the microneedle was flat, the needles were arranged uniformly, and the microneedles presented a pyramid shape with clear shape, consistent size and height. The length and width of the microneedle patch were 1 cm × 1 cm, the height of the needle body was 600 μm, the height of the backing was 1 mm, the tip diameter was 300 μm, the tip array was 12 × 12, and each patch had a total of 144 needles. As Figure 3 shown in B, the microneedles could successfully penetrate the skin of ex vivo rats, leaving a blue micropore array on the skin surface. The number of blue micropores in the array was close to the number of microneedle bodies, and the penetration efficiency was 97.68 ± 1.45%. As Figure 3As shown in C, both the tip and the shaft of the microneedles are rich in Portulaca oleracea exosomes stained with DiD, and they are distributed in dots and patches, indicating that the Portulaca oleracea exosomes have been successfully loaded into the microneedles. As Figure 3 As shown in D, the microneedles can rapidly dissolve and release Portulaca oleracea exosomes within 5 min.
[0143] Experimental Example 4. Therapeutic effect of the microneedle patch of the present invention on mice with atopic dermatitis model
[0144] 1. Test method
[0145] 1.1. Establish a mouse atopic dermatitis model
[0146] Select 15 - 17 g Balb / c female mice. After depilation on the back, the model group and the microneedle group were given 1% DNCB solution on the back, where the solvent was acetone: olive oil = 4:1. It was continuously applied for 3 d, and then no treatment was done for 4 d. On the morning of the 8th day, 4% SDS solution was applied, and 0.5% DNCB solution was applied in the afternoon. Starting from the next day, 0.375% DNCB was applied every morning for 11 d. On the afternoon of the 11th, 14th, 17th, and 20th days, the microneedle group applied the microneedles prepared in Preparation Example 4 (i.e., a microneedle patch with a length and width of 1 cm × 1 cm, a shaft height of 600 μm, a backing height of 1 mm, a tip diameter of 300 μm, and a tip array of 12 × 12) on the back, and fixed it with tape. The backing was removed after 30 min.
[0147] 1.2. Measure the scab area of the back skin
[0148] On the 21st day, the general condition of the skin of the mice in each test group was photographed and recorded, and the skin scab condition was calculated using image J.
[0149] 1.3. Detect the levels of inflammatory factors in the skin
[0150] On the 21st day, the mice were sacrificed, 100 mg of back skin was taken, 0.9 ml of PBS was added and homogenized, centrifuged at 5000 g for 10 min, and the supernatant was used to measure the levels of IFN - γ, IL - 13, IgE, and IL - 4 by the Elisa method.
[0151] 1.4. Detect the levels of oxidative factors in the skin
[0152] On the 21st day, the skin was taken, fixed in paraformaldehyde for 48 h, then paraffin - embedded, sectioned, and stained with 4 - HNE and immunofluorescently stained with 8 - OHDG.
[0153] 2. Test results
[0154] As Figure 4 As shown in A, the microneedle group of the present invention can significantly reduce the proportion of the skin scab area (P < 0.0001).
[0155] IL-4 and IL-13 are Th2-type cytokines, and IFN-γ is a Th1-type cytokine. As Figure 4 shown in Figure B, the microneedle group can significantly reduce the levels of IL-4, IL-13 and IgE in the skin of AD mice, and significantly increase the level of IFN-γ in the skin of AD mice, indicating that the microneedle patch of the present invention has significant anti-inflammatory and immunomodulatory effects. As Figure 4 shown in Figures C to 4E, the microneedle group significantly reduced the levels of the skin lipid peroxidation product 4-HNE and the DNA oxidative damage marker 8-OHDG, indicating that the microneedle has antioxidant ability.
[0156] Although specific embodiments of the present invention have been described, those skilled in the art should recognize that various changes and modifications can be made to the present invention without departing from the scope or spirit of the present invention. Thus, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.
Claims
1. A microneedle patch for treating atopic dermatitis, characterized in that, The microneedle patch is composed of soluble needles and a backing layer, and the soluble needles contain purslane exosomes and an antioxidant carrier material.
2. The microneedle patch according to claim 1, wherein The antioxidant carrier material is 4-amino-TEMPO modified hyaluronic acid.
3. The microneedle patch according to claim 1, wherein In the microneedle patch, the weight ratio of the protein content of purslane exosomes to the antioxidant carrier material is 1:(10 - 50).
4. The microneedle patch according to claim 1, wherein The preparation method of the purslane exosomes is as follows: purslane is separated by ultracentrifugation to obtain crude purslane exosomes, and then purified by size exclusion method or density gradient centrifugation to obtain the purslane exosomes.
5. The microneedle patch according to claim 4, wherein The size exclusion method uses an Exo-spin TM midi purification column for purification.
6. The microneedle patch according to claim 2, wherein, The antioxidant carrier material is prepared from hyaluronic acid and 4-amino-TEMPO in the presence of an acid-binding agent and a hydroxyl activating reagent.
7. The microneedle patch according to claim 1, wherein The backing layer is made of one or more selected from polylactic acid, polyvinylpyrrolidone, polyethylene glycol, and hydroxypropyl methylcellulose.
8. The microneedle patch according to claim 1, wherein, The length and width of the microneedle patch are both 0.5 - 2 cm, and the height of the backing layer is 0.5 - 1.5 mm.
9. The preparation method of the microneedle patch according to any one of claims 1-8, characterized in that, The preparation method comprises the following steps: (1) Prepare a soluble needle polymer solution: Take purslane exosomes, discard the supernatant after centrifugation, dissolve with water, mix the antioxidant carrier material with the purslane exosome solution to obtain a mixed solution, and centrifuge to remove air bubbles; (2) Prepare soluble needles: Inject the mixed solution obtained in step (1) into a microneedle mold, centrifuge to cover the mold, remove the excess solution, and dry the microneedle mold containing the soluble needles; (3) Prepare a backing layer polymer solution: Add the backing layer polymer material to water for dissolution, and centrifuge to remove air bubbles; (4) Prepare the microneedle patch: Aspirate the backing layer polymer solution prepared in step (3), inject it into the microneedle mold containing the soluble needles in step (2), centrifuge to cover the mold, dry, and peel it off from the microneedle mold to obtain the microneedle patch.
10. Use of the microneedle patch according to any one of claims 1 - 8 in the preparation of a medicament for treating inflammation.