Application of hypoxia Wharton jelly mesenchymal stem cell derivative in preparation for improving / treating atopic dermatitis

Abstract

The invention discloses an application of a low-oxygen Wharton jelly mesenchymal stem cell derivative in a preparation for improving / treating atopic dermatitis, and a gel agent which is developed from the low-oxygen Wharton jelly mesenchymal stem cell derivative and has an excellent treatment effect and is used for improving / treating the atopic dermatitis. The compound can stimulate the generation of cell derivatives comprising a large number of cytokines, growth factors and extracellular vesicles (EV) thereof, and through the synergistic effect of the growth factors, the cytokines and the extracellular vesicles thereof, multiple-effect improvement and treatment can be performed on AD through dual mechanisms of inflammation resistance and cell regeneration promotion.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to the application of hypoxic Wharton's jelly mesenchymal stem cell derivatives in preparations for improving / treating atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD), also known as atopic eczema, also known as "atopic dermatitis" and "genetic allergic dermatitis", is a chronic, recurrent, inflammatory skin disease characterized by dry skin, severe itching and eczematous rash. It often begins in infancy. The cause of the disease is still unclear. It often recurs, and the itching is severe at night, which can affect sleep. In severe cases, it affects daily life, work and study. Atopic dermatitis has different characteristics in different age groups and can be divided into three stages:

[0003] Infancy (birth to 2 years old), also known as "infant eczema", first appears on the cheeks, red spots, papules, papulovesicles, dense patches, polymorphic lesions, unclear boundaries, scratching, friction quickly form erosion, exudation and scabs, etc. Lesions can quickly spread to the scalp, forehead, neck, limbs, etc. Itching is severe, often causing babies to cry and sleep disturbed.

[0004] In childhood (2 to 12 years old), it can develop from infancy or develop directly. Lesions often occur in the antecubital fossa, popliteal fossa, wrist flexor (the back of the wrist), and can also be seen on the neck, face and eyelids. The lesions are dark red, with less exudation than infancy, often accompanied by scratches, and lichenification over time. If the itching is still severe, a vicious cycle of "itch-scratch-itch" can be formed.

[0005] Adolescents and adults (over 12 years old) may develop from childhood or develop directly, and are prone to occur in the elbows, popliteal fossa, front of the neck, etc., and can also be seen on the trunk, limbs, face, and eyelids. The skin lesions often show localized lichenification, sometimes acute or subacute eczematous changes, and some patients have skin lesions as generalized dry papules. Itching is severe, and scratches, blood crusts, scales, pigmentation, etc. can be seen around the lesions.

[0006] At present, the therapeutic effects of therapeutic preparations for atopic dermatitis are limited, such as the technical solution with Chinese patent publication number CN104173207A, which focuses on skin barrier repair and lipid supplementation, but is limited in deep skin inflammation and cell repair, only relieves symptoms, and cannot target potential cell dysfunction in AD, and has a single therapeutic effect. There are also treatments that use mesenchymal stem cells or their secretions for cell regeneration, but they are limited in the treatment of skin inflammation, and the above preparations are mostly achieved by injection, focusing on systemic or targeted biological delivery, and the treatment is slow to take effect. Summary of the invention

[0007] Therefore, based on the above background, the present application provides the use of hypoxic Wharton's jelly mesenchymal stem cell derivatives in preparations for improving / treating atopic dermatitis, and the development of a gel for improving / treating atopic dermatitis with excellent therapeutic effects, so as to provide a new direction for the treatment of atopic dermatitis.

[0008] The technical solution provided by the present invention is:

[0009] Application of hypoxic Wharton's jelly mesenchymal stem cell derivatives in improving / treating atopic dermatitis.

[0010] Furthermore, the hypoxic Wharton's jelly mesenchymal stem cell derivative is obtained by culturing Wharton's jelly mesenchymal stem cells in an environment of low oxygen content of 1-5vt%.

[0011] Based on the same inventive concept, the present invention also provides a gel for improving / treating atopic dermatitis, wherein the active ingredient is the hypoxic Wharton's jelly mesenchymal stem cell derivative prepared above.

[0012] Furthermore, the preparation of the gel comprises the following steps:

[0013] 1) encapsulating hypoxic Wharton's jelly mesenchymal stem cell derivatives in poly(lactic-co-glycolic acid) microspheres; 2) preparing a hydrogel;

[0014] 3) Add the poly(lactic acid-glycolic acid) microspheres prepared in step 1) into the hydrogel and stir evenly.

[0015] Further, the preparation operation of the hydrogel in step 2) is as follows:

[0016] After adding a certain amount of ultrapure water to hyaluronic acid and stirring evenly, polyethylene glycol is added according to the amount and stirred evenly, and then glycerol is slowly added and stirred evenly to prepare a hydrogel;

[0017] Alternatively, alginate is dissolved in purified water by stirring, and then a cross-linking agent is added and stirred to form a hydrogel;

[0018] Alternatively, a certain amount of ultrapure water is added to hyaluronic acid, the mixture is stirred evenly, polyvinyl alcohol is added according to the amount, the mixture is stirred evenly, and glycerol is slowly added and stirred evenly to prepare a hydrogel.

[0019] Furthermore, the cross-linking agent is a carboxyl-containing water-soluble polysaccharide and its derivatives.

[0020] Furthermore, the mass ratio of hyaluronic acid, polyvinyl alcohol or polyethylene glycol, and glycerol is 3:3:(1-2).

[0021] Furthermore, in step 3), the ratio of poly(lactic acid-glycolic acid) microspheres to hydrogel is (2-5):10.

[0022] Based on the same inventive concept, the present invention also provides a dressing for improving / treating atopic dermatitis, which is made of the gel for improving / treating atopic dermatitis.

[0023] Based on the same inventive concept, the present invention applies the above-mentioned hypoxic Wharton's jelly mesenchymal stem cell derivative and a gel for improving / treating atopic dermatitis to prepare a preparation for improving / treating any of the following diseases:

[0024] Such diseases include:

[0025] ①Ulcers and pressure sores caused by diabetes;

[0026] ② Wounds or burns caused by mechanical trauma;

[0027] ③Chronic inflammatory skin diseases including psoriasis.

[0028] The above technical solution has the following beneficial effects:

[0029] The present invention utilizes Wharton's jelly mesenchymal stem cells to be cultured in a hypoxic environment, which can stimulate them to generate a large number of cytokines, growth factors and cell derivatives of their extracellular vesicles (EVs), wherein the cytokines are mainly cytokines (such as IL-10) that can inhibit pro-inflammatory cytokines (such as TNF-α, IL-6), which can inhibit and reduce inflammation, and can effectively alleviate symptoms such as redness, swelling, irritation and wound inflammation associated with AD; growth factors mainly include VEGF and TGF-β that can promote angiogenesis to stimulate fibroblast activation and collagen synthesis, angiogenesis and collagen regeneration, so as to accelerate tissue regeneration and wound closure, targeting deeper structural damage; and extracellular vesicles can promote the communication and transmission of active molecules in the intercellular matrix, so that the active molecules reach the target cells, so that by culturing Wharton's jelly mesenchymal stem cell derivatives in a hypoxic environment, AD can be improved and treated through dual mechanisms of anti-inflammatory and cell regeneration;

[0030] In addition, the present invention can achieve sustained release of active ingredients and their protective effects through hydrogel preparations, ensure that active molecules retain their biological activity during the delivery process, prevent the common "sudden release" phenomenon in local medication, which may lead to ineffectiveness or side effects, and reduce the frequency of repeated dressing changes, thereby improving patient compliance: by encapsulating cell derivatives in degradable poly (lactic acid-glycolic acid) microspheres, it can protect sensitive component factors such as cytokines, growth factors and their extracellular vesicles from oxidative damage and degradation, leading to failure, and by embedding the microspheres in the hydrogel matrix, it can achieve sustained release of molecules within 24-72 hours, improve the therapeutic effect, and reduce the need for frequent replacement; the semi-permeable hydrogel matrix can form a barrier against external pathogens, reduce the possibility of secondary infection of chronic wounds or inflamed AD lesions (, while allowing gas exchange, reducing the risk of infection, and enhancing the oxygen required for tissue fiber repair, and it is also conducive to maintaining a moist healing environment, facilitating keratinocyte migration and epithelialization, accelerating tissue repair and preventing dryness. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] Example 1: Preparation of hypoxic Wharton's jelly mesenchymal stem cell derivatives. The specific operation is as follows:

[0035] The Wharton's jelly mesenchymal stem cells are extracted after being cultured in an environment of low oxygen content of 1-5vt%.

[0036] Specifically, passages 0-1 used a DMEM (low-glucose) medium containing 1-2 wt % fetal bovine serum (FBS); it used FBS for cell attachment.

[0037] Passages 0 to 2 used DMEM (low glucose) medium containing 5-10 wt % HPL (human platelet lysate) for cell expansion.

[0038] Culture parameters: 1-5vt% low oxygen environment, 37°C.

[0039] After the cells grow to 70-80% confluence, the cultured cells are washed with DPBS, and the culture medium is replaced with DMEM-LG phenol-free basal medium for further culturing for 48-72 hours; the used culture medium is collected and filtered with a 0.22 μm filter to obtain the OxyWharton's jelly mesenchymal stem cell derivative.

[0040] Example 2: A gel for improving / treating atopic dermatitis, the preparation of which comprises the following steps:

[0041] 1) Encapsulating hypoxic Wharton's jelly mesenchymal stem cell derivatives in poly(lactic-co-glycolic acid) microspheres;

[0042] 2) adding a certain amount of ultrapure water to hyaluronic acid, stirring evenly, adding polyethylene glycol according to the amount, stirring evenly, and then slowly adding glycerol, stirring evenly, to prepare a hydrogel;

[0043] The mass ratio of hyaluronic acid, polyethylene glycol and glycerin is 3:3:1-2.

[0044] 3) Add the poly(lactic acid-glycolic acid) microspheres prepared in step 1) into the hydrogel and stir evenly.

[0045] The ratio of poly(lactic acid-glycolic acid) microspheres to hydrogel is 3:10

[0046] Example 3: A gel for improving / treating atopic dermatitis, the preparation of which comprises the following steps:

[0047] 1) Encapsulating hypoxic Wharton's jelly mesenchymal stem cell derivatives in poly(lactic-co-glycolic acid) microspheres;

[0048] The specific operations of this step are as follows:

[0049] 1. Preparation of PLGA solution: PLGA is dissolved in a volatile organic solvent, such as dichloromethane or ethyl acetate, to form an organic phase. PLGA is dissolved in an organic solvent at a concentration of 2-5% (w / v) to effectively form microspheres.

[0050] 2. Add hypoxic Wharton's jelly mesenchymal stem cell derivatives: Mix the hypoxic Wharton's jelly mesenchymal stem cell derivatives into the PLGA solution under stirring to ensure uniform dispersion.

[0051] The weight ratio of PLGA to hypoxic Wharton's jelly mesenchymal stem cell derivative in this step was 10:1 to achieve optimal encapsulation efficiency while maintaining release kinetics.

[0052] 3. Add antioxidant additives; mix 0.5% sodium ascorbyl phosphate (SAP), the additive stabilizes the secretion derivatives, prevents environmental degradation and oxidative stress, and maintains biological activity for a longer time.

[0053] 4. Emulsion formation: Under high-speed homogenization or ultrasonic treatment, the organic phase is added dropwise to the aqueous phase containing a stabilizer such as polyvinyl alcohol (PVA) to form an oil-in-water emulsion.

[0054] PVA was used at a concentration of 1-3% (w / v) in the aqueous phase to stabilize the emulsion droplets.

[0055] 5. Solvent evaporation: The volatile solvent is allowed to evaporate while stirring, resulting in the solidification of the PLGA microspheres encapsulating the secretion body.

[0056] 6. Collection of microspheres: Wash the microspheres with distilled water to remove residual stabilizers and impurities, and then freeze-dry for storage.

[0057] Temperature: Keep the process at or slightly below room temperature to maintain the biological activity of the secretome (e.g., 4°C to 25°C).

[0058] pH: A neutral pH (approximately 7.0) is ideal to ensure stability during the encapsulation process.

[0059] Homogenization speed: Usually between 10,000 and 20,000 RPM for effective emulsification.

[0060] Solvent Removal: Evaporation is performed under mild vacuum or ambient conditions to prevent thermal degradation of bioactive molecules.

[0061] The volume ratio of aqueous phase to organic phase was 10:1 to ensure proper dispersion and emulsification.

[0062] 2) dissolving alginate in purified water by stirring to form a solution with a mass fraction of 0.1-10%, adding a cross-linking agent and stirring to form a hydrogel; the cross-linking agent is a carboxyl-containing water-soluble polysaccharide and its derivatives;

[0063] 3) Add the poly(lactic acid-glycolic acid) microspheres prepared in step 1) into the hydrogel and stir evenly.

[0064] The dosage ratio of poly(lactic acid-glycolic acid) microspheres to hydrogel is 3:10.

[0065] Example 4: A gel for improving / treating atopic dermatitis, the preparation of which comprises the following steps:

[0066] 1) Encapsulating hypoxic Wharton's jelly mesenchymal stem cell derivatives in poly(lactic-co-glycolic acid) microspheres;

[0067] 2) adding a certain amount of ultrapure water to hyaluronic acid, stirring evenly, adding polyvinyl alcohol according to the amount, stirring evenly, and then slowly adding glycerol, stirring evenly, to prepare a hydrogel;

[0068] The mass ratio of hyaluronic acid, polyvinyl alcohol and glycerin is 3:3:1-2.

[0069] 3) Add the poly(lactic acid-glycolic acid) microspheres prepared in step 1) into the hydrogel and stir evenly.

[0070] The dosage ratio of poly(lactic acid-glycolic acid) microspheres to hydrogel is 3:10.

[0071] 0.5 wt% sodium ascorbyl phosphate (SAP) was added in this step to stabilize the gel formulation by preventing oxidative stress and maintaining the stability of the bioactive ingredients.

[0072] Example 5: A patch for improving / treating atopic dermatitis, comprising a base film and a gel prepared in any one of Examples 2 to 4 applied on the base film.

[0073] The present invention and its implementation methods have been described, but such description is not restrictive and the actual methods are not limited thereto. In short, if ordinary technicians in the field are inspired by it and design implementation methods and embodiments similar to the technical solution without creative design without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. Application of hypoxic Wharton's jelly mesenchymal stem cell derivatives in the preparation for improving / treating atopic dermatitis.

2. The use according to claim 1, characterized in that: The hypoxic Wharton's jelly mesenchymal stem cell derivative is obtained by culturing the Wharton's jelly mesenchymal stem cells in an environment of low oxygen content of 1-5vt%.

3. A gel for improving / treating atopic dermatitis, characterized in that: The active ingredient is the hypoxic Wharton's jelly mesenchymal stem cell derivative prepared according to claim 2.

4. A gel for improving / treating atopic dermatitis according to claim 3, characterized in that: Its preparation comprises the following steps: 1) Encapsulating hypoxic Wharton's jelly mesenchymal stem cell derivatives in poly(lactic-co-glycolic acid) microspheres; 2) preparing a hydrogel; 3) Add the poly(lactic acid-glycolic acid) microspheres prepared in step 1) into the hydrogel and stir evenly.

5. A gel for improving / treating atopic dermatitis according to claim 4, characterized in that: The preparation operation of the hydrogel in step 2) is as follows: After adding a certain amount of ultrapure water to hyaluronic acid and stirring evenly, polyethylene glycol is added according to the amount and stirred evenly, and then glycerol is slowly added and stirred evenly to prepare a hydrogel; Alternatively, alginate is dissolved in purified water by stirring, and then a cross-linking agent is added and stirred to form a hydrogel; Alternatively, a certain amount of ultrapure water is added to hyaluronic acid, the mixture is stirred evenly, polyvinyl alcohol is added according to the amount, the mixture is stirred evenly, and glycerin is slowly added and stirred evenly to prepare a hydrogel.

6. A gel for improving / treating atopic dermatitis according to claim 5, characterized in that: The cross-linking agent is water-soluble polysaccharide containing carboxyl groups and its derivatives.

7. The gel for improving / treating atopic dermatitis according to claim 5, characterized in that: The mass ratio of hyaluronic acid, polyvinyl alcohol or polyethylene glycol, and glycerol is 3:3:(1-2).

8. The gel for improving / treating atopic dermatitis according to claim 4, characterized in that: In step 3), the ratio of poly(lactic acid-glycolic acid) microspheres to hydrogel is (2-5):

10.

9. A patch for improving / treating atopic dermatitis, characterized in that: The gel is prepared from the gel for improving / treating atopic dermatitis according to any one of claims 3 to 8.

10. Use of the hypoxic Wharton's jelly mesenchymal stem cell derivatives according to claims 1 to 2 and the gel for improving / treating atopic dermatitis according to any one of claims 3 to 8 in the preparation of a preparation for improving / treating any of the following diseases, characterized in that: Such diseases include: ①Ulcers and pressure sores caused by diabetes; ② Wounds or burns caused by mechanical trauma; ③Chronic inflammatory skin diseases including psoriasis.