Method for preparing small nucleic acid combination microneedle and its use for repairing skin photo-damage

Abstract

The present application relates to a small nucleic acid combination microneedle preparation method and its skin light damage repair purposes, specifically discloses a microneedle composition, including the complex of SNAP25 specific siRNA and nano selenium and hyaluronic acid, wherein the nucleotide sequence of the sense strand of siRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of siRNA is as shown in SEQ ID NO.2.The microneedle composition of the present application can realize the stable delivery of siRNA, inhibit the expression of UV-induced skin photoaging related genes, increase the repair of skin extracellular matrix, significantly improve the skin light damage, and is suitable for clinical transformation application.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and dermatology, specifically to a method for preparing small nucleic acid combination microneedles and their application in repairing photodamage to the skin. Background Technology

[0002] Ultraviolet (UV) radiation is a major environmental factor leading to photoaging, inflammation, and damage to the skin barrier function. It activates abnormal signaling pathways within skin fibroblasts, triggering pathological changes such as collagen degradation and increased oxidative stress. SNAP25 (synaptic-associated protein 25), a key regulator of neurotransmitter release and cell signaling, is highly expressed in UV-induced skin damage. It exacerbates skin damage by mediating the release of inflammatory factors and inhibiting fibroblast proliferation. Therefore, targeting and silencing SNAP25 has become an important therapeutic target for UV-induced skin damage.

[0003] Small interfering RNA (siRNA) can specifically silence target gene expression through RNA interference mechanisms, but its clinical application is limited by: 1) poor in vivo stability and easy degradation by nucleases; 2) weak cell penetration ability and low transfection efficiency; 3) systemic administration is prone to triggering immune responses; 4) different siRNAs have different inhibitory efficiencies; 5) even siRNAs with similar inhibitory efficiencies have differences in their delivery vector compatibility and delivery efficiency.

[0004] Currently, how to leverage the role of SNAP25 siRNA and achieve its efficient delivery and transfection for the treatment of UV-induced skin damage remains an urgent problem to be solved. Furthermore, there is a lack of SNAP25 siRNA sequences with high silencing efficiency that have been validated through cell screening, as well as a delivery system that can achieve efficient transfection of specific siRNAs with low toxicity.

[0005] Therefore, there is an urgent need for a method that can efficiently and stably deliver siRNA to effectively silence the SNAP25 gene in order to treat UV-induced skin damage. Summary of the Invention

[0006] In view of this, the main objective of this invention is to overcome the shortcomings of current SNAP25 siRNA, such as low delivery efficiency, poor stability, high carrier toxicity, and insufficient treatment methods for UV-induced skin damage. The invention provides a SNAP25 siRNA sequence with high silencing efficiency, and its combination with nano-selenium and hyaluronic acid (HA). The safety and therapeutic effect of the microneedles made by combining these sequences are verified, thereby achieving precise and efficient treatment of UV-induced skin damage.

[0007] Chemically derived selenium nanoparticles, as novel inorganic nanocarriers, possess low toxicity, good biocompatibility, and transfection-promoting capabilities. However, when delivering siRNA alone, they still suffer from insufficient targeting and poor in vitro circulation stability.

[0008] Microneedles, as transdermal drug delivery carriers, can penetrate the skin's stratum corneum barrier to achieve precise local drug delivery and reduce systemic side effects. Hyaluronic acid (HA), as a natural polysaccharide, has excellent biocompatibility, moisturizing properties, and degradability, making it an ideal substrate material for microneedles.

[0009] After screening out the SNAP25 siRNA 1 sequence with high silencing efficiency, the inventors combined it with selenium nanoparticles to verify the efficient transfection effect of selenium nanoparticles on siRNA, and finally made HA-based microneedles, thus completing the present invention.

[0010] The specific technical solution of the present invention is as follows.

[0011] The present invention provides a composition comprising a complex of SNAP25-specific siRNA and selenium nanoparticles and hyaluronic acid, wherein the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1 and the nucleotide sequence of the antisense strand of the siRNA is shown in SEQ ID NO.2.

[0012] In some embodiments, the siRNA is a modified siRNA, and the modification is selected from one or more of 2'-O-methyl and 2'-fluoro modifications.

[0013] In some implementations, the mass ratio of siRNA to selenium nanoparticles is 1:5 to 1:10.

[0014] In some implementations, the final concentration of siRNA is 30 nM to 100 nM.

[0015] In some implementations, the mass fraction of hyaluronic acid is 15% to 25%.

[0016] In some implementations, the microneedles are based on hyaluronic acid and loaded with a complex of siRNA and selenium nanoparticles.

[0017] In some embodiments, the final concentration of the complex is from 10 μg / mL to 20 μg / mL.

[0018] In some implementations, the microneedle has a needle height of 500 μm to 800 μm and a needle tip diameter of 10 μm to 20 μm.

[0019] The present invention also provides a method for preparing a microneedle composition, comprising the following steps:

[0020] Prepare a hyaluronic acid solution with a mass fraction of 15% to 25%;

[0021] SNAP25-specific siRNA and selenium nanoparticles were mixed at a mass ratio of 1:5 to 1:10 and incubated at room temperature in the dark for 30 min to form a complex of siRNA and selenium nanoparticles. The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the siRNA is shown in SEQ ID NO.2.

[0022] The complex was added to a hyaluronic acid solution at a final concentration of 10 μg / mL to 20 μg / mL and ultrasonically dispersed to form a premix of the composition; and

[0023] Optionally, the premixed liquid is injected into a PDMS microneedle mold, vacuum degassing is performed, and the mold is then refrigerated at 4°C for 12 hours before demolding to form hyaluronic acid-based microneedles.

[0024] The present invention also provides the use of microneedle compositions in the preparation of medicaments for treating UV-induced skin damage, wherein the composition is the microneedle composition of the present invention or a microneedle composition obtained according to the preparation method of the present invention.

[0025] The beneficial effects of this invention are:

[0026] On the one hand, the SNAP25 siRNA 1 sequence screened by this invention has high silencing efficiency and can specifically downregulate SNAP25 expression, and has the potential to inhibit UV-induced skin damage at the target level.

[0027] On the other hand, the use of nano-selenium to form a complex with the siRNA 1 of this application can not only improve the in vitro stability and cell transfection rate of the siRNA 1 (the transfection efficiency is comparable to that of Lipo3000 and there is no obvious cytotoxicity), but also synergize with the siRNA to exert a therapeutic effect.

[0028] Furthermore, this invention selects HA as the microneedle substrate, which achieves the best transfection efficiency for the complex compared to common substrates such as Cs and PVA.

[0029] On the other hand, by combining siRNA 1-nanose complex with HA substrate to form microneedles, the good biocompatibility and skin penetration of HA substrate microneedles can be utilized to achieve precise local delivery, avoiding the side effects of systemic drug administration. Moreover, the microneedle preparation process is simple, low-cost, and easy to scale up.

[0030] On the other hand, this invention demonstrates through in vivo experiments that the microneedles prepared by this invention can significantly improve UV-induced skin tissue damage, downregulate SNAP25 protein expression, and have a significantly better therapeutic effect than the blank HA microneedle group and the Lipo3000-siRNA group, showing good prospects for clinical application. Attached Figure Description

[0031] Figure 1 This is a graph showing the real-time quantitative fluorescence detection results of 100 SNAP25 siRNAs.

[0032] Figure 2 Skin phenotype of mice after 2 weeks of UV photoaging treatment.

[0033] Figure 3 HE staining of skin tissue from mice treated with UV photoaging for 2 weeks.

[0034] Figure 4 This is a fluorescence microscope image used to detect the transfection efficiency of siRNA by selenium nanoparticles.

[0035] Figure 5 This is a graph showing the qPCR results for detecting siRNA transfection efficiency in human skin fibroblasts aged by UV light.

[0036] Figure 6 Fluorescence microscopy images of different types of microneedle substrates used to detect siRNA transfection efficiency.

[0037] Figure 7 This image shows the preparation and morphology analysis of HA-based microneedles.

[0038] Figure 8 This is a graph showing the CCK8 cytotoxicity test results of the microneedles of the present invention.

[0039] Figure 9 This is a diagram showing the results of detecting live and dead cells using the microneedles of the present invention. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which this invention pertains. The following definitions supplement those in the art and relate to this application, but are not extrapolated to any relevant or unrelated circumstances, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0042] In this invention, the terms "comprising," "including," and "having" are open-ended descriptions that include the specified steps described, as well as other steps that do not substantially affect them, and are optional and not excluded.

[0043] As mentioned above, this invention was developed to address the problem of efficient delivery and transfection of SNAP25 siRNA for the treatment of UV-induced skin damage.

[0044] The present invention provides a microneedle composition comprising a complex of SNAP25-specific siRNA (denoted as siRNA 1) and selenium nanoparticles, and hyaluronic acid, wherein the nucleotide sequence of the sense strand of siRNA 1 is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of siRNA 1 is shown in SEQ ID NO.2.

[0045] SEQ ID NO.1:

[0046] 5'-UCCUUGUAACAAGUAGGUAC-3'

[0047] SEQ ID NO.2:

[0048] 5'-AGUACCUACUUGUUACAAGGACA-3'

[0049] In this invention, SNAP25-specific siRNA, namely siRNA 1, has high silencing efficiency and can effectively inhibit the expression of SNAP25, showing significant effects on the treatment of UV-induced skin damage; nano-selenium can serve as an siRNA carrier, enabling efficient and stable transfection of siRNA without toxicity to cells; hyaluronic acid can achieve precise delivery of the siRNA-nano-selenium complex, reducing the risk of side effects; combining the three can achieve synergistic therapeutic effects and more effectively treat UV-induced skin damage.

[0050] In this invention, HA was selected as the delivery carrier, which exhibits better performance compared to other carriers (such as polyvinyl alcohol (PVA), chitosan (Cs), etc.). This may be because HA has better advantages over Cs and PVA in terms of biocompatibility and skin functional adaptability: HA has both high viscosity and good fluidity; a 15%~25% HA solution, when made into microneedles, has moderate mechanical strength and dissolves rapidly upon contact with skin fluids. 1,2 Furthermore, HA is a natural component of the human body (found in skin connective tissue, synovial fluid, etc.), its molecular structure is highly consistent with the skin extracellular matrix, and it can be gradually degraded by hyaluronidase in the skin without any risk of residue. Although Cs is natural and environmentally friendly, it contains strongly positively charged amino groups, which easily bind to the negatively charged cell membranes of the skin, disrupting the barrier integrity. 1High concentrations may lead to cytotoxicity (fibroblast survival rate <85%). 3 PVA is a synthetic polymer. Although it has no obvious toxicity, its structure differs greatly from that of the skin's extracellular matrix, and long-term exposure may trigger a mild inflammatory reaction. 4 .

[0051] In some implementations, the siRNA is a modified siRNA. Appropriate modification of the siRNA's bases can enhance its resistance to nucleases and ribose stability, thereby increasing its affinity for target mRNA.

[0052] In some embodiments, the modification is a modification of a base. In some specific embodiments, the modification is selected from one or more of 2'-O-methyl and 2'-fluoro modifications. In some embodiments, the modification is a cholesterol modification.

[0053] In one specific implementation, all bases of siRNA 1 are modified with 2'-O-methyl (denoted as "m") or 2'-fluorine (denoted as "f"), and cholesterol (denoted as "Chol") is modified at the 3' end of the positive strand. The nucleotide sequence of the modified siRNA is shown below:

[0054] 5'-(mU)*(mC)*(mC)(mU)(mU)(mG)(fU)(mA)(fA)(fC)(fA)(mA)(mG)(mU)(mA)(mG)(mG)(mU)(mA)(mC)(mU)(Chol)-3'

[0055] 5'-(mA)*(fG)*(mU)(mA)(mC)(fC)(mU)(mA)(mC)(mU)(mU)(mG)(mU)(fU)(mA)(fC)(mA)(mA)(mG)(mG)(mA)*(mC)*(mA)-3'

[0056] In some embodiments, the final concentration of siRNA is from 30 nM to 100 nM. For example, the final concentration of siRNA can be 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. In one specific embodiment, the final concentration of siRNA is 50 nM.

[0057] In some embodiments, the mass ratio of siRNA to selenium nanoparticles is 1:5 to 1:10. For example, the mass ratio of siRNA to selenium nanoparticles can be 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In one specific embodiment, the mass ratio of siRNA to selenium nanoparticles is 1:8.

[0058] In some implementations, the 3' ends of the sense and / or antisense strands of siRNA are covalently linked to selenium nanoparticles to form siRNA-selenium nanoparticle complexes.

[0059] It is understood that selenium nanoparticles can be prepared by any suitable method in the art. For example, selenium nanoparticles can be prepared by the ascorbic acid reduction of sodium selenite method.

[0060] In some embodiments, the particle size of the selenium nanoparticles is from 50 nm to 100 nm. For example, the particle size of the selenium nanoparticles can be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In one specific embodiment, the particle size of the selenium nanoparticles is 80 nm.

[0061] In some embodiments, the mass fraction of hyaluronic acid is 15% to 25%. For example, the mass fraction of hyaluronic acid is 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%. In one specific embodiment, the mass fraction of hyaluronic acid is 20%.

[0062] In some implementations, the microneedles are based on hyaluronic acid and loaded with siRNA and a nano-selenium complex.

[0063] In this invention, the microneedles are composed of multiple micron-sized tiny needle tips connected in an array on a base. They can penetrate the stratum corneum in a directional manner to create micron-sized mechanical channels, allowing the drugs loaded on the microneedles to be placed directly in the epidermis or upper dermis without passing through the stratum corneum to participate in microcirculation and exert pharmacological reactions.

[0064] In some embodiments, the final concentration of the complex is from 10 μg / mL to 20 μg / mL. For example, the final concentration of the complex is 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, or 20 μg / mL. In one specific embodiment, the final concentration of the complex is 15 μg / mL.

[0065] In some implementations, the needle height of the microneedle is between 500 μm and 800 μm. For example, the needle height of the microneedle is 500 μm, 600 μm, 700 μm, or 800 μm. In one specific implementation, the needle height of the microneedle is 600 μm.

[0066] In some implementations, the tip diameter of the microneedle is 10 μm to 20 μm. For example, the tip diameter of the microneedle is 10 μm, 12 μm, 14 μm, 15 μm, 16 μm, 18 μm or 20 μm.

[0067] The present invention also provides a method for preparing a microneedle composition, comprising the following steps:

[0068] Prepare a hyaluronic acid solution with a mass fraction of 15% to 25%;

[0069] SNAP25-specific siRNA and selenium nanoparticles were mixed at a mass ratio of 1:5 to 1:10 and incubated at room temperature in the dark for 30 min to form a complex of siRNA and selenium nanoparticles. The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the siRNA is shown in SEQ ID NO.2.

[0070] The complex was added to a hyaluronic acid solution at a final concentration of 10 μg / mL to 20 μg / mL and ultrasonically dispersed to form a premix of the microneedle composition; and

[0071] Optionally, the premixed liquid is injected into a PDMS microneedle mold, vacuum degassing is performed, and the mold is then refrigerated at 4°C for 12 hours before demolding to form hyaluronic acid-based microneedles.

[0072] The present invention also provides the use of microneedle compositions in the preparation of medicaments for treating UV-induced skin damage, wherein the microneedle composition is the microneedle composition of the present invention or a microneedle composition obtained by the preparation method of the present invention.

[0073] Based on a general inventive concept, the preparation method of the present invention and the use of the microneedle composition of the present invention can adopt some or all of the technical solutions of the above-described embodiments of the microneedle composition of the present invention, and therefore have at least all the beneficial effects brought about by the technical solutions of the above-described embodiments, which will not be elaborated here.

[0074] The following describes preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. Reagents used, unless otherwise specified, are all commercially available products. When the method is not described in detail, any suitable method in the art can be used.

[0075] Example 1: Screening of SNAP25 siRNA sequences

[0076] Targeting the human SNAP25 gene (GenBank accession number: NM_003081.4), 233 candidate siRNA sequences were designed using siDirect software (AmbionDesign Tool). From these 233 sequences, 100 were selected and synthesized by Yaoyuan Biotechnology (Shanghai) Co., Ltd. Their silencing efficiency was verified using real-time quantitative PCR. Specifically, silencing efficiency was screened using human skin fibroblasts (HSF cells), as follows:

[0077] HSF cells were seeded in 6-well plates and cultured until confluence reached 70%-80%. Each candidate siRNA was mixed with Lipo3000 and transfected into HSF cells according to the transfection reagent instructions. A blank control group (NC, without siRNA) was set up. 48 hours after transfection, total RNA was extracted from the cells, and the expression level of SNAP25 mRNA in each group was detected by qRT-PCR. The relative expression levels of siRNA in each group are shown below. Figure 1 As shown.

[0078] The silencing efficiency is calculated using the following formula:

[0079] Silencing efficiency = (1 - relative mRNA expression level in experimental group / relative mRNA expression level in control group) × 100%.

[0080] Based on the calculation results, four siRNA sequences with a silencing efficiency ≥70% were selected (denoted as siRNA 1-4, with their sense strand sequences shown as SEQ ID NO.1, 3, 5, and 7, and their antisense strand sequences shown as SEQ ID NO.2, 4, 6, and 8, respectively, and corresponding to...). Figure 1 (Sequences 42, 43, 100 and sequence 12).

[0081] SEQ ID NO.1:

[0082] 5'-UCCUUGUAACAAGUAGGUAC-3'

[0083] SEQ ID NO.2:

[0084] 5'-AGUACCUACUUGUUACAAGGACA-3'

[0085] SEQ ID NO.3:

[0086] 5'-AAGGGAUGGACCAAAUCAATA-3'

[0087] SEQ ID NO.4:

[0088] 5'-UAUUGAUUUGGUCCAUCCCUUCC-3'

[0089] SEQ ID NO.5:

[0090] 5'-CCAGAUCGACAGGAUCAUGGA-3'

[0091] SEQ ID NO.6:

[0092] 5'-UCCAUGAUCCUGUCGAUCUGGCG-3'

[0093] SEQ ID NO.7:

[0094] 5'-GUGCAACAAAGAUGCUGGGAA-3'

[0095] SEQ ID NO.8:

[0096] 5'-UUCCCAGCAUCUUUGUUGCACGU-3'

[0097] The selected siRNA sequences were modified with 2'-O-methyl and 2'-fluorine, and the 3' end of the sense strand was modified with cholesterol. The nucleotide sequences of the modified siRNAs are shown below:

[0098] 5'-(mU)*(mC)*(mC)(mU)(mU)(mG)(fU)(mA)(fA)(fC)(fA)(mA)(mG)(mU)(mA)(mG)(mG)(mU)(mA)(mC)(mU)(Chol)-3';5' -(mA)*(fG)*(mU)(mA)(mC)(fC)(mU)(mA)(mC)(mU)(mU)(mG)(mU)(fU)(mA)(fC)(mA)(mA)(mG)(mG)(mA)*(mC)*(mA)-3';

[0099] 5'-(mA)*(mA)*(mG)(mG)(mG)(mA)(fU)(mG)(fG)(fA)(fC)(mC)(mA)(mA)(mA)(mU)(mC)(mA)(mA)(mU)(mA)(Chol)-3'; 5 '-(mU)*(fA)*(mU)(mU)(mG)(fA)(mU)(mU)(mU)(mG)(mG)(mU)(mC)(fC)(mA)(fU)(mC)(mC)(mC)(mU)(mU)*(mC)*(mC)-3'

[0100] 5'-(mC)*(mC)*(mA)(mG)(mA)(mU)(fC)(mG)(fA)(fC)(fA)(mG)(mG)(mA)(mU)(mC)(mA)(mU)(mG)(mG)(mA)(Chol)-3';5' -(mU)*(fC)*(mC)(mA)(mU)(fG)(mA)(mU)(mC)(mC)(mU)(mG)(mU)(fC)(mG)(fA)(mU)(mC)(mU)(mG)(mG)*(mC)*(mG)-3';

[0101] 5'-(mG)*(mU)*(mG)(mC)(mA)(mA)(fC)(mA)(fA)(fA)(fG)(mA)(mU)(mG)(mC)(mU)(mG)(mG)(mG)(mA)(mA)(Chol)-3';5' -(mU)*(fU)*(mC)(mC)(mC)(fA)(mG)(mC)(mA)(mU)(mC)(mU)(mU)(fU)(mG)(fU)(mU)(mG)(mC)(mA)(mC)*(mG)*(mU)-3'.

[0102] Example 2: Therapeutic effect of siRNA sequence in a mouse model of UV-induced skin damage

[0103] The four siRNA sequences (human-mouse homologous sequences) selected above were combined with Lipo3000 liposome reagent to obtain a complex.

[0104] BALB / c mice (n=20) were randomly divided into 5 groups of 4 mice each. The backs of the mice in each group were shaved to expose a 1.5cm×2cm skin area. The mice were then irradiated with a UVB lamp (wavelength 312nm) at a dose of 100 mJ / cm², 3 times a week for 2 consecutive weeks, to establish a UV-induced skin damage model.

[0105] The modified siRNA sequence and liposome complex were applied to the damaged skin area of ​​mice and gently pressed. After daily UV irradiation of the skin, the siRNA and liposome complex were applied for 2 weeks. The mice were then sacrificed, and the back skin tissue was taken for HE staining. The untreated siRNA and liposome complex was used as the model group (blank control).

[0106] Real-life images of the backs of mice in the model group and each experimental group after 1, 7, and 14 days of treatment are shown below. Figure 2As shown, the UV model group mice had deeper back injuries, while siRNA treatment with siRNAs 1-4 showed a trend of less damage overall compared to the control group, with siRNA1 treatment resulting in the least severe back injuries. HE staining results after mouse sacrifice are shown below. Figure 3 As shown, the model group exhibited epidermal thickening and dermal collagen fiber breakage; while after treatment with siRNA 1 sequence, the epidermal thickness returned to normal, collagen fibers were neatly arranged, and the damage repair effect was more significant; after treatment with siRNA 2-4, the structural damage effect on the mouse skin was more severe than that on the skin of mice treated with siRNA 1. Therefore, the siRNA 1 sequence was selected for subsequent experiments.

[0107] Example 3: Detection of different siRNA transfection efficiencies in UV-photoaged human skin fibroblasts

[0108] UV photoaging human skin fibroblasts were used at a rate of 5×10 4 Cells were seeded at a density of 1 cell per well in 24-well cell culture plates, and DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin antibiotics was added. The plates were then incubated at 37°C in a 5% CO2 incubator. Transfection was performed when the cell confluence reached 60%-70%.

[0109] According to Lipofectamine TM Follow the instructions for the 3000 transfection reagent. ① Preparation of transfection complex: Take appropriate amounts of siRNA (final concentrations of 30nM, 60nM, and 100nM respectively) and mix with Opti-MEM™ serum-free medium. Gently pipette 3-5 times to mix thoroughly and let stand at room temperature for 5 minutes. Simultaneously, take the corresponding volume of Lipofectamine. TM 3000 Transfection Reagent and Opti-MEM TM Mix the serum-free culture medium, gently pipette to mix, and let stand at room temperature for 5 min; gently mix the two mixtures above, and incubate at room temperature for 15 min to form the siRNA-liposome transfection complex. ② Transfection procedure: Discard the original culture medium in the 24-well plate, gently wash the cells twice with PBS buffer, and add 500 μL of Opti-MEM to each well. TM Serum-free medium was used, and then the prepared transfection complex was added to the corresponding group of cell wells one by one. The culture plate was gently shaken to distribute the complex evenly. ③ Subsequent culture: The culture plate was placed back in a 37℃, 5% CO2 incubator for 6 hours. The medium containing the transfection complex was then discarded and replaced with DMEM high-glucose complete medium containing 10% FBS. The plate was cultured for another 48 hours for subsequent qPCR detection.

[0110] In this embodiment, the successfully constructed UV photoaging human skin fibroblasts were randomly divided into 4 groups, with 3 replicates in each group. The specific groupings are as follows: ① Negative control group (NC group): transfected with NC-siRNA; ② siRNA-1 group: transfected with siRNA-1 targeting the target gene; ③ siRNA-2 group: transfected with siRNA-2 targeting the target gene; ④ siRNA-3 group: transfected with siRNA-3 targeting the target gene.

[0111] Test results as follows Figure 4 As shown in the qPCR results of this embodiment, there were significant differences in the relative expression levels of the target genes in each experimental group. Specifically, compared with the blank control group and the NC group, the relative expression levels of the target genes in the siRNA-1, siRNA-2, and siRNA-3 groups were significantly reduced (P<0.05), indicating that all three targeted siRNAs were successfully transfected into UV-aged human skin fibroblasts and effectively exerted gene silencing effects, i.e., all possessed transfection activity. Further comparison of the transfection effects of the three targeted siRNAs showed that siRNA-1 had the lowest relative expression level of the target gene, the highest gene silencing efficiency, and the best transfection efficiency; the relative expression levels of the target genes in the siRNA-2 and siRNA-3 groups were higher than those in the siRNA-1 group, with the second highest transfection efficiency, and there was no statistically significant difference between the two groups (P>0.05).

[0112] Example 4: Chemically synthesized selenium nanoparticles for siRNA transfection

[0113] The sodium selenite was reduced by ascorbic acid. 0.1 mol / L sodium selenite solution and 0.2 mol / L ascorbic acid solution were mixed at a volume ratio of 1:2 and reacted at 37°C in the dark for 2 h. After centrifugation at 8000 rpm, the precipitate was collected after 10 min and washed three times with ultrapure water to obtain nano-selenium particles with a particle size of about 80 nm.

[0114] The siRNA 1 with the best therapeutic effect selected in Example 2 was further modified by adding a CY3 group to evaluate transfection efficiency. The modified sequence is as follows:

[0115] 5'-(Cy3)(mU)*(mC)*(mC)(mU)(mU)(mG)(fU)(mA)(fA)(fC)(fA)(mA)(mG)(mU)(mA)(mG)(mG)(mU)(mA)(mC)(mU)(Chol)-3' ;5'-(mA)*(fG)*(mU)(mA)(mC)(fC)(mU)(mA)(mC)(mU)(mU)(mG)(mU)(fU)(mA)(fC)(mA)(mA)(mG)(mG)(mA)*(mC)*(mA)-3'

[0116] The modified siRNA and selenium nanoparticles were mixed at a mass ratio of 1:8 and incubated at room temperature in the dark for 30 minutes to form a stable siRNA-selenium nanoparticle complex through electrostatic interaction.

[0117] In this embodiment, in addition to the above-mentioned siRNA-selenium nanoparticle complex group, three other groups (three replicates per group) were set up, namely: ① blank control group (HSF cells only), ② Lipo3000-siRNA group (positive control, i.e., Lipo3000 was combined with modified siRNA, and the final concentration of siRNA was 50 nM), ③ siRNA-SeNP group-HA group (i.e., siRNA, selenium nanoparticles and hyaluronic acid were combined to obtain a complex according to the method in Example 4 below, and the final concentration of siRNA was 50 nM).

[0118] Each complex group was co-cultured with HSF cells. After 24 hours, the fluorescence intensity of CY3 was observed using electron microscopy, and the transfection efficiency was assessed. Transfection efficiency was analyzed using ImageJ software. First, the total fluorescence integral density of the siRNA fluorescence channel was calculated, and simultaneously, the total number of cells in the DAPI channel was counted. The average fluorescence intensity per unit cell was calculated using the formula: "Average fluorescence intensity per unit cell = Total fluorescence intensity ÷ Total number of cells." The relative level of this value reflected the transfection efficiency. Using the average fluorescence intensity of the DEME group (background) as a baseline, the relative transfection efficiencies of each group were: DEME group 12%, SeNP+siRNA group 60%, SeNP+HA+siRNA group 85%, and Lipo3000+siRNA group 52%.

[0119] Test results as follows Figure 5 As shown in the figure, and combined with the calculation results of relative transfection efficiency, it can be seen that the siRNA channel (green fluorescence) in the SeNP+siRNA group has a significant signal, and most of the blue cell nuclei (DAPI) in the Merge channel are co-localized with the green fluorescence, indicating that the selenium nanoparticles successfully delivered siRNA into the cells. The transfection efficiency of the SeNP+siRNA group (approximately 60%) is similar to that of the Lipo3000+siRNA group (approximately 52%), which shows that the siRNA delivery ability of the selenium nanoparticles is comparable to that of commonly used transfection reagents (Lipo3000). After the addition of the substrate HA, the green fluorescence signal of the siRNA channel is stronger, and the proportion of positive cells co-localized with the blue cell nuclei in the Merge channel increases from approximately 60% to approximately 85%, indicating that HA enhances the siRNA delivery ability of the selenium nanoparticles.

[0120] Example 5: Verification of transfection efficiency of different types of microneedles in siRNA-selenium nanocomplex

[0121] The siRNA-selenium nanocomplex was prepared using the same method as in Example 3.

[0122] To verify the potential impact of different microneedle substrates on the transfection efficiency of siRNA loaded with selenium nanoparticles, three commonly used microneedle substrates were selected for verification, as follows: HA (hyaluronic acid) and PVA (polyvinyl alcohol) were dissolved in ultrapure water; Cs (chitosan) was dissolved in a pre-prepared 1% acetic acid solution; all were prepared into 20% (w / w) solutions. Then, the siRNA-selenium nanoparticle complex was compounded with the above three substrates.

[0123] In this embodiment, a total of 4 groups (3 replicates per group) were set up: ① blank control group (siRNA only), ② SeNP-siRNA-Cs complex group, ③ SeNP-siRNA-HA complex group, ④ SeNP-siRNA-PVA complex group (the final concentration of siRNA in groups ②-④ was 50 nM).

[0124] Each complex was co-cultured with HSF cells, and the fluorescence intensity of CY3 was observed and the transfection efficiency was detected by electron microscopy after 24 hours.

[0125] Test results as follows Figure 6 As shown, when PVA is used as the substrate, the fluorescent group modified with siRAN is in a diffuse state. When HA is used as the substrate, its fluorescence intensity and cell state are in the best transfection state and the best activity state compared with PVA and Cs. Therefore, HA is chosen as the substrate for the fabrication of microneedles.

[0126] Example 6: Preparation and Morphology Analysis of HA-Based Microneedles

[0127] Weigh 2g of HA powder, add 10mL of PBS solution, and stir at 60℃ for 2h until completely dissolved to obtain a 20% HA solution;

[0128] The siRNA-selenium nanocomplex was prepared using the same method as in Example 3. The siRNA-selenium nanocomplex (final concentration 15 μg / mL) was added to the HA solution prepared above and ultrasonically dispersed at 300 W for 5 min to form a homogeneous and transparent premix.

[0129] The premixed liquid was injected into the PDMS mold, vacuum degassed for 10 min, refrigerated at 4℃ for 12 h, and then demolded to obtain conical microneedles.

[0130] Microneedles were characterized using electron microscopy.

[0131] The preparation and characterization results of the microneedles are as follows: Figure 7 As shown, the prepared microneedles are regular conical in shape, with a needle height of 600 μm, a needle tip diameter of 15 μm, and a smooth needle body surface without bubbles or cracks.

[0132] Example 7: In vitro toxicity testing of microneedles

[0133] Microneedles were prepared using the same method as in Example 6. The following microneedles were prepared in this example:

[0134] The following groups were selected: Con group (blank control), SeNP microneedle group (selenium nanoparticles only), siRNA+SeNP complex group, and siRNA+SeNP+HA group. The following experiments were conducted:

[0135] CCK8 assay: Microneedles (containing the complex) in each group were co-cultured with HSF cells for 24 h, with 3 replicates per group. CCK8 reagent was added and incubated for 2 h. The absorbance at 450 nm was measured and the cell viability was calculated.

[0136] The results are as follows Figure 8 As shown, after 24 hours of co-culture, the cell viability was ≥95%, which was not significantly different from the blank control group (P>0.05).

[0137] Dead and live cell staining experiment: After co-culturing each group of microneedles (containing the complex) with HSF cells for 24 h, the cells were incubated for 15 min using the Calcein-AM / PI double staining kit and observed under a fluorescence microscope.

[0138] The results are as follows Figure 9 As shown, each group exhibits a large amount of green fluorescence (live cells) and almost no red fluorescence (dead cells), indicating that the microneedles prepared in this invention have no obvious cytotoxicity.

[0139] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

[0140] [References]

[0141] 1. Wang Zhouyun et al. Preparation and characterization of soluble hyaluronic acid injections. Chinese Journal of Marine Drugs 43, 55-61 (2024). https: / / doi.org / 10.13400 / j.cnki.cjmd.2024.05.009

[0142] 2. Yan Naiqing. A composite gel inhibitor for preventing spontaneous combustion of coal in coal mines and its preparation method.

[0143] 3. Valachová, K. & Šoltés, L. Versatile Use of Chitosan andHyaluronan in Medicine. Molecules 26 (2021). https: / / doi.org / 10.3390 / molecules26041195

[0144] 4. Fang Zehui, Huang San, Liao Liqi, Wu Guoyu & Pan Yufang. Effects of matrix composition on the formation and penetration of soluble microneedles of the antitumor drug resveratrol. Journal of Guangdong Pharmaceutical University, 40, 108-114 (2024). https: / / doi.org / 10.16809 / j.cnki.2096-3653.2024042301.

Claims

1. A microneedle composition, characterized in that, The microneedle composition comprises a complex of SNAP25-specific siRNA and selenium nanoparticles, as well as hyaluronic acid. The nucleotide sequence of the positive strand of the siRNA is shown in SEQ ID NO.

1. The nucleotide sequence of the antisense strand of the siRNA is shown in SEQ ID NO.

2.

2. The microneedle composition according to claim 1, characterized in that, The siRNA is a modified siRNA, and the modification is selected from one or more of 2'-O-methyl and 2'-fluoro modifications.

3. The microneedle composition according to claim 1, characterized in that, The mass ratio of the siRNA to the nano-selenium is 1:5 to 1:

10.

4. The microneedle composition according to claim 1, characterized in that, The final concentration of the siRNA is 30 nM to 100 nM.

5. The microneedle composition according to claim 1, characterized in that, The hyaluronic acid has a mass fraction of 15% to 25%.

6. The microneedle composition according to any one of claims 1 to 5, characterized in that, The microneedles are based on hyaluronic acid and loaded with a complex of siRNA and nano-selenium.

7. The microneedle composition according to claim 6, characterized in that, The final concentration of the complex is from 10 μg / mL to 20 μg / mL.

8. The microneedle composition according to claim 6, characterized in that, The microneedle has a needle height of 500μm to 800μm and a needle tip diameter of 10μm to 20μm.

9. A method for preparing a microneedle composition, characterized in that, Includes the following steps: Prepare a hyaluronic acid solution with a mass fraction of 15% to 25%; SNAP25-specific siRNA and selenium nanoparticles were mixed at a mass ratio of 1:5 to 1:10 and incubated at room temperature in the dark for 30 min to form a complex of siRNA and selenium nanoparticles. The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the siRNA is shown in SEQ ID NO.

2. The complex was added to the hyaluronic acid solution at a final concentration of 10 μg / mL to 20 μg / mL and ultrasonically dispersed to form a premix of the microneedle composition. as well as Optionally, the premixed liquid is injected into a PDMS microneedle mold, vacuum degassed, and then refrigerated at 4°C for 12 hours before demolding to form hyaluronic acid-based microneedles.

10. The use of the microneedle composition in the preparation of a medicament for treating UV-induced skin damage, characterized in that, The microneedle composition is the microneedle composition according to any one of claims 1 to 8 or the microneedle composition obtained by the preparation method according to claim 9.

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