Nucleic acid transdermal agent, functional nucleic acid preparation and application thereof

Abstract

The invention provides a nucleic acid transdermal agent composition, a functional nucleic acid preparation and application of the nucleic acid transdermal agent composition and the functional nucleic acid preparation, and belongs to the technical field of nucleic acid transdermal absorption. The additive is one or more of 1, 4-dicarboxylate, dimethyl isosorbide DMI, dodecyl maltoside DDM, decapeptide-4, dimethyl sulfone, inositol, ethyoxyl diglycol, azone and polysorbate-40. The additive is one or more of the following components: 1, 4-dicarboxylate, dimethyl isosorbide DMI, dodecyl maltoside DDM, decapeptide-4, dimethyl sulfone, inositol, ethyoxyl diglycol, azone and polysorbate-40 The nucleic acid transdermal agent with a promoting effect on transdermal absorption of small nucleic acid drugs is obtained by utilizing double screening of qPCR and LC-MS / MS, and the transdermal absorption of small nucleic acid components can be remarkably promoted, so that the exerting effect of the effects of the small nucleic acid components is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid transdermal absorption, and in particular relates to a nucleic acid transdermal agent, a functional nucleic acid preparation and applications thereof. Background Art

[0002] Small nucleic acid drugs (including siRNA, antisense oligonucleotides, and miRNA inhibitors), as cutting-edge technologies in gene therapy, have shown great potential in the treatment of genetic diseases, inflammatory diseases, and tumors by targeting and regulating the expression of disease-related genes. The skin is an ideal target for localized gene therapy, as pathological conditions such as psoriasis, atopic dermatitis, and skin cancer are often directly linked to abnormal expression of specific genes. For example, skin aging is associated with overactivation of collagen degradation genes (such as MMP-1), UV-induced pigmentation is often accompanied by abnormal expression of the tyrosinase gene, and impaired skin barrier function is closely associated with insufficient synthesis of filaggrin. In the cosmetics field, small nucleic acid technology can achieve anti-aging, whitening, and barrier repair benefits by silencing pathogenic genes or regulating signaling pathways related to the skin microenvironment (such as the NF-κB pathway). Compared to traditional active ingredients such as retinol and vitamin C, small nucleic acid drugs offer advantages such as precise targeting and low dosage requirements, making them particularly suitable for the development of high-end functional skincare products. However, as products for daily use, the active ingredients of cosmetics must rely on transdermal absorption mechanisms to work. Invasive methods such as injections cannot be used, and it is necessary to ensure that the ingredients effectively reside in the epidermis. Due to the natural defense mechanism of the skin barrier, small nucleic acids with large molecular weight (usually 13-15kDa), strong hydrophilicity and negative charge are difficult to penetrate the lipid barrier, and the degradation of nucleases on the skin surface will significantly reduce the stability of small nucleic acids. Therefore, the development of a delivery system that is compatible with cosmetic matrices and has both efficient transdermal properties and mild safety is a key breakthrough in promoting the commercial application of small nucleic acid technology in the beauty field.

[0003] Compared with traditional injection or oral administration, transdermal delivery of small nucleic acids offers multiple advantages: First, local targeting allows precise action at the lesion site, reducing the risk of systemic exposure; second, non-invasive delivery significantly improves patient compliance, making it particularly suitable for chronic skin diseases requiring long-term treatment; third, patches or gels allow for flexible dosage control and sustained release, while avoiding gastrointestinal degradation and first-pass effects in the liver. However, existing technologies still face two major bottlenecks: First, conventional transdermal agents (such as ethanol and propylene glycol) have limited effects on the permeation of small nucleic acids, making it difficult to achieve effective therapeutic concentrations; second, while some chemical permeation enhancers (such as dimethyl sulfoxide (DMSO)) can improve permeation efficiency, they may cause skin irritation or disrupt barrier function. Although physical permeation enhancement technologies (such as microneedles and electroporation) can partially address this efficiency issue, their operational complexity, high cost, and potential risk of tissue damage limit their application in cosmetics or consumer products. In this context, screening for novel chemical permeation enhancers has become a key research direction to balance efficiency and safety.

[0004] Transdermal agents have diverse mechanisms of action and can be broadly categorized into the following: lipid-based solvents disrupt the dense structure of the stratum corneum by dissolving lipids; hydrophilic channel openers expand hydrophilic channels by enhancing stratum corneum hydration; surfactants promote drug distribution by reducing surface tension; natural extracts have both permeation-enhancing and soothing functions; and peptide carriers may assist in delivery via paracellular pathways or transcytosis. It is worth noting that the cosmetics sector has even stricter safety requirements for transdermal agents. Commonly used ingredients include low-concentration azone (<5%), ethoxydiglycol, DMI (dimethyl isosorbide), and natural terpenes (such as limonene). These ingredients have been widely used to promote the absorption of active ingredients such as vitamins and antioxidants, but their permeation-enhancing effects on small nucleic acids have yet to be systematically studied.

[0005] Researching the unique value of small nucleic acids in transdermal delivery will open up new paths for expanding the transdermal application of functional nucleic acid ingredients (such as anti-aging oligonucleotides) in the cosmetics field, and has significant industrialization prospects. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a nucleic acid transdermal agent, a functional nucleic acid preparation and its application; the present invention uses dual screening of qPCR and LC-MS / MS to obtain a nucleic acid transdermal agent that has a promoting effect on the transdermal absorption of small nucleic acid drugs, which can significantly promote the transdermal absorption of small nucleic acid components, thereby improving the efficacy of small nucleic acid components.

[0007] The present invention provides a nucleic acid transdermal agent, comprising one or more of bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate, dimethyl isosorbide DMI, dodecyl maltoside DDM, decapeptide-4, dimethyl sulfone, inositol, ethoxydiglycol, laurocapram, and polysorbate 40;

[0008] When choosing one of these;

[0009] The concentration (v / v) of bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate is 1 to 6%;

[0010] The concentration of dimethyl isosorbide DMI is 1 to 12% (v / v);

[0011] The concentration of dodecyl maltoside DDM is 1 to 12% (w / v);

[0012] The concentration of decapeptide-4 is 1 to 12% (w / v);

[0013] The concentration of dimethyl sulfone is 1 to 12% (w / v);

[0014] The concentration of inositol is 1-5% (w / v);

[0015] The concentration of ethoxydiglycol is 1 to 5% (v / v);

[0016] The concentration of laurocapram is 1-6% (v / v);

[0017] The concentration of polysorbate 40 is 1 to 6% (v / v).

[0018] Preferably, it comprises isosorbide dimethyl ether DMI and laurocapram; the concentration of the isosorbide dimethyl ether DMI is 1 to 12%, and the concentration of the laurocapram is 1 to 12%.

[0019] Preferably, the concentration of dimethyl isosorbide DMI is 2-7%, and the concentration of laurocapram is 2-7%.

[0020] The present invention provides application of the nucleic acid transdermal agent in preparing nucleic acid transdermal medicine.

[0021] The present invention provides the use of the nucleic acid transdermal agent in the preparation of nucleic acid transdermal cosmetics.

[0022] The present invention provides a functional nucleic acid miRNA1, the sequence of which is as follows:

[0023] ss chain:

[0024] mC*mU*mAmCmUmAfAmAfAfCfAmUmGmGmAmAmGmCmAmCmU mUmA-chol

[0025] as chain:

[0026] mU*fA*mAmGmUfGmCmUmUmCmCmAmUfGmUfUmUmUmAmGmU*mA*mG;

[0027] Among them, * represents the thio modification of the connection modification between two nucleic acids, the base after the lowercase m is methoxy-modified, the base after the lowercase f is fluorinated, and chol represents cholesterol modification.

[0028] The present invention provides a functional nucleic acid preparation, comprising the miRNA1 and the nucleic acid transdermal agent.

[0029] Preferably, the concentration of miRNA1 in the functional nucleic acid preparation is 10 ng / mL-20 mg / mL.

[0030] The present invention provides the use of the functional nucleic acid preparation in the preparation of medicines or cosmetics.

[0031] Preferably, the cosmetic is a cosmetic having the effects of treating hair loss, anti-aging or whitening.

[0032] Compared with the existing technology, the present invention has the following advantages: the nucleic acid transdermal agent provided by the present invention has been dual-screened by qPCR and LC-MS / MS, ensuring that its transdermal absorption-promoting effect is more reliable and can significantly promote the transdermal absorption of small nucleic acid drugs through the skin. The functional nucleic acid miRNA1 double-stranded product provided by the present invention has been modified through a series of sequence modifications, while ensuring the functional activity of miRNA1 and increasing its stability. The present invention combines the nucleic acid transdermal agent with the functional nucleic acid miRNA1 to prepare a functional nucleic acid preparation, which can improve the transdermal absorption of miRNA1 and thereby enhance the efficacy of miRNA1. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Standard curve for qPCR detection of miRNA1;

[0034] Figure 2 To detect the effect of each group of transdermal enhancers on the transdermal effect of miRNA1 by qPCR;

[0035] Figure 3 This is the peak diagram of LC-MS / MS detection of miRNA1;

[0036] Figure 4 Standard curve for detection of miRNA1 by LC-MS / MS method;

[0037] Figure 5The LC-MS / MS method was used to detect the effect of each group of transdermal enhancers on the transdermal effect of miRNA1 (1, 3, and 6 h);

[0038] Figure 6 The results of subcutaneous injection of the same dose of transdermal enhancers in each group were detected by L C-MS / MS method;

[0039] Figure 7 The effect of different concentrations of laurocapram on the transdermal effect of miRNA1;

[0040] Figure 8 The effect of different concentrations of dimethyl isosorbide DMI on the transdermal effect of miRNA1;

[0041] Figure 9 The effects of different transdermal agent combinations on the transdermal effect of miRNA1;

[0042] Figure 10 This is the transdermal effect of RNAi-1 to RNAi-5 through the combination of laurocapram and DMI. DETAILED DESCRIPTION

[0043] The present invention provides a nucleic acid transdermal agent, which comprises one or more of bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate, dimethyl isosorbide DMI, dodecyl maltoside DDM, decapeptide-4, dimethyl sulfone, inositol, ethoxydiglycol, laurocapram, and polysorbate 40.

[0044] In the present invention, when one of them is selected, the concentration of the bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate is preferably 1 to 6%, more preferably 4.5 to 5.5%, and most preferably 5%.

[0045] The concentration of dimethyl isosorbide DMI is preferably 1-12%, specifically 1-12% or 9%-11%, more preferably 10%.

[0046] The concentration of the dodecyl maltoside DDM is preferably 1-12%, more preferably 5-11%, and most preferably 10%;

[0047] The concentration of the decapeptide-4 is preferably 1 to 12%, more preferably 5 to 11%, and most preferably 10%;

[0048] The concentration of dimethyl sulfone is preferably 1-12%, more preferably 5-11%, and most preferably 10%;

[0049] The concentration of the inositol is preferably 1-5%, more preferably 3.5-4.5%, and most preferably 4%;

[0050] The concentration of the ethoxydiglycol is preferably 1-5%, more preferably 3.5-4.5%, and most preferably 4%;

[0051] The concentration of laurocapram is preferably 1-12%, more preferably 2-7%, and most preferably 5%;

[0052] The concentration of polysorbate 40 is preferably 1-6%, more preferably 2-5.5%, and most preferably 5%;

[0053] In the present invention, when two types are selected, preferably dimethyl isosorbide DMI and laurocapram are included; the concentration of dimethyl isosorbide DMI is preferably 1 to 12%, more preferably 2 to 7%, and most preferably 2.5%; the concentration of laurocapram is preferably 1 to 12%, more preferably 2 to 7%, and most preferably 2.5%.

[0054] When two are selected, another preferred scheme includes laurocapram and decapeptide-4; the concentration of the laurocapram is preferably 2-2.9%, more preferably 2.4-2.6%, and most preferably 2.5%; the concentration of the decapeptide-4 is preferably 8-12%, preferably 9-11%, and more preferably 10%.

[0055] Another preferred solution includes laurocapram and inositol; the concentration of the laurocapram is preferably 2-2.9%, more preferably 2.4-2.6%, and most preferably 2.5%; the concentration of the inositol is preferably 3-5%, preferably 3.5-4.5%, and more preferably 4%.

[0056] Another preferred solution includes isosorbide dimethyl ether DMI and decapeptide-4; the concentration of the isosorbide dimethyl ether DMI is preferably 2-2.9%, more preferably 2.4-2.6%, and most preferably 2.5%; the concentration of the decapeptide-4 is preferably 8-12%, preferably 9-11%, and more preferably 10%.

[0057] Another preferred solution includes isosorbide dimethyl ether DMI and inositol; the concentration of the isosorbide dimethyl ether DMI is preferably 2-2.9%, more preferably 2.4-2.6%, and most preferably 2.5%; the concentration of the inositol is preferably 3-5%, preferably 3.5-4.5%, and more preferably 4%.

[0058] The present invention provides application of the nucleic acid transdermal agent in preparing nucleic acid transdermal medicine.

[0059] The present invention provides the use of the nucleic acid transdermal agent in the preparation of nucleic acid transdermal cosmetics.

[0060] The present invention provides a functional nucleic acid miRNA1, the sequence of which is as follows:

[0061] ss chain:

[0062] mC*mU*mAmCmUmAfAmAfAfCfAmUmGmGmAmAmGmCmAmCmU mUmA-chol

[0063] as chain:

[0064] mU*fA*mAmGmUfGmCmUmUmCmCmAmUfGmUfUmUmUmAmGmU*mA*mG;

[0065] Among them, * represents the thio modification of the connection modification between two nucleic acids, the base after the lowercase m is methoxy-modified, the base after the lowercase f is fluorinated, and chol represents cholesterol modification.

[0066] The present invention also provides a functional nucleic acid preparation, comprising the miRNA1 and the nucleic acid transdermal agent.

[0067] In the present invention, the concentration of miRNA1 in the functional nucleic acid preparation is preferably 10 ng / mL to 20 mg / mL, more preferably 10 to 18 mg / mL, and even more preferably 15 mg / mL. In the present invention, the functional nucleic acid preparation is preferably prepared by mixing the miRNA1 with the nucleic acid transdermal agent.

[0068] The present invention provides the use of the functional nucleic acid preparation in the preparation of cosmetics.

[0069] In the present invention, the cosmetics are preferably cosmetics with hair loss treatment, anti-aging or whitening effects. The present invention has no particular restrictions on the type of cosmetics, including but not limited to shampoo and hair care products, lotions, emulsions, creams, facial masks, etc.

[0070] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0071] Example 1

[0072] Preparation of small nucleic acid miRNA1

[0073] Preparation process:

[0074] Monomers and reagents were purchased from Beijing Haijing Gaochuang Technology Co., Ltd. or other qualified companies. The target small nucleic acid miRNA1 sequence information is shown below, where * represents the thiolation modification of the connection modification between two nucleic acids, the base following the lowercase m is methoxy-modified, the base following the lowercase f is fluorinated, and chol represents cholesterol modification.

[0075] Target small nucleic acid miRNA1 sequence information:

[0076] ss chain:

[0077] mC*mU*mAmCmUmAfAmAfAfCfAmUmGmGmAmAmGmCmAmCmU mUmA-chol

[0078] as chain:

[0079] mU*fA*mAmGmUfGmCmUmUmCmCmAmUfGmUfUmUmUmAmGmU*mA*mG;

[0080] Preparation process:

[0081] Step 1. Synthesis was performed using a 12-channel synthesizer (HJ-12, Beijing Haijing Gaochuang Technology Co., Ltd.): including deprotection, coupling, oxo / thio modification, capping, etc., and was commissioned by Beijing Haijing Gaochuang Technology Co., Ltd.

[0082] Specifically:

[0083] Prepare a DCA toluene solution as a deprotecting agent, 0.25M ETT (0.25M 5-ethylthiotetrazolyl N / A acetonitrile) as a coupling agent, a 0.05M iodine pyridine / water solution as an oxidant, hydrogenated xanthan gum as a thiolation agent, and a CH3CN / imidazole solution containing 20% ​​acetyl chloride as a capping agent, and place them in the designated reagent position corresponding to the HJ-12 synthesizer. Set the synthesis program to enter the specified oligonucleotide base sequence and start cyclic oligonucleotide synthesis. The coupling time for each step is 6 minutes, and the coupling time for the galactose ligand corresponding to the L and S monomers is 15 minutes. After automatic cycling, the oligonucleotide solid phase synthesis is completed.

[0084] Step 2. Ammonolysis.

[0085] Aminolysis was performed at 160 mL / mmol AMA at 60°C for 3 h. The system was diluted with purified water, the pH of the system was adjusted to 7.0 with aqueous acetic acid, and the solid was collected by centrifugation.

[0086] Step 3. Purification.

[0087] Agilent HPLC purification system, 45 ° C hot ethanol dissolved sample, mobile phase A: 200 mM TEAA in H2O, mobile phase B: prepared acetonitrile, elution gradient: 20-80% 90 min, linear flow rate: 160-200 cm / h (10 mL / min), purification column using C8 (with cholesterol, use C8) or C18 column (without cholesterol, use C18), column temperature: 40 ° C.

[0088] Step 4. Desalting and freeze-drying.

[0089] Purified water was used for ultrafiltration desalination, and a 3KDa TFF membrane was used for ultrafiltration desalination. Finally, double-stranded small nucleic acids were prepared by annealing and lyophilization. Annealing buffer: pure water; Annealing: equimolar guide strand and passenger strand RNA solutions were mixed, and an equal volume of 2× annealing buffer (15-50μM) was added. The mixture was annealed according to the following procedure: incubation at 95°C for 3 minutes, incubation at 37°C for 30 minutes, and incubation at 25°C for 60 minutes. Lyophilization: Lyophilization was performed using a Ningbo Xinzhi lyophilizer (model SCIENTZ-18N / C).

[0090] Example 2

[0091] Standard curve establishment for quantitative qPCR methodology

[0092] The absolute quantification method of miRNA can clearly determine the number of molecules, accurately measure the actual copy number or concentration of miRNA in the sample, and directly give the specific content of miRNA in the sample. Relative quantification is based on the expression of the internal reference gene to calculate the relative expression of the target miRNA. The expression of the internal reference gene in different samples may vary, which will affect the comparability of the relative quantification results between different samples. Since absolute quantification gives a specific value, it can be directly compared between different samples (such as samples from different individuals, different tissue types, and different experimental conditions), so an absolute quantification standard curve for the target small nucleic acid is established.

[0093] The mature chain of the chemically synthesized target miRNA1 was used as a standard, and a quantitative standard curve was drawn after dilution at more than 5 gradient points.

[0094] The standard synthesis sequence of the target small nucleic acid miRNA1 (synthesized by Jima Bio) is: 5'-UAAGUGCUUCCAUGUUUUAGUAG-3' (SEQ ID NO.1)

[0095] The synthesized standard was dissolved in NF-H2O to a 100 μM stock solution and diluted according to the serial dilution method. The copy number was calculated based on the molecular weight as shown in Table 1.

[0096] Table 1 Copy number of standard products

[0097] Standards copies / reation S7 1.20E+06 S6 1.20E+07 S5 1.20E+08 S4 1.20E+09 S3 1.20E+10 S2 1.20E+11 S1 1.20E+12

[0098] Mir-X miRNA First-Strand Synthesis Kit (Takara, Cat. No. 638313) was used to perform cDNA synthesis reaction of the gradient standard. The reaction system is shown in Table 2.

[0099] Table 2 cDNA synthesis reaction system

[0100]

[0101]

[0102] The solution of the system was mixed and placed in a PCR instrument. The reaction was carried out at 37°C for 1 hour and 85°C for 5 seconds, and then the reverse transcribed cDNA sample was stored at -20°C for a long time.

[0103] According to the detection principle of miRNA tailing method, the primer sequences for qPCR detection of target small nucleic acids were designed as shown in Table 3. The reverse primer of the qPCR reaction was provided by the kit.

[0104] Table 3 Primer sequences

[0105]

[0106] The cDNA was diluted 10-fold and qPCR reaction was performed using Adlay qPCR reagent (Cat. No. PC3301) according to the system described in Table 4 below.

[0107] Table 4 qPCR reaction system

[0108] Components Volume (μl) 2xSybrqPCRmix 10 miRNA1-specific primer (10 μM) 0.4 mRNA 3' Primer (10 μM) 0.4 cDNA (diluted 10 times) 2 <![CDATA[H2O]]> 7.2 Total 20

[0109] The qRT-PCR reaction program was as follows: 95°C for 10 min; 95°C for 15 s, 60°C for 35 s, 40 cycles.

[0110] The results are shown in Table 5.

[0111] Table 5 qRT-PCR detection results

[0112]

[0113]

[0114] Standard curve Figure 1 As shown, specifically:

[0115] Ct = -3.653 × log (conc) + 48.759, the correlation coefficient is 0.9965, and the linearity is good.

[0116] Example 3

[0117] Preliminary screening of transdermal enhancers

[0118] The transdermal enhancers and their concentrations that were initially screened are shown in Table 6 below.

[0119] Table 6 Types and concentrations of transdermal enhancers

[0120]

[0121]

[0122] The transdermal agent is first prepared into a solution of corresponding concentration (volume concentration or mass concentration), and then used as a solvent for miRNA1 to prepare a small nucleic acid drug to be applied. The dosage for each mouse is 1.5 mg miRNA1 in a volume of 100 μL.

[0123] After arriving at the laboratory, 8-week-old C57 mice (purchased from Weitonglihua) were adaptively fed for 1 week, with 5 mice in each group. They were depilated with rosin paraffin 24 hours before the experiment to obtain a 2 cm × 2 cm hairless area. On the day of the experiment, each group of mice was smeared with the target small nucleic acid drug corresponding to different transdermal agents, 1.5 mg per mouse, and gently smeared until the drug was absorbed. Note that laboratory gloves need to be changed between each group. Dermal tissue was obtained 3 hours after smearing, RNA was extracted, and after reverse transcription into cDNA, QPCR detection was performed. Mice smeared with normal saline after depilation were used as the control group for data analysis.

[0124] The content of target small nucleic acid drugs in a fixed amount of dermal tissue of each mouse was calculated to evaluate the transdermal effect of small nucleic acid in each group.

[0125] The steps for RNA extraction are as follows:

[0126] 1) Grind 50 mg of dermal tissue with liquid nitrogen, add 500 μl of Trizol and mix thoroughly. Then transfer the suspension into a 1.5 ml RNase-free centrifuge tube.

[0127] 2) Add 100 μl of chloroform to each tube and shake thoroughly for 15 seconds, then let it stand at room temperature for 3 minutes;

[0128] 3) Set the refrigerated centrifuge to 4°C in advance, centrifuge at 12,000 rpm for 15 minutes, and carefully transfer the supernatant to a new 1.5 ml RNase-free centrifuge tube.

[0129] 4) Add 250 μl of isopropanol to the supernatant to precipitate RNA. Mix thoroughly and let stand at room temperature for about 10 minutes. Centrifuge again at 12,000 rpm for 10 minutes.

[0130] 5) After discarding the supernatant, add 1 ml of 80% ethanol solution to the pellet and wash the RNA by inverting it;

[0131] 6) Centrifuge again at 7500 rpm at 4°C for 5 min;

[0132] 7) Discard the supernatant, let it stand at room temperature for 3-5 minutes to evaporate the ethanol, and add 20 μl of DEPC water to dissolve the RNA;

[0133] 8) The concentration and quality of the extracted RNA samples were detected by NanoDrop 2000 (when the concentration was measured by NanoDrop 2000, RNA with 260 / 280 = 1.9-2.0 and 260 / 230 > 2 had good purity, indicating less residual protein and phenolic substances).

[0134] The experimental steps and reaction system for reverse transcription of RNA into cDNA and qPCR detection refer to Example 2.

[0135] The results of qPCR detection of the transdermal effect of each group of transdermal agents are as follows Figure 2 As shown, from the experimental results, it can be seen that group 1 (10% bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate), group 6 (5% dodecyl maltoside DDM), group 8 (5% decapeptide-4), group 10 (5% dimethyl sulfone), group 12 (2% inositol), group 15 (5% N,N-dimethylacetamide), group 16 (5% polyvinyl alcohol), group 17 (10% poloxamer), group 19 (2 % polysorbate-20), group 20 (2.5% decyl maltoside), group 21 (1.5% lauryl maltoside), group 22 (2.5% octyl-beta-D-glucoside), group 23 (5% cocoyl glucoside), group 24 (1.25% p-hydroxybenzyl glucosinolate), group 25 (7.5% lauryl maltoside), group 26 (5% oleic acid), group 27 (2% menthol ), group 28 (2.5% limonene) and the control NC group had no significant difference, indicating that these transdermal agents could not promote the absorption of the target small nucleic acid drug in the skin at this experimental concentration; while group 2 (5% bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate), group 3 (10% dimethyl isosorbide DMI), group 4 (5% dimethyl isosorbide DMI), group 5 (10% dodecyl maltoside DDM), group 7 (10% decapeptide-4), group 9 (10% dimethyl sulfone), group 11 (4% inositol), group 13 (5% ethoxydiglycol), group 14 (5% laurocapram), and group 18 (2.5% polysorbate-40) had the effect of promoting the absorption of the target small nucleic acid in the skin, among which 10% DMI, 5% ethoxydiglycol and 5% laurocapram had the best effect in promoting transdermal absorption.

[0136] Since qPCR has high requirements for sample quality, the integrity of the RNA in the sample will affect the efficiency of reverse transcription and amplification. If the RNA in the sample is degraded, the target miRNA may not be effectively amplified, thereby affecting the accuracy of the test results; because biological samples often contain various PCR inhibitors, such as hemoglobin, polysaccharides, bile salts, etc., these inhibitors will inhibit the activity of DNA polymerase, interfere with the PCR reaction, resulting in reduced amplification efficiency or inability to amplify, making the quantitative results inaccurate; and the detection range of qPCR is usually limited by the amplification efficiency and the linear range of the fluorescent signal. When the concentration of the target miRNA in the sample is too high or too low, it may lead to inaccurate quantification. For high-concentration samples, amplification saturation may occur, so that the fluorescent signal no longer increases linearly with the increase in the concentration of the target miRNA; for low-concentration samples, the presence of the target miRNA may not be accurately detected due to low amplification efficiency or background noise. Therefore, the present invention also developed a more specific LC-MS / MS experimental platform.

[0137] Example 4

[0138] Standard Curve Development for LC-MS / MS Methodology

[0139] LC-MS / MS combines the separation capability of liquid chromatography with the detection capability of mass spectrometry. Liquid chromatography can separate different components in complex biological samples based on the physicochemical properties of small nucleic acids (such as size, polarity, etc.), effectively separating the target small nucleic acids from other impurities. Mass spectrometry, on the other hand, identifies and quantifies small nucleic acids based on their precise molecular weight, fragment ion information, and other molecular structural characteristics; and each small nucleic acid has its own unique molecular weight and fragmentation pattern, which is as unique as a fingerprint. Even small nucleic acids with similar sequences will appear differently in mass spectrometry, so LC-MS / MS can accurately distinguish and quantify different small nucleic acid molecules with extremely high specificity.

[0140] Experimental instrument: AB SCIEX 6500+

[0141] Chromatographic conditions:

[0142] Chromatographic column: reverse phase chromatography column (WatersACQUITYUPLC BEH C18 Column, 1.7μm, 2.1mm×50mm)

[0143] Mobile phase:

[0144] Phase A is an aqueous solution containing 0.2% HFIP + 0.1% TEA + 5 μM EDTA.

[0145] Phase B is a methanol solution containing 0.2% HFIP + 0.1% TEA + 5 μM EDTA.

[0146] The elution conditions are shown in Table 7.

[0147] Table 7 Elution program settings

[0148] Time A(％) B(％) Time 0.0 90 10 0.0 0.4 90 10 0.4 1.7 50 50 1.7 2.8 50 50 2.8 3.0 90 10 3.0 6.0 90 10 6.0

[0149] Flow rate: 0.4 mL / min

[0150] Column temperature: 50°C

[0151] Mass spectrometry conditions:

[0152] Ion source: Electrospray ionization (ESI) source, negative ion mode;

[0153] Scanning mode: Multiple reaction monitoring (MRM) mode, selecting characteristic ion pairs of miRNA1 and internal standard for monitoring.

[0154] Table 8 Mass spectrometry parameter settings

[0155]

[0156] Detection mode: Scan(-) or SIM(-)

[0157] Injection volume: 10 μL

[0158] Parameters such as capillary voltage, cone voltage, and collision energy are shown in Table 9.

[0159] Table 9 Parameter settings

[0160]

[0161]

[0162] Standard stock solution: Dissolve the synthesized miRNA1 standard in RNase-free water to prepare a 1000 ng / mL stock solution and store at -80°C.

[0163] Serial standard solutions: Prepare at least six standard solutions of different concentrations by serial dilution of the standard stock solution with RNase-free water: 1000 ng / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, and 10 ng / mL.

[0164] Internal standard solution: Prepare the internal standard solution at a concentration of 100 ng / mL in RNase-free water.

[0165] Establishment of standard curve:

[0166] Standard solutions of different concentrations were mixed with 100 ng / mL internal standard solution and then subjected to LC-MS / MS analysis, and the peak areas of miRNA1 and internal standard were recorded.

[0167] A standard curve was drawn with the ratio of the peak area of ​​the antisense strand of miRNA1 to the peak area of ​​the internal standard as the ordinate and the concentration of the standard solution to the concentration of the internal standard solution as the abscissa.

[0168] Using linear regression analysis, the regression equation and correlation coefficient (R 2 ), generally requires R 2 Greater than 0.99.

[0169] The results are as follows Figure 4 As shown, the LC-MS / MS standard curve is y=0.000892x+0.00101, with a correlation coefficient of 0.9951, indicating good linearity.

[0170] Example 5

[0171] Further screening of transdermal enhancers

[0172] The 10 transdermal enhancers screened in Example 3 were further screened using LC-MS / MS, as shown in Table 10.

[0173] Table 10 Types and concentrations of transdermal enhancers

[0174]

[0175] The transdermal agent is first prepared into a solution of corresponding concentration (volume concentration or mass concentration), and then used as a solvent for miRNA1 to prepare a small nucleic acid drug to be applied. The dosage for each mouse is 1.5 mg miRNA1 in a volume of 100 μL.

[0176] Eight-week-old C57 mice (purchased from Weitonglihua) were brought to the laboratory and adaptively fed for one week. There were five mice in each group. They were depilated with rosin paraffin 24 hours before the experiment to obtain a 2 cm × 2 cm hairless area. On the day of the experiment, each group of mice was smeared with the target small nucleic acid drug corresponding to different transdermal agents, 1.5 mg per mouse, and gently applied until the drug was absorbed. After 1 hour, 3 hours, and 6 hours, the dermal tissue was collected and stored in liquid nitrogen. The content of the target small nucleic acid drug in a fixed amount of dermal tissue of each mouse was detected by LC-MS / MS. Mice smeared with normal saline after depilation were used as the control group for data analysis.

[0177] In addition, an injection group was set up, and the dermal tissues of mice were collected and stored in liquid nitrogen 1 h, 3 h, and 6 h after the injection of 1.5 mg miRNA1 (dissolved in 100 μL saline) into the dermis of mice (5 mice at each time point).

[0178] The pre-treatment process of dermal tissue samples is as follows:

[0179] 1) Accurately weigh 30 mg of dermal sample into a 1.5 mL EP tube, add 20 μL of internal standard (100 ng / mL, prepared in methanol), and add 400 μL of methanol-water (V:V = 4:1);

[0180] 2) Add two small steel balls, pre-cool in a -20°C refrigerator for 2 minutes, and grind in a grinder (60 Hz, 2 minutes);

[0181] 3) Ultrasonic extraction in an ice-water bath for 10 min, followed by standing at -20°C for 30 min;

[0182] 4) Centrifuge for 10 min (13,000 rpm, 4°C), take 300 μL of the supernatant and transfer to an LC-MS injection vial for evaporation;

[0183] 5) Redissolve in 300 μL of methanol-water (V:V = 1:4), add 10 μL of internal standard (Lyso PC17:0, 0.1 mg / mL, prepared in methanol), vortex for 30 seconds, and sonicate for 3 minutes;

[0184] 6) Stand at -20°C for 2 hours;

[0185] 7) Centrifuge for 10 min (13,000 rpm, 4°C), and transfer 150 μL of the supernatant into an LC-MS injection vial with a foot-lined tube for analysis;

[0186] 8) Quality control samples (QC) were prepared by mixing equal volumes of extracts from all samples. The volume of QC was the same as that of the samples.

[0187] The experimental results are as follows Figure 5 and Figure 6As shown, the lipid-based solvent-based laurocapram (Group A) and DMI (Group B) showed high antisense strand concentrations at 1 and 3 hours, and detectable antisense strands at 6 hours, reaching approximately 3% of the miRNA1 expression level in the injected group. These two transdermal agents demonstrated superior transdermal penetration compared to the other transdermal agents. Bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate, decapeptide-4, inositol, and natural plant enhancers showed high antisense strand concentrations at 1 hour, but lower levels at 3 hours. With the exception of laurocapram and DMI, no antisense strands were detected in the dermis at 6 hours in the other groups. Taking all factors into consideration, laurocapram and DMI demonstrated the best transdermal performance. Furthermore, Group J (natural plant enhancer), a commercial transdermal agent combination containing tetrahydropiperine, inositol, and hydroxypropyl cyclodextrin, only achieved approximately half the levels of Groups A and B at 1 hour, indicating that these combinations did not synergistically enhance the transdermal penetration of small nucleic acids.

[0188] The content of these two agents in cosmetics cannot exceed 3%, so the two most effective transdermal agents were tested at different concentrations, namely 2.5%, 5%, and 10% of laurocapram and DMI for the transdermal effect of miRNA1. There were 10 mice in each group, and the mice were adaptively fed for 1 week. They were depilated with rosin paraffin 24 hours before the experiment to obtain a 2cm×2cm hairless area. On the day of the experiment, each group of mice was smeared with the target small nucleic acid drug corresponding to the different transdermal agents, 1.5 mg per mouse, and gently smeared until the drug was absorbed. After 1h, 3h, and 6h, the dermal tissue was taken and stored in liquid nitrogen. The tissue pretreatment was performed as described above, and the content of the target small nucleic acid drug in the dermal tissue of each mouse was detected by LC-MS / MS. The mice smeared with normal saline after depilation were used as the control group for data analysis. The experimental results are as follows: Figure 7 and Figure 8 As shown, in the azone group, compared with the concentrations of 2.5% and 10%, the 5% azone had the best transdermal effect. This may be because azone achieved efficient penetration enhancement at a relatively low concentration of 5% through a unique "lipid fluidization" mechanism, avoiding the irritation caused by high concentrations; and compared with the 2.5% and 5% DMI groups, the 10% DMI had the best transdermal effect. As a commonly used solvent in cosmetics, DMI at a high concentration of 10% can effectively destroy the lipid barrier of the stratum corneum while maintaining good biocompatibility.

[0189] Example 6

[0190] Screening of transdermal enhancer combinations

[0191] However, since the dosage of DMI and Azone in cosmetics cannot exceed 3%, a series of combination tests were conducted. The specific combination information is as follows:

[0192] Table 11 Combinations of transdermal enhancers

[0193] Group Transdermal combination solvent a Laurocapram (2.5%) + DMI (2.5%) Normal saline b Laurocapram (2.5) + Decapeptide-4 (10%) Normal saline c Laurocapram (2.5%) + Inositol (4%) Normal saline d DMI (2.5%) + decapeptide-4 (10%) Normal saline e DMI (2.5%) + Inositol (4%) Normal saline

[0194] Different combinations of transdermal agents were pre-prepared into solutions of corresponding concentrations (volume concentration or mass concentration), and then used as solvents for miRNA1 to prepare small nucleic acid drugs to be applied. The dosage for each mouse was 1.5 mg miRNA1 in a volume of 100 uL.

[0195] After 8-week-old C57 mice arrived at the laboratory, they were adaptively fed for 1 week, with 10 mice in each group. 24 hours before the experiment, they were depilated with rosin paraffin to obtain a 2 cm × 2 cm hairless area. On the day of the experiment, each group of mice was smeared with the target small nucleic acid drug corresponding to different transdermal agents, 1.5 mg per mouse, and gently applied until the drug was absorbed. After 1 hour, 3 hours, and 6 hours, the dermal tissue was collected and stored in liquid nitrogen. After tissue pretreatment as described in Example 5, the content of the target small nucleic acid drug in a fixed amount of dermal tissue of each mouse was detected by LC-MS / MS. Mice smeared with normal saline after depilation were used as the control group for data analysis.

[0196] The experimental results are shown in Figure 9. The five combinations of transdermal agents tested in the experiment all had good transdermal effects. The content of miRNA1 antisense chain could be detected in the skin tissue at 6 hours. Among them, group a of 2.5% lauryl azone + 2.5% DMI had the best transdermal effect on miRNA1. Among them, 2.5% DMI can effectively destroy the lipid barrier of the stratum corneum, and 2.5% lauryl azone plays a role in promoting penetration. Moreover, the two have a synergistic effect on enhancing the transdermal effect of miRNA1. In summary, it can be concluded that the transdermal effect is better when lipid solvent-based lauryl azone and DMI are added simultaneously.

[0197] Example 7

[0198] Application of transdermal agent combinations on other small nucleic acids

[0199] The best small nucleic acid transdermal agent combination screened out above, namely 2.5% laurocapram + 1.5% DMI, was extended to other small nucleic acid drugs. The sequences of FDA-approved RNAi therapeutic drugs were selected to test the transdermal absorption-promoting effect of this combination. The synthetic sequences of the selected RNAi therapeutic drugs are:

[0200] RNAi-1:

[0201] Chain of Justice:

[0202] 5'-CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm-L96-3'

[0203] Antisense strand:

[0204] 3'-UmsGmsGmUfCmUfUmUfCmUfCmAfCmAfGmAfGmUfAmGfAfsAfsUm-5'

[0205] RNAi-2:

[0206] Chain of Justice:

[0207] 5'-GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm-L96-3'

[0208] Antisense strand:

[0209] 3'-AmsCmsCmUmGmAmAmAfGmUfAmGmGmAmCfCfUmUfUmAmUmsAfsUm-5'

[0210] RNAi-3:

[0211] Chain of Justice:

[0212] 5'-CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmUm-L96-3'

[0213] Antisense strand:

[0214] 3'-AmsAmsGmAmUmCfUmGfGmAfCmAfAmAfAmCfGmAfAfAfAmsCfsAm-5'

[0215] RNAi-4:

[0216] Chain of Justice:

[0217] 5'-UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAAmGmAm-L96-3'

[0218] Antisense strand:

[0219] 3'-CmsUmsAmCmCmCmUmAfAmAfGmUmAmCmAfUmUmGfGmUmUmsCfsUm-5'

[0220] RNAi-5:

[0221] Chain of Justice:

[0222] 5'-AmsUmGfUmUfGmUmCfCfUfUfUmUfUmAfUmCfUmGmAmGmCmAmGmCm-Cm-AdemG-AdemA-AdemA-AdemA-GmGmCmUmGmCm-3'

[0223] Antisense strand:

[0224] 3'-GmsGmsUmAmCfAmAfCmAfGmGmAmAfAfAmAfGmAfGfsAfsCfsUm*-5'

[0225] Wherein: A, adenosine; AdemA, GalNAc amino sugar conjugated adenosine; AdemG, GalNAc amino sugar conjugated guanosine; Af, 2'-fluoroadenosine; Am, 2'-O-methyladenosine; C, cytidine; Cf, 2'-fluorocytidine; Cm, 2'-O-methylcytidine; dT, thymidine; G, guanosine; Gf, 2'-fluoroguanosine; Gm, 2'-O-methylguanosine; s, phosphorothioate; U, uridine; Uf, 2'-fluorouridine; Um, 2'-O-methyluridine; Um*, 2'-O-methyl-4'-O-((methoxy)phosphoryl)methyluridine.

[0226] A solution of 2.5% laurocapram + 1.5% DMI was prepared in advance and then used as a solvent for the above RNAi nucleic acid sequence to prepare a small nucleic acid drug to be applied. The dosage for each mouse was 1.5 mg in a volume of 100 μL.

[0227] After 8-week-old C57 mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) arrived at the laboratory, they were adaptively fed for 1 week, with 10 mice in each group. 24 hours before the experiment, they were depilated with rosin paraffin to obtain a 2 cm × 2 cm hairless area. On the day of the experiment, each group of mice was smeared with the target small nucleic acid drug corresponding to different transdermal agents, 1.5 mg per mouse, and gently applied until the drug was absorbed. After 1 hour, 3 hours, and 6 hours, the dermal tissue was collected and stored in liquid nitrogen. After tissue pretreatment as described in Example 5, the content of the target small nucleic acid drug in a fixed amount of dermal tissue of each mouse was detected by LC-MS / MS for data analysis.

[0228] The experimental results are as follows Figure 10 As shown, it can be seen that in the five groups of experiments, the antisense chain of the nucleic acid can be detected in the dermal tissues sampled at three time points, and as time goes by, the content of the antisense chain shows a trend of first increasing and then decreasing. The antisense chain content detected at 3 hours is the highest, which may be related to the time when different nucleic acid drugs take effect and their stability, indicating that the transdermal agent combination of 2.5% laurocapram + 2.5% DMI can also play a role in nucleic acid drugs of other sequences.

[0229] It can be seen from the above examples that the nucleic acid transdermal agent provided by the present invention has undergone dual screening by qPCR and LC-MS / MS to ensure that its transdermal absorption promotion effect is more reliable and can significantly promote the transdermal absorption of small nucleic acid drugs on the skin; especially the combination of 2.5% laurocapram + 2.5% DMI, which has a significant transdermal promotion effect and can be used as a transdermal agent in different small nucleic acid drugs.

[0230] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nucleic acid transdermal agent, characterized in that: Including one or more of bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate, isosorbide dimethyl ether DMI, dodecyl maltoside DDM, decapeptide-4, dimethyl sulfone, inositol, ethoxydiglycol, laurocapram, and polysorbate-40; When choosing one of these; The concentration (v / v) of bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate is 1 to 6%; The concentration of dimethyl isosorbide DMI is 1 to 12% (v / v); The concentration of dodecyl maltoside DDM is 1 to 12% (w / v); The concentration of decapeptide-4 is 1 to 12% (w / v); The concentration of dimethyl sulfone is 1 to 12% (w / v); The concentration of inositol is 1-5% (w / v); The concentration of ethoxydiglycol is 1 to 5% (v / v); The concentration of laurocapram is 1-12% (v / v); The concentration of polysorbate 40 is 1 to 6% (v / v).

2. The nucleic acid transdermal agent according to claim 1, wherein The invention comprises isosorbide dimethyl ether DMI and laurocapram; the concentration of the isosorbide dimethyl ether DMI is 1-12%, and the concentration of the laurocapram is 1-12%.

3. The nucleic acid transdermal agent according to claim 2, characterized in that The concentration of the isosorbide dimethyl ether DMI is 2-7%, and the concentration of the laurocapram is 2-7%.

4. Use of the nucleic acid transdermal agent according to any one of claims 1 to 3 in the preparation of nucleic acid transdermal drugs.

5. Use of the nucleic acid transdermal agent according to any one of claims 1 to 3 in the preparation of nucleic acid transdermal cosmetics.

6. A functional nucleic acid miRNA1, characterized in that The sequence is as follows: ss chain: mC*mU*mAmCmUmAfAmAfAfCfAmUmGmGmAmAmGmCmAmCmU mUmA-chol as chain: mU*fA*mAmGmUfGmCmUmUmCmCmAmUfGmUfUmUmUmAmGmU*mA*mG; Among them, s represents thio modification, m represents 2'-methoxyethoxy modification, chol represents cholesterol modification, and LNA represents locked nucleic acid.

7. A functional nucleic acid preparation, characterized in that: The invention comprises the miRNA1 according to claim 6 and the nucleic acid transdermal agent according to any one of claims 1 to 3.

8. The functional nucleic acid preparation according to claim 7, characterized in that The concentration of miRNA1 in the functional nucleic acid preparation is 10 ng / mL-20 mg / mL.

9. Use of the functional nucleic acid preparation according to claim 7 or 8 in the preparation of medicines or cosmetics.

10. The use according to claim 9, characterized in that The cosmetics are cosmetics with the effects of treating hair loss, anti-aging or whitening.

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