WO2021130541A1

WO2021130541A1 - Transdermal sirna delivery composition and use therefor

Info

Publication number: WO2021130541A1
Application number: PCT/IB2020/020080
Authority: WO
Inventors: 陈铭; 梁雪娇
Original assignee: 厦门大学
Priority date: 2019-12-24
Filing date: 2020-12-24
Publication date: 2021-07-01
Also published as: CN110882219B; CN110882219A

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

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 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 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

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

(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%.

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.

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.

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

100

(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

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

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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. 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. 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. 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.

The 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. Use of the siRNA transdermal delivery composition of any one of claims 1 to 5 in gene knockout.

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.