CN116850279A - Reductive adjuvant nano system for relieving adaptive dermatitis and application - Google Patents

Abstract

The invention provides a reductive adjuvant nano system capable of relieving adaptive dermatitis and application thereof, wherein the reductive adjuvant nano system comprises an oil phase: the water phase is 0.01% -50%. The nano system controls the activation mode and the immune function of DCs in focus microenvironment by loading the strong-excitation adjuvant for stimulating the DCs in a carrier containing an active oxygen scavenger, so as to regulate the differentiation of downstream helper T cells, radically reduce the pathological phenomena of excessive differentiation and local accumulation of pathogenic Th2, and realize the treatment effect of relieving the adaptive dermatitis. The nano system is applied locally through nano preparation, or micro needle or aqueous adhesive dressing is applied transdermally for a long time without pain, or is applied to prepare DCs vaccine, and the healthy and stable auxiliary T cell immune balance is reestablished.

Description

Reductive adjuvant nano system for relieving adaptive dermatitis and application

Technical Field

The invention belongs to the field of pharmacy, and relates to a reductive adjuvant nano system for relieving adaptive dermatitis and application thereof. The nanometer preparation can be used for regulating the dendritic cell function in vivo and in vitro and preventing and treating the adaptive dermatitis, can reduce the pathological phenomena of excessive differentiation and local accumulation of downstream pathogenic Th2 in inflammatory skin suffering from the adaptive dermatitis, reestablish the immune balance based on Th1/Th2, realize the effect of relieving and treating the adaptive dermatitis, namely relieving and treating the abnormal itching, moss-like keratosis, red swelling, scars and other adaptive dermatitis syndromes of patients suffering from the severe and recurrent adaptive dermatitis, improve the physiological and economic burden of the patients and the family members of the patients, and can be applied to the treatment of skin diseases.

Background

Adaptive dermatitis, also known as adaptive eczema, is a heterogeneous autoimmune disease, and is characterized clinically by recurrent eczematoid lesions (including erythema accompanied by exudative erythema, early blisters and crusting, late desquamation, cracking and bryoid cuticle thickening) and intolerable severe itching, which currently afflict 15% to 30% of children and 10% of adults in European and American countries, and seriously affect human health and quality of life. Although it is not fatal in itself, it can occur at any age, cause lesions on all The skin throughout The body, repeatedly occur, have a high incidence of life-long, and have severely disturbed barrier functions, and The like, is highly likely to cause psychological disorders and decline in self-esteem of patients, and often leads to an increased risk of patients suffering from other inflammatory diseases such as arthritis and inflammatory bowel diseases, and thus has been considered as a non-fatal dermatological disease with highest personal and socioeconomic burden and medical burden [ The Lancet,2020,396 (10247):345-360 ]. Thus, it is conceivable that effective control/alleviation of the atopic dermatitis syndrome is of vital importance for maintaining the physiological and psychological health stability of patients and families as an essentially controllable chronic/acute inflammatory disease. However, despite the urgent need and the wide range of patients, unfortunately, many safe and specific drugs for treating atopic dermatitis are not approved for sale worldwide.

In fact, as an autoimmune abnormality-induced skin inflammation, its immunopathological mechanism essentially involves: abnormal immune imbalance in the diseased area, i.e., excessive differentiation and local accumulation of T helper Th2 cells in the focal skin, results in a Th2 cell response that overwhelms the Th1 cell response. That is, at the site of onset of atopic dermatitis, dendritic Cells (DCs) aggregate in large amounts in the epidermis, dermis, and even the stratum corneum, and specifically trigger a large activation of Th2 in this diseased region, and locally cause type 2 active cytokine storms (such as interleukin-4 (IL-4), IL-13, immunoglobulin E (IgE), thymic stromal lymphopoietin (thymic stromal lymphopoietin, TSLP)), resulting in a significant increase in expression of downstream pruritin [ Nat Immunol,2018,19:1286-1298]. On the other hand, inflammatory skin sites have a large number of diffuse reactive oxygen species (Reactive Oxygen Species, ROS), and abnormally increased ROS further negatively impact the wound healing process by modulating a range of physiological and pathological processes, including inflammatory reactions, cell proliferation, angiogenesis, granulation, and extracellular matrix formation. In addition, excessive ROS-mediated oxidative stress can further cause mechanical sensitization of cells, thereby enhancing the inflammatory response of cells under scratching, and synergistically promoting the release of a variety of active cytokines of type 2, including IL-4, promoting the formation of itching-scratching vicious circle with further impairment of the skin barrier and proliferation of locally pathogenic microorganisms [ Nat Commun,2023,14:2478]. Thus, it would be an effective means to alleviate or even cure the atopic dermatitis syndrome if it were possible to simultaneously 1) modulate DCs in a local dermatitis-based microenvironment to manipulate Th cell differentiation and reconstitute immune balance, and 2) inhibit ROS-based abnormal oxidative stress to prevent the worsening of inflammation and further impairment of epidermal barrier function.

However, although Th2 immune response storms caused by oxidative stress and aberrant activation of DCs are all in a significant position during the occurrence, development and recurrent attacks of atopic dermatitis, there are still few drugs or clinical and laboratory inhibition means for this pathway that are currently on the market, and inhibition of oxidative stress and modulation of Th1/Th2 local immune balance based on DCs are not of sufficient importance in alleviating atopic eczema. Currently FDA approved drugs for the treatment or alleviation of the indication dermatitis syndrome are mainly divided into two main categories: 1) Monoclonal antibodies to type 2 cytokines and type 2 cytokine receptors, such as IL-4 receptor alpha antagonist Dupu Li Youshan antibody, IL-13 Ralpha receptor antagonist Lestuzumab, to prevent heterodimerization of the IL-4 Ralpha/IL-13 Ralpha 1 subunit to antagonize development of adaptive dermatitis mediated by pathogenic factor signaling [ J.Dermatol,2021,48:152-157], 2) topical glucocorticoids and oral immunosuppressants, such as topical dexamethasone creams, and oral Janus kinase inhibitor Barittinib [ Sci Adv,2021,7:eabe2888], to locally or systematically suppress DCs and T cell based immune responses. However, both of these major classes of therapeutic agents have some predictable side effects. In the case of monoclonal antibody drugs, since monoclonal antibodies are expensive and can only target the signaling of a certain pathogenic cytokine, the symptoms of recurrent dermatitis adaptive syndrome can only be relieved by treating the symptoms, and the economic and medical burden of patients can be easily further increased. For immunosuppressants containing steroid hormones, which are prone to drug resistance based on glucocorticoid resistance, hormonal dermatitis response after withdrawal and other adverse effects, and problems of safety and biocompatibility remain to be ignored since complete immunosuppression is likely to cause interference of pathogenic bacteria in damaged skin barriers and complications of dermatitis symptoms. In conclusion, expensive monoclonal antibodies and broad-spectrum immunosuppressive therapy may not be the best means to treat atopic dermatitis.

Therefore, it is important to construct a drug which has high biocompatibility and is suitable for alleviating and treating severe and recurrent atopic dermatitis syndromes. In fact, modulation of adjuvant signals that stimulate DCs is indeed the most upstream event in regulating Th differentiation, and furthermore, how to reverse aberrant Th2 differentiation based on DCs, compared to Th1/Th2 in an immunocompetent state in normal skin, to re-establish the balance between Th1 and Th2 immunity, is probably an effective measure against opportunistic infections by external pathogenic bacteria and to treat atopic dermatitis. On the other hand, considering the importance of ROS-based abnormal oxidative stress in promoting the progression of adaptive dermatitis, simultaneously regulating DCs immune function and eliminating local excessive ROS is expected to restore Th1/Th2 immune balance of focal skin, and finally effectively treat or relieve symptoms of adaptive dermatitis, possibly bringing benefit to clinical million dermatitis patients.

Disclosure of Invention

The invention aims to provide a reductive adjuvant nano system for relieving adaptive dermatitis, which consists of an oil phase and a water phase, wherein the oil phase is composed of phospholipid (5-70%, w/w), triglyceride (5-70%, w/w) and other membrane material components, and components such as an oxidative stress inhibitor (or ROS scavenger) (0.01-30%, w/w), an oil-soluble strong-agitation adjuvant (0.01-30%, w/w) and the like, the water phase is composed of deionized water solution (0.01-30%, w/w) of a water-soluble strong-agitation adjuvant, or deionized water solution (0.01-30%, w/w) of a water-soluble oxidative stress inhibitor, or pure deionized water.

The oil-water ratio (oil phase: water phase) of the nano system is 0.01% -50%, w/w.

The nano system can be nano emulsion, liposome, nano micelle or other nano preparations such as lipid nanoparticles, and the preparation method can be a single emulsion method, a reverse emulsion method, a film dispersion method, a rotary evaporation method and the like, so as to obtain the corresponding drug-carrying nano system.

(1) In addition to the functional oxidative stress inhibitors (or ROS scavengers) and the strongly-active adjuvants that regulate DCs function, other oil phase components (or major membrane components) can be replaced or added with other lipid components and polymer components that are good in drug properties, good in biocompatibility, high in safety, weak in immunogenicity, easy to obtain and low in production cost, including lecithin, phosphatidylglycerol, cephalin, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, cholesterol, soybean oil, olive oil, squalane, squalene, glyceryl stearate, long-chain fatty acid esters, short-chain fatty acid esters, medium-chain fatty acid esters, sucrose fatty acid esters, PLGA, PEG-PLA, DSPE-PEG, DOPE and the like.

(2) The liposoluble oxidative stress inhibitor or ROS scavenger can be vitamin E or its isomer or racemate (such as alpha-tocopherol), vitamin E acetate, astaxanthin, tea polyphenols, beta-carotene, prussian blue, mnO _2-x 、Fe ₃ O ₄ Polydopamine, N-acetyl-L-cysteine, N' -dimethylthiourea, isofraxidin, fulvic acid, cerium oxide (CeO) and CeO ₂ ) Nanoenzyme, non-steroidal anti-inflammatory drugs, fullerenes, necroX-7, 2-methacryloxyethyl phosphorylcholine, salidroside, superoxide dismutase, catalase, glutathione peroxidase, 4-benzoic acid, porphyrin chloride, 4-hydroxy-2, 6-tetramethylpiperidine 1-oxy, triphenylphosphine, dihydronicotinamide, NAD (P) H mimics, sitagliptin, resveratrol monomers, dimers thereof, and isofluoxetine, and the like. After the oil-soluble reducing ROS scavenger is uniformly mixed with other film material components, an oil phase mother solution of the nano preparation can be formed, so that a certain method is convenient to prepare the corresponding medicine-carrying nano preparation later. The water-soluble oxidative stress inhibitor or ROS scavenger can be vitamin C, polydopamine hydrochloride, L-kynurenine, etc., and can be dissolved in deionized water to form an aqueous phase. Can be directly combined on the surface of the nano preparation in the modes of probe ultrasonic or electrostatic adsorption and the like, or directly wrapped in an internal water phase of the preparation to be loaded.

(3) The fat-soluble strongly-agonistic adjuvant may be Toll-like receptor (TLR) -4 agonist monophosphoryl lipid A, neoseptin 3, TLR-7 and/or TLR-8 agonist raschimod, telraolimod, imiquimod, DSR-6434, gardiquimod diTFA, GSK2245035, loxoribine, AZD8848, CL264, CL097, TMX-201, 1V209, CL097 hydrochloride, guretolimod, SM-324405, UC-1V150, TLR-9 agonist CU-CPT17e, TLR-1 and/or TLR-2 agonist dipivocim and its derivative dipivocim-X, pam CSK4 TFA, dectin-1 agonist curdlan, and various natural adjuvants including ginsenoside (ginsenoside Rh2, ginsenoside Rg2, ginsenoside Rc, ginsenoside Rd, ginsenoside Rh1, ginsenoside Rg3, ginsenoside Rf, etc.), etc. Similar to the oil-soluble reducing ROS scavenger, the fat-soluble strong-excitation adjuvant, other membrane components and the oil-soluble reducing ROS scavenger can be uniformly mixed to form an oil phase mother solution of the nano preparation, so that the corresponding medicine-carrying nano preparation can be conveniently obtained by adopting a certain preparation process in the follow-up, wherein the adjuvant accounts for 0.1-50% of the total mass of the oil phase. In another aspect, water soluble TLR-4 agonist lipopolysaccharide, kdo 2-lipid A ammonium, TLR-7 and/or TLR-8 agonists imiquimod hydrochloride, LHC-165, AXC-715hydro chloride, imiquimod maleate, 1V209 polysaccharide conjugates, TLR-9 agonist Oligodeoxynucleotide (ODN) CpG 1826, cpG ODN 1018, cpG ODN 1668, ODN 7909, ODN M326, agatolimod, ODN 2395, ODN 2216, ODN M362, vidutolimod, lefitolimod, TLR-3 agonists Poly (I: C) and sodium and potassium salts and the like. The water-soluble strong-agitation adjuvant can be dissolved in deionized water to form a water phase (can simultaneously contain water-soluble ROS scavenging components), wherein the water-soluble adjuvant component accounts for 0.1-50% of the total mass of the water phase, can be directly combined on the surface of a nano preparation in a probe ultrasonic or electrostatic adsorption mode or can be loaded in an inner water phase of a carrier in a self-assembly, encapsulation mode and the like, and the preparation method is similar to the drug loading mode of the water-soluble ROS scavenging agent.

It is another object of the present invention to provide the use of the reductive adjuvant nanosystems in the manufacture of a medicament for the alleviation and treatment of recurrent and severe atopic dermatitis.

The application of the invention is realized by the following three main ways:

(1) The nano system is self-prepared medicine for direct local delivery, the administration concentration is 0.1 mug/kg-10 mg/kg, the administration route is local smearing and transdermal, and the preparation can control the differentiation and the number of the downstream helper T cells by directly regulating and controlling the activation mode and the immune function of DCs in the focus microenvironment of the adaptive dermatitis, thereby fundamentally reducing the pathological phenomena of excessive differentiation and local accumulation of pathogenic Th2 in inflammatory skin and reestablishing the immune balance of healthy, stable and normal helper T cells (Th 1/Th 2).

(2) The preparation method of the reductive adjuvant nanometer system comprises the steps of continuously manufacturing a microneedle patch or a water-based adhesive dressing in a micro-die after the preparation, and directly applying the microneedle patch or the water-based adhesive dressing to an affected part with the drug administration concentration of 0.1 mug/kg-10 mg/kg for local long-acting drug release, thereby rapidly relieving severe type adaptive dermatitis.

(3) The nanometer system is used for preparing the DCs vaccine for preventing and treating the adaptive dermatitis by treating DCs in vitro, adjusting allogeneic or autologous initial DCs which are subjected to in vitro induced differentiation into cell vaccines which can be used for adoptive feedback, and the administration concentration of the in vitro cell level is 1ng/mL-50mg/mL, and treating the DCs for 1-48 hours by the drug-loaded nanometer preparation.

The invention prevents further aggravation of skin inflammation and continuous destruction of epidermis barrier function by simultaneously regulating and controlling DCs activation mode and immune function based on local dermatitis microenvironment and inhibiting abnormal oxidative stress based on ROS in dermatitis progression, so as to control Th cell differentiation and reconstruct local microenvironment immune balance, and finally is hopeful to realize alleviation and even cure of the adaptive dermatitis syndrome. Mechanically, firstly, oxidative stress inhibitors or reductive ROS scavengers can effectively scavenge the ROS accumulated in large amounts in skin inflammation sites, preventing them from initiating a series of pathological processes which are detrimental to wound healing and epidermal barrier re-establishment and promoting itch-scratching malignant circulation pathways by acting as regulatory factors, thereby improving inflammatory responses caused by adaptive dermatitis to some extent. Meanwhile, the Th1 promoting adjuvant component can act on DCs accumulated in the horny layer, epidermis layer and even dermis layer of diseased skin, and through twisting the DCs from pathogenic Th2 activated DCs phenotype to Th1 activated DCs phenotype, the differentiation, in situ development and local accumulation of sensitized Th2 are radically reduced, the release and accumulation of various 2-type active cytokines are reduced, the Th1/Th2 immune response in focus imbalance is reversed, and the Th1/Th2 immune balance similar to healthy skin is rebuilt. It is worth noting that the method is not similar to two main stream treatment modes developed in clinic at present, and neither type 2 active cytokines and receptors thereof are used as targets, nor broad-spectrum immunosuppression strategies are adopted, so that systemic immune disorder or overall immunity reduction is avoided, local or systemic opportunistic infection of pathogenic bacteria is avoided, and finally safe and effective adaptive dermatitis relief is realized.

The invention has the innovation that the preparation method prepares the reductive adjuvant nano system which is convenient to produce, does not contain drug resistance, has low cost, wide audience, easy popularization, small side effect, stable property, high safety and good biocompatibility, and can be regarded as a passive targeting preparation of DCs when being locally administrated by relying on the strong affinity and phagocytic capacity of DCs as the strongest antigen presenting cell family to lipid nano particles, thereby having better production and application prospects. Adaptive dermatitis is a kind of skin inflammation based on autoimmunity, the immunological nature of which is to gather DCs at focus sites of excessive accumulation of ROS, induce excessive development accumulation of local pathogenic Th2 and secrete a large amount of pathogenic type 2 cytokines, so that the disease progress and recurrent attacks are caused, and therefore, the invention simultaneously eliminates or improves two culprits of worsening dermatitis progress: ROS and overwhelming Th2 immune responses eventually restore Th1/Th2 immune balance gradually at the lesions and strengthen the damaged skin barrier, effecting relief and treatment of severe and recurrent adaptive dermatitis.

Drawings

Fig. 1 is a drug-loaded nanoemulsion particle size (prescription 9).

Fig. 2 is a drug-loaded nanoemulsion transmission electron microscope modality (prescription 9).

Fig. 3 is the particle size uniformity (prescription 9) for different batches of drug-loaded nanoemulsions.

Fig. 4 is the PDI uniformity (prescription 9) for different batches of drug-loaded nanoemulsions.

Fig. 5 is cytotoxicity of drug-loaded nanoemulsion 24h against human normal keratinocytes (HACAT).

Figure 6 is cytotoxicity of drug-loaded nanoemulsion 24h against primary mouse DCs.

Figure 7 shows that the uptake level of the drug-loaded nanoemulsion by the primary DCs was higher than that of human normal keratinocytes.

FIG. 8 is a graph showing the activation of DCs by drug-loaded nanoemulsion PV-NE into the Th1 activated DCs phenotype.

FIG. 9 is a graph showing that drug-loaded nanoemulsion PV-NE enhances the ability of DCs to secrete Th1 activating cytokines.

FIG. 10 shows the morphology of the drug-loaded nanoliposome RT-lipo transmission electron microscope.

FIG. 11 is a graph showing the ability of drug-loaded liposome RT-lipo to confer on DCs the ability to induce Th1 differentiation and reduce secretion of environmental type 2 factors.

FIG. 12 is a transmission electron microscope morphology of drug-loaded nanoliposomes CA-lipo.

FIG. 13 is the reducibility of drug-loaded nanoliposomes CA-lipo.

FIG. 14 is ROS scavenging capacity of drug-loaded nanoliposomes CA-lipo.

FIG. 15 is a transmission electron microscope morphology of drug-loaded nanoemulsions PT-NE and P-NE.

Fig. 16 shows that drug-loaded nanoemulsion PT-NE was effective in alleviating the progression of recurrent adaptive dermatitis.

Figure 17 shows that drug-loaded nanoemulsion PT-NE was effective in reducing the adaptive dermatitis marker, serum IgE.

FIG. 18 shows that drug-loaded nanoemulsion PT-NE is effective in reducing the adaptive dermatitis marker, namely type 2 cytokine IL-4 in the skin.

FIG. 19 is a drug loaded reducing PLGA nanoparticle MS@PLGA transmission electron microscope morphology.

FIG. 20 is a graph showing the effective activation of DCs into Th1 activated DCs phenotypes by drug loaded reducing PLGA nanoparticles MS@PLGA.

Fig. 21 is a graph showing that the drug-loaded nanoemulsion CDT-micolle of fig. 21 is effective in alleviating the progression of severe type of atopic dermatitis.

Fig. 22 shows that the drug-loaded nanoemulsion CDT-micolle of fig. 22 is effective in alleviating type 2 immune responses in severe atopic dermatitis.

Fig. 23 is a graph showing that the novel microneedle patch RAC-micro-needle is effective in alleviating atopic dermatitis.

Figure 24 is a novel microneedle patch RAC-micro-needle effective in reducing type 2 pathogenic agents in atopic dermatitis.

FIG. 25 is a view of a novel drug-loaded iron-manganese nanoparticle Rh2@MnFeNP transmission electron microscope morphology.

FIG. 26 is a transmission electron microscope morphology of the novel drug-loaded solid lipid nanoparticle CUVe@NP.

FIG. 27 is a phenotype of Th1 activated DCs vaccine for adoptive reinfusion.

FIG. 28 shows the Th1/Th2 ratio in a controlled microenvironment of a Th 1-activated DCs vaccine for adoptive feedback

Figure 29 is that Th 1-activated DCs vaccine for adoptive reinfusion was effective in alleviating recurrent atopic dermatitis.

Detailed Description

The invention is further described with reference to the drawings and examples of implementation.

Example 1 prescription screening of reductive adjuvant-loaded nanoemulsions, drug loading and cellular uptake

(1) Prescription screening and physicochemical property characterization of drug-loaded nanoemulsion

Table one: prescription composition and fumbling of drug-loaded nanoemulsion

First, a reductive adjuvant-loaded nanoemulsion (PV-NE) was prepared by inverse emulsification-electrostatic adsorption-ultrasound and subjected to prescription screening. By changing the type of phosphatidylcholine (PL 100M and/or E-80), the ratio of each lipid component and the content of the water phase, a prescription with higher stability and proper particle size and potential is screened, namely, prescription 9 is used for subsequent study when PL100M is taken as a membrane material. The particle size of the drug-loaded nanoemulsion under the prescription is about 100-150nm (detected based on a dynamic light scattering method) (see figure 1); an emulsion morphology that is typically spherical and relatively uniform in overall particle size is presented under Transmission Electron Microscopy (TEM) (see fig. 2); and formulations between different production batches also have stability in particle size and PDI (see fig. 3 and 4).

(2) Cytotoxicity experiment of drug-loaded nanoemulsion

According to the selected drug administration concentration (0-30 mug/mL) in the gradient range, the 24h cytotoxicity to human normal keratinocyte (HACAT) and primary DCs cells is tested, and the safety and biocompatibility are explored. Cytotoxicity test at 24h of gradient administration found negligible cytotoxicity even at high concentrations (fig. 5). Meanwhile, the toxicity to DCs is also smaller (figure 6), and the drug-carrying preparation is proved to have high safety and can be used for subsequent experiments at the cell level and the animal level.

(3) Cellular uptake of drug-loaded nanoemulsions

Nanoemulsions were fluorescently labeled with the fluorescent dye DID and the uptake levels of DCs and normal somatic cells (keratinocytes) on reductive adjuvant loaded nanoemulsions were compared. Uptake of the prepared drug-loaded nanoemulsion within 24 hours was examined using in vitro differentiated primary DCs and keratinocytes (HACAT) as model cells. As a result of fluorescent inverted microscope imaging, in view of the functional characteristics of DCs themselves, which are the most powerful antigen presenting cells, have powerful uptake and internalization characteristics, and can uptake nanoparticles higher than other normal somatic cell populations within 24 hours (fig. 7), indicating that the drug-loaded nanoemulsion may be considered as a passive targeting agent for adipose tissue to some extent, and there is a great possibility that the nanoparticles after local administration may be taken up by DCs accumulated in focal regions and then function to modulate DCs after administration on animal level.

Example 2 validation of drug loaded nanoformulations to activate DCs into Th 1-induced DCs

Taking example 1, prescription 9 as an example, model antigen OVA and drug-loaded nanoformulation PV-NE were incubated with primary DCs differentiated in vitro for 24h to investigate whether DCs could be activated to a Th1 inducible DCs phenotype. Wherein Poly (I: C) is an anionically charged TLR3 agonist. First, we validated activation after 24h treatment with the nanoformulation PV-NE by flow cytometry, stained each set of DCs with the appropriate amount of fluorescein-labeled CD11c, CD80, MHC II flow antibodies according to the usage amounts indicated in the instructions, followed by incubation at 37 ℃ for 45 minutes, washing 2-3 DCs with fresh ice-cold PBS, resuspending 100 tens of thousands of DCs in 500 μl PBS, and observing expression representing Th 1-induced double positivity of MHC II molecules and CD80 molecules in a subpopulation of DCs analyzed for cd11c+ by polychromic flow meter. The results showed that DCs treated with the drug-loaded nanofabric exhibited a Th 1-activated phenotype (see fig. 8). Subsequently, we examined cytokine secretion of DCs after 24h treatment with the reductive adjuvant loaded nanoagent PV-NE by ELISA, where IFN- α and IL-12 were critical for Th1 stimulation, proliferation and differentiation. We have tested with the sandwich method according to the instructions of the specification and found that DCs treated with the drug-loaded nanofabric PV-NE are capable of secreting the Th1 activating cytokines IFN- α and IL-12 (see FIG. 9).

Example 3 prescription screening and physicochemical Property characterization of reductive adjuvant loaded drug loaded liposomes

And (II) table: prescription composition of drug-loaded liposome and fumbling

Leixomadine is liposoluble, is an agonist of the pattern recognition receptors TLR7 and TLR8 of DCs, can effectively induce the release of cytokines (such as TNF-alpha and IFN-alpha), and can induce DCs into Th 1-induced DCs. Fat-soluble alpha-tocopherol is a powerful antioxidant and is the most reducing vitamin E isomer, thus playing an effective role in scavenging ROS. Firstly, the components are dissolved in chloroform according to a proportion, then a film dispersion method-probe ultrasonic method is used for preparing the drug-loaded liposome RT-lipo loaded with strong agonistic adjuvant rassimol and ROS scavenging component alpha-tocopherol, and prescription screening is carried out on the drug-loaded liposome RT-lipo. The prescription with higher stability and proper particle size, namely the lipid and oil phase ratio of the prescription 4, is screened out for subsequent study by changing the proportion of the egg yolk lecithin S100, other lipid components and alpha-tocopherol and the oil-soluble adjuvant raschimod. The reductive adjuvant loaded drug loaded liposome RT-lipo under the prescription has a particle size of about 100-150nm and a double-layer lipid spherical appearance (see figure 10).

Example 4 reducing adjuvant drug loaded liposomes confer Th1 Induction and Th1/Th2 modification ability to DCs

Taking prescription 4 of example 3 as an example, it was investigated whether reducing adjuvant loaded drug loaded liposomes could induce DCs to a Th1 promoting phenotype in vitro and alter the ratio of Th1 to Th2 in vitro. First, we treated primary DCs obtained by in vitro differentiation with a reducing adjuvant-loaded liposome RT-lipo with OVA antigen for 24h, then incubated primary spleen T lymphocytes isolated in advance with the treated DCs at a ratio of 20:1 for 72h, and then examined the supernatant for secretion of the Th 1-type cytokines IFN- γ and Th 2-type cytokine IL-4 by ELISA. The results indicate that, following treatment of DCs with liposomal RT-lipo loaded with the adjuvant raschimote and the reducing component α -tocopherol, T cells can be efficiently induced to Th 1-type cells, promoting release of type 1 cytokines, and reducing secretion of type 2 cytokines in the environment (see fig. 11). It is demonstrated that liposomal RT-lipo, which together carries the reducing liposoluble component alpha-tocopherol and the liposoluble adjuvant component ramotimod, has the ability to stimulate DCs to Th 1-differentiated DCs in vitro and reduce Th2 differentiation.

Example 5 preparation of drug-loaded liposomes and exploration of their powerful ROS scavenging Properties

Drug-loaded liposome formulation:

DOPE 50-80mg

DSPE-PEG 0.2-2mg

cholesterol 20-30mg

DOTAP 0.1-10mg

Astaxanthin 1-3mg

CpG ODN 1018 0.1-5mg

1mL of water.

CpG ODN 1018 is a water-soluble oligodeoxynucleotide with anions, is a strong agonist of TLR9, is commonly used as a vaccine adjuvant, and can induce DCs into Th1 induced DCs. On the other hand, fat-soluble astaxanthin is a powerful ROS scavenger. Firstly, the components are dissolved in chloroform or ethanol according to a certain proportion, then a film which is uniformly mixed with ROS scavenging component astaxanthin is obtained through a film dispersion method, then a deionized water hydration film without RNase and DNase is used, a probe ultrasonic method is adopted to obtain liposome A-lipo containing astaxanthin, the water without RNase and DNase containing CpG ODN 1018 and the water with leaving out of A-lipo are incubated for 15 minutes at 37 ℃ according to a certain proportion, and the liposome CA-lipo with the surface adsorbed with Jiang Xiaozuo agents of CpG ODN 1018 and ROS scavenging component and with the particle size of about 150-200nm is obtained, and a TEM electron microscope result shows that the liposome A-lipo has a remarkable lipid bilayer spherical appearance (see figure 12). The reduction ability was measured using the probe DPPH for measuring the reduction property of the solution, and the result showed that the liposome CA-lipo was able to effectively reduce the purple DPPH to yellow, compared with the control group, indicating that it has strong antioxidant property (see FIG. 13). Furthermore, we pre-treated normal keratinocytes with 500. Mu.M hydrogen peroxide for 3 hours, treated with CA-lipo for 24 hours, stained cells with ROS probe DCFH-DA for 30 minutes at 37℃and photographed with a high resolution inverted fluorescence microscope to show their intracellular ROS scavenging effect. The results indicate that CA-lipo is effective in scavenging ROS in HACAT cells and in resisting oxidation and inhibiting oxidative stress compared to the control group (see FIG. 14).

Example 4 drug-loaded nanoemulsion PT-NE was effective in alleviating progression of recurrent adaptive dermatitis in mice

Drug-loaded nanoemulsion PT-NE prescription:

yolk lecithin E80-150 mg

DOTAP 40-60mg

70-100mg of long chain triglyceride

Alpha-tocopherol 50-80mg

Poly(I:C)8-30mg

1mL of water.

Poly (I: C) is a water-soluble double-stranded RNA analog of a charged anion, is a potent agonist of TLR3, is commonly used as a vaccine adjuvant and immunopotentiator in clinical trials, and can induce DCs into Th 1-induced DCs. While fat-soluble alpha-tocopherol is effective in scavenging ROS to aid in the protection against the progression of skin inflammation. Firstly, E80, DOTAP and long chain triglyceride are proportionally dissolved in ethanol, and then the reducing cationic nanoemulsion T-NE is obtained by an inverse emulsion method-probe ultrasonic method, and the cationic nanoemulsion B-NE without alpha-tocopherol is obtained. Then incubating the water without leaving ions of RNase and DNase with Poly (I: C) with T-NE or B-NE at 37 ℃ for 15 min to obtain nanoemulsion PT-NE or P-NE with strong-excitation adjuvant Poly (I: C) adsorbed on the surface with particle diameter of about 150-200nm and with or without ROS scavenging component alpha-tocopherol, wherein the TEM electron microscope result shows that PT-NE or P-NE has a remarkable solid spherical appearance (see figure 15). Subsequently we repeatedly stimulated balb/c mice with 1% DNCB for one week, followed by local administration of mice with PT-NE or P-NE, and during this period kept repeatedly stimulated balb/c mice at a frequency of 0.5% DNCB once every 4 days. During this time, we found that either PT-NE or P-NE was effective in alleviating the progression of recurrent dermatitis in mice, and in particular PT-NE with a strongly reducing component α -tocopherol, exhibited a more effective treatment of dermatitis with adaptability than untreated controls (see fig. 16). And the drug-loaded nanoemulsion PT-NE can effectively reduce the adaptive dermatitis markers, namely serum IgE (see figure 17) and type 2 cytokine IL-4 in skin (see figure 18). The data show that the drug-loaded nanoemulsion PT-NE which is loaded with the reducing component alpha-tocopherol and the strong DCs agonist adjuvant component Poly (I: C) can finally and effectively play a role in relieving the progress of recurrent adaptive dermatitis through the regulation and control of DCs immunological functions and the differentiation of downstream Th2 cells and the concentration of type 2 pathogenic cytokines.

Example 5 preparation of hydrogel dressing for adjuvant-loaded reductive drug delivery System

Taking the prescription of the drug-loaded nano system PT-NE mentioned in example 4 as an example, the hydrogel dressing with longer local residence time and high biocompatibility is prepared, so as to achieve the aim of long-acting slow-release treatment of the adaptive dermatitis. The hydrogel dressing is hyaluronic acid with better skin affinity. 1.5g of poloxamer F127 is dissolved in 8.5mL of ultrapure water, then 1mL of the solution is used for redissolving PT-NE prepared in advance for freeze-drying, and after being fully mixed and vortexed, the temperature-sensitive hydrogel which can be smeared on an adaptive skin affected part can be prepared, and the hydrogel has good biocompatibility, and can form a layer of protective film on the affected part, so that opportunistic infection of pathogenic bacteria such as external staphylococcus aureus can be resisted, and recovery of the adaptive dermatitis is further accelerated.

EXAMPLE 6 drug loaded reducing PLGA nanoparticles can effectively activate DCs into Th1 inducible DCs

Drug loaded reducing PLGA nanoparticle formulation:

PLGA300-400mg

monophosphoryl lipid A40-50mg

Superoxide dismutase 10-18mg

1mL of water.

Monophosphoryl lipid a is a TLR-4 agonist of greater molecular weight, is a lipopolysaccharide modifier of lower toxicity and greater safety, and is effective in activating DCs, stimulating DCs to a DCs phenotype that induces Th 1. Superoxide dismutase can be used as reactive ROS reductase with larger molecular weight, and can effectively help the removal of ROS. We prepared PLGA nanoparticles MS@PLGA coated with monophosphoryl lipid A and superoxide dismutase by a single emulsion method, and TEM electron microscopy results show that the particle size of the nanoparticles is about 500nm (see figure 19). Subsequently, we treated primary mouse DCs obtained by in vitro differentiation with drug-loaded reducing PLGA nanoparticles ms@plga and OVA antigen for 24h, and detected the proportion of cd80+mhc ii+ DCs at the cd11c+ index, i.e. Th 1-induced DCs phenotype, by flow analysis. We found that DCs after ms@plga treatment exhibited a higher proportion of MHC II and CD80 double positive expression phenotype than the control group (see figure 20).

Example 7 preparation and use of PEG-PLA micelles effective in alleviating severe atopic dermatitis

Composition of drug-loaded PEG-PLA micelle:

200-360mg of polyethylene glycol (PEG) -polylactic acid (PLA)

Vitamin C120-300 mg

Tea polyphenols 0.1-8mg

Diprovocim 0.01-20mg

1mL of water

Vitamin C and tea polyphenols can be used as water-soluble and fat-soluble reducing ROS scavenger respectively, and can achieve auxiliary effect of relieving inflammation by eliminating excessive ROS at skin inflammation sites after topical administration. Diprovocim acts as an effective TLR-1 and TLR-2 agonistic adjuvant that regulates Th1 and Th2 differentiation based on DCs. To prepare drug-loaded PEG-PLA micelles, we first added DMSO in which Dipivocim and tea polyphenol were dissolved to DMSO of a commercially available PEG-PLA conjugate to form a mixed solution, then added this oil phase component drop-wise to a buffer in which an appropriate amount of reducing vitamin C was dissolved, and stirred for 16-24 hours. Then, after dialysis for 16-24 hours by ultrapure water, PEG-PLA micelle CDT-micellar loaded with vitamin C, tea polyphenol and an adjuvant Dipivocim is prepared, and then the objective micelle solution is obtained by dissolving the PEG-PLA micelle CDT-micelle with ultrapure water (1-2 mL). ROS scavenger vitamin C, tea polyphenol and TLR-1 and TLR-2 agonist Dipivocim can be used as active ingredients thereof, and plays roles in regulating and controlling the balance of Th cells based on the immune function of DCs to regulate microenvironment and further relieving skin inflammation by utilizing a reducing component to treat the adaptive dermatitis. To verify the efficacy of PEG-PLA micelles CDT-micelle in alleviating severe atopic dermatitis, we first stimulated balb/c mice with 1% DNCB every two days for one week, then kept repeatedly stimulating balb/c mice with 0.5% DNCB at a frequency of once every 4 days, and after 21 days a mouse model with severe atopic dermatitis was obtained. We treated balb/C mice with atopic dermatitis with PEG-PLA micelles CDT-micelle coated with vitamin C, tea polyphenol and adjuvant dipivocim, PEG-PLA micelles D-micelle loaded with adjuvant dipivocim only, PEG-PLA micelles CT-micelle loaded with reduced vitamin C, tea polyphenol, and mice with reduced atopic dermatitis during dosing, recorded and observed. We found that the adjuvant and ROS scavenger loaded nanoformulations CDT-micelle exhibited a greater efficacy in alleviating severe atopic dermatitis (see figure 21) and clearly played a key role in restoring Th1/Th2 balance in the skin sites of mice compared to the nanoformulations D-micelle loaded with a potent adjuvant ingredient alone and the reduced ROS scavenger alone CT-micelle (see figure 22).

Example 8 preparation and use of novel microneedle Patch containing drug-loaded nanoemulsion for alleviating adaptive dermatitis

Nanoemulsion formulation:

phosphatidylserine 45-55mg

Cholesterol 0.2-3mg

Sucrose fatty acid ester 15-20mg

Beta-carotene 0.1-12mg

Astaxanthin 10-16mg

Raximote 10-28mg

1mL of water.

After the drug-loaded nanoemulsion containing the adjuvant rassimol for regulating and controlling the function of DCs and the liposoluble reducing agent ROS scavenger beta-carotene and astaxanthin is prepared by a reverse emulsion method, the RAC-NE is subjected to the preparation of a novel microneedle patch by using a silica gel micro-mould to relieve the adaptive dermatitis. Specifically, each needle cavity of the silicone micro-mold was selected from 200 μm×200 μm quadrangular base, and was tapered upward with a total height of 600 μm. The target microneedles were arranged in a 15 x 15 array with a 500 μm center-to-center spacing. First, we deposited 10% (w/w) hyaluronic acid solution of the prepared drug-loaded nanoemulsion RAC-NE into the needles, followed by drying the cavity under vacuum for 15 minutes, followed by depositing 100 μl of hyaluronic acid and collagen tripeptide as matrix materials to fill the needle cavities of the microneedles, after removing the excess solution, the micro-mold was stored overnight at the dry place to form hydrogels (room temperature). Subsequently, 500. Mu.L of hyaluronic acid solution was deposited on the microneedles and kept in air at room temperature for 4 hours, and after complete drying, the microneedles of the drug-loaded nanoemulsion were separated from the micro-mold to obtain RAC-NE. The prepared microneedle patch RAC-microacupuncture needle (RAC-micro-needle) can be directly placed on affected skin with adaptive dermatitis. We found that after three weeks, the inflamed skin was greatly improved in mice treated with the microneedle patch RAC-micro-needle (FIG. 23), and ELISA results showed that the concentration of the type 2 pathogenic factor TSLP in the skin was greatly reduced after treatment with the novel microneedle patch RAC-micro-needle (FIG. 24), indicating that the novel microneedle patch RAC-micro-needle was effective in alleviating and even treating refractory atopic dermatitis.

Example 9 preparation and application of novel manganese-iron nanoparticles for relieving adaptive dermatitis

Composition of drug-loaded manganese-iron nanoparticles:

FeCl ₃ ·6H ₂ O 1.5g

MnCl ₂ ·4H ₂ O 5g

FeCl ₂ ·4H ₂ O 4g

MnSO ₄ proper amount of solution

10-1000mg of ginsenoside Rh 2.

Fe-containing ginsenoside Rh2 ₃ O ₄ The manganese nano preparation is prepared by a coprecipitation method and thenCan be used for relieving the treatment of the adaptive dermatitis. Firstly, divalent manganese itself can be used as a reducing agent for consuming extracellular matrix and intracellular excess ROS, while subsequently acting as a STING agonist in the cells of activated DCs to induce release of type I interferon, promoting subsequent Th1 differentiation. On the other hand, ginsenoside Rh2 can be used as an effective active ingredient of the traditional Chinese medicine ginseng extract to play a role in regulating and controlling the activation and immune functions of DCs. Therefore, the novel manganese-iron nanoparticle Rh2@MnFeNP loaded with ginsenoside can be used for relieving the application of the adaptive dermatitis. To prepare Rh2@MnFeNP, first, an appropriate amount of Rh2, mnCl is added ₂ ·4H ₂ O and FeCl ₂ ·4H ₂ O is dissolved in advance to dissolve FeCl ₃ ·6H ₂ The total molar ratio of divalent manganese to iron was made 0.3 in the ultrapure water solution. Subsequently, 5% polyethylene glycol was added ₂₀₀₀ (PEG ₂₀₀₀ ) Slowly adding the (w/w) solution into the metal chloride solution, fully mixing by ultrasonic stirring, dripping 2.5mol/L KOH solution into the mixed solution at 55 ℃, and stirring to cause coprecipitation of manganese-iron metal ions. The manganese-iron-Rh 2 coprecipitate obtained later is aged at 55 ℃, and the operations of separation, washing, drying and the like are completed and are ready for use. Dispersing 1.0g of the dried sample in PEG under ultrasonic stirring ₂₀₀₀ To the mixture is then added MnSO ₄ Solution (100 mL 0.04 mol/L) and KMnO ₄ The solution (0.045 mol/L85 mL) was heated to 55deg.C and stirred to form ginsenoside Rh 2-loaded Fe/MnO ₂ The nano-particle Rh2@MnFeNP was purified by magnetic separation means, followed by washing with ultra pure water and drying overnight. We examined Rh2@MnFeNP by means of TEM, and found that this method was able to form nanoparticles with a particle size of 100-200nm (FIG. 25). Wherein Fe/MnO ₂ The components can be used for scavenging ROS, activating SING to promote the release of Th1 induced type one interferon, rh2 as an effective DCs activating adjuvant further promotes the formation of Th1 induced DCs, regulates the differentiation and relative proportion of Th1 and Th2 in dermatitis microenvironment, and finally relieves the occurrence and development of adaptive dermatitis caused by pathogenic Th2 cell excess.

Example 10 preparation and use of novel self-assembled micelles for alleviation of atopic dermatitis

Composition of drug-loaded self-assembled micelles:

polydopamine 45-60mg

Furillic acid 31-75mg

Gardiquimod 0.1-10mg。

The micelle loaded with the powerful TLR-7 agonist adjuvant Gardinquimod is prepared based on the conjugation of the amino group of the polydopamine serving as a reducing component and the carboxyl group of the fulvic acid serving as a reducing component, and is used for improving the inflammatory environment of inflammatory sites, regulating and controlling the DCs function, improving the balance of Th1/Th2 at the sites of the adaptive dermatitis and relieving the progress of the adaptive dermatitis. Preparation method briefly, an appropriate amount of fulvic acid, which can be used as a ROS scavenger, was dissolved in solvent THF, N, N' -dicyclohexylcarbodiimide and N-hydroxysuccinimide were added, and activated at room temperature for 8 hours. Subsequently, ice-cold n-hexane was added to the above mixture to precipitate activated fulvic acid, followed by drying at 40 ℃. The polydopamine and activated fulvic acid were co-incubated in dichloromethane for 15-24 hours to form the fulvic acid-polydopamine conjugate, and the target conjugate was dried by rotary evaporation. Dissolving by dilute hydrochloric acid, precipitating by ice-cold acetone, mixing by ultrapure water, filtering, and freeze-drying to obtain the fulvic acid-polydopamine conjugate. An appropriate amount of Gardiquimod was dispersed in a mixed solution of triethylamine (0.1 mL) and anhydrous DMSO (1 mL), and activated with equal amounts of N, N' -dicyclohexylcarbodiimide and N-hydroxysuccinimide at room temperature for 2 hours. The fulvic acid-polydopamine (3:1, 100 mg) prepared in advance was added to 25mL of ultrapure water, and then diluted with methanol and stirred to obtain an optically clear solution, and the activated Gardiquimod solution was added dropwise. The mixture was stirred at room temperature under nitrogen for 24 hours, dialyzed against phosphate buffered saline at pH 7 to eliminate excess unreacted substrate, dialyzed against ultrapure water, isolated by lyophilization to self-assemble the component polymers, which were subsequently reconstituted into the corresponding solutions or subsequently made into microneedles or topically applicable gels. In the self-assembled micelle, polydopamine and fulvic acid can be used as ROS scavengers, and fat-soluble Gardiquimod can be used as an adjuvant, so that the severity of the adaptive dermatitis is improved.

Example 11 preparation and use of novel solid lipid nanoparticles for relieving adaptive dermatitis

Composition of drug-loaded solid lipid nanoparticles:

0.2-17mg of glyceryl monostearate,

PEG ₂₀₀₀ 0.1-20mg，

vitamin E acetate 0.01-5mg,

CU-CPT17e 0.01-5mg。

the novel solid lipid nanoparticle loaded with the liposoluble adjuvant CU-CPT17E and the antioxidant vitamin E acetate can be prepared by a solvent diffusion method, the TLR-9 receptor of DCs is activated by the CU-CPT17E to induce the DCs to Th1 triggering DCs, and the inhibition effect of oxidative stress is carried out by the vitamin E acetate, so that the development of recurrent type and severe type adaptive dermatitis is realized. First, CU-CPT17E and vitamin E acetate are co-dissolved in a mixture containing appropriate amounts of glycerol monostearate and PEG ₂₀₀₀ The resulting organic solution was then rapidly dispersed in poloxamer 188 solution (0.1%, w/v) with mechanical stirring at 400rpm for 5 minutes (70 ℃). Then, after cooling the molten lipid drop pre-emulsion to room temperature, dialyzing the dialysis membrane with 10% polyvinylpyrrolidone K30 solution for 48 hours, and concentrating to obtain solid lipid nanoparticle CUVe@NP loaded with CU-CPT17E and vitamin E acetate, wherein the method is capable of forming nanoparticle CUVe@NP with the particle size of 100-200nm (figure 26), and has the effect of treating the atopic dermatitis.

EXAMPLE 12 adoptive feedback of skin lysate activation for adaptive dermatitis and drug-loaded nanoformulation-treated DCs vaccine

Drug-loaded nano-formulation prescription:

egg yolk lecithin S100-80 mg

DSPE-PEG 0.1-3mg

Alpha-tocopherol 0.1-20mg

DOTAP 0.04-20mg

CpG ODN 7909D10 0.01-10mg

1mL of water.

The skin lysate of the adaptive dermatitis accounts for 0.1% -50% (w/w) of the normal DCs culture medium.

Considering that atopic dermatitis is an autoimmune disease in nature, direct adoptive infusion of DCs vaccine treated with the skin lysate of the lesion of atopic dermatitis and the drug-loaded nano-formulation may be an effective measure for preventing atopic dermatitis. And extracting mononuclear cells/DCs precursor cells in peripheral blood or bone marrow, and performing in vitro expansion and differentiation to obtain DCs cells to be used. Subsequently, after 24h of pulse treatment of DCs with a proportion of the skin lysate of the lesion of atopic dermatitis and the adjuvant-loaded reductive drug loaded nano-preparation CpGT-NE, DCs were obtained which captured and presented to some extent the antigen associated with atopic dermatitis. After detection by flow analysis means, the CPGT-NE plus lesion skin lysate treated DCs were found to exhibit a Th1 activated DCs phenotype (FIG. 27), and after incubation of the treated DCs with spleen T lymphocytes at a ratio of 1:20 for 72 hours, to examine whether the reducing drug loaded nanoformulation CpGT-NE could increase the Th1/Th2 ratio of T cells in the microenvironment. The results indicate that nano-formulation CpGT-NE treated DCs reduced relative differentiation of Th2 and regulated Th1/Th2 ratio in the microenvironment compared to DCs treated with skin lysate of only the focal site of atopic dermatitis (corresponding to DCs used to mimic the focal site of atopic dermatitis) (FIG. 28). Subsequently, we have succeeded in infusing back the DCs vaccine treated in this way into mice suffering from atopic dermatitis, and found that the nano-formulation CpGT-NE + atopic dermatitis focus skin lysate-treated DCs vaccine was effective in alleviating the symptoms of recurrent atopic dermatitis (fig. 29).

Claims (5)

1. A reductive adjuvant nano-system capable of relieving adaptive dermatitis is characterized by comprising an oil phase and a water phase, wherein the oil phase is selected from 5-70% of phospholipid, 5-70% of triglyceride, 0.01-30% of oxidative stress inhibitor, 0.01-30% of oil-soluble strong-agitation adjuvant, and the water phase is selected from 0.01-30% of deionized water solution of water-soluble strong-agitation adjuvant, or 0.01-30% of deionized water solution of water-soluble oxidative stress inhibitor, or single deionized water.

2. The reducing adjuvant nanosystem of claim 1, wherein the nanosystem comprises an oil phase: the water phase is 0.01-50%:1%, w/w.

3. The nanosystem of claim 1, wherein the nanosystem is a liposome, nanoemulsion, micelle or lipid nanoparticle, and is prepared by a single emulsion method, a reverse emulsion method, a thin film dispersion method or a rotary evaporation method.

4. Use of a reductive adjuvant nanosystem according to claim 1 for the manufacture of a medicament for the alleviation and treatment of recurrent and severe form of atopic dermatitis.

5. The use according to claim 4, wherein the pharmaceutical formulation and route of administration is:

(1) The nano system is self-prepared medicine for direct local delivery, the administration concentration is 0.1 mug/kg-10 mg/kg, and the administration route is local smearing and transdermal;

(2) The nanometer system is continuously manufactured into a microneedle patch or a water-based adhesive dressing in a micro-die, and the drug administration concentration is 0.1 mug/kg-10 mg/kg, and the microneedle patch or the water-based adhesive dressing is directly applied to an affected part;

(3) The nano system is prepared into DCs vaccine and injected for administration.