WO2021130541A1 - Transdermal sirna delivery composition and use therefor - Google Patents

Transdermal sirna delivery composition and use therefor Download PDF

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WO2021130541A1
WO2021130541A1 PCT/IB2020/020080 IB2020020080W WO2021130541A1 WO 2021130541 A1 WO2021130541 A1 WO 2021130541A1 IB 2020020080 W IB2020020080 W IB 2020020080W WO 2021130541 A1 WO2021130541 A1 WO 2021130541A1
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sirna
skin
liposomes
cfl
flexible
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PCT/IB2020/020080
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French (fr)
Chinese (zh)
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陈铭
梁雪娇
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厦门大学
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02Inorganic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/02Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14Liposomes; Vesicles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/25Silicon; Compounds thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60Sugars; Derivatives thereof
    • A61K8/606Nucleosides; Nucleotides; Nucleic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin

Definitions

  • a siRNA transdermal delivery composition and its use Technical Field belongs to the technical field of transdermal drug delivery preparations, and specifically relates to a siRNA transdermal delivery composition and its use. Background technique
  • RNA interference is a specific gene silencing mechanism triggered by small interfering RNA (siRNA), which uses short double-stranded RNA with 21 to 23 nucleotides in a highly sequence-specific manner Inhibit gene expression.
  • siRNA small interfering RNA
  • RNAi therapy has been proven to treat skin diseases caused by abnormal gene expression, including hair loss, psoriasis, allergic skin disease, skin cancer, congenital pneumonia and hyperpigmentation, etc.
  • the local application of RNAi therapy can avoid the first-pass metabolism and side effects involved in systemic administration, while local administration can directly act on the skin lesions, improving patient compliance, etc. .
  • siRNA is a hydrophilic and negatively charged biological macromolecule ( ⁇ 13 kDa)
  • the first obstacle to delivery is the outermost stratum corneum (SC) of the skin, mainly due to the "brick and cement" structure and lipophilicity of the stratum corneum.
  • SC stratum corneum
  • various penetration enhancement strategies and technologies have been developed and utilized, such as microneedles, chemical penetration enhancers and nanocarrier systems.
  • SHS as a new type of silicon microneedles, can be used alone or in combination with flexible liposomes to improve the transdermal absorption of hydrophilic macromolecules.
  • SHS can penetrate the stratum corneum of the skin and create a large number of long-lasting microchannels (up to 72 h) in the stratum corneum. Applying 10 mg of SHS at 1.77 cm 2 can produce about 850 micropores per mm 2.
  • the second obstacle to the local delivery of siRNA is the skin cell membrane. Many methods have been adopted to overcome this challenge and promote the internalization of siRNA, such as electroporation Pores, lipid complexes and heat shock, etc.
  • encapsulating the siRNA into a nanocarrier is a strategy to protect the siRNA from degradation to the greatest extent.
  • a siRNA transdermal delivery composition including sponge spicules, flexible liposomes and the siRNA.
  • the flexible liposome and the siRNA are in the form of flexible liposomes carrying siRNA, or a mixed form of flexible liposomes and siRNA, that is, non-carrying form, preferably flexible liposomes carrying siRNA form.
  • the sponge spicules are bee sponge spicules, which are derived from bee sponge Haliclonasp.
  • the purity of the sponge spicule is preferably not less than 90%.
  • the sponge spicules are in the form of a sponge spicule solution, which is prepared by buffer, deionized water, double distilled water or physiological saline, wherein the mass concentration of the sponge spicules is 0.01 ⁇ 100%.
  • the flexible liposomes include ordinary flexible liposomes and cationic flexible liposomes.
  • the flexible liposome of the present invention is prepared by adding surface active substances (such as sodium cholate, sodium deoxycholate, Tween, Span, polyoxyethylene oleyl ether, etc.) during the preparation of ordinary liposomes , Has a high degree of deformability.
  • Flexible liposomes can be classified into cationic flexible liposomes, anionic flexible liposomes, and neutral flexible liposomes according to the charge they carry.
  • the cationic flexible liposomes of the present invention are positively charged flexible liposomes.
  • the cationic flexible liposomes of the present invention can be prepared by phospholipids with cations ((2,3-dioleoyl-propyl)-trimethylamine (DOTAP), 2,3-dioleoyloxypropyl-l-bromo At least one of trimethylamine (DOTMA), dimethyl dioctadecyl ammonium bromide (DDAB), dioleoylphosphatidylethanolamine (DOPE), etc.) and a surfactant.
  • DOTAP 2,3-dioleoyl-propyl)-trimethylamine
  • DOTMA 2,3-dioleoyloxypropyl-l-bromo
  • DOTMA trimethylamine
  • the ordinary flexible liposomes described in the present invention are other flexible liposomes other than cationic flexible liposomes, for example, they may be neutral flexible liposomes.
  • the ordinary flexible liposomes of the present invention can be prepared by soybean lecithin, egg yolk lecithin, etc. and surfactants.
  • the phospholipid concentration of the ordinary flexible liposome is 3 ⁇ 5%.
  • the membrane material of the ordinary flexible liposome includes soybean lecithin and a surfactant.
  • the mass ratio of soybean lecithin and surfactant is 4:1 ⁇ 1.5.
  • the phospholipid concentration of the cationic flexible liposome is 0.02% ⁇ 1.5%, preferably 0.04% ⁇
  • the membrane material of the cationic flexible liposome includes DOTAP and a surfactant.
  • the mass ratio of DOTAP and surfactant is 1:1 ⁇ 1.5.
  • the surfactant is polyoxyethylene 20 oleyl ether.
  • the particle size of the ordinary flexible liposome is 90 ⁇ 110 nm, (; the potential is -10 ⁇ 0 mV, and the potential decreases after carrying siRNA.
  • the cationic flexible liposome The particle size is 90 ⁇ 110 nm, and the potential is 30 ⁇ 40 mV. After carrying siRNA, the G potential is reduced to 20 ⁇ 30 mV or -35 ⁇ -25 mV.
  • the third technical solution adopted by the present invention to solve its technical problems is: The use of an siRNA transdermal delivery composition in gene knockout.
  • the fourth technical solution adopted by the present invention to solve its technical problems is: The use of an siRNA transdermal delivery composition in the preparation of transdermal absorption preparations or cosmetics.
  • the transdermal absorption preparation or cosmetic is directly made of sponge spicules, flexible liposomes and siRNA, for example, spongy spicules, flexible liposomes and siRNA are prepared separately according to the method provided by the present invention, Or prepared according to other medical, pharmaceutical or cosmetic related processes, or prepared by mixing sponge spicules, flexible liposomes and siRNA according to medical, pharmaceutical or cosmetic related processes; or, the process Skin absorption preparations or cosmetics are sponge spicules, flexible liposomes and siRNA prepared by adding auxiliary materials respectively according to relevant medical, pharmaceutical or cosmetic processes, or mixing spongy spicules, flexible liposomes, siRNA and auxiliary materials , Prepared according to medical, pharmaceutical or cosmetic related processes.
  • the excipients of the present invention should be medically, pharmaceutically or cosmetically acceptable excipients that comply with relevant laws and regulations, such as diluents, solvents, excipients, absorbents, wetting agents, binders, and disintegrants , Lubricants, solubilizers, emulsifiers, suspending agents, surfactants, film-forming agents, propellants, antioxidants, flavors, fragrances, fungicides, preservatives, etc.
  • This technical solution has the following advantages:
  • the present invention combines SHS with different lipid vesicles, ordinary flexible liposomes (FL) and cationic flexible liposomes (CFL) to greatly enhance siRNA Skin permeability outside the body.
  • FIG. 1 is the liposome characterization in Example 2, where: a. liposome size, b. liposome potential, c. liposome flexibility.
  • Figure 2 shows the penetration rate of GAPDH-siRNA in the skin under different topical treatments in the in vitro transdermal penetration experiment of Example 3 (all liposomes in the figure carry siRNA) (* means p ⁇ 0.05, ** means p ⁇ 0.0l, *** means /? ⁇ 0.001 ).
  • Figure 3 is a fluorescence image of siRNA skin delivery under a confocal microscope in the in vitro transdermal penetration experiment of Example 3 (all liposomes in the figure carry siRNA), in which: (8) control group; (b) SHS massage; (c) Topically use CFL (l %)@siRNA; (d) Topically use CFL (0.05%)@siRNA; (e) Topically use SHS and FL@ siRNA; (f) Topically use SHS and CFL (l%)@siRNA ; (g) Local combined use of dermaroller and CFL (0.05%)@siRNA; (h) Local combined use of SHS and CFL (0.05%)@siRNA.
  • Figure 4 is the fluorescence image of liposome-carrying FAM-siRNA transfection in L929 cells in Example 4.
  • CFL(0.05%)@siRNA A, a): Add 1.5 ( J CFL(0.05%) and 30 pmol FAM-siRNA mixture to each well
  • CFL(l%)@siRNA(B, b) Add 1.5 ( JCFL (1 %) and 30 pmol FAM-siRNA mixture to each well
  • FL@ siRNA (C, c) Add 1.5 ( J FL and 30 pmol FAM-siRNA mixture to each well
  • siRNA( D, d) 30 pmol FAM-siRNA.
  • FIG. 5 shows the effect of liposomes carrying FAM-siRNA on L929 cells in Example 4 GAPDH protein knockout rate (*** means /? ⁇ 0.001).
  • Figure 6 shows the cytotoxicity of liposomes to L929 cells in Example 4, where: (8)Adding different doses After CFL (0.05%), CFL (1%) and FL, the cell growth inhibition rate.
  • (b) Cell growth status after adding different doses of liposomes.
  • Figure 7 shows the knockout rate of GAPDH protein in the local treatment in vivo in Example 5. Among them: A: 3D simulation diagram of GAPDH protein knockout rate; B: Top view of Figure A. The final concentration of GAPDH-siRNA administration in all groups was 25nmol/ml.
  • Injection + CFL(0_05%)@siRNA This group is 100 ( J GAPDH-siRNA (3.75nmol) mixture, the group tested the GAPDH protein knockout rate in the skin of three different treatment positions, namely the skin at the injection center, the skin 0.5 cm from the injection center, and 1.0 cm from the injection center Skin.
  • SHS + CFL(0.05 %)@siRNA This group is 1000 GAPDH-siRNA
  • Example 1 Preparation of cationic flexible liposomes (CFL) from liposomes
  • the preparation of cationic flexible liposomes (CFL) adopts a film hydration method: 1% DOTAP ((2, 3 -dioleoyl -Propyl) -trimethylamine) + 1.2% surfactant (BRU® O20, polyoxyethylene 20 oleyl ether) in a round-bottomed flask in a solvent commonly used to prepare flexible liposomes (such as chloroform, ether, etc.) After steaming, a lipid film is formed on the inner wall of the round-bottomed fla
  • the hydration solution is passed through the lipid film with a 100 nm pore diameter polycarbonate film.
  • CFL(l%)@siRNA Use CFL(1%) as a solvent to dissolve siRNA, and then mix with ultrasound (20 min) to prepare siRNA-carrying CFL(1%), denoted as CFL(l%)@siRNA.
  • CFL(0.05%)@ siRNA Use CFL(0.05%) as a solvent to dissolve siRNA, and then mix with ultrasound (20 min) to prepare CFL(0_05%)@ siRNA.
  • Preparation of FL@ siRNA Use FL as a solvent to dissolve siRNA, then mix with ultrasound (20 min) to prepare FL@ siRNA.
  • Example 2 Characterization of liposomes Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments, UK) was used to characterize the particle size and potential of cationic flexible liposomes and ordinary flexible liposomes. Under a pressure of 0.25 MPa, 1 ml of liposomes were extruded through a 100 nm polycarbonate membrane to determine the deformability of different lipid vesicles.
  • CFL@ siRNA shows a similar particle size distribution, but the potential of CFL(0.05%)@siRNA becomes electronegative (-31.4+1.1 mV) c ordinary flexible liposomes (FL or FL@
  • the average diameter of siRNA, Figure la) is about 102.5 nm, and the G potential is neutral, about -2.2 mV.
  • Lipid Body flexibility results show that the optimized CFL (0.05%, 94.08% ⁇ 1.01%) has better deformability than the original CFL (1%, 56.03% ⁇ 1.98%) and FL (4%, 61.79% ⁇ 0.48%) (/? ⁇ 0.001).
  • Example 3 In vitro transdermal permeation of siRNA. This example uses isolated pig skin as a skin model for experiments.
  • the pig skin needs to be pre-treated, and the subcutaneous fat tissue on the pig skin is carefully removed with a scalpel, and then the hair on the pig skin is shaved with an electric shaver to make the length less than 5 mm.
  • Use ultra-pure water Wash the pigskin after the above treatment and store it at -20 ° (: for later use. Thaw the skin at room temperature before use.
  • Use a round punch with a diameter of 40 mm to drill a pig skin of the same diameter Installed on the Franz diffusion transdermal device, using the isolated skin percutaneous resistance test to measure the electrical conductance of the skin to ensure the integrity of the skin barrier.
  • the effective penetration area of the Franz diffusion cell is 1.77 cm 2 , and the receptor volume is 12 ml.
  • PBS pH 7.4, 0.2 M
  • SHS is: through a household electric massager (Codos KP-3000) (Applying about 0.3N force, rotating speed about 300 rpm/min) SHS (5 mg / cm 2 ) was applied topically to the skin for 2 minutes. Then the skin was washed 3 times with PBS (0.2 M), In order to remove the residual SHS on the skin.
  • the drug penetration time of each group is 16 hours. Each group is repeated at least three times.
  • Example 4 Liposome cytotoxicity and in vitro cell transfection. L929 cells were seeded into a 96-well plate and cultured in a cell incubator for 24 h (5% CO 2 , 37 ° C). Remove the medium in each well and replace with 100M fresh medium.
  • the cells were incubated with different doses of liposomes (5.0M/well, 2.5M/well, 1.0ul/well, 0.50/well, 0.10/well) at 37 ° C for another 24 hours. Then the MTT method was used to determine the survival rate of the cells under different liposome doses. Inoculate L929 cells into a 24-well plate and culture in a cell incubator for 24 h (5% CO 2 , 37 ° C). When the cell density is about 70%-80%, perform cell transfection experiments.
  • this example also measured the expression of GAPDH protein in L929 cells to further verify the cell transfection effect (Figure 5).
  • CFL(0.05%)@siRNA can lead to GAPDH protein knockout rate of 41.09% ⁇ 5.14% after 68 hours of transfection, which is much higher than CFL(l%)@siRNA (8.38% ⁇ 2.08%), FL@ siRNA (3.48) % ⁇ 2.25%) and siRNA alone (6.07% ⁇ 1.62%) knockout rate of GAPDH protein.
  • the relationship between the cytotoxicity of CFL (0.05%), CFL (1%) and FL and the additive dose was determined by MTT method.
  • mice were anesthetized by intraperitoneal injection of 1500 chloral hydrate (4%), the hair on the back of the mice was trimmed, and a hollow cylinder with an area of 1.77 cm 2 was glued on the exposed skin area of the back of the mice using 3M Vetbond Then use SHS (5 mg/cm 2 ) to massage the bare skin for 2 minutes under an applied force of 0.3 N, and finally wash the treated area 3 times with PBS (0.2 M) to remove SHS. Then, for local combined application 100
  • the mixed solution of the solution (see Example 1 for the preparation method) was applied to the treatment area non-sealed.
  • 150 The siRNA (3.75 nmol) solution is applied to the treatment area in a closed manner.
  • 150 pi negative control siRNA (3.75 nmol) solution was applied to the treatment area in a closed manner.
  • the mice were sacrificed by overexposure to pyrolysis. The mouse skin tissue was collected from the treatment area, and the KDalert GAPDH assay kit was used to determine the GAPDH protein expression level, and calculate the protein knockout effect in vivo (each group contains 3 replicates).
  • the combined use of SHS and CFL shows the best transdermal penetration enhancement effect on siRNA in vitro; CFL (0.05%) has lower toxicity to cells and higher protein knockout rate; in vivo results show The combined use of SHS and CFL (0.05%) can have a comparable protein knockout rate with the skin of the subcutaneous injection center, but it has a better area.
  • CFL (0.05%) is the optimal concentration to cooperate with SHS, and the combined use of SHS and CFL (0.05%) is the most effective combination method for local application of RNAi, and it is expected to become a promising delivery system for RNAi therapy. .
  • the above are only preferred embodiments of the present invention, so the scope of implementation of the present invention cannot be limited accordingly.
  • the present invention discloses a siRNA transdermal delivery composition and its use.
  • the present invention combines bee sponge spicules with The combination of ordinary flexible liposomes and cationic flexible liposomes can enhance the skin permeability of siRNA in vitro.
  • the combined use of bee sponge spicules and cationic flexible liposomes shows the best transdermal penetration enhancement effect for siRNA, and is expected to become a promising delivery system for RNAi therapy, opening up new ideas for local delivery of siRNA for the treatment of skin diseases Opportunities, have good industrial usability.

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Abstract

A transdermal siRNA delivery composition and use therefor. In the composition, sponge Haliclona sp. spicules is used in combination with common flexible liposomes and cationic flexible liposomes, thus allowing the in vitro skin permeation of siRNA to be strengthened. Joint use of sponge Haliclona sp. spicules and cationic flexible liposomes shows the best transdermal permeation enhancement effect for siRNA, potentially being able to become a promising delivery system for siRNA therapy, and opening new opportunities for applying local delivery of siRNA in treating skin diseases.

Description

一种 siRNA经皮递送组合物及其用途 技术 领域 本发明属于经皮给药制剂技术领域, 具体涉及一种 siRNA 经皮递送组合物及 其用途。 背景 技术 A siRNA transdermal delivery composition and its use Technical Field The present invention belongs to the technical field of transdermal drug delivery preparations, and specifically relates to a siRNA transdermal delivery composition and its use. Background technique
RNA 干扰 (RN A interference, RNAi)是由小干扰 RNA ( small interfering RNA, siRNA) 引发的特定基因沉默机制, 即利用具有 21至 23个核苷酸的短双链 RNA以 高度序列特异性的方式抑制基因表达。 RNAi疗法已被证明可以治疗由异常的基因表 达引起的皮肤疾病, 包括脱发, 牛皮癖, 过敏性皮肤病, 皮肤癌, 先天性肺炎和色 素沉着等等。对这些皮肤病区域中的特定基因抑制, RNAi治疗的局部应用可以避免 全身给药涉及的首过代谢和副作用, 同时局部给药可以直接在皮肤病变部位发挥作 用, 提高患者的依从性, 等等。 由于 siRNA是亲水性的且带负电荷的生物大分子 (~13 kDa) , 因此其向活体 皮肤细胞的局部递送主要存在 两个障碍。 递送第一个障碍是皮肤的最外层角质层 (SC), 主要是由于角质层是“砖和水泥”构造和亲脂性。 为了克服这种皮肤障碍, 已经开发和利用了各种渗透增强策略和技术, 例如微针, 化学渗透增强剂和纳米载 体系统等。 之前的研究, 我们已经证明蜂海绵骨针 (SHS) 作为新型硅微针, 单独 或与柔性脂质体结合使用可用来改善亲水性大分子的经皮吸收。 SHS 可以穿透皮 肤角质层, 在角质层上产生大量持久的微通道 (持续时间可达 72 h), 在 1.77 cm2 施加 lOmg SHS, 可在每 mm2上产生约 850个微孔。 siRNA局部递送的第二个障碍 皮肤细胞膜。 采用了许多方法来克服这一挑战, 促进 siRNA的内在化, 例如电穿 孔,脂质复合物和热休克等。为了使裸露的 siRNA局部应用后保持其稳定,将 siRNA 封装到纳米载体中, 是一种最大程度保护 siRNA免受降解的策略。 发 明内容 本发明的目的在于克服现有技术的不足之处, 提供了一种 siRNA经皮递送组合 物及其用途。 本发明解决其技术问题所采用的技术方案之一是: 一种 siRNA经皮递送组合物, 包括海绵骨针、 柔性脂质体和所述 siRNA。 一 实施例中: 所述柔性脂质体与所述 siRNA为柔性脂质体携载 siRNA形式, 或 为柔性脂质体与 siRNA的混合形式即非携载形式,优选柔性脂质体携载 siRNA形式。 一 实施例中: 所述海绵骨针为蜂海绵骨针, 采源于蜂海绵 Haliclonasp .。 海绵骨 针的纯度最好不低于 90%。 一 实施例中:所述海绵骨针为海绵骨针溶液的形式,该海绵骨针溶液为缓冲液、 去离子水、 双蒸水或生理盐水配制而成, 其中海绵骨针的质量浓度为 0.01〜 100%。 一 实施例中: 所述柔性脂质体包括普通柔性脂质体与阳离子柔性脂质体。 本发明所述的柔性脂质体是在普通脂质体的制备过程中加入表面活性物质 (例 如胆酸钠、 去氧胆酸钠、 吐温、 司盘、 聚氧乙烯油醚等) 制备得到, 具有高度的变 形能力。 柔性脂质体按照所带的电荷可以分为阳离子柔性脂质体、 阴离子柔性脂质 体和中性柔性脂质体。 本发明所述的阳离子柔性脂质体即为带正电荷的柔性脂质体。 本发明所述的阳 离子柔性脂质体可以通过带阳离子的磷脂 ( (2,3 -二油酰基-丙基) -三甲胺 (DOTAP)、 2,3-二油酰氧丙基-l-溴化三甲胺(DOTMA)、 二甲基双十八烷基溴化铵 (DDAB)、 二油 酰磷脂酰乙醇胺 (DOPE)等中的至少一种) 与表面活性剂制备得到。 本发明所述的普通柔性脂质体为除阳离子柔性脂质体之外的其他柔性脂质体, 例如可以为中性柔性脂质体。 本发明所述的普通柔性脂质体可以通过大豆卵磷脂、 蛋黄卵磷脂等与表面活性剂制备得到。 一 实施例中: 所述普通柔性脂质体的磷脂浓度为 3〜 5%。 一 实施例中: 所述普通柔性脂质体的膜材包括大豆卵磷脂和表面活性剂。 一 实施例中: 大豆卵磷脂和表面活性剂的质量比为 4:1〜 1.5。 一 实施例中: 所述阳离子柔性脂质体的磷脂浓度为 0.02%〜 1.5%, 优选 0.04%〜RNA interference (RN A interference, RNAi) is a specific gene silencing mechanism triggered by small interfering RNA (siRNA), which uses short double-stranded RNA with 21 to 23 nucleotides in a highly sequence-specific manner Inhibit gene expression. RNAi therapy has been proven to treat skin diseases caused by abnormal gene expression, including hair loss, psoriasis, allergic skin disease, skin cancer, congenital pneumonia and hyperpigmentation, etc. For specific gene suppression in these skin disease areas, the local application of RNAi therapy can avoid the first-pass metabolism and side effects involved in systemic administration, while local administration can directly act on the skin lesions, improving patient compliance, etc. . Since siRNA is a hydrophilic and negatively charged biological macromolecule (~13 kDa), there are two main obstacles to its local delivery to living skin cells. The first obstacle to delivery is the outermost stratum corneum (SC) of the skin, mainly due to the "brick and cement" structure and lipophilicity of the stratum corneum. In order to overcome this skin barrier, various penetration enhancement strategies and technologies have been developed and utilized, such as microneedles, chemical penetration enhancers and nanocarrier systems. In previous studies, we have demonstrated that SHS, as a new type of silicon microneedles, can be used alone or in combination with flexible liposomes to improve the transdermal absorption of hydrophilic macromolecules. SHS can penetrate the stratum corneum of the skin and create a large number of long-lasting microchannels (up to 72 h) in the stratum corneum. Applying 10 mg of SHS at 1.77 cm 2 can produce about 850 micropores per mm 2. The second obstacle to the local delivery of siRNA is the skin cell membrane. Many methods have been adopted to overcome this challenge and promote the internalization of siRNA, such as electroporation Pores, lipid complexes and heat shock, etc. In order to keep the naked siRNA stable after topical application, encapsulating the siRNA into a nanocarrier is a strategy to protect the siRNA from degradation to the greatest extent. SUMMARY OF THE INVENTION The purpose of the present invention is to overcome the shortcomings of the prior art and provide a siRNA transdermal delivery composition and its use. One of the technical solutions adopted by the present invention to solve its technical problems is: a siRNA transdermal delivery composition, including sponge spicules, flexible liposomes and the siRNA. In one embodiment: the flexible liposome and the siRNA are in the form of flexible liposomes carrying siRNA, or a mixed form of flexible liposomes and siRNA, that is, non-carrying form, preferably flexible liposomes carrying siRNA form. In one embodiment: the sponge spicules are bee sponge spicules, which are derived from bee sponge Haliclonasp. The purity of the sponge spicule is preferably not less than 90%. In one embodiment: the sponge spicules are in the form of a sponge spicule solution, which is prepared by buffer, deionized water, double distilled water or physiological saline, wherein the mass concentration of the sponge spicules is 0.01 ~ 100%. In one embodiment: The flexible liposomes include ordinary flexible liposomes and cationic flexible liposomes. The flexible liposome of the present invention is prepared by adding surface active substances (such as sodium cholate, sodium deoxycholate, Tween, Span, polyoxyethylene oleyl ether, etc.) during the preparation of ordinary liposomes , Has a high degree of deformability. Flexible liposomes can be classified into cationic flexible liposomes, anionic flexible liposomes, and neutral flexible liposomes according to the charge they carry. The cationic flexible liposomes of the present invention are positively charged flexible liposomes. The cationic flexible liposomes of the present invention can be prepared by phospholipids with cations ((2,3-dioleoyl-propyl)-trimethylamine (DOTAP), 2,3-dioleoyloxypropyl-l-bromo At least one of trimethylamine (DOTMA), dimethyl dioctadecyl ammonium bromide (DDAB), dioleoylphosphatidylethanolamine (DOPE), etc.) and a surfactant. The ordinary flexible liposomes described in the present invention are other flexible liposomes other than cationic flexible liposomes, for example, they may be neutral flexible liposomes. The ordinary flexible liposomes of the present invention can be prepared by soybean lecithin, egg yolk lecithin, etc. and surfactants. In one embodiment: The phospholipid concentration of the ordinary flexible liposome is 3~5%. In one embodiment: The membrane material of the ordinary flexible liposome includes soybean lecithin and a surfactant. In one embodiment: the mass ratio of soybean lecithin and surfactant is 4:1~1.5. In an embodiment: the phospholipid concentration of the cationic flexible liposome is 0.02%~1.5%, preferably 0.04%~
0.06% c 一 实施例中: 所述阳离子柔性脂质体的膜材包括 DOTAP和表面活性剂。 一 实施例中: DOTAP和表面活性剂的质量比为 1:1〜 1.5。 一 实施例中: 所述表面活性剂为聚氧乙稀 20油醚。 一 实施例中: 所述普通柔性脂质体的粒径为 90〜 110 nm, (;电位为 -10〜 0 mV, 携载 siRNA后 电位降低。 一 实施例中:所述阳离子柔性脂质体的粒径为 90〜 110 nmj电位为 30〜 40 mV, 携载 siRNA后 G电位降低, 降低至 20〜 30 mV或 -35〜 -25 mV。 本发明解决其技术问题所采用的技术方案之二是: 一种 siRNA经皮递送组合物的使用方法, 先将所述海绵骨针施加于皮肤, 然后 将所述柔性脂质体和所述 siRNA施加于皮肤。或者将该 siRNA经皮递送组合物直接 施加于皮肤。 一 实施例中:将所述海绵骨针施加于皮肤时,可以配合手动或电动的按摩方式。 一 实施例中: 在皮肤上施用所述柔性脂质体和所述 siRNA之前, 需要先洗去残 留的海绵骨针。 一 实施例中: 所述海绵骨针在皮肤上的施用量为 2〜 8 mg / cm2。 本发明解决其技术问题所采用的技术方案之三是: 一种 siRNA经皮递送组合物在基因敲除中的用途。 本发明解决其技术问题所采用的技术方案之四是: 一种 siRNA经皮递送组合物在制备经皮吸收制剂或化妆品中的用途。 一 实施例中: 所述经皮吸收制剂或化妆品为海绵骨针、 柔性脂质体和 siRNA直 接制成, 例如将海绵骨针、 柔性脂质体和 siRNA分别按照本发明提供的方法制备得 到, 或者按照其他医学上、 药学上或化妆品上的相关工艺制备得到, 或将海绵骨针、 柔性脂质体和 siRNA 混合后按照医学上、 药学上或化妆品上的相关工艺制备得到; 或者, 该经皮吸收制剂或化妆品为海绵骨针、 柔性脂质体和 siRNA分别添加辅料后 按照医学上、 药学上或化妆品上的相关工艺制备得到, 或将海绵骨针、柔性脂质体、 siRNA和辅料混合, 按照医学上、 药学上或化妆品上的相关工艺制备得到。 本发明所述的辅料应为医学上、 药学上或化妆品上可接受的、 符合相关法规的 辅料, 例如包括稀释剂、 溶剂、 赋形剂、 吸收剂、 润湿剂、 黏合剂、 崩解剂、 润滑 剂、 增溶剂、 乳化剂、 助悬剂、 表面活性剂、 成膜剂、 抛射剂、 抗氧剂、 矫味剂、 芳香剂、 杀菌剂、 防腐剂等。 本技术方案与背景技术相比, 它具有如下优点: 本发明将 SHS与不同的脂质囊泡, 普通柔性脂质体 (FL) 和阳离子柔性脂质体 (CFL)结合使用,以大大增强 siRNA在体外的皮肤渗透性。此外, SHS和 CFL(0.05%) 的联合使用可产生协同作用, 以将 GAPDH-siRNA传递至皮肤细胞, 从而显著抑制 BALB/c雌性小鼠中 GAPDH蛋白的表达, 此外, 将 SHS和 CFL的局部联合使用可 适应于其他目标部位或皮肤病变部位, 为局部递送 siRNA应用于皮肤病的治疗以及 新型功能化妆品领域开辟新的机会。 附 图说明 下面结合附图和实施例对本发明作进一步说明。 图 1为实施例 2中脂质体表征, 其中: a.脂质体大小, b.脂质体电位, c.脂质体 柔性。 图 2为实施例 3 的体外经皮渗透实验中不同局部治疗下皮肤的 GAPDH-siRNA 渗透率 (图中所有的脂质体均携载了 siRNA) (*表示 p<0.05, **表示 p<0.0l, *** 表示 /?<0.001 )。 图 3为实施例 3的体外经皮渗透实验中共聚焦显微镜下 siRNA皮肤递送荧光图 (图中所有脂质体均携载了 siRNA) , 其中: ⑻对照组; (b)SHS按摩; (c)局部使用 CFL(l %)@siRNA ; (d)局部使用 CFL(0.05 %)@siRNA ; (e)局部联合使用 SHS 和 FL@ siRNA; (f)局部联合使用 SHS和 CFL(l%)@siRNA; (g)局部联合使用 dermaroller 和 CFL(0.05%)@siRNA; (h)局部联合使用 SHS和 CFL(0.05%)@ siRNA。 图 4 为实施例 4 中脂质体携载 FAM-siRNA在 L929细胞转染荧光图。 其中: CFL(0.05 % )@siRNA (A , a) : 向每个孔中添加 1.5(J CFL(0.05 % )和 30 pmol FAM-siRNA混合物 ; CFL(l%)@siRNA(B, b) : 向每个孔中添加 1.5(JCFL(1 %)和 30 pmol FAM-siRNA混合物; FL@ siRNA (C, c) : 向每个孔中添加 1.5(J FL和 30 pmol FAM -siRNA混合物; siRNA(D, d) : 30 pmol FAM -siRNA。 对照组 (E, e) : 30 pmol 阴性对照 siRNA。 图中标尺为 20pm。 图 5为实施例 4中脂质体携载 FAM-siRNA对 L929细胞中 GAPDH蛋白敲除率 (*** 表示 /?<0.001 )。 图 6为实施例 4 中脂质体对 L929细胞的细胞毒性, 其中: ⑻加入不同剂量的 CFL(0.05%), CFL(1%)和 FL 后, 细胞的生长抑制率。 (b)添加不同剂量脂质体后的 细胞生长状态。 图 7为实施例 5中体内局部治疗 GAPDH蛋白的敲除率。 其中: A: GAPDH蛋 白敲除率 3D模拟图; B : 图 A的俯视图。 所有组的 GAPDH-siRNA给药终浓度均为 25nmol /ml。 注射 + CFL(0_05%)@siRNA: 该组为
Figure imgf000007_0001
100(J GAPDH-siRNA (3.75nmol) 混合液, 该组检测了三个不同治疗位置皮肤中 GAPDH 蛋白敲除率, 即注射中心点的皮肤、 距离注射中心 0.5 cm的皮肤, 距离注射中心 1.0 cm的皮肤。 SHS + CFL(0.05 %)@siRNA: 该组为
Figure imgf000007_0002
1000 GAPDH-siRNA
0.06% c In an embodiment: The membrane material of the cationic flexible liposome includes DOTAP and a surfactant. In one embodiment: the mass ratio of DOTAP and surfactant is 1:1~1.5. In one embodiment: the surfactant is polyoxyethylene 20 oleyl ether. In an embodiment: the particle size of the ordinary flexible liposome is 90~110 nm, (; the potential is -10~0 mV, and the potential decreases after carrying siRNA. In an embodiment: the cationic flexible liposome The particle size is 90~110 nm, and the potential is 30~40 mV. After carrying siRNA, the G potential is reduced to 20~30 mV or -35~-25 mV. The second technical solution adopted by the present invention to solve its technical problems Yes: A method of using the siRNA transdermal delivery composition, first applying the sponge spicule to the skin, and then applying the flexible liposome and the siRNA to the skin. Or the siRNA transdermal delivery composition Apply directly to the skin. In one embodiment: when applying the sponge spicules to the skin, manual or electric massage can be used. In one embodiment: before applying the flexible liposomes and the siRNA on the skin , Need to wash away the remaining sponge spicules. In one embodiment: the application amount of the sponge spicules on the skin is 2-8 mg/cm 2 . The third technical solution adopted by the present invention to solve its technical problems is: The use of an siRNA transdermal delivery composition in gene knockout. The fourth technical solution adopted by the present invention to solve its technical problems is: The use of an siRNA transdermal delivery composition in the preparation of transdermal absorption preparations or cosmetics. In an embodiment: the transdermal absorption preparation or cosmetic is directly made of sponge spicules, flexible liposomes and siRNA, for example, spongy spicules, flexible liposomes and siRNA are prepared separately according to the method provided by the present invention, Or prepared according to other medical, pharmaceutical or cosmetic related processes, or prepared by mixing sponge spicules, flexible liposomes and siRNA according to medical, pharmaceutical or cosmetic related processes; or, the process Skin absorption preparations or cosmetics are sponge spicules, flexible liposomes and siRNA prepared by adding auxiliary materials respectively according to relevant medical, pharmaceutical or cosmetic processes, or mixing spongy spicules, flexible liposomes, siRNA and auxiliary materials , Prepared according to medical, pharmaceutical or cosmetic related processes. The excipients of the present invention should be medically, pharmaceutically or cosmetically acceptable excipients that comply with relevant laws and regulations, such as diluents, solvents, excipients, absorbents, wetting agents, binders, and disintegrants , Lubricants, solubilizers, emulsifiers, suspending agents, surfactants, film-forming agents, propellants, antioxidants, flavors, fragrances, fungicides, preservatives, etc. Compared with the background technology, this technical solution has the following advantages: The present invention combines SHS with different lipid vesicles, ordinary flexible liposomes (FL) and cationic flexible liposomes (CFL) to greatly enhance siRNA Skin permeability outside the body. In addition, the combined use of SHS and CFL (0.05%) can produce a synergistic effect to deliver GAPDH-siRNA to skin cells, thereby significantly inhibiting the expression of GAPDH protein in BALB/c female mice. In addition, the localization of SHS and CFL The combined use can be adapted to other target sites or skin lesions, for the local delivery of siRNA for the treatment of skin diseases and The field of new functional cosmetics opens up new opportunities. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be further described below in conjunction with the drawings and embodiments. Figure 1 is the liposome characterization in Example 2, where: a. liposome size, b. liposome potential, c. liposome flexibility. Figure 2 shows the penetration rate of GAPDH-siRNA in the skin under different topical treatments in the in vitro transdermal penetration experiment of Example 3 (all liposomes in the figure carry siRNA) (* means p<0.05, ** means p< 0.0l, *** means /?<0.001 ). Figure 3 is a fluorescence image of siRNA skin delivery under a confocal microscope in the in vitro transdermal penetration experiment of Example 3 (all liposomes in the figure carry siRNA), in which: ⑻ control group; (b) SHS massage; (c) Topically use CFL (l %)@siRNA; (d) Topically use CFL (0.05%)@siRNA; (e) Topically use SHS and FL@ siRNA; (f) Topically use SHS and CFL (l%)@siRNA ; (g) Local combined use of dermaroller and CFL (0.05%)@siRNA; (h) Local combined use of SHS and CFL (0.05%)@siRNA. Figure 4 is the fluorescence image of liposome-carrying FAM-siRNA transfection in L929 cells in Example 4. Among them: CFL(0.05%)@siRNA (A, a): Add 1.5 ( J CFL(0.05%) and 30 pmol FAM-siRNA mixture to each well; CFL(l%)@siRNA(B, b): Add 1.5 ( JCFL (1 %) and 30 pmol FAM-siRNA mixture to each well; FL@ siRNA (C, c): Add 1.5 ( J FL and 30 pmol FAM-siRNA mixture to each well; siRNA( D, d): 30 pmol FAM-siRNA. Control group (E, e): 30 pmol negative control siRNA. The scale in the figure is 20pm. Figure 5 shows the effect of liposomes carrying FAM-siRNA on L929 cells in Example 4 GAPDH protein knockout rate (*** means /?<0.001). Figure 6 shows the cytotoxicity of liposomes to L929 cells in Example 4, where: ⑻Adding different doses After CFL (0.05%), CFL (1%) and FL, the cell growth inhibition rate. (b) Cell growth status after adding different doses of liposomes. Figure 7 shows the knockout rate of GAPDH protein in the local treatment in vivo in Example 5. Among them: A: 3D simulation diagram of GAPDH protein knockout rate; B: Top view of Figure A. The final concentration of GAPDH-siRNA administration in all groups was 25nmol/ml. Injection + CFL(0_05%)@siRNA: This group is
Figure imgf000007_0001
100 ( J GAPDH-siRNA (3.75nmol) mixture, the group tested the GAPDH protein knockout rate in the skin of three different treatment positions, namely the skin at the injection center, the skin 0.5 cm from the injection center, and 1.0 cm from the injection center Skin. SHS + CFL(0.05 %)@siRNA: This group is
Figure imgf000007_0002
1000 GAPDH-siRNA
(3.75nmol) 混合液。 SHS按摩; 在 siRNA局部给药区域随机选择了三个位置的皮 肤检测了 GAPDH 蛋白的敲除率。图中显示的数值表示方式均是平均值土标准差 (n = 3)。 具体 实施方 式 下面通过实施例具体说明本发明的内容: 实施例 1: 脂貭体制作 阳离子柔性脂质体 (CFL) 的制备采用薄膜水化法: 1% DOTAP ((2, 3 -二油酰 基-丙基) -三甲胺) + 1.2% 表面活性剂 (BRU® O20, 聚氧乙烯 20油醚) 在制备柔 性脂质体常用的溶剂 (例如氯仿、 乙醚等) 中置于圆底烧瓶内旋蒸后, 在圆底烧瓶 内壁形成一层脂膜, 然后用 Tris-HCl (0.2 M, pH=4) 水化, 最后, 将水合溶液都通 过带有孔径为 100 nm聚碳酸酯薄膜的脂质体挤出器挤压过膜 21次, 获得磷脂浓度 为 1%的阳离子柔性脂质体记为 CFL(1%) ; 然后将过膜后的 CFL(1%)用纯水稀释 20 倍, 获得磷脂浓度为 0.05%的阳离子柔性脂质体, 记为 CFL(0.05%)。 普通 柔性脂质体 (FL)的制备采用簿膜水化法: 4% 90G (大豆卵磷脂) + 1.2% 表面活性剂 (BRU® 020) 在制备柔性脂质体常用的溶剂 (例如氯仿、 乙醚等) 中 置于圆底烧瓶内旋蒸后,在圆底烧瓶内壁形成一层脂膜,然后用 PBS (0.2 M,pH=7.4) 水化, 最后, 将水合溶液都通过带有孔径为 100 nm聚碳酸酯薄膜的脂质体挤出器挤 压过膜 21次, 获得普通柔性脂质体 (FL), 磷脂浓度为 4%。 (3.75nmol) mixed solution. SHS massage; Randomly selected three skin locations in the siRNA local administration area to detect the knockout rate of GAPDH protein. The values shown in the figure are all expressed in mean value ± standard deviation (n = 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the content of the present invention in detail: Example 1: Preparation of cationic flexible liposomes (CFL) from liposomes The preparation of cationic flexible liposomes (CFL) adopts a film hydration method: 1% DOTAP ((2, 3 -dioleoyl -Propyl) -trimethylamine) + 1.2% surfactant (BRU® O20, polyoxyethylene 20 oleyl ether) in a round-bottomed flask in a solvent commonly used to prepare flexible liposomes (such as chloroform, ether, etc.) After steaming, a lipid film is formed on the inner wall of the round-bottomed flask, which is then hydrated with Tris-HCl (0.2 M, pH=4). Finally, the hydration solution is passed through the lipid film with a 100 nm pore diameter polycarbonate film. The body extruder squeezed the membrane 21 times, and the cationic flexible liposome with a phospholipid concentration of 1% was obtained as CFL (1%) ; then the CFL (1%) after the membrane was diluted 20 times with pure water to obtain Cationic flexible liposomes with a phospholipid concentration of 0.05% are designated as CFL (0.05%). Ordinary flexible liposomes (FL) are prepared by thin-film hydration: 4% 90G (soy lecithin) + 1.2% Surfactant (BRU® 020) is placed in a round-bottomed flask in a solvent commonly used to prepare flexible liposomes (such as chloroform, ether, etc.), and then a layer of lipid film is formed on the inner wall of the round-bottomed flask, and then PBS ( 0.2 M, pH=7.4) for hydration. Finally, the hydrated solution was extruded 21 times through a liposome extruder with a polycarbonate film with a pore size of 100 nm to obtain ordinary flexible liposomes (FL) , Phospholipid concentration is 4%.
CFL(l%)@siRNA 的制备:将 CFL(1%)作为溶剂溶解 siRNA, 然后超声混合 (20 min) 制备出携载 siRNA的 CFL(1%), 记为 CFL(l%)@siRNA。 Preparation of CFL(l%)@siRNA: Use CFL(1%) as a solvent to dissolve siRNA, and then mix with ultrasound (20 min) to prepare siRNA-carrying CFL(1%), denoted as CFL(l%)@siRNA.
CFL(0.05%)@ siRNA的制备: 将 CFL(0.05%)作为溶剂溶解 siRNA, 然后超声混 合 (20 min) 制备出 CFL(0_05%)@ siRNA。 Preparation of CFL(0.05%)@ siRNA: Use CFL(0.05%) as a solvent to dissolve siRNA, and then mix with ultrasound (20 min) to prepare CFL(0_05%)@ siRNA.
FL@ siRNA的制备: 将 FL作为溶剂溶解 siRNA, 然后超声混合 (20 min) 制备 出 FL@ siRNA。 实施例 2: 脂质体表征 使 用 Malvern Zetasizer Nano ZS90仪器 (Malvern Instruments , UK) , 对阳离子 柔性脂质体, 普通柔性脂质体的粒径和 ^电位进行了表征。 在 0.25MPa的压力下将 lml的脂质体通过 100nm的聚碳酸酯膜挤出来确定不同脂质囊泡的可变形性。 水、 CFL 和 FL穿过膜的时间分别记录为水的 Tw, Tc和 Tf, CFL与 FL的可变形性计算 如下: 丄 $ 水穿过膜所需要的时间 (Jw) 月旨质体柔性 = - * 100% 脂质体穿过膜所需要的时间{Tc/Tf) 脂质体表征结果 : 阳离子柔性脂质体 (CFL) 平均直径为 101.4±l.l nm, G电位为 35.7±0.4 mV (图 la&b )。 当与 siRNA 结合后, CFL@ siRNA 表现出相似的粒径分 布, 但是 CFL(0.05%)@siRNA的 电位变为电负性 (-31.4+1.1 mV) c 普通柔性脂质体 (FL或 FL@ siRNA, 图 la) 平均直径均约为 102.5 nm, G电位显中性, 约为 -2.2 mV。 脂质 体柔性结果显示优化后的 CFL (0.05%, 94.08%±1.01%) 具有比原始 CFL (1%, 56.03%±1.98%) 和 FL (4%, 61.79%±0.48%) 更好的可变形性 (/?<0.001)。 实施例 3: siRNA体外经皮渗透 本实施例使用离体猪皮作为皮肤模型进行实验。 首先需要对猪皮进行预处理, 用手术刀小心地除去猪皮上的皮下脂肪组织, 然后使用电动剃须刀将猪皮上的毛发 剃除, 使其长度在 5 mm以下, 使用超纯水清洗经上述处理后的猪皮, 并将其储存 在 -20 °(:下备用。 使用前将皮肤在室温下解冻。 用直径为 40 mm的圆形打孔器钻取 同样直径大小的猪皮, 安装至 Franz扩散透皮装置上, 采用离体皮肤经皮电阻试验 测定皮肤的电导以确保皮肤屏障的完整性。 Franz扩散池的有效穿透面积为 1.77 cm2, 受体体积为 12 ml。 将皮肤放置在垂直 Franz扩散池的顶部, 并用 PBS (pH 7.4, 0.2 M) 填充受体池, 然后将装置置于 37±0.5°C的水浴中。 SHS的使用方法为: 通过家 用电动按摩器 (Codos KP-3000) (施加约 0.3N的力, 转速约 300 rpm/min) 将 SHS (5 mg / cm2) 局部施用于皮肤 2分钟。 随后用 PBS (0.2 M) 对皮肤洗涤 3次, 以 去除皮肤上残留的 SHS。 将几组药物制剂 (给药体积为 150 ^1, 其中混有 50M的脂 质体和 100[jl的 siRNA (3.75nmol), siRNA给药终浓度各组均为 25 nmol/ml), 溶剂 为 DEPC处理水, 制备方法参照实施例 1) 非封闭地施加在皮肤表面上。 不与 SHS 联合的组则直接将药物施加在皮肤表面上, 无需 SHS处理。 siRNA组直接施加 25 nmol/ml siRNA (给药体积为 150 ^1, 溶剂为 PBS(0_2 M, pH=4)) ; SHS+siRNA组的 siRNA给药方式为 SHS按摩后,直接施加用 DEPC溶解的 siRNA溶液 (25 nmol/ml, 150(^1) ; 微针 (dermaroller) 联合 CFL(0.05%)@siRNA组是将滚轮微针按照 “米” 字形在皮肤上滚动一次, 然后施加 CFL(0.05%)@siRNA (给药体积为 150^1, siRNA 浓度为 25 nmol/ml)。 各组药物渗透时间为 16小时。 每个组至少重复三次。 取出受 体池中的液体, 测定渗透至皮下 siRNA的含量, 然后用胶带剥离法测定各皮层皮肤 内 siRNA的含量。 将猪 皮用冷冻切片机切除厚度为 20 pm的组织, 然后在共聚焦显微镜下成像, 定性判断 siRNA在各皮层下的渗透量。 实验结果: 为了克服 siRNA局部递送的第一个障碍, 即皮肤的角质层屏障, 本实施例中使 用了不同的实验组别来检验 GAPDH-siRNA在皮肤中的递送效果。 与所有其他组相 比, 局部联合使用 SHS与 CFL(0.05%)@siRNA显示出最高的 siRNA的皮肤渗透性 (图 2), 透皮 16 h后, GAPDH-siRNA的皮肤吸收率高达 61.18%±2.49%, 显著高 于 SHS 联合 CFL(l%)@siRNA 组 ( 48.19 % ±7_21 %, /?<0_05 ) 及 SHS 联合 FL(4%)@siRNA组 (37.99%±1.52%, /?<0.001)。 该结果表明, 携载 siRNA的脂质 体在经 SHS预先处理按摩后,将 siRNA递送至皮肤的能力似乎与其变形能力成正相 关关系。 CFL(0.05%)@siRNA与 SHS联合使用显著高于 CFL(0.05%)@siRNA与传 统滚轮微针 Dermaroller联合使用对 siRNA经皮促渗效果。共聚焦显微镜下成像也显 示出类似的结果 (图 3)。 实施例 4: 脂质体细胞毒性及体外细胞转染 将 L929细胞接种至 96孔板中, 在细胞培养箱中培养 24 h (5% C02, 37°C )。 除去每个孔中的培养基, 并用 100M 新鲜的培养基代替。 然后将细胞与不同剂量的 脂质体 (5.0M/孔, 2.5M/孔, l.Oul/孔, 0.50/孔, 0.10/孔)在 37°C下再孵育 24小时。 然后采用 MTT 法测定不同脂质体剂量下细胞的存活率。 将 L929细胞接种至 24孔板中, 在细胞培养箱中培养 24 h (5% C02, 37°C )。 细胞密度约为 70%-80%时, 进行细胞转染实验。 将 1.5 pi 的不同脂质体和 3M 的 FAM-siRNA ( 10 nmol / ml) 分别添加至 25 的无血清培养基 opti-mem中, 然后再 将两者参照实施例 1 的制备方法充分混合, 最后将混合物在室温下静置 5分钟后添 加到每个孔中, 然后继续将细胞在培养箱中培养 6 小时后, 使用共聚焦显微镜进行 转染成像。 或将转染混合物添加到每个孔中继续孵育 68小时后, 用 KDalert GAPDH 检测试剂盒测定 GAPDH蛋 白表达水平, 并计算蛋白敲除率 (每组重复六次)。 实验结果: 为了克服 siRNA局部递送的第二个障碍, 即细胞膜屏障, 本实施例评估了三种 脂质体 CFL(0.05%), CFL(1 %)和 FL将 siRNA递送进入细胞降低蛋白表达的能力。 通过共聚焦拍摄 FAM- siRNA在 L929细胞中分布情况 (图 4), 结果显示, 与其他 组相比, CFL(0.05%)可以诱导 FAM-siRNA进入细胞, 细胞内有显著的荧光 (图 4A, a), 其余组的细胞内并没有出现显著的荧光现象, 说明 CFL(0.05%)相较于 CFL(1 %) 和 FL能够显著促进 siRNA进入细胞。 随后, 本实施例又测定了 L929细胞中的 GAPDH蛋白表达情况, 进一步验证细 胞转染效果 (图 5)。 CFL(0.05%)@siRNA在转染 68 h后可导致 GAPDH蛋白敲除率 为 41.09%±5.14%, 远高于 CFL(l%)@siRNA (8.38%±2.08% ) , FL@ siRNA (3.48% ± 2.25% ) 和单独 siRNA (6.07%±1.62% ) 对 GAPDH蛋白的敲除率。 通过 MTT法测定了 CFL(0.05%), CFL(1 %)和 FL的细胞毒性与添加剂量之间 的关系。 MTT测定表明, CFL(0.05 % ), CFL( 1 % )和 FL的细胞生长 IC50值分别为 6.634 #1/孔, 0.626 孔和 0.428 孔 (图 6a)。 随着脂质体添加量减少, 细胞状态逐渐恢 复 (图 6b)。 这些结果均表明, 适当剂量的 CFL(0.05%)更适合于将 siRNA传递至细 胞, 而且显示出对细胞较低的毒性, 所以又进一步用 CFL(0.05%)验证了其在体内的 蛋白敲除效果。 实施例 5: 体内蛋白敲除 本实施例研究了 CFL(0.05%)在体内诱导 GAPDH蛋白表达降低的功效。 一共设 计了四组实验: 皮下注射 CFL(0.05%)@siRNA作为阳性对照; 局部联合施用 SHS与 CFL(0.05%)@siRNA; 局部联合施用 SHS和 siRNA; 对照组。 对于 皮下注射组, 将 50(J的 CFL(0.05%)与 100(J的 siRNA (3.75nmol) 溶液参 照实施例 1的制备方法充分混合, 然后将混合物皮下注射到小鼠中。 对于其余组, 首先给小鼠腹腔注射 1500水合氯醛 (4%) 进行麻醉, 修剪小鼠 背部的毛发, 并使用 3M Vetbond 组织粘合剂在小鼠背部裸露皮肤区域粘上面积为 1.77 cm2的空心圆柱体, 然后使用 SHS (5 mg / cm2) 在 0.3 N的施加力下在裸露的 皮肤上按摩 2分钟, 最后用 PBS (0.2 M) 将治疗部位洗 3次以去除 SHS。 随后, 对 于局部联合施
Figure imgf000012_0001
100
Figure imgf000012_0002
Preparation of FL@ siRNA: Use FL as a solvent to dissolve siRNA, then mix with ultrasound (20 min) to prepare FL@ siRNA. Example 2: Characterization of liposomes Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments, UK) was used to characterize the particle size and potential of cationic flexible liposomes and ordinary flexible liposomes. Under a pressure of 0.25 MPa, 1 ml of liposomes were extruded through a 100 nm polycarbonate membrane to determine the deformability of different lipid vesicles. Water, CFL FL and time are recorded through the membrane is water Tw, Tc and Tf, CFL and FL deformability is calculated as follows: $ Shang water through time (of Jw) film require a flexible body LIPID = month -* The time required for 100% liposomes to pass through the membrane {Tc/Tf) Liposome characterization results: Cationic flexible liposomes (CFL) have an average diameter of 101.4±11 nm and a G potential of 35.7±0.4 mV (Figure la&b ). When combined with siRNA, CFL@ siRNA shows a similar particle size distribution, but the potential of CFL(0.05%)@siRNA becomes electronegative (-31.4+1.1 mV) c ordinary flexible liposomes (FL or FL@ The average diameter of siRNA, Figure la) is about 102.5 nm, and the G potential is neutral, about -2.2 mV. Lipid Body flexibility results show that the optimized CFL (0.05%, 94.08%±1.01%) has better deformability than the original CFL (1%, 56.03%±1.98%) and FL (4%, 61.79%±0.48%) (/?<0.001). Example 3: In vitro transdermal permeation of siRNA. This example uses isolated pig skin as a skin model for experiments. First, the pig skin needs to be pre-treated, and the subcutaneous fat tissue on the pig skin is carefully removed with a scalpel, and then the hair on the pig skin is shaved with an electric shaver to make the length less than 5 mm. Use ultra-pure water Wash the pigskin after the above treatment and store it at -20 ° (: for later use. Thaw the skin at room temperature before use. Use a round punch with a diameter of 40 mm to drill a pig skin of the same diameter , Installed on the Franz diffusion transdermal device, using the isolated skin percutaneous resistance test to measure the electrical conductance of the skin to ensure the integrity of the skin barrier. The effective penetration area of the Franz diffusion cell is 1.77 cm 2 , and the receptor volume is 12 ml. Place the skin on the top of the vertical Franz diffusion cell and fill the receptor cell with PBS (pH 7.4, 0.2 M), and then place the device in a 37±0.5 ° C water bath. The use of SHS is: through a household electric massager (Codos KP-3000) (Applying about 0.3N force, rotating speed about 300 rpm/min) SHS (5 mg / cm 2 ) was applied topically to the skin for 2 minutes. Then the skin was washed 3 times with PBS (0.2 M), In order to remove the residual SHS on the skin. Several groups of pharmaceutical preparations (administration volume of 150 ^ 1, mixed with 50M liposomes and 100 [j l siRNA (3.75nmol), the final concentration of siRNA administration in each group 25 nmol/ml), the solvent is DEPC-treated water, and the preparation method refers to Example 1) applied on the skin surface in a non-sealed manner. The group not combined with SHS applied the drug directly on the skin surface without SHS treatment. The siRNA group is directly applied with 25 nmol/ml siRNA (administration volume is 150 ^1, the solvent is PBS (0_2 M, pH=4)); the siRNA administration method of the SHS+siRNA group is after SHS massage, directly applied and dissolved with DEPC SiRNA solution (25 nmol/ml, 150 ( ^1); microneedle (dermaroller) combined with CFL (0.05%) @siRNA group is to roll the roller microneedle in the shape of a "meter" on the skin once, and then apply CFL (0.05 %)@siRNA (administration volume is 150^1, siRNA concentration is 25 nmol/ml). The drug penetration time of each group is 16 hours. Each group is repeated at least three times. Measure the content of siRNA that penetrates the subcutaneous fluid in the body pool, and then measure the content of siRNA in each cortical skin by tape stripping method. The pig skin was cut with a cryostat to cut the tissue with a thickness of 20 pm, and then imaged under a confocal microscope to qualitatively determine the amount of siRNA penetration under each cortex. Experimental results: In order to overcome the first obstacle to the local delivery of siRNA, that is, the stratum corneum barrier of the skin, different experimental groups were used in this example to test the delivery effect of GAPDH-siRNA in the skin. Compared with all other groups, the topical combination of SHS and CFL (0.05%)@siRNA showed the highest skin permeability of siRNA (Figure 2). After 16 hours of transdermal penetration, the skin absorption rate of GAPDH-siRNA was as high as 61.18%± 2.49%, significantly higher than SHS combined with CFL(l%)@siRNA group (48.19% ±7_21%, /?<0_05) and SHS combined with FL(4%)@siRNA group (37.99%±1.52%, /?<0.001 ). This result indicates that the ability of liposomes carrying siRNA to deliver siRNA to the skin after being pre-treated and massaged by SHS seems to be positively correlated with their deformability. The combined use of CFL(0.05%)@siRNA and SHS is significantly higher than the combination of CFL(0.05%)@siRNA and the traditional roller microneedle Dermaroller on the percutaneous penetration of siRNA. Imaging under a confocal microscope also showed similar results (Figure 3). Example 4: Liposome cytotoxicity and in vitro cell transfection. L929 cells were seeded into a 96-well plate and cultured in a cell incubator for 24 h (5% CO 2 , 37 ° C). Remove the medium in each well and replace with 100M fresh medium. Then the cells were incubated with different doses of liposomes (5.0M/well, 2.5M/well, 1.0ul/well, 0.50/well, 0.10/well) at 37 ° C for another 24 hours. Then the MTT method was used to determine the survival rate of the cells under different liposome doses. Inoculate L929 cells into a 24-well plate and culture in a cell incubator for 24 h (5% CO 2 , 37 ° C). When the cell density is about 70%-80%, perform cell transfection experiments. Combine 1.5 pi of different liposomes and 3M FAM-siRNA (10 nmol/ml) was added to 25% serum-free medium opti-mem, and then the two were thoroughly mixed according to the preparation method of Example 1, and finally the mixture was allowed to stand at room temperature for 5 minutes before adding Go to each well, and then continue to culture the cells in an incubator for 6 hours, then use a confocal microscope for transfection imaging. Or add the transfection mixture to each well and continue to incubate for 68 hours, then use the KDalert GAPDH detection kit to determine the GAPDH protein expression level, and calculate the protein knockout rate (repeat six times per group). Experimental results: In order to overcome the second obstacle to the local delivery of siRNA, the cell membrane barrier, this example evaluated three liposomes CFL (0.05%), CFL (1%) and FL to deliver siRNA into cells to reduce protein expression. ability. The distribution of FAM-siRNA in L929 cells was captured by confocal (Figure 4). The results showed that compared with other groups, CFL (0.05%) could induce FAM-siRNA to enter the cells, and there was significant fluorescence in the cells (Figure 4A, a). There was no significant fluorescence in the cells of the remaining groups, indicating that CFL (0.05%) can significantly promote siRNA to enter cells compared to CFL (1%) and FL. Subsequently, this example also measured the expression of GAPDH protein in L929 cells to further verify the cell transfection effect (Figure 5). CFL(0.05%)@siRNA can lead to GAPDH protein knockout rate of 41.09%±5.14% after 68 hours of transfection, which is much higher than CFL(l%)@siRNA (8.38%±2.08%), FL@ siRNA (3.48) % ± 2.25%) and siRNA alone (6.07% ± 1.62%) knockout rate of GAPDH protein. The relationship between the cytotoxicity of CFL (0.05%), CFL (1%) and FL and the additive dose was determined by MTT method. MTT assay showed that the cell growth IC 50 values of CFL (0.05%), CFL (1%) and FL were 6.634 #1/well, 0.626 well and 0.428 well, respectively (Figure 6a). As the amount of liposomes added decreased, the cell state gradually recovered (Figure 6b). These results all show that the appropriate dose of CFL (0.05%) is more suitable for delivering siRNA to cells, and shows lower toxicity to cells, so CFL (0.05%) was further used to verify its protein knockout in vivo effect. Example 5: In vivo protein knockout This example investigated the effect of CFL (0.05%) in inducing the reduction of GAPDH protein expression in vivo. A total of four groups of experiments were designed: subcutaneous injection of CFL (0.05%)@siRNA as a positive control; local combined application of SHS and CFL (0.05%)@siRNA; local combined application of SHS and siRNA; control group. For the subcutaneous injection group, 50 (J of CFL (0.05%) and 100 (J of siRNA (3.75 nmol)) were thoroughly mixed according to the preparation method of Example 1, and then the mixture was injected subcutaneously into mice. For the remaining groups, First, the mice were anesthetized by intraperitoneal injection of 1500 chloral hydrate (4%), the hair on the back of the mice was trimmed, and a hollow cylinder with an area of 1.77 cm 2 was glued on the exposed skin area of the back of the mice using 3M Vetbond Then use SHS (5 mg/cm 2 ) to massage the bare skin for 2 minutes under an applied force of 0.3 N, and finally wash the treated area 3 times with PBS (0.2 M) to remove SHS. Then, for local combined application
Figure imgf000012_0001
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Figure imgf000012_0002
(3.75 nmol) 溶液的混合溶液 (制备方法参照实施例 1)非封闭地应用于治疗区域。 对于局部联合施用 SHS和 siRNA组, 将 150
Figure imgf000012_0003
siRNA (3.75 nmol) 溶液封闭地应用 于治疗区域。对于对照组,无需 SHS按摩处理,将 150 pi阴性对照 siRNA (3.75 nmol) 溶液封闭地应用于治疗区域。 72小时后, 通过二氧化破过度暴露处死小鼠。 从治疗 区域收集小鼠皮肤组织, 并使用 KDalert GAPDH测定试剂盒, 测定 GAPDH蛋白表 达水平, 计算体内蛋白敲除效果 (每组包含 3个重复)。 实验结果: 根 据体外皮肤渗透、 体外细胞转染以及体外细胞毒性研究结果表明, SHS 和 CFL(0.05%)的联合使用是局部应用 RNAi的最有效的组合方式。 因此, 随后我们用 雌性 BALB/C 小鼠 (7-8 周), 测定体内局部联合施用 SHS 和 CFL(0.05 % )将 GAPDH-siRNA 递送到皮肤中的能力 。 通过皮下注射及 SHS 按摩处理将携载 GAPDH-siRNA 的 CFL(0.05%)递送到小鼠皮肤中。 皮下注射组的结果显示, 注射中 心 (26.17%±6.25%) 和距注射中心 0.5 cm位置 (26.38%±7.40% ) GAPDH蛋白的 敲除率没有显着差异, 但在距注射中心 1 cm位置处皮肤 GAPDH蛋白敲除率迅速下 降 (6.45%±5.87% ) (图 7)。 相比之下, 在小鼠皮肤上局部联合施用 SHS 按摩与 CFL(0.05%)可引起整个局部应用区域的 GAPDH敲除率高达为 29.21%±1.41%, 显 著高于单独进行 SHS按摩处理的结果 (5.48%±7.97%, p<0.01) , 并且与皮下注射 中心区域具 有相当的蛋白敲除结果 0? = 0.458)(图 7)。 这些结果表明, 将 SHS 与 CFL(0.05 % )联合使用相较于注射在治疗面积上更具有优势, 所以该组合在体内局部 应用 RNAi治疗是一种很有前途的递送系统。 结论 : 脂质体 浓度会影响脂质体的柔性和对细胞的毒性。 阳离子柔性脂质体与 SHS联 合将 siRNA递送进入皮肤与脂质体柔性存在正相关关系。 SHS和 CFL(0.05%)联合使 用在体外显示出对 siRNA具有最好的经皮促渗效果; CFL(0.05%)对细胞有较低的毒 性和较高的蛋白敲除率; 体内结果显示出 SHS 和 CFL(0.05%)的联合使用可与皮下 注射 中心点皮肤具有相当的蛋 白敲除率, 但在面积上具有更好的优势。 综上, CFL(0.05%)是与 SHS配合的最佳浓度, SHS和 CFL(0.05%)的联合使用是局部应用 RNAi 的最有效的组合方式, 有望成为 RNAi治疗一种很有前途的递送系统。 以上所述, 仅为本发明较佳实施例而已, 故不能依此限定本发明实施的范围, 即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的 范 围内。 工 业实用性 本发明公开了一种 siRNA经皮递送组合物及其用途。 本发明将蜂海绵骨针与 普通柔性脂质体、 阳离子柔性脂质体结合使用, 能够增强 siRNA在体外的皮肤渗 透性。 蜂海绵骨针与阳离子柔性脂质体联合使用显示出对 siRNA最好的经皮促渗 效果, 有望成为 RNAi治疗一种很有前途的递送系统, 为局部递送 siRNA应用于 皮肤病的治疗开辟新的机会, 具有良好的工业使用性。
(3.75 nmol) The mixed solution of the solution (see Example 1 for the preparation method) was applied to the treatment area non-sealed. For the local combined application of SHS and siRNA group, 150
Figure imgf000012_0003
The siRNA (3.75 nmol) solution is applied to the treatment area in a closed manner. For the control group, without SHS massage treatment, 150 pi negative control siRNA (3.75 nmol) solution was applied to the treatment area in a closed manner. After 72 hours, the mice were sacrificed by overexposure to pyrolysis. The mouse skin tissue was collected from the treatment area, and the KDalert GAPDH assay kit was used to determine the GAPDH protein expression level, and calculate the protein knockout effect in vivo (each group contains 3 replicates). Experimental results: According to the results of in vitro skin penetration, in vitro cell transfection and in vitro cytotoxicity studies, the combined use of SHS and CFL (0.05%) is the most effective combination method for topical RNAi. Therefore, we subsequently used female BALB/C mice (7-8 weeks) to determine the ability of local combined application of SHS and CFL (0.05%) in vivo to deliver GAPDH-siRNA to the skin. It will be carried by subcutaneous injection and SHS massage treatment The CFL (0.05%) of GAPDH-siRNA was delivered to the skin of mice. The results of the subcutaneous injection group showed that there was no significant difference in the knockout rate of GAPDH protein between the injection center (26.17%±6.25%) and 0.5 cm from the injection center (26.38%±7.40%), but at 1 cm from the injection center The knockout rate of skin GAPDH protein decreased rapidly (6.45%±5.87%) (Figure 7). In contrast, the local combined application of SHS massage and CFL (0.05%) on the skin of mice can cause the GAPDH knockout rate of the entire topical application area to be as high as 29.21%±1.41%, which is significantly higher than the result of SHS massage treatment alone. (5.48%±7.97%, p<0.01), and the protein knockout result is equivalent to that of the central area of the subcutaneous injection 0 ? = 0.458) (Figure 7). These results indicate that the combined use of SHS and CFL (0.05%) has an advantage over injection in the treatment area, so the combination is a promising delivery system for local application of RNAi therapy in the body. Conclusion: The concentration of liposomes will affect the flexibility of liposomes and their toxicity to cells. The combination of cationic flexible liposomes and SHS to deliver siRNA into the skin has a positive correlation with the flexibility of liposomes. The combined use of SHS and CFL (0.05%) shows the best transdermal penetration enhancement effect on siRNA in vitro; CFL (0.05%) has lower toxicity to cells and higher protein knockout rate; in vivo results show The combined use of SHS and CFL (0.05%) can have a comparable protein knockout rate with the skin of the subcutaneous injection center, but it has a better area. In summary, CFL (0.05%) is the optimal concentration to cooperate with SHS, and the combined use of SHS and CFL (0.05%) is the most effective combination method for local application of RNAi, and it is expected to become a promising delivery system for RNAi therapy. . The above are only preferred embodiments of the present invention, so the scope of implementation of the present invention cannot be limited accordingly. That is, equivalent changes and modifications made according to the scope of the patent of the present invention and the contents of the specification should still be covered by the present invention. Within range. Industrial Applicability The present invention discloses a siRNA transdermal delivery composition and its use. The present invention combines bee sponge spicules with The combination of ordinary flexible liposomes and cationic flexible liposomes can enhance the skin permeability of siRNA in vitro. The combined use of bee sponge spicules and cationic flexible liposomes shows the best transdermal penetration enhancement effect for siRNA, and is expected to become a promising delivery system for RNAi therapy, opening up new ideas for local delivery of siRNA for the treatment of skin diseases Opportunities, have good industrial usability.

Claims

权 利 要 求 Rights request
1. 一种 siRNA 经皮递送组合物, 其特征在于: 包括海绵骨针、 柔性脂质体和 所述 siRNA; 所述柔性脂质体为阳离子柔性脂质体;所述阳离子柔性脂质体的磷脂在柔性脂质体 中的浓度为 0.04%〜 0.06%。 1. A composition for transdermal delivery of siRNA, characterized in that: comprising sponge spicules, flexible liposomes and the siRNA; the flexible liposomes are cationic flexible liposomes; the cationic flexible liposomes The concentration of phospholipids in flexible liposomes is 0.04% to 0.06%.
2. 根据权利要求 1 所述的 siRNA经皮递送组合物, 其特征在于: 所述柔性脂 质体与所述 siRNA为柔性脂质体携载 siRNA形式, 或为柔性脂质体与 siRNA的混 合形式。 2. The siRNA transdermal delivery composition according to claim 1, characterized in that: the flexible liposomes and the siRNA are in the form of flexible liposomes carrying siRNA, or a mixture of flexible liposomes and siRNA form.
3. 根据权利要求 1或 2所述的 siRNA经皮递送组合物, 其特征在于: 所述阳 离子柔性脂质体的膜材包括 DOTAP和表面活性剂。 3. The siRNA transdermal delivery composition according to claim 1 or 2, wherein the membrane material of the cationic flexible liposome comprises DOTAP and a surfactant.
4. 一种权利要求 1或 2或 3所述的 siRNA经皮递送组合物的使用方法, 其特 征在于: 先将所述海绵骨针施加于皮肤, 然后将所述柔性脂质体和所述 siRNA施加 于皮肤。 4. A method of using the siRNA transdermal delivery composition of claim 1 or 2 or 3, characterized in that: the sponge spicules are first applied to the skin, and then the flexible liposomes and the siRNA is applied to the skin.
5. 根据权利要求 4所述的 siRNA经皮递送组合物的使用方法, 其特征在于: 所述海绵骨针在皮肤上的施用量为 2〜 8 mg / cm2The method of using the siRNA transdermal delivery composition according to claim 4, characterized in that: the application amount of the sponge spicule on the skin is 2-8 mg/cm 2 .
6. 一种权利要求 1 至 5 中任一项所述的 siRNA经皮递送组合物在基因敲除中 的用途。 6. Use of the siRNA transdermal delivery composition of any one of claims 1 to 5 in gene knockout.
7. 一种权利要求 1至 5中任一项所述的 siRNA经皮递送组合物在制备经皮吸收 制剂或化妆品中的用途。 7. A use of the siRNA transdermal delivery composition of any one of claims 1 to 5 in the preparation of transdermal absorption preparations or cosmetics.
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