阅读说明：本技术 一种用于表皮脱敏治疗的相变同轴纳米纤维膜及其制备方法和应用 (Phase-change coaxial nanofiber membrane for epidermal desensitization treatment and preparation method and application thereof ) 是由 丁杨 周建平 汪瑱 于 2021-10-27 设计创作，主要内容包括：本发明属于生物医药技术领域,公开了一种负载过敏原-佐剂组合物的同轴纳米纤维膜及其制备方法和应用。所述同轴纳米纤维采用静电纺丝技术制备,以亲水性聚合物为外壳,以疏水相变材料为核心。所述发明涉及过敏原-佐剂组合物,以同轴纳米纤维壳层负载过敏原,芯层负载佐剂。所述发明可利用加热装置实现芯层材料固-液转变过程,促进佐剂释放和皮肤角质层渗透性增加。所述纳米纤维膜通过在疗程内施用于患者皮肤,实现表皮脱敏治疗,可以由患者自行使用和监控。(The invention belongs to the technical field of biological medicines, and discloses a coaxial nanofiber membrane loaded with an allergen-adjuvant composition, and a preparation method and application thereof. The coaxial nanofiber is prepared by adopting an electrostatic spinning technology, and takes a hydrophilic polymer as a shell and a hydrophobic phase-change material as a core. The invention relates to an allergen-adjuvant composition, wherein a coaxial nanofiber shell layer is used for loading allergen, and a core layer is used for loading adjuvant. The invention can realize the solid-liquid conversion process of the core layer material by utilizing the heating device, and promote the release of the adjuvant and the increase of the permeability of the skin cuticle. The nanofiber membrane achieves epidermal desensitization therapy by application to the skin of a patient within a course of treatment, and can be self-administered and monitored by the patient.)

1. A preparation method of a phase-change coaxial nanofiber membrane for epidermal desensitization treatment is characterized by comprising the following steps:

step 1, mixing a hydrophilic polymer with deionized water, and stirring under a heating condition until the hydrophilic polymer is dissolved to prepare a hydrophilic polymer solution; cooling the hydrophilic polymer solution to room temperature, mixing with the allergen, and fully stirring until the mixture is dissolved to obtain a shell layer solution of coaxial electrostatic spinning;

adding the phase transition material and the immunologic adjuvant into a mixed solution of dichloromethane and ethanol, and ultrasonically dissolving to obtain a core layer solution of coaxial electrostatic spinning;

preferably, the volume ratio of the dichloromethane to the ethanol in the mixed solution is 4: 1-1: 4;

and 2, respectively pouring the shell layer solution and the core layer solution into an injector with a coaxial needle, and preparing the coaxial nanofiber membrane by a coaxial electrostatic spinning process.

2. The method as set forth in claim 1, wherein the concentration of the hydrophilic polymer in the shell solution of step 1 is 5% to 20% (w/v);

the hydrophilic polymer is selected from one or more of polyvinylpyrrolidone, polyvinyl alcohol, polyethylene oxide, gelatin, collagen, silk fibroin, sodium alginate and hyaluronic acid.

3. The method for preparing a skin patch according to claim 1, wherein the concentration of allergen in the shell solution of step 1 is 0.1-2% (w/v);

the allergen is selected from one or more of respiratory, food or skin allergens; preferably, the allergen is selected from one or more of house dust mite, artemisia annua pollen, artemisia argyi pollen, ragweed pollen, milk, peanut, egg, soybean and cashew nut;

preferably, step 1 is mixing the allergen after preparation into an extract, leachate, peptide, recombinant or synthetic product with a hydrophilic polymer solution.

4. The method according to claim 1, wherein the core layer solution of step 1 has a concentration of the phase change material of 10% to 40% (w/v);

the phase transition material is fatty acid, fatty alcohol and/or binary eutectic mixture thereof, and preferably, the phase transition material is selected from one or more of capric acid, lauric acid, palmitic acid, stearic acid, myristic acid and tetradecanol.

5. The method according to claim 1, wherein the concentration of the immunoadjuvant in the core layer solution of step 1 is 0.1-20% (w/v);

the immunological adjuvant is selected from monophosphoryl lipid A, CRX-675, R837, AZD8848, R848, VTX-1463, CpG ODN, 1018ISS, QbG10, and Vitamin D₃One or more of (a).

6. The preparation method of claim 1, wherein the coaxial electrospinning process in step 2 is performed at a voltage of 15-25kV, a shell flow rate of 0.5-1.5mL/h, a core flow rate of 0.05-0.5mL/h, a receiving distance of 10-20cm, a roller rotation speed of 0-150rpm, an ambient temperature of 20-30 ℃ and a humidity of 20-70%.

7. The coaxial nanofiber membrane prepared by the preparation method of any one of claims 1 to 6.

8. The coaxial nanofiber membrane of claim 7, comprising a shell layer and a core layer, the shell layer being a hydrophilic polymer loaded with an allergen and the core layer being a phase change material loaded with an immunoadjuvant;

preferably, the diameter of the coaxial nanofiber is 100 nm-1 μm;

the coaxial nanofiber membrane comprises 50-75% of hydrophilic polymer, 0.1-10% of allergen, 5-30% of phase transition material and 0.1-10% of immunologic adjuvant by mass percentage,

the dose of the coaxial nanofiber membrane loaded allergen is 1-500 mu g/cm²The dose of the adjuvant is 1-500 mu g/cm²。

9. Use of the coaxial nanofiber membrane of claim 7 in the preparation of a medicament for epidermal desensitization therapy.

10. Use according to claim 9, wherein the coaxial nanofibrous membrane is heated in use; preferably, the heating temperature is 34-43 ℃.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a phase-transition coaxial nanofiber membrane for epidermal desensitization treatment as well as a preparation method and application thereof.

Background

Allergy (allergy) is a type of recurrent hypersensitivity induced by the body's immune mechanisms. It is a world-wide public health phenomenon, with 30% -40% of the world population ever or suffering from allergic disease.

Desensitization therapy (also known as allergen-specific immunotherapy), which utilizes repeated exposure of patients to increasing doses of allergens to control or alleviate allergic symptoms, is considered the only "causal therapy" that may alter the natural course of allergic disease.

The World Health Organization (WHO), the World Allergy Organization (WAO), fully confirm the clinical efficacy of desensitization therapy, with subcutaneous immunotherapy (SCIT) being the gold standard and widely used. Although expensive and requiring the involvement of a specialized physician for each injection, it is currently considered the standard desensitization approach. Slight adverse reactions such as swelling of an injection part and the like can be seen by subcutaneous injection of the allergen, and the risk of systemic severe adverse reactions such as urticaria, asthma attack, allergy and the like is accompanied, and the incidence rate of the systemic adverse reactions accounts for 5.9 percent of the total number of patients; the estimated mortality rate was 1 out of 250 million injections, with an average of 3-4 deaths per year.

The French biotechnological company DBV Technology developed epidermal-based immunotherapy (epicutaneous all allergy i)mmunitotherpy, EPIT), demonstrating the possibility of desensitization therapy via the epidermal route. By repeated application of the allergen to the skin, directed against potent antigen presenting cells in the epidermis, Langerhans Cells (LC), causes the allergen to diffuse into the avascular epidermal layer of the skin, generally without any significant transdermal penetration. Allergens delivered to the epidermis are captured and processed by LC, migrate to draining lymph nodes, and present antigens to CD4⁺T cells. Regulatory T cells (CD 4)⁺CD25⁺Treg) after differentiation, cytokines such as TGF-beta, IL-10 and the like are generated to play a desensitization treatment role: 1. directly inhibiting the activation and degranulation of mast cells and basophils and inhibiting the activity of eosinophils; 2. direct inhibition of Th 2-associated cellular activity; 3. modulation of IL-4, IL-5 induced B cell activity, allows B cell switching from IgE secretion to antigen specific IgG1, IgG4, IgA production.

However, the effect of Viaskin is influenced by the skin condition, e.g. by application to the damaged skin surface of the stratum corneum, which causes a Th 2-directed immune response, i.e. a local or systemic allergic reaction. Secondly, Viaskin acts as a closed system, hydrates the skin, dissolves allergens and interacts with epidermal immune cells, however, the existence of the natural barrier of the stratum corneum greatly limits the antigen uptake of epidermal LC, and sweat generated by the skin at the application part is difficult to volatilize out through a patch, and finally red swelling, eczema and even inflammation of the skin are caused. Therefore, by designing a safe and comfortable transdermal drug delivery system and simultaneously delivering the allergen and the immunologic adjuvant, the problems of safety and compliance faced by the current desensitization treatment are solved, and the epidermal desensitization treatment effect is enhanced, so that the transdermal drug delivery system is the most promising research direction of allergic symptoms in the field of treatment.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention discloses a coaxial nanofiber membrane loaded with an allergen-adjuvant composition, and a preparation method and application thereof. The coaxial nanofiber is prepared by adopting an electrostatic spinning technology, takes a hydrophilic polymer as a shell layer, loads an allergen, takes a phase transition material as a core layer, and loads an adjuvant.

The technical scheme of the invention is as follows:

the first object of the invention is to provide a preparation method of a coaxial nanofiber membrane for epidermal desensitization treatment, which comprises the following steps:

step 1, mixing a hydrophilic polymer with deionized water, stirring the mixture under a heating condition until the hydrophilic polymer is dissolved to prepare a hydrophilic polymer solution, cooling the hydrophilic polymer solution to room temperature, mixing the hydrophilic polymer solution with allergen, and fully stirring the mixture until the hydrophilic polymer is dissolved to obtain a shell solution of coaxial electrostatic spinning;

adding the phase transition material and the immunologic adjuvant into a mixed solution of dichloromethane and ethanol, and ultrasonically dissolving to obtain a core layer solution of coaxial electrostatic spinning;

preferably, the volume ratio of the dichloromethane to the ethanol in the mixed solution is 4: 1-1: 4 (v/v);

and 2, respectively pouring the shell layer solution and the core layer solution into an injector with a coaxial needle, and preparing the coaxial nanofiber membrane by a coaxial electrostatic spinning process.

Further, in the shell solution in the step 1, the concentration of the hydrophilic polymer is 5-20% (w/v);

the hydrophilic polymer is selected from one or more of polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene oxide (poly (ethylene oxide), PEO), gelatin (gelatin), collagen (collagen), Silk Fibroin (SF), sodium alginate (NaAlg), Hyaluronic Acid (HA).

Further, in the shell solution in the step 1, the concentration of the allergen is 0.1-2% (w/v).

The allergen is selected from one or more of respiratory, food or skin allergens;

preferably, the allergen is selected from one or more of house dust mite, artemisia annua pollen, artemisia argyi pollen, ragweed pollen, milk, peanut, egg, soybean and cashew nut;

preferably, step 1 is mixing the allergen after preparation into an extract, leachate, peptide, recombinant or synthetic product with a hydrophilic polymer solution.

Further, in the core layer solution in the step 1, the concentration of the phase transition material is 10% -40% (w/v);

the phase transition material is fatty acid, fatty alcohol and/or binary eutectic thereof, preferably, the phase transition material is selected from one or more of Capric Acid (CA), Lauric Acid (LA), Palmitic Acid (PA), Stearic Acid (SA), Myristic Acid (MA), and Tetradecanol (TD).

Further, in the core layer solution in the step 1, the concentration of the immunologic adjuvant is 0.1-20% (w/v); in certain specific embodiments, the concentration of the immunoadjuvant is 0.1-10% (w/v);

the immunological adjuvant is selected from monophosphoryl lipid A (MPL A, TLR2 agonist), CRX-675(MPL analogue, TLR2 agonist), R837 (imiquimod, TLR7 agonist), AZD8848(TLR7 agonist), R848 (Racemate, TLR7/8 agonist), VTX-1463(TLR8 agonist), CpG ODN (TLR9 agonist), 1018ISS (TLR9 agonist), QbG10, Vitamin D₃(vitamin D derivative).

Further, the coaxial electrostatic spinning process in the step 2 is that the voltage is 15-25kV, the flow rate of a shell layer is 0.5-1.5mL/h, the flow rate of a core layer is 0.05-0.5mL/h, the receiving distance is 10-20cm, the rotating speed of a roller is 0-150rpm, the ambient temperature is 20-30 ℃, and the humidity is 20-70%.

The second purpose of the invention is to provide the coaxial nanofiber membrane prepared by the preparation method;

preferably, the coaxial nanofiber membrane comprises a shell layer and a core layer, wherein the shell layer is a hydrophilic polymer loaded with allergen, the core layer is a phase transition material loaded with immune adjuvant;

preferably, the diameter of the coaxial nanofiber is 100 nm-1 μm;

preferably, the coaxial nanofiber membrane prepared by the preparation method comprises, by mass, 50-75% (w/w) of a hydrophilic polymer, 0.1-10% (w/w) of an allergen, 5-30% (w/w) of a phase transition material, and 0.1-10% (w/w) of an immunoadjuvant.

The dose of the coaxial nanofiber membrane loaded allergen is 1-500 mu g/cm²The dose of the adjuvant is 1-500 mu g/cm²。

The third purpose of the invention is to provide the application of the coaxial nanofiber membrane in preparing the medicines for the epidermal desensitization treatment.

The coaxial nanofiber membrane allows for the delivery of respiratory, food, skin allergens to the skin, achieving epidermal desensitization treatment by repeated application of the nanofiber membrane on the patient's skin; the nanofiber membrane application site may be a different area of the body, preferably above the back or on the inside of the arm; the application time, frequency and treatment course of the nanofiber membrane can be 6-48h every time, 1-15 days apart, and the nanofiber membrane can be continuously used for 1-36 months.

Preferably, the coaxial nanofiber membrane is heated in use, and the heating device can be used for realizing the solid-to-liquid conversion of the core layer of the coaxial nanofiber membrane, promoting the adjuvant release process and simultaneously promoting the permeability of the stratum corneum to the drugs and the adjuvants to be increased.

More preferably, the heating temperature is 34 to 43 ℃. Preferably, the core layer of the coaxial nanofibers is capable of transforming from a solid state to a liquid state in a heated state.

The nanofiber membrane is applied to the skin of a patient, and the diffusion of allergen and adjuvant in the membrane promotes the migration and activation of Langerhans cells in the epidermis, thereby inducing a desensitization reaction; the epidermal route does not require the use of means to disrupt the stratum corneum integrity of the skin; the epidermal route avoids allergens entering the blood circulation.

The nanofiber membrane can be used and monitored by a patient, the allergen dosage can be adjusted by applying membranes with different areas in the treatment process, and the frequency or the application time can be flexibly adjusted according to the degree of immune response.

Compared with the prior art, the invention has the advantages that:

1. the nanofiber membrane prepared by the electrostatic spinning technology has excellent air permeability, and is beneficial to eliminating side effects of local eczema, inflammation and the like caused by poor air permeability of the traditional membrane.

2. The combined use of allergen and immunoadjuvant can change the property of skin to allergen inflammatory reaction, namely change from Th2 type to Th1 type, and induce synergistic effect in vivo to enhance immune response. By using immune adjuvants such as toll-like receptor (TLR) ligands for inducing Th1 or Treg cellular immune response, the occurrence of anaphylactic reaction can be reduced, the efficacy of immunotherapy can be enhanced, the treatment time can be shortened, and safe and efficient desensitization treatment can be realized.

3. Warming is used to control drug release and affect stratum corneum penetration properties: (1) promote the solid-liquid phase change of the core of the coaxial nano fiber, and enhance the capability of releasing the medicine from the membrane and diffusing the medicine to the skin; (2) structural changes are brought to the skin, thereby reversibly changing the permeability of the stratum corneum; (3) the distribution and diffusion of the drug in the different skin layers can be improved.

4. The nanofiber membrane can be used and monitored by a patient, the allergen dosage can be adjusted by applying membranes with different areas in the treatment process, and the frequency or the application time can be flexibly adjusted according to the degree of immune response.

Drawings

FIG. 1 is a photograph and Scanning Electron Microscope (SEM) image of a coaxial nanofiber membrane; wherein, (a) is a coaxial nanofiber membrane photograph, (b) is a coaxial nanofiber membrane SEM image, and (c) is a coaxial nanofiber membrane diameter distribution diagram.

FIG. 2 is a laser confocal microscope (CLSM) view of coaxial nanofibers;

FIG. 3 is an infrared (FT-IR) plot of nanofibers;

FIG. 4 is a Differential Scanning Calorimetry (DSC) curve of a coaxial nanofiber;

FIG. 5 is an in vitro release profile of a coaxial nanofiber membrane; wherein (a) the in vitro release profile of the adjuvant and (b) the in vitro release profile of the allergen.

FIG. 6 is an in vivo safety evaluation of coaxial nanofiber membranes;

FIG. 7 is an in-skin distribution study of coaxial nanofiber membranes; wherein, (a) a mouse dorsal fluorescence image, (b) a skin slice fluorescence image;

FIG. 8 lymph node migration study of coaxial nanofiber membranes; wherein, (a) an intra-lymph node Cy5 fluorescence image; (b) quantification of Cy5 fluorescence in lymph nodes; (c) DiI fluorescence image within lymph nodes; (d) quantification of DiI fluorescence within lymph nodes;

FIG. 9 pharmacodynamic evaluation of coaxial nanofiber membranes; wherein, (a) Penh value; (b) allergen-specific IgE concentration in serum; (c) total number of cells in alveolar lavage fluid; (d) proportion of eosinophils in alveolar lavage fluid.

Detailed Description

The present invention provides some specific examples, but the present invention is not limited by these examples.

The invention discloses a coaxial nanofiber membrane loaded with an allergen-adjuvant composition, and a preparation method and application thereof. The coaxial nanofiber membrane is prepared by adopting an electrostatic spinning method, and takes a hydrophilic polymer as a shell and a hydrophobic phase-change material as a core. The invention relates to an allergen-adjuvant composition, wherein the coaxial nanofiber membrane shell layer is loaded with allergen, and the core layer is loaded with adjuvant. The coaxial nanofiber membrane can realize the solid-liquid conversion process of the core layer material by utilizing a heating device, and the release of an adjuvant and the increase of the permeability of the skin stratum corneum are promoted. The nanofiber membrane allows for the delivery of respiratory, food, skin allergens to the skin, and epidermal desensitization treatment is achieved by repeated application of the nanofiber membrane to the skin of a patient.

Preparation of coaxial nanofiber membrane

The preparation steps of the coaxial nanofiber membrane are as follows:

step 1:

mixing a hydrophilic polymer with deionized water, stirring under a heating condition until the hydrophilic polymer is dissolved to prepare a hydrophilic polymer solution, cooling the hydrophilic polymer solution to room temperature, mixing with an allergen, and fully stirring until the hydrophilic polymer solution is dissolved to obtain a shell layer solution of coaxial electrostatic spinning;

adding the phase transition material and the immunologic adjuvant into a mixed solution of dichloromethane and ethanol, and ultrasonically dissolving to obtain a core layer solution of coaxial electrostatic spinning;

step 2:

and respectively pouring the shell layer solution and the core layer solution into an injector with a coaxial needle head, and preparing the coaxial nanofiber membrane by a coaxial electrostatic spinning process.

The amounts and proportions of the reactants are detailed in table 1.

TABLE 1 formulation composition of coaxial nanofiber membranes

TABLE 2 coaxial electrospinning process parameters

Two-axis and coaxial nanofiber membrane property research

Subsequent studies were conducted using the formulation shown in working example 2, with the coaxial nanofiber membrane constructed under the process shown in working example 12.

1. Coaxial nanofiber diameter and morphology

The surface morphology of the nanofibers was observed using a Scanning Electron Microscope (SEM), and the fiber diameters and diameter distributions were calculated. The SEM result is shown in figure 1, the diameter of the prepared drug-loaded nano-fiber is (205.1 +/-89.8) nm, the diameter distribution is uniform, the shape is good, and no adhesion exists.

2. Coaxial nanofiber membrane drug distribution

Fluorescence-labeled nanofibers in which the allergen was labeled with Cy5, the adjuvant was replaced with DiI, and fluorescence distribution of the drug in the nanofibers was analyzed using a Confocal Laser Scanning Microscope (CLSM), as shown in fig. 2, the red fluorescence of the allergen and the green fluorescence of the adjuvant were both distributed in the nanofibers, the allergen in situ was in the shell, and the adjuvant was distributed in the core.

The composition of the nanofiber membrane was analyzed using a fourier transform infrared spectrometer (FT-IR), and the results are shown in fig. 3, which demonstrates successful loading of allergen and adjuvant in the coaxial nanofiber membrane from characteristic peaks.

The prepared coaxial nanofiber membrane is subjected to component analysis, and the result shows that: comprises 66.45% (w/w) of hydrophilic polymer, 6.64% (w/w) of allergen, 26.58% (w/w) of phase transition material, and 0.3% (w/w) of immunologic adjuvant, wherein the coaxial nanofiber membrane is loaded with the allergen with the dose of 100 mu g/cm²Adjuvant dose of 5 μ g/cm²。

3. Coaxial nanofiber membrane phase transition property investigation

Phase Change Materials (PCM) and phase transition temperature of the coaxial nanofiber membrane were tested by Differential Scanning Calorimeter (DSC), all tests were performed under nitrogen atmosphere (50 mL/min). The experimental temperature range is 0-100 ℃, and the heating rate is 5 ℃/min.

The results of the experiment are shown in fig. 4, where LA/SA PCM forms a dibasic fatty acid eutectic mixture, and shows a single eutectic peak in DSC measurement, which has the lowest phase transition temperature of 40.7 ℃. For the coaxial nanofiber, the phase transition temperature is 40.3 ℃, which can meet the requirements of realizing phase transition and promoting drug release at 41 ℃.

4. In vitro transmembrane release of coaxial nanofiber membranes

The area is 1cm²The coaxial nanofiber membrane of (2) was placed over a cellulose acetate semipermeable membrane with a pore size of (0.22 μm), and then a layer of filter paper was placed over the system. The sample was sandwiched between the supply and receiving cells of a Franz diffusion cell. The receiving cell was added with 7mL of PBS buffer (pH 7.4). Franz diffusion cells were placed on a transdermal delivery apparatus at a constant temperature of 32 + -1 deg.C and 500rpm for in vitro delivery experiments. 0.2mL of the release solution was removed at the set time point and then supplemented with an equal volume of fresh solution. The drug concentration was determined by HPLC and the cumulative amount released was calculated.

Considering that the phase transition material PCM material of the coaxial nanofiber core could complete the phase transition at 41 ℃ and promote the release of the adjuvant, the nanofiber membrane was heated at 41 ℃ for 20min to observe the release behavior of the drug in the nanofibers, and the results are shown in fig. 5. It can be observed that after 2h, the adjuvant release was about 26% after 20min heating at 41 ℃, while the unheated group was only 10%, indicating that heat can promote the coaxial nanofiber core solid-liquid phase change and promote the release of adjuvant. After 12h of in vitro release, about 50% of the adjuvant was released from the nanofibers, 48h reached 70% or more, and the adjuvant was released at 32 ℃ to less than 20% (fig. 5 a). For the allergen, the release was essentially in equilibrium after 24h and could reach more than 80% after 48h (fig. 5 b).

5. Evaluation of in vivo safety

The experiment was divided into 3 groups: (1) a subcutaneous injection group, (2) a nanofiber membrane group, and (3) a nanofiber membrane +41 ℃ group.

Mice were injected subcutaneously (containing 100. mu.g allergen) or coaxial nanofiber membranes (about 1 cm) respectively²Containing 100 μ g allergen) was applied to the back of a previously depilated mouse, and the nanofiber membrane +41 ℃ treatment group was heated to 41 ℃ with an external heat source after attachment of the nanofiber membrane. Sera were prepared by orbital bleeds of mice at 0h, 2h, 6h, 12h, 24h and 48h, and the safety of epidermal desensitization treatment was evaluated by quantifying allergen concentrations in serum samples using ELISA kits.

The concentration of the allergen in the serum after administration is shown in fig. 6, and the allergen administered by subcutaneous injection is easy to enter blood circulation, thereby causing the occurrence of systemic severe adverse reactions such as allergy and the like; the desensitization treatment of the two nanofiber membrane treatment groups by the skin route has basically unchanged allergen concentration in blood whether being heated or not in the administration process, which indicates that the epidermal desensitization treatment has higher safety compared with the traditional subcutaneous desensitization treatment.

6. In vivo experiments and in-skin distribution of coaxial nanofiber membranes

The experiment was divided into 3 groups: (1) blank control group, (2) nanofiber membrane group, (3) nanofiber membrane +41 ℃ group.

The blank control group was left untreated, and two nanofiber membrane treatment groups were applied to a nanofiber membrane (about 1 cm)²) Applied to the back of a pre-depilated mouse, and the nanofiber membrane is treated at 41 ℃ to form a patchAfter the nanofiber membrane is attached, the temperature is heated to 41 ℃ by adopting an external heat source. After 48h, detecting the distribution condition of the back fluorescence by using a living body imager; and the skin under the collecting membrane was collected, cryosectioned, DAPI stained, and used for fluorescence imaging. Wherein the allergen is labelled with Cy5 and the adjuvant is replaced with DiI.

At 48h post-administration, the fluorescence distribution in the back and in the skin of the mice is shown in fig. 7, and significant fluorescence was detected in the back of the mice of both nanofiber membrane treated groups, indicating successful release of the allergen and adjuvant from the membrane and into the skin (fig. 7 a). After heating at 41 ℃ for 20min, the mouse active epidermal layer can detect obvious fluorescence distribution of allergen (Cy5) and adjuvant (DiI), while the unheated group mostly retains the drug in the horny layer (FIG. 7b), which shows that the warm heat obviously promotes the release of the drug from the nanofiber and the capability of penetrating the horny layer.

7. Coaxial nanofiber membrane for promoting Langerhans cell migration capability investigation to lymph node

The experiment was divided into 3 groups: (1) blank control group, (2) nanofiber membrane group, (3) nanofiber membrane +41 ℃ group.

The blank control group was left untreated, and two nanofiber membrane treatment groups were applied to a nanofiber membrane (1 cm)²) Applied to the back of a pre-depilated mouse, and the nanofiber membrane +41 ℃ treatment group is heated to 41 ℃ by an external heat source after being attached with the nanofiber membrane. After 48h, the mice were sacrificed, the underarm and inguinal lymph nodes were removed and observed for lymph node tendency in a live fluorescence imager. Wherein the allergen is labelled with Cy5 and the adjuvant is replaced with DiI.

After 48h of administration, lymph node ex vivo fluorescence imaging results are shown in FIG. 8, and epidermal Langerhans cells migrated to skin draining lymph nodes after uptake of Cy 5-allergen and adjuvant (DiI). The fluorescence intensity of Cy 5-allergen in mouse lymph nodes was significantly increased after 20min warming at 41 ℃ indicating that heat increased the distribution of Cy 5-allergen within the epidermis and promoted Langerhans cell migration (FIGS. 8a, b). In the adjuvant (DiI), the heat significantly promoted the release of the drug from the nanofibers and the ability to penetrate the stratum corneum, and the fluorescence intensity of Cy 5-allergen in lymph nodes was significantly enhanced after the adjuvant was added, which was caused by the adjuvant promoting keratinocytes in the epidermis to secrete cytokines such as TNF- α, IL-1 β, etc., and increasing langerhans cell migration (fig. 8c, d).

8. Evaluation of pharmacodynamics

The experiment was divided into 5 groups: (1) the preparation method comprises the following steps of (1) preparing a non-molding group, (2) preparing a blank control group, (3) preparing a subcutaneous injection group, (4) preparing a nanofiber membrane group, and (5) preparing a nanofiber membrane +41 ℃ group.

Wherein (1) the non-established model is a normal mouse and is not treated;

(2) the placebo group was asthmatic mice, not receiving treatment;

(3) the subcutaneous group was asthmatic mice, receiving subcutaneous injection therapy (100 μ g of allergen + 1mg of aluminium hydroxide gel) 1 time per week for 8 times;

(4) the nanofiber membrane group was asthmatic mice, which received nanofiber membrane treatment (about 1 cm) after back depilation²100 ug of allergen and 5 ug of adjuvant) 1 time a week for 48 hours each time for 8 times;

(5) the nanofiber membrane +41 ℃ group was asthmatic mice, which received nanofiber membrane treatment (about 1 cm) after back depilation²100 mug allergen and 5 mug adjuvant) after the nanofiber membrane is attached, the temperature is heated to 41 ℃ by adopting an external heat source, 1 time per week, 48 hours each time, and 8 times in total.

8.1 mouse airway hyperreactivity assay

And detecting the change of the airway hyperreactivity of the mouse by using a mouse noninvasive lung function detector. When the measurement is carried out, the ambient environment is kept quiet, solutions of methacholine with various concentrations (3.125mg/mL, 6.25mg/mL, 12.5mg/mL, 25mg/mL and 50mg/mL) are prepared, the measurement is carried out on mice respectively, and the Penh value is used as an index for reflecting the airway reactivity to evaluate the curative effect of the nanofiber membrane.

As shown in fig. 9(a), after 8 weeks of treatment, the Penh values of the mice in the treated group were decreased to different degrees compared with those in the blank control group. The Penh value of the nanofiber membrane at the temperature of +41 ℃ is reduced most obviously, and the nanofiber membrane is close to that of an unfinished module, so that the treatment effect is best; the Penh values of the nanofiber membrane group and the subcutaneous injection group are obviously reduced compared with that of a blank control group, but are higher than those of the nanofiber membrane +41 ℃.

8.2 mouse allergen-specific IgE assay

Blood is taken from mouse orbit to prepare serum, and allergen specific IgE is quantitatively determined through an ELISA kit and used for evaluating the treatment effect of the nanofiber membrane.

The specific IgE concentration reflects the treatment effect, as shown in fig. 9(b), after 8 weeks of treatment, the IgE value of the nanofiber membrane +41 ℃ group is reduced most obviously, and the treatment effect is the best; the IgE values of the nanofiber membrane group and the subcutaneous injection group are obviously reduced compared with that of a blank control group, but are higher than those of the nanofiber membrane group and the 41 ℃ group.

8.3 Total bronchoalveolar lavage fluid (BALF) cells and differential counts

Mice were sacrificed and fixed in supine position, trachea intubated, lavage 3 times with 0.5mL alveolar lavage with 0.01M PBS, repeated slow aspiration 3 times per lavage, recovery recorded (should be greater than 80%), bronchoalveolar lavage fluid recovered, 1000r/min at 4 ℃, centrifuged 3min, pellet resuspended, total cell number recorded, and smears prepared. Staining the smear by Liu's staining method, and counting the proportion of eosinophilic granulocyte.

The total number of cells in BALF and the proportion of eosinophils reflect the inflammatory condition of the lung. As shown in fig. 9(c, d), the total number of inflammatory cells and the proportion of eosinophils in the mice in the treated group were decreased to a different extent compared to the blank control group, wherein the total number of cells and the proportion of eosinophils in the nanofiber membrane +41 ℃ group were close to those in the non-model group, and the treatment effect was the best; the total cell count and eosinophil fraction were significantly reduced in the nanofiber membrane group and the subcutaneous injection group, but were higher than in the nanofiber membrane +41 ℃ group.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art may apply the above-mentioned technical details to other fields of equivalent embodiments with equivalent changes or modifications, but all simple modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention may still fall within the protection scope of the technical solution of the present invention.