CN110882219A

CN110882219A - siRNA transdermal delivery composition and application thereof

Info

Publication number: CN110882219A
Application number: CN201911347585.3A
Authority: CN
Inventors: 陈铭; 梁雪娇
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-03-17
Anticipated expiration: 2039-12-24
Also published as: WO2021130541A1; CN110882219B

Abstract

The invention discloses a siRNA transdermal delivery composition and application thereof. The invention combines the bee sponge spicule with the common flexible liposome and the cation flexible liposome for use, and can enhance the skin permeability of siRNA in vitro. The bee sponginum needles and the cationic flexible liposome are combined to use to show the best percutaneous penetration promoting effect on siRNA, are expected to become a promising delivery system for RNAi treatment, and open up a new opportunity for local delivery of siRNA to be applied to treatment of skin diseases.

Description

siRNA transdermal delivery composition and application thereof

Technical Field

The invention belongs to the technical field of transdermal administration preparations, and particularly relates to a siRNA transdermal delivery composition and application thereof.

Background

RNA interference (RNAi) is a specific gene silencing mechanism triggered by small interfering RNAs (sirnas), i.e. the inhibition of gene expression in a highly sequence-specific manner using short double-stranded RNAs of 21 to 23 nucleotides. RNAi therapy has been demonstrated to treat skin diseases caused by aberrant gene expression, including alopecia, psoriasis, allergic skin diseases, skin cancer, congenital pneumonia and pigmentation, among others. For specific gene suppression in these dermatological areas, topical application of RNAi therapy can avoid first-pass metabolism and side effects involved with systemic administration, while topical administration can act directly at the site of the skin lesion, improving patient compliance, and so forth.

Since siRNA is a hydrophilic and negatively charged biomacromolecule (-13 kDa), its local delivery to living skin cells presents two major obstacles. The first barrier to delivery is the outermost Stratum Corneum (SC) of the skin, primarily due to the stratum corneum's "brick and cement" construction and lipophilicity. To overcome this skin barrier, various permeation enhancement strategies and techniques have been developed and utilized, such as microneedles, chemical permeation enhancers, and nanocarrier systems, among others. Previous studies, we have demonstrated that bee sponge spicules (SHS), as novel silicon microneedles, alone or in combination with flexible liposomes can be used to improve transdermal absorption of hydrophilic macromolecules. SHS can penetrate the stratum corneum of skin to generate a large number of durable microchannels (with duration up to 72h) on the stratum corneum, and the length of the microchannels is 1.77cm²Applying 10mg SHS, which may be per mm²Resulting in about 850 micropores. The second obstacle to local delivery of siRNA is the skin cell membrane. A number of approaches have been taken to overcome this challenge, facilitating the internalization of siRNA, such as electroporation, lipid complexes and heat shock. In order to keep naked siRNA stable after local application, the siRNA is encapsulated into a nano-carrier, which is a strategy for protecting siRNA from degradation to the maximum extent.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a siRNA transdermal delivery composition and application thereof.

One of the technical schemes adopted by the invention for solving the technical problems is as follows:

a siRNA transdermal delivery composition comprises a sponginum bone needle, a flexible liposome and the siRNA.

In one embodiment: the flexible liposome and the siRNA are in a form that the flexible liposome carries the siRNA, or in a mixed form of the flexible liposome and the siRNA, namely a non-carrying form, and the flexible liposome carries the siRNA is preferred.

In one embodiment: the sponge spicule is a bee sponge spicule which is derived from bee sponge Haliclona sp. The purity of the spongy bone needles is preferably not less than 90%.

In one embodiment: the sponge spicule is in the form of a sponge spicule solution, and the sponge spicule solution is prepared from a buffer solution, deionized water, double distilled water or normal saline, wherein the mass concentration of the sponge spicule is 0.01-100%.

In one embodiment: the flexible liposome comprises common flexible liposome and cationic flexible liposome.

The flexible liposome is prepared by adding surface active substances (such as sodium cholate, sodium deoxycholate, tween, span, polyoxyethylene oleyl ether and the like) in the preparation process of a common liposome, and has high deformability. The flexible liposome can be divided into cationic flexible liposome, anionic flexible liposome and neutral flexible liposome according to the carried charges.

The cation flexible liposome is a flexible liposome with positive charges. The cationic flexible liposome can be prepared from phospholipid with cation (at least one of (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP), 2, 3-dioleoyloxypropyl-1-trimethylamine bromide (DOTMA), dimethyldioctadecylammonium bromide (DDAB), Dioleoylphosphatidylethanolamine (DOPE)) and a surfactant.

The common flexible liposome of the invention is other flexible liposome besides cationic flexible liposome, for example, neutral flexible liposome. The common flexible liposome can be prepared from soybean lecithin, yolk lecithin and the like and a surfactant.

In one embodiment: the phospholipid concentration of the common flexible liposome is 3-5%.

In one embodiment: the membrane material of the common flexible liposome comprises soybean lecithin and a surfactant.

In one embodiment: the mass ratio of the soybean lecithin to the surfactant is 4: 1-1.5.

In one embodiment: the phospholipid concentration of the cationic flexible liposome is 0.02-1.5%, preferably 0.04-0.06%.

In one embodiment: the membrane material of the cationic flexible liposome comprises DOTAP and a surfactant.

In one embodiment: the mass ratio of the DOTAP to the surfactant is 1: 1-1.5.

In one embodiment: the surfactant is polyoxyethylene 20 oleyl ether.

In one embodiment: the particle size of the common flexible liposome is 90-110 nm, the zeta potential is-10-0 mV, and the zeta potential is reduced after carrying siRNA.

In one embodiment: the particle size of the cationic flexible liposome is 90-110 nm, the zeta potential is 30-40 mV, and the zeta potential is reduced to 20-30 mV or-35-25 mV after carrying siRNA.

The second technical scheme adopted by the invention for solving the technical problems is as follows:

a method of using a siRNA transdermal delivery composition by first applying the spongosine needles to the skin and then applying the flexible liposomes and the siRNA to the skin. Or applying the siRNA transdermal delivery composition directly to the skin.

In one embodiment: when the spongy bone needles are applied to the skin, a manual or electric massage mode can be matched.

In one embodiment: the remaining spongiosa needles need to be washed away prior to applying the flexible liposomes and the siRNA to the skin.

In one embodiment: the application amount of the sponge spicules on the skin is 2-8 mg/cm²。

The third technical scheme adopted by the invention for solving the technical problems is as follows:

use of a transdermal siRNA delivery composition for gene knock-out.

The fourth technical scheme adopted by the invention for solving the technical problems is as follows:

use of siRNA transdermal delivery composition in preparing transdermal absorption preparation or cosmetic is provided.

In one embodiment: the percutaneous absorption preparation or the cosmetic is prepared directly from spongiform spicules, flexible liposome and siRNA, for example, the spongiform spicules, the flexible liposome and the siRNA are respectively prepared according to the method provided by the invention, or are prepared according to other related processes in medicine, pharmacy or cosmetics, or are prepared according to related processes in medicine, pharmacy or cosmetics after the spongiform spicules, the flexible liposome and the siRNA are mixed; or the percutaneous absorption preparation or the cosmetic is prepared by adding auxiliary materials into the spongonella needles, the flexible liposome and the siRNA respectively and then preparing the spongonella needles, the flexible liposome, the siRNA and the auxiliary materials according to related medical, pharmaceutical or cosmetic processes, or is prepared by mixing the spongonella needles, the flexible liposome, the siRNA and the auxiliary materials according to related medical, pharmaceutical or cosmetic processes.

The auxiliary materials of the invention are pharmaceutically, pharmaceutically or cosmetically acceptable auxiliary materials which meet relevant regulations, and comprise, for example, diluents, solvents, excipients, absorbents, wetting agents, adhesives, disintegrants, lubricants, solubilizers, emulsifiers, suspending agents, surfactants, film forming agents, propellants, antioxidants, flavoring agents, fragrances, bactericides, preservatives and the like.

Compared with the background technology, the technical scheme has the following advantages:

the present invention uses SHS in combination with different lipid vesicles, common Flexible Liposomes (FL) and Cationic Flexible Liposomes (CFL), to greatly enhance the skin permeability of siRNA in vitro. Furthermore, the combined use of SHS and CFL (0.05%) can produce a synergistic effect to deliver GAPDH-siRNA to skin cells, thereby significantly inhibiting GAPDH protein expression in BALB/c female mice, and furthermore, the local combined use of SHS and CFL can be adapted to other target sites or skin lesion sites, opening up new opportunities for local delivery of siRNA for the treatment of skin disorders and new functional cosmetic fields.

Drawings

The invention is further illustrated by the following figures and examples.

Figure 1 is a representation of liposomes from example 2, wherein: a. liposome size, b liposome potential, c liposome flexibility.

Figure 2 shows GAPDH-siRNA permeabilities of the skin under different topical treatments in the in vitro transdermal permeation experiment of example 3 (all liposomes in the figure carry siRNA) (. indicates p <0.05,. indicates p <0.01,. indicates p < 0.001).

FIG. 3 is a fluorescence image of siRNA skin delivery under confocal microscopy in vitro transdermal permeation experiments of example 3 (all liposomes in the image carry siRNA), wherein: (a) a control group; (b) SHS massage; (c) CFL (1%) @ siRNA was used topically; (d) CFL (0.05%) @ siRNA was used topically; (e) local combined use of SHS and FL @ siRNA; (f) topical combined use of SHS and CFL (1%) @ siRNA; (g) topical combined use of dermaroller and CFL (0.05%) @ siRNA; (h) SHS and CFL (0.05%) were used locally in combination as @ siRNA.

FIG. 4 is the fluorescence image of transfection of L929 cells with FAM-siRNA carried by liposomes in example 4. Wherein: CFL (0.05%) @ siRNA (a, a): add 1.5. mu.l CFL (0.05%) and 30pmol FAM-siRNA mixture to each well; CFL (1%) @ siRNA (B, B): add 1.5. mu.l CFL (1%) and 30pmol FAM-siRNA mixture to each well; FL @ siRNA (C, C): add 1.5. mu.l FL and 30pmol FAM-siRNA mixture to each well; siRNA (D, D): 30pmol FAM-siRNA. Control group (E, E): 30pmol of negative control siRNA. The scale in the figure is 20 μm.

Fig. 5 shows the GAPDH protein knockout rate in L929 cells of example 4 with FAM-siRNA carried liposomes (p < 0.001).

FIG. 6 is the cytotoxicity of L929 cells by liposomes of example 4 wherein: (a) growth inhibition of cells after addition of different doses of CFL (0.05%), CFL (1%) and FL. (b) Cell growth status after addition of different doses of liposomes.

FIG. 7 shows the knockdown rate of the GAPDH protein in the in vivo topical treatment of example 5. Wherein: a: 3D simulation graph of GAPDH protein knockout rate; b: fig. a is a top view. GAPDH-siRNA was administered to all groups at a final concentration of 25 nmol/ml. Injection + CFL (0.05%) @ siRNA: the group, which was a 50. mu.l CFL and 100. mu.l GAPDH-siRNA (3.75nmol) cocktail, examined GAPDH protein knockdown in skin at three different treatment sites, i.e., skin at the center of injection, skin 0.5cm from the center of injection, and skin 1.0cm from the center of injection. SHS + CFL (0.05%) @ siRNA: the group was 50. mu.l CFL (0.05%) and 100. mu.l GAPDH-siRNA (3.75nmol) mixed. SHS massage; the knockdown rate of GAPDH protein was examined in the skin in which three sites were randomly selected in the region where siRNA was topically administered. The values shown in the figure are all mean values ± standard deviation (n ═ 3).

Detailed Description

The present invention will be described in detail with reference to the following examples:

example 1: liposome preparation

The preparation of Cationic Flexible Liposome (CFL) adopts a thin film hydration method: 1% DOTAP ((2, 3-dioleoyl-propyl) -trimethylamine) + 1.2% surfactant ((D-methyl-ethyl-L-methyl-L-propyl-L-dimethyl-N-propyl

O20, polyoxyethylene 20 oleyl ether) in a solvent (e.g. chloroform, diethyl ether, etc.) commonly used for preparing flexible liposomes, placing the solvent in a round-bottomed flask for rotary evaporation, forming a lipid membrane on the inner wall of the round-bottomed flask, then hydrating with Tris-HCl (0.2M, pH 4), and finally, extruding the hydrated solution through a liposome extruder with a polycarbonate film with a pore size of 100nm for 21 times to obtain cationic flexible liposomes with a phospholipid concentration of 1%, which is noted as CFL (1%); the membrane-passed extrusion was then diluted 20-fold with purified water to give cationic flexible liposomes with a phospholipid concentration of 0.05%, noted CFL (0.05%).

The preparation of the common Flexible Liposome (FL) adopts a thin film hydration method: 4% 90G (soya lecithin) + 1.2% surfactant: (

O20) is placed in round-bottomed flask in solvent (such as chloroform, diethyl ether, etc.) commonly used for preparing flexible liposome, and is subjected to rotary evaporation to form a lipid film on the inner wall of round-bottomed flask, and then the lipid film is usedPBS (0.2M, pH 7.4) was hydrated and finally the hydrated solution was extruded through the membrane 21 times through a liposome extruder with a 100nm pore polycarbonate membrane to obtain common Flexible Liposomes (FL) with a phospholipid concentration of 4%.

Preparation of CFL (1%) @ siRNA: CFL (1%) was used as a solvent to dissolve siRNA, followed by ultrasonic mixing (20min) to prepare CFL (1%) carrying siRNA, denoted CFL (1%) @ siRNA.

Preparation of CFL (0.05%) @ siRNA: CFL (0.05%) was used as a solvent to dissolve siRNA, followed by ultrasonic mixing (20min) to prepare CFL (0.05%) @ siRNA.

Preparation of FL @ siRNA: FL was used as a solvent to dissolve siRNA, followed by ultrasonic mixing (20min) to prepare FL @ siRNA.

Example 2: liposome characterization

Cationic flexible liposomes, normal flexible liposomes, were characterized for particle size and zeta potential using a Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments, UK). The deformability of the different lipid vesicles was determined by extruding 1ml of liposomes through a 100nm polycarbonate membrane under a pressure of 0.25 MPa. The time for water, CFL and FL to cross the membrane was recorded as Tw, Tc and Tf, respectively, of the water, and the deformability of CFL and FL was calculated as follows:

liposome characterization results:

cationic Flexible Liposomes (CFL) had a mean diameter of 101.4. + -. 1.1nm and a zeta potential of 35.7. + -. 0.4mV (FIGS. 1a & b). CFL @ siRNA exhibited a similar particle size distribution when bound to siRNA, but the zeta potential of CFL (0.05%) @ siRNA became electronegative (-31.4. + -. 1.1 mV). Common flexible liposomes (FL or FL @ siRNA, FIG. 1a) all had an average diameter of about 102.5nm and a zeta potential of neutrality of about-2.2 mV. Liposome flexibility results show that the optimized CFL (0.05%, 94.08% ± 1.01%) has better deformability (p <0.001) than the original CFL (1%, 56.03% ± 1.98%) and FL (4%, 61.79% ± 0.48%).

Example 3: external transdermal permeation of siRNA

This example uses ex vivo pigskin as a skin model for the experiment. The method comprises pretreating Corii Sus Domestica, carefully removing subcutaneous adipose tissue with a scalpel, shaving hair with an electric shaver to length of 5mm or less, cleaning the treated Corii Sus Domestica with ultrapure water, and storing at-20 deg.C. The skin was thawed at room temperature prior to use. The same size diameter pigskin was drilled with a circular punch of 40mm diameter and mounted on a Franz diffusion transdermal device and the conductance of the skin was measured using the ex vivo skin percutaneous resistance test to ensure skin barrier integrity. The effective penetration area of Franz diffusion cell is 1.77cm²The volume of the receptor was 12 ml. The skin was placed on top of a vertical Franz diffusion cell and the receptor cell was filled with PBS (pH 7.4, 0.2M) and the device was then placed in a water bath at 37 ± 0.5 ℃. The using method of the SHS comprises the following steps: SHS (5 mg/cm) was applied by a household electric massager (Codos KP-3000) applying a force of about 0.3N at a speed of about 300rpm/min²) Topical application to skin was carried out for 2 minutes. The skin was then washed 3 times with PBS (0.2M) to remove residual SHS on the skin. Several groups of pharmaceutical preparations (administration volume of 150. mu.l with 50. mu.l of liposomes and 100. mu.l of siRNA (3.75nmol) mixed therein, final concentration of siRNA administration of 25nmol/ml for each group), solvent DEPC-treated water, preparation method with reference to example 1) were applied non-occlusive to the skin surface. The group not associated with SHS applied the drug directly to the skin surface without SHS treatment. siRNA group was directly administered with 25nmol/ml siRNA (administration volume 150 μ l, solvent PBS (0.2M, pH 4)); the administration of siRNA to the SHS + siRNA group was carried out by applying a solution of siRNA dissolved with DEPC (25nmol/ml, 150. mu.l) directly after SHS massage; microneedle (dermaroller) in combination with CFL (0.05%) @ siRNA group the roller microneedles were rolled once over the skin in a "rice" pattern, followed by application of CFL (0.05%) @ siRNA (administration volume 150 μ l, siRNA concentration 25 nmol/ml). The drug permeation time for each group was 16 hours. Each group was repeated at least three times. The liquid in the receptor pool was removed, the amount of siRNA permeating to the subcutaneous skin was measured, and then the amount of siRNA in each skin layer was measured by tape stripping.

The pig skin was cut out of a tissue having a thickness of 20 μm with a cryomicrotome, and then imaged under a confocal microscope to qualitatively judge the amount of siRNA permeation under each cortex.

The experimental results are as follows:

to overcome the first obstacle to local delivery of siRNA, i.e., the stratum corneum barrier of the skin, different experimental groups were used in this example to examine the delivery effect of GAPDH-siRNA in the skin. Compared with all other groups, the topical combination of SHS with CFL (0.05%) @ siRNA showed the highest skin permeability of siRNA (fig. 2), and the skin absorption rate of GAPDH-siRNA was as high as 61.18% ± 2.49% after transdermal 16h, significantly higher than SHS in combination with CFL (1%) @ siRNA group (48.19% ± 7.21%, p <0.05) and SHS in combination with FL (4%) @ siRNA group (37.99% ± 1.52%, p < 0.001). This result indicates that the ability of siRNA-carrying liposomes to deliver siRNA to the skin after pre-treatment massage with SHS appears to be directly correlated with their deformability. The combined use of CFL (0.05%) @ siRNA and SHS is significantly higher than the combined use of CFL (0.05%) @ siRNA and traditional roller microneedle Dermaroller, and the percutaneous penetration promoting effect of siRNA is achieved. Confocal microscopy imaging also showed similar results (figure 3).

Example 4: liposome cytotoxicity and in vitro cell transfection

L929 cells were seeded into 96-well plates and cultured in a cell incubator for 24h (5% CO)₂At 37 ℃ C. The medium in each well was removed and replaced with 100 μ l of fresh medium. The cells were then incubated with different doses of liposomes (5.0. mu.l/well, 2.5. mu.l/well, 1.0. mu.l/well, 0.5. mu.l/well, 0.1. mu.l/well) for a further 24 hours at 37 ℃. The viability of the cells was then determined using the MTT method at different liposome doses.

L929 cells were seeded into 24-well plates and cultured in a cell incubator for 24h (5% CO)₂At 37 ℃ C. Cell transfection experiments were performed at cell densities of about 70% -80%. Mu.l of different liposomes and 3. mu.l of FAM-siRNA (10nmol/ml) were added to 25. mu.l of serum-free medium opti-mem, respectively, and then the two were mixed well according to the preparation method of example 1, and finally the mixture was left to stand at room temperature for 5 minutes and added to each well, and then the cells were cultured in an incubator for 6 hours, followed by using a confocal methodThe transfection was imaged by microscopy. Or after adding the transfection mixture to each well for a further 68 hours of incubation, GAPDH protein expression levels were determined using a KDalert GAPDH detection kit and protein knock-out rates were calculated (six replicates per group).

The experimental results are as follows:

to overcome the second obstacle to local delivery of siRNA, i.e., the cell membrane barrier, this example evaluated the ability of three liposomal CFLs (0.05%), CFLs (1%), and FLs to deliver siRNA into cells to reduce protein expression. The distribution of FAM-siRNA in L929 cells was photographed by confocal photography (fig. 4), and the results showed that CFL (0.05%) could induce FAM-siRNA to enter cells, significant fluorescence was observed in cells (fig. 4A, a) and no significant fluorescence was observed in cells of the other groups, compared to other groups, indicating that CFL (0.05%) significantly promoted siRNA entry into cells compared to CFL (1%) and FL.

Subsequently, this example further measured the expression of GAPDH protein in L929 cells, and further verified the transfection effect (fig. 5). CFL (0.05%) @ siRNA resulted in a knockdown rate of GAPDH protein of 41.09% + -5.14% after transfection for 68h, which was much higher than CFL (1%) @ siRNA (8.38% + -2.08%), FL @ siRNA (3.48% + -2.25%) and siRNA alone (6.07% + -1.62%) for GAPDH protein.

The relationship between cytotoxicity of CFL (0.05%), CFL (1%) and FL and the additive dose was determined by the MTT method. MTT assay showed cell growth IC for CFL (0.05%), CFL (1%) and FL₅₀Values were 6.634. mu.l/well, 0.626. mu.l/well and 0.428. mu.l/well, respectively (FIG. 6 a). The cell state gradually recovered as the amount of liposome added was decreased (fig. 6 b). These results all show that CFL (0.05%) at the appropriate dose is more suitable for delivering siRNA to cells and shows lower toxicity to cells, so its protein knock-out effect in vivo was further verified with CFL (0.05%).

Example 5: in vivo protein knock-out

This example investigates the efficacy of CFL (0.05%) in inducing a decrease in GAPDH protein expression in vivo. A total of four sets of experiments were designed: CFL (0.05%) @ siRNA was injected subcutaneously as a positive control; topical co-administration of SHS with CFL (0.05%) @ siRNA; local co-administration of SHS and siRNA; and (4) a control group.

For the subcutaneous injection group, 50. mu.l of CFL (0.05%) and 100. mu.l of siRNA (3.75nmol) solution were mixed well according to the preparation method of example 1, and then the mixture was injected subcutaneously into mice.

For the remaining groups, mice were first anesthetized by intraperitoneal injection of 150 μ l chloral hydrate (4%), the hair on the back of the mice was trimmed, and the mice were taped to a bare skin area of 1.77cm on the back using 3M Vetbond tissue adhesive²Then using SHS (5 mg/cm)²) The treatment site was finally washed 3 times with PBS (0.2M) to remove SHS by massaging on bare skin for 2 minutes under an applied force of 0.3N. Subsequently, for the group of topical co-administration of SHS with CFL (0.05%) @ siRNA, a mixed solution of 50. mu.l CFL (0.05%) and 100. mu.l siRNA (3.75nmol) solutions (preparation method reference example 1) was applied non-blocked to the treatment area. For the topical combined administration of SHS and siRNA group, 150 μ l siRNA (3.75nmol) solution was applied blocked to the treatment area. For the control group, 150 μ l of negative control siRNA (3.75nmol) solution was applied blocked to the treatment area without SHS massage treatment. After 72 hours, mice were sacrificed by carbon dioxide overexposure. Mouse skin tissue was collected from the treatment area and the in vivo protein knock-out effect was calculated using a KDalert GAPDH assay kit to measure GAPDH protein expression levels (each group contains 3 replicates).

The experimental results are as follows:

the combined use of SHS and CFL (0.05%) was shown to be the most effective combination of topical RNAi based on in vitro skin penetration, in vitro cell transfection and in vitro cytotoxicity studies. Thus, we subsequently used female BALB/C mice (7-8 weeks) to determine the ability of in vivo topical combined administration of SHS and CFL (0.05%) to deliver GAPDH-siRNA into the skin. CFL (0.05%) carrying GAPDH-siRNA was delivered into the skin of mice by subcutaneous injection and SHS massage treatment. The results of the subcutaneous injection group showed no significant difference in the knockdown rate of GAPDH protein at the injection center (26.17% + -6.25%) and at a position 0.5cm from the injection center (26.38% + -7.40%), but the skin GAPDH protein knockdown rate rapidly decreased (6.45% + -5.87%) at a position 1cm from the injection center (FIG. 7). In contrast, topical application of SHS massage in combination with CFL (0.05%) on the mouse skin resulted in GAPDH knockdown rates as high as 29.21% ± 1.41% over the topically applied area, significantly higher than the SHS massage treatment alone (5.48% ± 7.97%, p < 0.01), and comparable protein knockdown results (p ═ 0.458) in the subcutaneous central region (fig. 7). These results indicate that the combination of SHS and CFL (0.05%) is superior to injection in terms of treatment area, so that the combination is a promising delivery system for in vivo local application of RNAi therapy.

And (4) conclusion:

liposome concentration affects liposome flexibility and toxicity to cells. The combination of cationic flexible liposomes and SHS for siRNA delivery into the skin is in a positive correlation with liposome flexibility. SHS and CFL (0.05%) in combination showed the best transdermal penetration enhancing effect on siRNA in vitro; CFL (0.05%) has lower toxicity to cells and higher protein knockout rate; in vivo results show that the combined use of SHS and CFL (0.05%) can have comparable protein knock-out rates to subcutaneous injection into the central skin, but with better area advantages. In conclusion, CFL (0.05%) is the optimal concentration for matching with SHS, and the combined use of SHS and CFL (0.05%) is the most effective combination mode for local application of RNAi, and is expected to be a promising delivery system for RNAi treatment.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Claims

1. A siRNA transdermal delivery composition, comprising: comprises a spongy spicule, a flexible liposome and the siRNA.

2. The siRNA transdermal delivery composition of claim 1, wherein: the flexible liposome and the siRNA are in a form that the flexible liposome carries the siRNA, or in a mixed form of the flexible liposome and the siRNA.

3. The siRNA transdermal delivery composition of claim 1, wherein: the flexible liposome comprises common flexible liposome and cationic flexible liposome.

4. The siRNA transdermal delivery composition of claim 3, wherein: the phospholipid concentration of the cationic flexible liposome is 0.02-1.5%.

5. The siRNA transdermal delivery composition of claim 4, wherein: the phospholipid concentration of the cationic flexible liposome is 0.04-0.06%.

6. The siRNA transdermal delivery composition of claim 4, wherein: the membrane material of the cationic flexible liposome comprises DOTAP and a surfactant.

7. A method of using the siRNA transdermal delivery composition of any of claims 1-6, characterized in that: the spongiosa spicules are first applied to the skin, and then the flexible liposomes and the siRNA are applied to the skin.

8. The method of using a siRNA transdermal delivery composition according to claim 7, wherein: the application amount of the sponge spicules on the skin is 2-8 mg/cm²。

9. Use of the siRNA transdermal delivery composition of any one of claims 1 to 6 for gene knock-out.

10. Use of the siRNA transdermal delivery composition according to any one of claims 1 to 6 for preparing a transdermal absorption preparation or a cosmetic.