Soluble nano microneedle as well as preparation method and application thereof

Abstract

The invention relates to a soluble nano microneedle for treating atopic dermatitis, the soluble nano microneedle comprises a substrate layer and a needle tip layer, the needle tip layer is formed by a bletilla striata polysaccharide loaded nano drug si slc31a1 / CaP, and the nano drug si slc31a1 / CaP comprises Slc31a1 siRNA, calcium phosphate nanoparticles and bovine serum albumin. The soluble nano microneedle si-Slc31a1 / CaP coated BSP provided by the invention can provide a new direction for safe, effective, simple and convenient treatment of specific dermatitis.

Description

Technical Field

[0001] The present invention relates to a soluble nano-microneedle and a preparation method and application thereof, belonging to the field of medicine. Background Art

[0002] Atopic dermatitis is one of the most common chronic inflammatory skin diseases. According to statistics, its prevalence is 10% in adults and as high as 15-20% in children. Clinical symptoms of atopic dermatitis include redness, swelling, exfoliation, and lichenification, often accompanied by exudation and dryness, which seriously affect patients' quality of life. The pathogenesis of atopic dermatitis is complex and diverse, and is generally closely related to an abnormal inflammatory response mediated by helper T cells and elevated eosinophil and serum immunoglobulin E levels. Targeting this mechanism, topical corticosteroids and immunosuppressants are currently the first-line treatment options for atopic dermatitis. However, patients often develop drug resistance and corresponding side effects after long-term use of these drugs. Therefore, in order to improve the quality of life of patients with atopic dermatitis, there is an urgent need to identify new therapeutic targets and establish a treatment strategy that effectively inhibits skin inflammation.

[0003] Atopic dermatitis (AD) and the gene SLC31A1 may be indirectly linked through pathways such as copper ion metabolism and immune regulation. The inventors found that solute carrier family 31, member 1 (SLC31A1) is highly expressed in the epidermis of patients with AD and rodent models. Epidermal SLC31A1-mediated copper death can promote keratinocyte pyroptosis, thereby affecting the development and progression of AD. Therefore, the development of drugs targeting SLC31A1 as a specific target has broad clinical application prospects.

[0004] With the continuous elucidation and improvement of the structure and function of DNA and RNA, gene therapy has become a safer and more effective new therapeutic approach. Its principle is to selectively weaken or enhance disease-related target genes. Small interfering RNA (siRNA) is one of the tools used in gene therapy. It can directly inhibit the expression of target transcripts. Due to its versatility, selectivity, and specificity, it has attracted widespread attention in recent years. Knockdown of SLC31A1 using Slc31a1 siRNA significantly inhibits SLC31A1 expression, thereby treating atopic dermatitis. However, this approach faces many challenges in practical application. First, free siRNA is easily digested by various nucleases in serum, resulting in premature drug inactivation and low local drug retention. Second, siRNA has poor bioavailability, making it difficult to penetrate the stratum corneum and directly transfect cells in lesion sites to interfere with the expression of target transcripts, resulting in poor therapeutic efficacy.

[0005] In summary, the development of a topical drug that can maintain the activity of Slc31a1 siRNA while increasing its bioavailability has great clinical application value and potential in the treatment of atopic dermatitis. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to construct an siRNA delivery system for treating atopic dermatitis.

[0007] To achieve the above objectives, the present invention first provides a soluble nano-microneedle comprising a basal layer and a tip layer. The tip layer is composed of a bletilla striata polysaccharide-loaded nanodrug si slc31a1 / CaP, which contains Slc31a1 siRNA, calcium phosphate nanoparticles, and bovine serum albumin. This soluble nano-microneedle is a nano-microneedle for treating atopic dermatitis.

[0008] Preferably, the soluble nano-microneedle has a length of 500-600 μm, and the side length of the base layer is 8-10 mm.

[0009] Preferably, the needle tip is obtained by mixing and drying a solution of Bletilla striata polysaccharide and a nano drug si slc31a1 / CaP;

[0010] Preferably, the nanodrug si slc31a1 / CaP is calcium phosphate nanoparticles generated under the mediation of Slc31a1 siRNA and bovine serum albumin.

[0011] The present invention also provides a method for preparing the above-mentioned soluble nano-microneedles, which specifically comprises the following steps:

[0012] S1. Dissolve Slc31a1 siRNA in DEPC water; then disperse the Slc31a1 siRNA solution and bovine serum albumin in sugar-free DMEM and stir evenly;

[0013] S2. Add CaCl2 solution to the mixed solution obtained in step S1, incubate at 37°C, centrifuge, collect the precipitate, and re-dissolve it by ultrasonication to obtain si slc31a1 / CaP nanoparticles;

[0014] S3. Disperse the si slc31a1 / CaP nanoparticles obtained in step S2 into the Bletilla striata polysaccharide solution, fill the resulting solution into a microneedle mold, centrifuge and vacuum, and then dry in a constant temperature and humidity chamber for 4 hours to solidify the microneedle tip.

[0015] S4. Inject the hyaluronic acid solution into the microneedle mold with the needle tip solidified as a base layer, centrifuge and dry, and demold to obtain soluble nano-microneedles.

[0016] Preferably, the concentration of the Slc31a1 siRNA solution in S1 is 2-2.5 μmol / L.

[0017] Preferably, the concentration of the CaCl2 solution in S2 is 1-1.5 mol / L.

[0018] Preferably, the concentration of the Bletilla striata polysaccharide solution in S3 is 0.2-0.3 mg / mL.

[0019] Preferably, the concentration of the hyaluronic acid solution in S4 is 0.4-0.5 mg / mL, and the molecular weight of the hyaluronic acid is 10 KDa.

[0020] The present invention also provides the use of the soluble nano-microneedles in preparing drugs for treating / preventing atopic dermatitis.

[0021] the term

[0022] Synergistic effect: When two drugs are used simultaneously or sequentially, the original efficacy can be enhanced, which is called synergistic effect, including addition, enhancement and sensitization;

[0023] Microneedles: A microneedle patch is a microneedle array (50 to 1500 microns in length) made of soluble or insoluble materials that can penetrate the stratum corneum to painlessly deliver drugs or vaccines.

[0024] Soluble microneedles: The needle body is made of a drug-loaded polymer that dissolves and releases the drug after insertion (such as insulin delivery).

[0025] Nanodelivery carrier: refers to a type of tiny carrier system (usually in the range of 1-100 nanometers) designed based on nanotechnology, which is used to efficiently and accurately deliver drugs, genes, proteins or other therapeutic substances to specific parts of the body (such as diseased tissues, cells or organelles).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention uses soluble nanoneedles to introduce Slc31a1 siRNA during the biomineralization process of CaP nanoparticles, forming si-Slc31a1 / CaP nanoparticles. This method effectively prevents the premature degradation of Slc31a1 siRNA and significantly improves local drug retention and utilization. In addition, the nanoparticles, as carriers, can efficiently transduce Slc31a1 siRNA into keratinocytes in the epidermis of lesions, enhancing their inhibitory effect, thereby effectively inhibiting the expression of epidermal SLC31A1 and ultimately achieving a therapeutic effect on atopic dermatitis.

[0028] (2) The present invention uses a soluble nanoneedle delivery system to deliver si-Slc31a1 / CaP@BSP nanoparticles in situ to the site of atopic dermatitis skin lesions via a local, highly efficient delivery method. This method can effectively reduce the interference of Slc31a1 siRNA with surrounding normal tissues or organs, and the carrier components are all non-toxic substances with good biocompatibility and degradability. In addition, the Bletilla striata polysaccharide contained in the microneedle tip itself has an anti-inflammatory effect, which synergizes with Slc31a1 siRNA to achieve the therapeutic effect of atopic dermatitis.

[0029] (3) The preparation process of the soluble nano-microneedles for treating atopic dermatitis provided by the present invention is fast and simple, the reaction process is controllable and green and environmentally friendly, no additional auxiliary materials and organic solvents are required, and no other impurities are introduced.

[0030] The soluble nano-microneedle si-Slc31a1 / CaP@BSP provided by the present invention can provide a new direction for the treatment of atopic dermatitis as a safe, effective and simple method. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 TEM image of si Slc31a1 / CaP nanoparticles prepared in Example 1 (scale 200 nm);

[0032] Figure 2 is the particle size distribution diagram of si Slc31a1 / CaP nanoparticles prepared in Example 1;

[0033] Figure 3 Element distribution diagram of si Slc31a1 / CaP nanoparticles prepared in Example 1;

[0034] Figure 4 This is a diagram of cellular uptake of si Slc31a1 / CaP nanoparticles prepared in Example 1;

[0035] Figure 5 The general morphology of the si Slc31a1 / CaP@BSP microneedles prepared in Example 1;

[0036] Figure 6 This is an optical microscope image of the si Slc31a1 / CaP@BSP microneedle prepared in Example 1;

[0037] Figure 7 Fluorescence microscopy image of si Slc31a1 / CaP@BSP microneedles prepared in Example 1;

[0038] Figure 8 This is a stress test diagram of the si Slc31a1 / CaP@BSP microneedle prepared in Example 1;

[0039] Figure 9 The si Slc31a1 / CaP@BSP prepared in Example 1 dissolves over time in mouse skin;

[0040] Figure 10 This is an AD-like image of the ear skin of a mouse model on the 14th day of an animal experiment on the therapeutic effect of si Slc31a1 / CaP@BSP prepared in Example 1 on atopic dermatitis;

[0041] Figure 11 The EASI total score of the mouse ear skin on the 14th day of the animal experiment on the therapeutic effect of si Slc31a1 / CaP@BSP prepared in Example 1 on atopic dermatitis (mean±SD, n=6);

[0042] Figure 12 This is a HE staining image of the mouse model ear skin on the 14th day of the animal experiment on the therapeutic effect of si Slc31a1 / CaP@BSP prepared in Example 1 on atopic dermatitis (scale bar 100 μm).

[0043] In the picture:

[0044] Significance was determined by Wilcoxon rank sum test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0045] The present invention will be described in further detail and in full below in conjunction with the following embodiments. The following embodiments are exemplary and are only used to explain the present invention and are not to be construed as limiting the present invention.

[0046] The experimental methods in the following examples, unless otherwise specified, generally followed conventional conditions such as those described in the Manual of Pharmacology, 3rd edition, Science Press, 2002. Calcipotriol (MC903) was used to construct an atopic dermatitis model based on published literature (Reference: 1. Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis model).

[0047] 2. The AhR-Ovol1-Id1 regulatory axis in keratinocytes promotes sepidormal and immune homeostasis in atopic dermatitis-like skin inflammation.) or according to the conditions recommended by the manufacturer. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products and can be purchased through commercial channels.

[0048] The main materials involved in the embodiments of the present invention are as follows:

[0049] 1. Experimental Animals

[0050] Specific pathogen-free (SPF) BAL B / C male mice, 6-8 weeks old and weighing 20-23 g, were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. and maintained at the Central South University Laboratory Animal Center. This study has passed the Central South University Animal Welfare Ethics Review. The "3R" principles were adhered to throughout the experiment to minimize animal pain and discomfort.

[0051] 2. Experimental Reagents

[0052] BSA (Wuhan Sevier Biotechnology Co., Ltd.), CaCl2 (Shanghai Aladdin Biochemical Technology Co., Ltd.), DMEM (Wuhan Punosai Life Science Co., Ltd.), Slc31a1 siRNA (Ribo Biotechnology), Bletilla striata polysaccharide (Shanghai Yuanye Biotechnology Co., Ltd.), hyaluronic acid (Shanghai Yuanye Biotechnology Co., Ltd.), PBS (Shanghai Xiaopeng Biotechnology Co., Ltd.), MC903 (MedChemExpress Biotechnology Co., Ltd., USA), hematoxylin staining solution, hematoxylin bluing solution, hematoxylin differentiation solution (Sevier Biotechnology Co., Ltd.), mounting medium (Wuxi Jiangyuan Industrial Technology and Trade Corporation), environmentally friendly transparent dewaxing solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.)

[0053] Example

[0054] 1. Preparation of si Slc31a1 / CaP

[0055] 1. Dissolve 60 nmol of si Slc31a1 in 600 μL of diethyl pyrocarbonate (DEPC)-treated water. Dissolve this solution and 300 mg of bovine serum albumin (BSA) in 30 mL of sugar-free DMEM medium and stir well.

[0056] 2. Add 300 μL of 1 mol / L calcium chloride (CaCl2) solution to the mixture obtained in step 1. Incubate at 37°C for 24 hours and centrifuge at 12,000 rpm for 15 minutes to obtain si Slc31a1 / CaP.

[0057] 2. Preparation of soluble nano-microneedles si Slc31a1 / CaP@BSP

[0058] 1. A microneedle patch was fabricated using a polydimethylsiloxane (PDMS) mold with a conical pore array. The microneedles were 9 mm × 9 mm in size, with 100 tips in the array, each with a 250 μm base diameter and 550 μm length.

[0059] 2. Add 0.25g BSP to 1mL pure water to prepare a 25% Bletilla striata polysaccharide solution, and heat in a 60℃ water bath to dissolve for 30min.

[0060] 3. Dissolve the si Slc31a1 / CaP nanoparticles prepared in step 1 in the solution in step 2 at a volume ratio of 1:6, stir and mix to obtain a BSP solution containing si Slc31a1 / CaP nanoparticles, inject the solution into the microneedle mold, centrifuge, and dry in a constant temperature and humidity chamber at 37°C for 4 hours to form the microneedle tip;

[0061] 4. Take 100 μL of 50% hyaluronic acid solution and inject it into the microneedle mold as the base layer. After centrifugation, dry it in a constant temperature and humidity chamber at 37°C overnight. After demolding, soluble nano-microneedles are obtained.

[0062] 3. Evaluation of si Slc31a1 / CaP Characteristics

[0063] The physicochemical properties, in vitro and in vivo pharmacokinetic and pharmacodynamic characteristics of the si Slc31a1 / CaP nanoparticles obtained in this example were tested, including microscopic morphology, particle size, elemental composition and distribution, and in vitro cell transfection efficiency, as follows.

[0064] (1) Observation of the morphology of si Slc31a1 / CaP nanoparticles

[0065] A small amount of sample was dropped onto a 400-mesh zinc grid covered with a carbon film, and then placed in a desiccator to dry. After it dried naturally, the morphology of the nanoparticles was observed under a transmission electron microscope Titan G2-F20.

[0066] The results are as follows Figure 1 As shown, si Slc31a1 / CaP nanoparticles are amorphous polymers under transmission electron microscopy.

[0067] (2) Determination of the particle size of si Slc31a1 / CaP nanoparticles

[0068] The si Slc31a1 / CaP sample solution was placed in a Marlven Nano ZS instrument, and the dynamic light scattering method was used to detect the particle size of the nanoparticles.

[0069] The results are as follows Figure 2 As shown, the particle size of si Slc31a1 / CaP was approximately 102.8±7.7 nm.

[0070] (3) Observation of surface element distribution of si Slc31a1 / CaP nanoparticles

[0071] The sample was dropped onto a 400-mesh zinc grid covered with a carbon film, placed in a drying oven, and observed under a transmission electron microscope Titan G2-F20 after it was naturally dried.

[0072] The results are as follows Figure 3 As shown, C, O, N, P and Ca elements are distributed on the surface of nanoparticles.

[0073] (4) Investigating the transfection efficiency of si Slc31a1 / CaP

[0074] Human keratinocytes were used as model cells, and the experimental groups were Slc31a1 siRNA, si Slc31a1 / CaP, and si Slc31a1 / Lipo2000. After 48 hours of incubation, cells were digested and centrifuged, washed twice with PBS, and resuspended. Cell fluorescence intensity was measured by flow cytometry to determine the efficacy of Slc31a1 siRNA transfection with different vectors.

[0075] The results are as follows Figure 4 As shown, Cap exhibited a transfection efficiency similar to that of the commercial vector Lipo2000.

[0076] IV. Characterization of Soluble Nanoneedles si Slc31a1 / CaP@BSP

[0077] The apparent characteristics of si Slc31a1 / CaP@BSP were detected, including micromorphology, mechanical properties, and degradation rate, as follows.

[0078] (1) Observe the gross morphology of si Slc31a1 / CaP@BSP microneedles using a digital camera: Place si Slc31a1 / CaP@BSP on a table to observe the morphology of the microneedles.

[0079] The results are as follows Figure 5 As shown, the si Slc31a1 / CaP@BSP needle tips are orderly arranged in the basal layer, forming a regular and orderly microneedle array as a whole.

[0080] (2) Observation of the morphology of si Slc31a1 / CaP@BSP microneedles under an ordinary optical microscope: The morphology of the microneedles was observed under an upright microscope.

[0081] The results are as follows Figure 6 As shown, the tip width of si Slc31a1 / CaP@BSP is 10 μm and the needle length is 550 μm.

[0082] (3) Observation of the morphology of si Slc31a1 / CaP@BSP microneedles under a fluorescence microscope: The morphology of the microneedles was observed under an upright fluorescence microscope.

[0083] The results are as follows Figure 7 As shown, si Slc31a1 / CaP (green fluorescence) accumulated at the microneedle tip.

[0084] (4) Testing the mechanical properties of si Slc31a1 / CaP@BSP using an electronic universal testing machine

[0085] The microneedle (tip facing up) was placed on the test table. The mechanical sensor probe automatically compressed the microneedle at a speed of 0.5 mm / min. After reaching the maximum load (set to 55 N), it automatically stopped running and recorded the applied force to draw a force-displacement curve.

[0086] The results are as follows Figure 8 As shown in the figure, the drug-loaded microneedles did not break even under a pressure of 0.55N / needle, indicating that the microneedles have excellent mechanical strength, exceeding the 0.2N / needle required to penetrate the skin, and can penetrate the skin's stratum corneum to achieve effective drug delivery.

[0087] (5) Detection of si Slc31a1 / CaP@BSP dissolution rate

[0088] si Slc31a1 / CaP@BSP was applied to the back skin of Balb / c mice. After continuous pressing with the thumb for 5 minutes, 10 minutes, 30 minutes and 60 minutes, the microneedles were placed under an ordinary optical microscope to observe their dissolution.

[0089] The results are as follows Figure 9 As shown in the figure, si Slc31a1 / CaP@BSP gradually dissolved with the prolonged residence time in the skin and was basically dissolved after 60 min.

[0090] 5. Animal Experimentation

[0091] A mouse atopic dermatitis model was constructed to evaluate the in vivo therapeutic effect of the soluble nano-microneedle si Slc31a1 / CaP@BSP obtained in this example on atopic dermatitis.

[0092] 1. Establishment of a mouse atopic dermatitis model: Six-week-old male Balb / c mice were randomly divided into a normal group and an atopic dermatitis model group. Calcipotriol (MC903) was used to establish the atopic dermatitis model.

[0093] 2. Effect of si Slc31a1 / CaP@BSP in treating atopic dermatitis:

[0094] Mice with atopic dermatitis were randomly divided into four groups and treated with different microneedle treatments: Vehicle, siNT / CaP@BSP, siSlc31a1@BSP, and siSlc31a1 / CaP@BSP. The specific treatments are shown in Table 1. The mice were observed and recorded daily. The skin lesions, EASI scores, and HE staining of the mice were evaluated on the 14th day. The specific experimental results are as follows.

[0095]

[0096] (1) Skin lesions in mice

[0097] Figure 10 The following is a comparison of the gross appearance of the skin of the normal group and the atopic dermatitis model mice treated with different microneedle treatments. Figure 10 As shown in the figure, after treatment with si Slc31a1 / CaP@BSP, the scaling and thickening of the atopic dermatitis model in mice were significantly alleviated, approaching the skin of normal mice.

[0098] (2) The eczema area and severity index (EASI score) was used to score the severity of inflammation in the skin lesions of mice.

[0099] The results are as follows Figure 11 As shown in the figure, compared with other model groups, the EASI score of mice in the si Slc31a1 / CaP@BSP group was significantly reduced.

[0100] (3) Collect mouse skin for HE staining

[0101] like Figure 12 As shown, compared with other model groups, the epidermis of the mice in the si Slc31a1 / CaP@BSP group was thinner at the modeling site.

[0102] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Any technician familiar with this profession, without departing from the scope of the technical solution of this application, can make slight changes, modifications, substitutions, combinations, and simplifications using the above-disclosed technical content, which should be equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A soluble nano-microneedle, characterized in that: The soluble nano-microneedle comprises a basal layer and a needle tip layer, wherein the needle tip layer is composed of bletilla striata polysaccharide loaded nano-drug si slc31a1 / CaP, and the nano-drug sislc31a1 / CaP comprises Slc31a1 siRNA, calcium phosphate nanoparticles and bovine serum albumin.

2. The nano-microneedle according to claim 1, characterized in that The length of the soluble nano-microneedle is 500-600 μm, and the side length of the base layer is 8-10 mm.

3. The nano-microneedle according to claim 1, characterized in that The needle tip is obtained by mixing and drying bletilla striata polysaccharide and nano drug sislc31a1 / CaP solution.

4. The nano-microneedle according to claim 1, characterized in that The nano drug si slc31a1 / CaP is calcium phosphate nanoparticles generated under the mediation of Slc31a1 siRNA and bovine serum albumin.

5. The method for preparing the nano-microneedle according to any one of claims 1 to 4, characterized in that: The specific steps include: S1. Dissolve Slc31a1 siRNA in DEPC water; then disperse the Slc31a1 siRNA solution and bovine serum albumin in sugar-free DMEM and stir evenly; S2. Add CaCl2 solution to the mixed solution obtained in step S1, incubate at 37°C, centrifuge, collect the precipitate, and re-dissolve it by ultrasonication to obtain si slc31a1 / CaP nanoparticles; S3. Disperse the si slc31a1 / CaP nanoparticles obtained in step S2 into the Bletilla striata polysaccharide solution, fill the resulting solution into a microneedle mold, centrifuge and vacuum, and then dry in a constant temperature and humidity chamber for 4 hours to solidify the microneedle tip. S4. Inject the hyaluronic acid solution into the microneedle mold with the needle tip solidified as a base layer, centrifuge and dry, and demold to obtain soluble nano-microneedles.

6. The method for preparing nano-microneedles according to claim 5, characterized in that: The concentration of the Slc31a1 siRNA solution in the S1 is 2-2.5 μmol / L.

7. The method for preparing nano-microneedles according to claim 5, characterized in that: The concentration of the CaCl2 solution in the S2 is 1-1.5 mol / L.

8. The method for preparing nano-microneedles according to claim 5, characterized in that: The concentration of the Bletilla striata polysaccharide solution in S3 is 0.2-0.3 mg / mL.

9. The method for preparing nano-microneedles according to claim 5, characterized in that: The concentration of the hyaluronic acid solution in the S4 is 0.4-0.5 mg / mL, and the molecular weight of the hyaluronic acid is 10 KDa.

10. Use of the nano-microneedle according to any one of claims 1 to 4 or the nano-microneedle obtained by the preparation method according to any one of claims 5 to 9 in preparing a drug for treating / preventing atopic dermatitis.

Images