WO2014029143A1 - Plant extract-mediated drug transdermal introducing system and transdermal method thereof - Google Patents

Description

 Plant extract-mediated drug transdermal introduction system and transdermal method thereof

 Technical field

 The present invention relates to a plant extract-mediated drug transdermal delivery system and a transdermal method therefor. Background technique

 Abnormal pigmentation of the skin due to excessive and uneven secretion of melanin may cause spots or plaques on the face and back of the hand, such as: pregnancy spots, acne marks, birthmarks, moles, freckles, sunburn and age spots.

 Centuries ago, people used a variety of topical ointments to whiten the skin and eliminate pigmentation. In recent years, people have used hydroquinone (HQ), L-ascorbyl-2-phosphate magnesium (MAP), hydroquinone monomethyl ether, N-acetyl-4-S-cysteine, and arbutin. Chemicals to eliminate pigmentation, but these substances can cause side effects such as local discoloration, dermatitis, and even cancer and brownish yellow, forcing people to find alternatives (Parvez S et al, Phytother Res. 2006, 20 ( 11 ): 921-34).

 A transdermal absorption enhancer refers to a substance that promotes penetration of a drug through the stratum corneum and diffusion of the epidermis. Promoters are chemically classified into surfactants, lipid solvents, hydrophilic solvents, and macrocyclic compounds (Pfister W R et al, Pharm Tech, 1990, 14 (9): 132). At present, transdermal enhancers that have been researched and applied more include natural accelerators and synthetic accelerators (Fang Shiping, Chinese Journal of Hospital Pharmacy, 2000, 20(12): 750-752)

Natural accelerators include: 1) terpenoids. Such as mint (Menthol (Krishnaiah YS et al, Pharm DevTech nol, 2002, 7 (3): 305), menthol, peppermint oil), borneol (Zhu Jianping, Chinese Journal of Pharmaceutical Sciences, 1990, 34 (2): 104 ), Duchenene; 2) Volatile oil or essential oil, such as eucalyptus oil (Chen Hongqing, Progress in Pharmacy, 2000, 24(4): 235), Cinnamon oil (Shen Qi et al, Chinese Journal of Hospital Medicine, 2001, 21 (4) ): 197), Cnidium volatile oil, Fengxiang oil (Li Wei, Journal of Shanghai Medical University, 1998, 25(5): 365), turpentine (Li Juan et al., Journal of China Pharmaceutical University, 1999, 30(5): 343 ), galangal oil (Shen Qi et al, Chinese herbal medicine, 2000, 23 (11): 697), clove volatile oil (Zhao K et al, J Control Release, 1998, 55 (2-3): 253-260), Angelica volatile oil 3) Alkaloids, such as Fritillaria alkaloids (Ma Yunshu et al., Asia-Pacific Traditional Medicine, 2011 (4): 15-18); 4) Lactones, such as ligustilide in chuanxiong ether extract, _sen kyu _n0 lide, snake bed lactone, butenyl furosemide lactone, a new snake bed lactones have skin penetration, etc. (Tsuneo Namba, country Medical Chinese medicine volumes, 1993, 15 (1): 41).

 Natural penetration enhancers are natural plant extracts, have a good penetration-enhancing effect, and are less irritating, and have great value for development and utilization, and have become a hot spot in the research of penetration enhancers in recent years.

RN A interference (RNAi) technology is a natural harmful protein in the cell itself. The qualitative mechanism was discovered by American scientists Andrew Fire and Craig Mello, and won the Nobel Prize in Medicine in 2006. The mechanism of RNAi is that when normal cells are threatened or injured, small interfering RNA (siRNA) can trigger the gene silencing pathway, and the cells "silence" the harmful genes through the RNAi pathway. In this natural cellular defense process, double-stranded RNA (dsRNA) is digested with Dicer into 20-25 nucleotide siRNA, and siRNA is combined with RNA-induced silencing complex (RISC). Melting, one of the chains binds to the target gene mRNA, degrading the gene, so that the harmful target protein cannot be synthesized for therapeutic purposes.

 There has been no report that a plant extract is used as a transdermal agent to successfully deliver a macromolecular drug such as a nucleic acid to the dermis layer of the skin. For the first time, we have applied plant extracts such as angelica volatile oil as transdermal agents to the transdermal delivery system of biomacromolecules such as nucleic acids, and successfully enabled nucleic acid molecules to pass through the natural barrier of the skin, especially the stratum corneum, to the skin base. The bottom layer thus plays a therapeutic role. Summary of the invention

 The technical problem to be solved by the present invention is to provide a plant extract-mediated drug transdermal introduction system and a transdermal method, which enable biomacromolecules to pass through the skin's natural barrier (stratum corneum) to the basal layer of the skin to exert its therapeutic effect. effect.

 In order to achieve the above object, the present invention provides a plant extract-mediated drug transdermal delivery system, comprising: 1) a transdermal agent prepared from at least one plant extract; 2) a biomacromolecules Or its preparation.

 Preferably, the plant extract is a vegetable volatile oil.

 Further, the plant volatile oil is a mixture of any one or more of angelica volatile oil, eucalyptus oil, eucalyptus oil, clove oil, cinnamon oil, turpentine oil and citric oil.

 Preferably, the plant extract is a terpenoid.

 Further, the terpenoid is a mixture of any one or more of peppermint oil, borneol and menthol. Preferably, the transdermally-introducing agent comprises 0 to 98 parts by weight of jojoba oil, 1 to 100 parts by weight of angelica volatile oil,

0.2 to 6 parts by weight of rose essential oil, 0 to 5 parts by weight of lavender essential oil, 0 to 10 parts by weight of mastic oil, 0 to 4 parts by weight of tea tree oil and 0 to 0.5 parts by weight of peppermint essential oil.

 Preferably, the biomacromolecule is a nucleic acid molecule, a protein molecule, a polypeptide molecule or an insulin molecule. Further, the biomacromolecule is a nucleic acid molecule, and the biomacromolecular preparation is a nucleic acid preparation. Further, the nucleic acid preparation includes a nucleic acid molecule and a preparation compatible therewith.

Further, the preparation compatible with the nucleic acid molecule is a lipid or an alcohol. Further, the nucleic acid molecule is a mixture of functional RNA and any one or more of the structural forms or mimetics and DNA comprising the modification and the modified structural form or mimetic thereof.

 Further, the functional RNA is a mixture of any one or more of siRNA, miRNA, small ligand RNA (sliRNA) and RNA aptamer.

 Further, the nucleic acid preparation is a cosmetic emulsion comprising a nucleic acid molecule, comprising the following components in weight percent:

 Olivem 1000 2.5-4%;

 Oliwax IC 0.5-2%;

 Hexadecane 1- 6%

 Polydimethylsiloxane 垸 2- 4%

 Hydrogenated olive oil ethyl hexyl ester 1.5~8%;

 PEG-100 stearate 0.5~4%

 Stearic acid 0.5-3%;

 C15-19垸 3-6%;

 Hydrogenated polydecene 2-7%;

 Butylated hydroxytoluene 0.1-0.3%;

 Butanediol 5-7%;

 Glycerin 8.5~12%;

 Xanthan gum 0.2~0.35%;

 Hyaluronic acid 0.01~0.05%;

 EDTA-disodium 0.1-0.3%;

 siRNA 0.001-0.02%;

 Fragrance 0.1-0.5%;

Preservative 0~1% _;

 Ion-free water is added to 100%.

 Further, the nucleic acid preparation is a makeup containing a nucleic acid molecule

Components:

 Olivem 1000 3-4%;

 Oliwax IC 1-2%;

Stearic acid 1~3% _; Cetyl alcohol 1~6% _;

 PEG-100 stearate 2.5~3%;

Hydrogenated polydecene 2~7% _;

Caprylic/capric triglyceride 2~6% _;

 Avocado tree fruit fat 1~4%

 Isopropyl palmitate 2~4%

 Hydrogenated olive oil ethyl hexyl ester 1~7%

 Butylated hydroxytoluene 0.2~0.25%

 Butylene glycol 5~7%;

 Glycerin 8.5~12%;

 Xanthan gum 0.25~0.35%;

 Hyaluronic acid 0.01-0.05%;

 EDTA-disodium 0.1-0.3%;

 siRNA 0.001-0.02%;

 Fragrance 0.1-0.5%;

 Preservative 0-1%;

 Ion-free water is added to 100%.

 The invention also provides a plant extract-mediated drug transdermal method, characterized in that the above-mentioned plant extract-mediated drug transdermal introduction system is used, and the specific steps are as follows: 1) using the transdermal introduction first The agent is permeabilized, and then the biomacromolecule or a preparation thereof is transdermally; or 2) the biomacromolecule or a preparation thereof is uniformly mixed with the transdermal agent, and then a composite formulation is prepared to be transparent. skin.

 Preferably, the composite formulation in the method 2) comprises the following components in weight percent: Angelica essential oil 1-20%

 Olivem 1000 2.5-4%

 Oliwax IC 0.5-2%

 Hexadecane 1- 6%

 Polydimethylsiloxane 垸 2- 4%

 Hydrogenated olive oil ethyl hexyl ester 1.5-8%

 PEG-100 stearate 0.5-4%

Stearic acid 0.5-3% C15-19 alkane 3-6%;

Hydrogenated polydecene 2-7%;

Butylated hydroxytoluene 0.1-0.3%;

Butanediol 5-7%;

Glycerin 8.5-12%;

Xanthan gum 0.2-0.35%;

Hyaluronic acid 0.01-0.05%;

 EDTA-disodium 0.1-0.3%;

siRNA 0.001-0.02%;

Fragrance 0.1-0.5%;

Preservative 0-1%;

Ionized water to make up to 100%

Preferably, the composite formulation in the method 2) comprises the following components in weight percent: Angelica essential oil 1-20%;

Olivem 1000 3-4%;

Oliwax IC 1- 2%;

Stearic acid 1-3%;

Hexadeca octa alcohol 1- 6%;

PEG-100 stearate 2.5-3%;

Hydrogenated polydecene 2-7%;

Caprylic/capric triglyceride 2- 6%;

Avocado fruit jelly 1- 4%;

Isopropyl palmitate 2- 4%;

Hydrogenated olive oil ethyl hexyl ester 1-7%;

Butylated hydroxytoluene 0.2-0.25%;

Butanediol 5-7%;

Glycerin 8.5-12%;

Xanthan gum 0.25-0.35%;

Hyaluronic acid 0.01-0.05%;

EDTA-disodium 0.1-0.3%; siRNA 0.001-0.02%;

 Fragrance 0.1-0.5%;

 Preservative 0-1%;

 Ionized water to make up to 100% o

 The invention also provides the use of the above plant extract mediated drug transdermal delivery system for the preparation of a skin health product.

 Preferably, the skin health product is a cosmetic.

 Further, the cosmetic is a cosmetic having whitening and freckle function.

 Further, the cosmetic is a whitening and freckle makeup containing siRNA regulating the expression of a melanin-related gene.

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 The invention also provides the use of the above plant extract mediated drug transdermal method for the preparation of a skin health product. Preferably, the skin health product is a cosmetic.

 Further, the cosmetic is a cosmetic having whitening and freckle function.

 Further, the cosmetic is a whitening and freckle makeup containing siRNA regulating the expression of a melanin-related gene.

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 The plant extract-mediated drug transdermal introduction system and the transdermal method thereof provided by the invention can be applied to skin health products, such as cosmetics, so that biomacromolecules penetrate into the skin to achieve a better therapeutic effect. DRAWINGS

 Figure 1 is a transdermal photograph of a mouse;

 Figure 2 is a comparison diagram of the effect of transdermal delivery of different transdermal agents;

 Figure 3 is a comparison of the effects of different concentrations of Angelica sinensis oil and jojoba oil compound formula on nucleic acid transdermal; Figure 4 is a comparison of the effect of the composite formula transdermal agent combined with the nucleic acid emulsion formula I on the transdermal effect of nucleic acid; Comparison of the effects of transdermal delivery of the composite formula transdermal agent with the nucleic acid cream formulation I; Figure 6 is a comparison of the effect of the transdermal formulation of the composite formula with the nucleic acid emulsion formula II for transdermal delivery of nucleic acids; Comparison of the effects of transdermal drug introducer with nucleic acid cream formulation II for transdermal delivery of nucleic acid; Figure 8 is a comparison of the effects of different transdermal drug delivery agents combined with nucleic acid emulsion formulation I for transdermal delivery of nucleic acids;

Figure 9 is a comparison diagram of the effect of transdermal administration of a composite formulation (transdermal introducer + nucleic acid molecule / nucleic acid preparation). detailed description

 In order to make the present invention more apparent, the preferred embodiments are described in detail with reference to the accompanying drawings. Example 1

Animal skin penetration test

 Main instruments, reagents and materials

 1.1 ICR mice: Experimental Animal Center of Nantong University, 4 to 6 weeks old, weighing 18~20g, male and female random.

 1.2 Fluorescently labeled siRNA: Cy3-labeled siRNA (Cy3-siRNA) (Beaucom Biotech Co., Ltd.).

1.3 Instruments: Frozen slicer (Leica CM1900, Leica, USA); upright fluorescence microscope (X3, Olympus, Japan).

 1.4 Reagents: 10% (m/V) chloral hydrate (Zhongguo Group Chemical Reagent Co., Ltd.); Medical Glue (Guangzhou Baiyun Medical Adhesive Co., Ltd.); Sodium Chloride Injection (0.9%) (Shandong Corning Pharmaceutical Co., Ltd.) ; 0.1M PBS (Beauty Biotech Co., Ltd.); Angelica essential oil (Jiangxi Cedar Natural Pharmaceutical Oil Co., Ltd.); Azone (Aladdin Reagent (China) Co., Ltd.).

 1.5 Materials: lmL - secondary syringe, 5mL - secondary syringe (Jiangyan Xinsu Medical Devices Co., Ltd.), disposable cotton swab, 5mL centrifuge tube, ophthalmic scissors, medical gauze, etc.

 2. Experimental steps

 2.1 Preparation of transdermal agent

 2.1.1 Transdermal delivery agent mediated by Angelica essential oil

 2.1.2 The PBS solution was used as a transdermal agent as a control group.

 2.2 Pretreatment:

In the two groups of ICR mice, 8 (^L 10% chloral hydrate was injected into the peritoneal cavity for anesthesia. After the mice were completely anesthetized, the mice were placed flat on medical gauze, and the back of the mouse was cut with an eye clipping skin of about 1.5. _C mxl.5cm, can not cut the skin of the mouse. Cut the bottom of the centrifuge tube at 1cm along the 5mL centrifuge tube, take the end of the tube of the centrifuge tube, and repair the edge of the tube. Drain the lOuL medical glue and apply it to the 5mL centrifuge tube. At the edge, the tube was then pressed against the back skin of the mouse for 10 s, as shown in Figure 1.

2.3 Transdermal introducer to promote penetration:

 Use a cotton swab to draw the transdermal agent and evenly spread it on the skin of the mouse in the centrifuge tube, and continue to apply it with a cotton swab for lmin. After 10 min, repeat the application once and apply for 3 times.

After the third application, the interval is 10min. Wipe the area with the transdermal agent with a clean cotton swab to remove the skin. A residual transdermal introducer of the surface. A 5 mL centrifuge tube cap was capped, and 200 μM of the preparation was aspirated from a small hole of a 5 mL centrifuge tube cap with an ImL syringe to cover the mouse skin. The mice were placed in the dark place and observed every half an hour. Observe whether the centrifuge tube and the mouse skin are firmly adhered, whether there is leakage, and if there is leakage, re-stick the leak, make up the preparation, and record.

2.4 Take the skin observation:

 After 6 hours, the remaining preparation in the centrifuge tube was aspirated, placed in a new centrifuge tube, and stored at -80 °C. The centrifuge tube of the back of the mouse was removed, and the skin of the mouse was washed with physiological saline, and washed at least 10 times each time with 1 mL. The mice were sacrificed by the neck-breaking method, and the skin of the mice was carefully lifted with an ophthalmologist. The skin of the mice was cut along the lines of the centrifuge tube, and the water was removed and stored at -80 ° C until use.

2.5 Frozen slices:

 Frozen sections of skin tissue were prepared according to the method of the American Leica cryostat, and the skin was cut to a thickness of 5 μm.

2.6 Observe under a fluorescence microscope and take a picture. Example 2

Animal skin transdermal experiment

 Main instruments, reagents and materials

 1.1 ICR mice: Experimental Animal Center of Nantong University, 4 to 6 weeks old, weighing 18~20g, male and female random.

 1.2 Fluorescently labeled siRNA: Cy3-labeled siRNA (Cy3-siRNA) (Beaucom Biotech Co., Ltd.).

 1.3 Instruments: Frozen slicer (Leica CM1900, Leica, USA); upright fluorescence microscope (X3, Olympus, Japan).

 1.4 Reagents: 10% (m/V) chloral hydrate (Zhongguo Group Chemical Reagent Co., Ltd.); Medical Glue (Guangzhou Baiyun Medical Adhesive Co., Ltd.); Sodium Chloride Injection (0.9%) (Shandong Corning Pharmaceutical Co., Ltd.) ; 0.1M PBS (Beauty Biotech Co., Ltd.); Angelica essential oil (Jiangxi Cedar Natural Pharmaceutical Oil Co., Ltd.); Azone (Aladdin Reagent (China) Co., Ltd.).

 1.5 Materials: ImL - secondary syringe, 5mL - secondary syringe (Jiangyan Xinsu Medical Devices Co., Ltd.), disposable cotton swab, 5mL centrifuge tube, ophthalmic scissors, medical gauze, etc.

 2. Experimental steps

 2.1 Preparation of Angelica sinensis-mediated drug transdermal delivery system

2.1.1 Preparation method of transdermal enhancer is the same as step 2.1 in embodiment 1. 2.1.2 Dissolve 4 mg of siRNA in 10 mL of 0.1% PBS containing 25% glycerol and mix for later use.

2.2 transdermal

 The mice were randomly divided into 3 groups of 2 mice each:

 Table 1

 The procedure is the same as in steps 2.2-2.6 of Example 1.

 3. Experimental results

 As shown in Fig. 2, a comparison of the effect of transdermal delivery of nucleic acids for different transdermal agents, the white part of which is the distribution of Cy3 labeled siRNA in the cortex, wherein a and b are PBS negative control groups, c, d For the Angelica essential oil penetration group, e, f is the azone-promoting group. As can be seen from Fig. 2, the siRNA of the PBS negative control group was deposited on the surface of the skin, and the Angelica sinensis oil penetration group and the azone exposure group showed significant siRNA entry into the dermis layer. Example 3

Transdermal concentration-dependent experiment of transdermal drug molecule

 Main instruments, reagents and materials

 1.1 ICR mice: Experimental Animal Center of Nantong University, 4 to 6 weeks old, weighing 18~20g, male and female random.

 1.2 Fluorescently labeled siRNA: Cy3-labeled siRNA (Cy3-siRNA) (Baimax Biotech Co., Ltd.).

1.3 Instruments: Frozen slicer (Leica CM1900, Leica, USA); Upright fluorescence microscope (X3, Olympus, Japan).

 1.4 Reagents: 10% (m/V) chloral hydrate (Zhongguo Group Chemical Reagent Co., Ltd.); Sodium Chloride Injection (0.9%) (Shandong Corning Pharmaceutical Co., Ltd.); 0.1M PBS (Baimax Biotechnology) Ltd.); Angelica essential oil (Jiangxi Cedar Natural Pharmaceutical Oil Co., Ltd.); Jojoba Oil (淄博林森生物制品有限公司) and so on.

1.5 Materials: lmL - secondary syringe, 5mL - secondary syringe (Jiangyan Xinsu Medical Devices Co., Ltd.), disposable cotton swab, 5mL centrifuge tube, ophthalmic scissors, medical gauze, etc.

 2. Experimental steps

 2.1 Preparation of Angelica sinensis oil and Jojoba oil-mediated drug transdermal introduction system

2.1.1 The preparation method of the transdermal enhancer is the same as the step 2.1 in the first embodiment, except that the angelica essential oil and the jojoba oil are used. The mixture replaces Angelica essential oil.

 2.1.2 The preparation method of the nucleic acid preparation is the same as the step in the embodiment 2.1.2

2.2 transdermal

 The mice were randomly divided into 6 groups, 2 mice each, with jojoba oil as the base oil, and 10%, 20%, 50% and 100% of the essential oils of Angelica essential oil:

 Table 2

 The operation steps are the same as those in step 2 in Embodiment 2.

 3. Experimental results

 As shown in Fig. 3, the effect of nucleic acid transdermal coating for different concentrations of angelica essential oil and jojoba oil compound formulation is shown in Fig. 3. The white part of the figure shows the distribution of Cy3 labeled siRNA in the cortex. Among them, a and b are PBS negative control group, c and d are 100% jojoba oil to promote the control group, e, f is 10% angelica essential oil +90% jojoba oil penetration group, g, h is 20 % Angelica essential oil + 80% jojoba oil promoted through the group, i, j is 50% Angelica essential oil + 50% jojoba oil promoted through group, k, 1 is 100% Angelica essential oil to promote the group. As can be seen from Fig. 3, the siRNA of the PBS negative control group was deposited on the skin surface, and the 100% jojoba oil-permeation control group did not differ from the PBS negative control group, and the depth of siRNA entering the skin increased with the increase of the concentration of angelica essential oil. This indicates that the penetration of siRNA is concentration dependent on Angelica essential oil. Example 4

Transdermal test with compound transdermal agent combined with nucleic acid emulsion and cream preparation

Main instruments, reagents and materials

 1.1 ICR mice: Experimental Animal Center of Nantong University, 4 to 6 weeks old, weighing 18~20g, male and female random.

1.2 0.2 mg/mL fluorescently labeled siRNA cosmetic formula:

1.2.1 Nucleic acid emulsion I formula

table 3 Serial number component

Olivem 1000 (Cetearyl alcoholic acid)

1 2-5g ester, sorbitan olive oil)

 Oliwax IC (Cetyl Palmitate, Yamanashi

2 0.5g tancolipid, sorbitol olive oil)

3 hexadecanol (cetyl alcohol, stearyl alcohol) ig

4 polydimethylsiloxane oxime 2g oil phase

 5 hydrogenated olive oil ethyl hexyl ester 1.5g

6 PEG-100 stearate 0.5g

7 stearic acid 0.5g

8 C15-19垸 3g

9 hydrogenated polydecene 2g

10 butylated hydroxytoluene o.ig

11 Butanediol 5g

12 Glycerin 8.5g

13 xanthan gum 0.25g water phase

 14 hyaluronic acid 0.05g

15 EDTA disodium o.ig

16 ion-free water to make up to lOOg

17 siRNA 0.02g

18 Fragrance 0.5g

19 Preservative 0.2g Formula I

 Table 4

5 无离子水 补足至 100g

5 without ionized water to 100g

1.3 compound transdermal agent (%, by weight)

 table 5

 1.4 Instruments: Frozen slicer (Leica CM1900, Leica, USA); Upright fluorescence microscope (X3, Olympus, Japan).

 1.5 Reagents: 10% ( m/V ) chloral hydrate (Zhongguo Group Chemical Reagent Co., Ltd.); Sodium Chloride Injection (0.9%)

(Shandong Corning Pharmaceutical Co., Ltd.); 0.1M PBS (Bao Ao Mai Biotechnology Co., Ltd.) and so on.

1.6 Materials: lmL - secondary syringe, 5mL - secondary syringe (Jiangyan Xinsu Medical Devices Co., Ltd.), disposable cotton swab, 5mL centrifuge tube, ophthalmic scissors, medical gauze, etc.

 2. Experimental steps

 2.1 Preparation of Angelica sinensis oil and Jojoba oil-mediated drug transdermal introduction system

 2.1.1 The preparation method of the transdermal enhancer is the same as the step 2.1 in the first embodiment, except that the mixture of ingredients in 1.3 is used instead of the angelica essential oil.

2.1.2 Preparation of nucleic acid preparations

2.1.2.1 Preparation of nucleic acid emulsions I

The oil phase and the water phase in Table 3 were respectively heated to 85 °C, homogenized, and kept for 30 minutes _{; the} temperature was lowered to 75 °C, and the oil phase was added to the water phase under stirring, and stirred uniformly; rapid stirring, under vacuum Homogenize at 75 °C for 3 min _; cool to 45 °C. Add the components numbered 17, 18, 19 in Table 3, stir for 20 min, and cool to room temperature.

2.1.2.2 Preparation of Nucleic Acid Cream I

 The non-ionized water is heated to 85 ° C, the temperature is kept for 30 minutes, then cooled to 45 ° C, and the serial number is added to Table 4 under stirring.

3, 4 components, stir into a paste.

2.2 transdermal

 The mice were randomly divided into 8 groups, each treating 2 mice:

1 PBS溶液 核酸乳液配方 I Table 6

1 PBS solution nucleic acid emulsion formula I

 2 BW-01 Nucleic Acid Emulsion Formula I

 3 BW-02 Nucleic Acid Emulsion Formula I

 4 BW-03 Nucleic Acid Emulsion Formula I

 5 PBS solution Nucleic acid cream formula I

 6 BW-01 Nucleic Acid Cream Formula I

 7 BW-02 Nucleic Acid Cream Formula I

 8 BW-03 Nucleic Acid Cream Formula I

In each group of ICR mice, 8 (^L 10% chloral hydrate was intraperitoneally injected for anesthesia. After the mice were completely anesthetized, the mice were placed flat on medical gauze, and the back of the mouse was cut with an eye clipping skin of about 1.5 _C. Mxl.5cm, can not cut the skin of the mouse. Cut the bottom of the centrifuge tube at 1cm along the 5mL centrifuge tube, take the end of the tube of the centrifuge tube, and repair the edge of the tube. Drain the lOuL medical glue and apply it to the mouth of the 5mL centrifuge tube. Edge, then press the tube onto the back skin of the mouse for 10 s.

2.3 Transdermal introducer to promote penetration:

 Use a cotton swab to draw the transdermal agent and evenly spread it on the skin of the mouse in the centrifuge tube, and continue to apply it with a cotton swab for lmin. After 10 min, repeat the application once and apply for 3 times.

 After the third application, the interval was 10 min. Wipe the area where the transdermal agent was applied with a clean cotton swab to remove the residual transdermal agent on the skin surface. Apply a 0.2 mg/ml fluorescently labeled siRNA emulsion to the back skin with a clean cotton swab and apply evenly. The mice were then placed in the dark.

2.4 Take the skin observation:

 After 6 hours, the remaining preparation in the centrifuge tube was aspirated, placed in a new centrifuge tube, and stored at -80 °C. The centrifuge tube of the back of the mouse was removed, and the skin of the mouse was washed with physiological saline, and washed at least 10 times each time with 1 mL. The mice were sacrificed by the neck-breaking method, and the skin of the mice was carefully lifted with an ophthalmologist. The skin of the mice was cut along the lines of the centrifuge tube, and the water was removed and stored at -80 ° C until use.

2.5 Frozen slices:

 Frozen sections of skin tissue were prepared according to the method of the American Leica cryostat, and the skin was cut to a thickness of 5 μm.

2.6 Observe under a fluorescence microscope and take a picture.

3. Experimental results

Figure 4 is a comparison diagram of the effects of a compound transdermal agent combined with a nucleic acid emulsion I for transdermal delivery of nucleic acids, wherein a and b are In the PBS negative control group, cd was the BW-01 penetration group, ef was the BW-02 penetration group, and gh was the BW-03 penetration group. Figure 5 is a comparison diagram of the effects of the compound transdermal agent combined with the nucleic acid cream I for transdermal nucleic acid, wherein ab is

In the PBS negative control group, c d was the BW-01 penetration-promoting group, e f was the BW-02 penetration-promoting group, and g h was the BW-03 penetration-promoting group.

 As can be seen from Fig. 4-5, the white part of the figure shows the distribution of Cy3-labeled siRNA in the cortex, and the compound transdermal introducers BW-01 BW-02 and BW-03 can effectively promote the absorption of siRNA. Among them, the nucleic acid emulsion I promotes the transdermal penetration depth of the nucleic acid is superior to the nucleic acid cream I. Example 5

Transdermal test with compound transdermal agent combined with nucleic acid emulsion and cream preparation

Main instruments, reagents and materials

 1.1 ICR mice: Experimental Animal Center of Nantong University, 4 6 weeks old, weighing 18 20g, male and female random.

1.2 0.2 mg/mL fluorescently labeled siRNA cosmetic formula:

1.2.1 Nucleic acid emulsion I formulation, same as Table 3 in Example 4

1.2.2 Nucleic Acid Cream II Formulation

 Table 7

12 丁二醇 5g

12 Butanediol 5g

 13 Glycerin 8.5g

 14 xanthan gum 0.25g water phase

 15 hyaluronic acid 0.05g

 16 EDTA disodium O. lg

 17 ion-free water to make up to lOOg

18 siRNA 0.02g

 19 Fragrance 0.5g

 20 preservative 0.2g

 1.3 compound transdermal agent (%, by weight)

 Table 8

 1.4 Instruments: Frozen slicer (Leica CM1900, Leica, USA); Upright fluorescence microscope (X3, Olympus, Japan).

 1.5 Reagents: 10% ( m/V ) chloral hydrate (Zhongguo Group Chemical Reagent Co., Ltd.); Sodium Chloride Injection (0.9%)

(Shandong Kangning Pharmaceutical Co., Ltd.); 0.1M PBS (Baimax Biotech Co., Ltd.).

 1.6 Materials: lmL - secondary syringe, 5mL - secondary syringe (Jiangyan Xinsu Medical Devices Co., Ltd.), disposable cotton swab, 5mL centrifuge tube, ophthalmic scissors, medical gauze, etc.

 2. Experimental steps

 2.1 Preparation of Angelica sinensis oil and Jojoba oil-mediated drug transdermal introduction system

 2.1.1 The preparation method of the transdermal enhancer is the same as the step 2.1 in the first embodiment, except that the mixture of ingredients in 1.3 is used instead of the angelica essential oil.

 2.1.2 Preparation of nucleic acid preparations

 2.1.2.1 Preparation of nucleic acid emulsions I

 The preparation method is the same as the step in the embodiment 4. 2.1.2.1

 2.1.2.2 Preparation of Nucleic Acid Cream II

Do not heat the oil and water phases in Table 7 to 85 °C, and homogenize, keep warm for 30 min; cool down to 75 °C, stir. In the state, add the oil phase to the water phase and stir evenly; stir rapidly, homogenize at 75 ° C for 3 min under vacuum _; cool to 45 ° C and add the components numbered 18, 19, 20 in Table 7, stir for 20 min. , cool to room temperature.

2.2 transdermal

 The mice were randomly divided into 8 groups, and 2 mice were treated in each group:

 Table 9

 The operation steps are the same as those in step 2 in Embodiment 2.

3. Experimental results

 Fig. 6 is a comparison diagram of the effects of a compound transdermal agent combined with a nucleic acid emulsion I for transdermal nucleic acid, wherein a and b are PBS negative control groups, c and d are BW-04 penetration groups, and e and f are BW-05. Promote the group, g, h is the BW-06 promoting group.

 7 is a comparative diagram of the effect of a compound transdermal agent combined with a nucleic acid cream II for transdermal nucleic acid, wherein a and b are PBS negative control groups, c and d are BW-04 promoting groups, and e and f are BW- 05 promoted the group, g, h for the BW-06 promoting group.

 As can be seen from Fig. 6-7, the white part of the figure shows the distribution of Cy3-labeled siRNA in the cortex. The compound transdermal agents BW-04, BW-05, and BW-06 can effectively promote the absorption of siRNA. Among them, the nucleic acid emulsion formula I promotes the transdermal depth of the nucleic acid to be superior to the nucleic acid cream formulation. Example 6

Transdermal experiment of animals with different transdermal agents

Main instruments, reagents and materials

 1.1 ICR mice: Experimental Animal Center of Nantong University, 4 to 6 weeks old, weighing 18~20g, male and female random.

 1.2 0.2 mg/mL fluorescently labeled siRNA emulsion: Nucleic acid emulsion formulation I (same as Example 11).

1.3 Instruments: Frozen slicer (Leica CM1900, Leica, USA); Upright fluorescence microscope (X3, Japan) Olympus company) and so on.

 1.4 Reagents: 10% (m/V) chloral hydrate (Zhongguo Group Chemical Reagent Co., Ltd.); Sodium Chloride Injection (0.9%)

(Shandong Corning Pharmaceutical Co., Ltd.); 0.1M PBS (Bao Ao Mai Biotechnology Co., Ltd.) and so on.

1.5 transdermal introducer: eucalyptus oil, eucalyptus oil, clove oil, cinnamon oil, turpentine, peppermint oil, citric acid oil, angelica essential oil, borneol (5% borneol dissolved in 75% ethanol), menthol (5% Menthol is dissolved in 75% ethanol) (Jiangxi Cedar Natural Pharmaceutical Oil Co., Ltd.).

 1.6 Materials: lmL - secondary syringe, 5mL - secondary syringe (Jiangyan Xinsu Medical Devices Co., Ltd.), disposable cotton swab, 5mL centrifuge tube, ophthalmic scissors, medical gauze, etc.

 2. Experimental steps

2.1 Pretreatment:

ICR mice were intraperitoneally injected with 8 (^L 10% chloral hydrate for anesthesia. After the mice were completely anesthetized, the mice were placed flat on medical gauze, and the back of the mouse was cut with an ophthalmic clipping skin of about 1.5 _C mxl .5 cm. Can not cut the skin of mice. 2.2 Transdermal introduction agent to promote penetration:

 Use a cotton swab to draw the introduction agent (and its control PBS solution) and evenly spread it on the skin of the mouse in the centrifuge tube, and continue to apply lrnin with a cotton swab. Repeat the application once every 10 minutes and apply it for 3 times.

2.3 Transdermal:

 After the third application, the interval was 10 min. Wipe the area where the introduction agent (and its control PBS solution) was applied with a clean cotton swab to remove the residual introduction agent (and its control PBS solution) on the skin surface. Apply a 0.2 mg/ml fluorescently labeled siRNA emulsion to the back skin with a clean cotton swab and apply evenly. The mice were then placed in the dark.

2.4 Take the skin observation:

 After 6 hours of transdermal administration, the remaining nucleic acid preparation in the centrifuge tube was aspirated, placed in a new centrifuge tube, and stored at -80 °C. The centrifuge tube on the back of the mouse was removed, and the skin of the mouse was washed with physiological saline, and washed at least 10 times each time with 1 mL. The mice were sacrificed by cervical dislocation. The skin of the mice was carefully lifted with ophthalmology. The skin of the mice was cut along the lines of the centrifuge tube, and the water was removed and stored at -80 °C until use.

2.5 Frozen slices:

 Frozen sections of skin tissue were prepared according to the method of the American Leica cryostat, and the skin was cut to a thickness of 5 μm.

2.6 Observe under a fluorescence microscope and take a picture.

 3. Experimental results

As shown in FIG. 8, the effect of transfecting nucleic acid emulsions with different transdermal agent-incorporating nucleic acid emulsion formula I is shown in FIG. The middle white part is the distribution of Cy3 labeled siRNA in the cortex, wherein ab is the eucalyptus oil promoting group, cd is the eucalyptus oil promoting group, ef is the clove oil promoting group, gh is the cinnamon oil promoting group, ij For the turpentine oil-promoting group, k 1 is the peppermint oil promoting group, mn is the citric oil-promoting group, op is the angelica essential oil promoting group, qr is the borneol promoting group, st is the menthol promoting group, u V is PBS negative control group. It can be seen from Fig. 8 that the compound transdermal agent can effectively promote the absorption of siRNA, and all of the ten transdermal agents have a transdermal effect, wherein Angelica sinensis, eucalyptus oil, and peppermint oil have better permeation effect. Example 7

Compound formula (transdermal agent + nucleic acid molecule / nucleic acid preparation) transdermal test

Main instruments, reagents and materials

 1.1 ICR mice: Experimental Animal Center of Nantong University, 4 6 weeks old, weighing 18 20g, male and female random.

1.2 0.2 mg/mL fluorescently labeled siRNA cosmetic formula:

1.2.1 Nucleic acid emulsion compound formula

 Table 10

13 甘油 8.5g

13 Glycerin 8.5g

14 xanthan gum 0.25g

15 hyaluronic acid 0.05g

16 EDTA disodium O.lg

17 ion-free water to make up to lOOg

18 siRNA 0.02g

19 Angelica essential oil 2g

20 Preservative 0.2g Nucleic Acid Cream Complex Formula

No. Component

Olivem 1000 (cete stearyl oleate, sorbitan

1 3g olive oil ester)

 Oliwax IC (Cetyl Palmitate, Yamanite Palmitate,

2 ig sorbitol olive oil)

 3 stearic acid ig

4 cetostearyl alcohol (cetyl alcohol, stearyl alcohol) 3g oil phase 5 PEG-100 stearate 2-5g

 6 hydrogenated polydecene 2g

7 octanoic acid / citrate triglyceride 2g

8 Avocado Tree (BUTYROSPERMUM PARKII) Fruit Fat ig

9 isopropyl palmitate 2g

10 hydrogenated olive oil ethyl hexyl ester ig

11 Butylated hydroxytoluene 0.2g

12 Butanediol 5g

13 Glycerin 8.5g Water phase

 14 xanthan gum 0.25g

15 hyaluronic acid 0.05g 16 EDTA disodium o. ig

 17 no ionized water, make up to 100g

18 siRNA 0.02g

 19 Angelica essential oil 2g

 20 preservative 0.2g

 1.4 Instruments: Frozen slicer (Leica CM1900, Leica, USA); Upright fluorescence microscope (X3, Olympus, Japan).

 1.5 Reagents: 10% ( m/V ) chloral hydrate (Zhongguo Group Chemical Reagent Co., Ltd.); Sodium Chloride Injection (0.9%)

(Shandong Kangning Pharmaceutical Co., Ltd.); 0.1M PBS (Baimax Biotech Co., Ltd.).

1.6 Materials: lmL - secondary syringe, 5mL - secondary syringe (Jiangyan Xinsu Medical Devices Co., Ltd.), disposable cotton swab, 5mL centrifuge tube, ophthalmic scissors, medical gauze, etc.

2. Experimental steps

 The mice were randomly divided into 4 groups of 3 mice each:

 Table 12

 2.1 Preparation of composite formula transdermal introduction system

2.1.1 Preparation of compound nucleic acid emulsion

The oil phase and the water phase in Table 10 were respectively heated to 85 °C, and homogenized, and kept for 30 minutes _{; the} temperature was lowered to 75 °C, and the oil phase was added to the water phase under stirring, and stirred uniformly; rapid stirring, under vacuum Homogenize at 75 °C for 3 min _; cool to 45 °C. Add the components numbered 18, 19, 20 in Table 10, stir for 20 min, and cool to room temperature.

2.1.2 Preparation of compound nucleic acid cream

The oil phase and the water phase in Table 11 were respectively heated to 85 ° C, and homogenized, kept for 30 min _{; the} temperature was lowered to 75 V, and the oil phase was added to the water phase under stirring, and stirred uniformly; rapid stirring, under vacuum 75 °C was homogenized for 3 min _; cooled to 45 °C. The components numbered 18, 19, 20 in Table 11 were added, stirred for 20 min, and cooled to room temperature.

2.1.3 Preparation of nucleic acid emulsions I

The preparation method is the same as step 2.1.2.1 in the fourth embodiment. 2.1.4 Preparation of nucleic acid emulsionΠ

 The nucleic acid emulsion prepared in Example 5 was used.

2.2 Pretreatment:

ICR mice were intraperitoneally injected with 8 (^L 10% chloral hydrate for anesthesia. After the mice were completely anesthetized, the mice were placed flat on medical gauze, and the back of the mouse was cut with an eye clipping skin of about 1.5 _C mxl.5 cm. Can not cut the skin of mice.

2.3 Transdermal:

 Apply a proper amount of the nucleic acid-containing emulsion compound formula and the nucleic acid cream composite formulation to the back skin with a clean cotton swab, and apply evenly, while using the nucleic acid emulsion formula I (same as in Example 11) and the nucleic acid emulsion formula II (same as in the example 12) as a negative control. Operate as described above. The mice were placed in the dark after treatment.

2.4 Take the skin observation:

 After 6 hours of transdermal application, the remaining preparation in the centrifuge tube was aspirated, placed in a new centrifuge tube, and stored frozen at -80 °C. The centrifuge tube of the back of the mouse was removed, and the skin of the mouse was washed with physiological saline, and washed at least 10 times each time with 1 mL. The mice were sacrificed by cervical dislocation, and the skin of the mice was carefully lifted with an ophthalmology. The skin of the mice was cut along the lines of the centrifuge tube, and the water was removed and stored at -80 ° C until use.

2.5 Frozen slices:

 Frozen sections of skin tissue were prepared according to the method of the American Leica cryostat, and the skin was cut to a thickness of 5 μm.

2.6 Observe under a fluorescence microscope and take a picture.

3. Experimental results

 As shown in Fig. 9, a comparison of the effects of transdermal administration of a composite formulation (transdermal introducer + nucleic acid molecule/nucleic acid preparation), the white part of which is the distribution of Cy3-labeled siRNA in the cortex, wherein a, b are nucleic acids In the emulsion compound formula group, c and d are the nucleic acid cream compound formula group, e and f are the nucleic acid emulsion formula I control group, and g and h are the nucleic acid emulsion formula II control group. As can be seen from Fig. 9, both the nucleic acid emulsion composite formulation and the nucleic acid cream composite formulation are effective for transdermal siRNA.

Claims

Rights request:  A plant extract-mediated drug transdermal introduction system, comprising: 1) a transdermal agent prepared from at least one plant extract; 2) a biomacromolecule or a preparation thereof.  The plant extract-mediated drug transdermal delivery system according to claim 1, wherein the plant extract is a plant volatile oil.  The plant extract-mediated drug transdermal introduction system according to claim 2, wherein the plant volatile oil is angelica volatile oil, eucalyptus oil, eucalyptus oil, clove oil, cinnamon oil, turpentine oil and sim Any one or a mixture of fine oils.  The plant extract-mediated drug transdermal delivery system according to claim 1, wherein the plant extract is an anthraquinone compound.  The plant extract-mediated drug transdermal delivery system according to claim 4, wherein the terpenoid compound is a mixture of any one or more of peppermint oil, borneol and menthol. The plant extract-mediated drug transdermal introduction system according to claim 1, wherein the transdermal agent is composed of 0 to 98 parts by weight of jojoba oil and 1 to 100 parts by weight. Angelica volatile oil, 0.2~6 parts by weight of rose essential oil, 0~5 parts by weight of lavender essential oil, 0~10 parts by weight of frankincense oil, 0~4 parts by weight of tea tree oil and 0~0.5 parts by weight of peppermint essential oil.  The plant extract-mediated drug transdermal delivery system according to claim 1, wherein the biomacromolecule is a nucleic acid molecule, a protein molecule, a polypeptide molecule or an insulin molecule.  The plant extract-mediated drug transdermal introduction system according to claim 7, wherein the biomacromolecule is a nucleic acid molecule, and the biomacromolecular preparation is a nucleic acid preparation.  The plant extract-mediated drug transdermal delivery system according to claim 8, wherein the nucleic acid preparation comprises a nucleic acid molecule and a preparation compatible therewith.  The plant extract-mediated drug transdermal introduction system according to claim 9, wherein the preparation compatible with the nucleic acid molecule is a lipid or an alcohol.  The plant extract-mediated drug transdermal introduction system according to any one of claims 7 to 10, wherein the nucleic acid molecule is a functional RNA and a modified structural form or mimetic thereof And a mixture of DNA and any one or more of the structural forms or mimetics thereof. The plant extract-mediated drug transdermal introduction system according to claim 11, wherein the functional RNA is any one of siRNA, miRNA, small ligand RNA, and RNA aptamer. Or a mixture of several. 13. The plant extract mediated drug transdermal delivery system according to claim 8, wherein the nucleic acid preparation is a cosmetic emulsion comprising a nucleic acid molecule, comprising the following components by weight: Olivem 1000 2.5-4%; Oliwax IC 0.5-2%; Hexadecanol 1~6% Polydimethylsiloxane 垸 2~4% Hydrogenated olive oil ethyl hexyl ester 1.5~8% _; PEG-100 stearate 0.5~4% Stearic acid 0.5-3% C15-19垸 3-6% Hydrogenated polydecene 2-7% Butylated hydroxytoluene 0.1-0.3% Butanediol 5-7%; Glycerin 8.5-12%; Xanthan gum 0.2-0.35%; Hyaluronic acid 0.01-0.05%; EDTA-disodium 0.1-0.3%; siRNA 0.001-0.02%; Fragrance 0.1-0.5%; Preservative 0~1% _; Ion-free water is added to 100%.  14. The plant extract mediated drug transdermal delivery system according to claim 8, wherein the nucleic acid preparation is a cosmetic cream comprising a nucleic acid molecule, comprising the following components by weight: Olivem 1000 3-4%; Oliwax IC 1-2%; Stearic acid 1~3% _; Cetyl alcohol 1~6% _; PEG-100 stearate 2.5~3%; Hydrogenated polydecene 2~7% _; Caprylic/capric triglyceride 2~6% _; Avocado tree fruit fat 1~4% Isopropyl palmitate 2~4% Hydrogenated olive oil ethyl hexyl ester 1~7% Butylated hydroxytoluene 0.2~0.25%; Butylene glycol 5~7%; Glycerin 8.5~12%; Xanthan gum 0.25~0.35%; Hyaluronic acid 0.01~0.05%; EDTA-disodium 0.1-0.3%; siRNA 0.001-0.02%; Fragrance 0.1-0.5%; Preservative 0~1% _; Ion-free water is added to 100%.  A plant extract-mediated drug transdermal method, characterized by comprising the plant extract-mediated drug transdermal introduction system according to any one of claims 1 to 14, wherein the specific steps are: 1) First, transdermally transducing with the transdermal agent, and then transfecting the biomacromolecule or a preparation thereof; or 2) mixing the biomacromolecule or a preparation thereof with the transdermal agent, In a compound formulation, it is transdermal.  16. The plant extract mediated drug transdermal method according to claim 15, wherein the composite formulation in the method 2) comprises the following components in weight percent: Angelica essential oil 1~20% Olivem 1000 2.5-4% Oliwax IC 0.5-2% Hexadecanol 1~6% Polydimethylsiloxane 垸 2~4% Hydrogenated olive oil ethyl hexyl ester 1.5~8% PEG-100 stearate 0.5~4% Stearic acid 0.5~3% C15-19院 3-6% Hydrogenated polydecene 2~7% _; Butylated hydroxytoluene 0.1-0.3%; Butanediol 5-7%; Glycerin 8.5-12%; Xanthan gum 0.2-0.35%; Hyaluronic acid 0.01-0.05%;  EDTA-disodium 0.1-0.3%; siRNA 0.001-0.02%; Fragrance 0.1-0.5%; Preservative 0-1%; Ion-free water is added to 100%.  The plant extract-mediated drug transdermal method according to claim 15, wherein the method comprises 10-20% essential oil of Angelica sinensis;  Olivem 1000 3-4%;  Oliwax IC 1-2%; Stearic acid 1-3%; Hexadecane alcohol 1-6%;  PEG-100 stearate 2.5-3%; Hydrogenated polydecene 2-7%; Caprylic/capric triglyceride 2-6%; Avocado tree fruit fat 1-4%; Isopropyl palmitate 2-4%; Hydrogenated olive oil ethyl hexyl ester 1-7%; Butylated hydroxytoluene 0.2-0.25%; Butanediol 5-7%; Glycerin 8.5-12%; Xanthan gum 0.25-0.35%; Hyaluronic acid 0.01-0.05%;  EDTA-disodium 0.1-0.3%; siRNA 0.001-0.02%; Fragrance 0.1-0.5%; Preservative 0~1% _; Ion-free water is added to 100%.  18. Use of a plant extract mediated drug transdermal delivery system according to any of claims 1-14 for the preparation of a skin health product.  19. Use of a plant extract mediated drug transdermal delivery system according to claim 18 for the preparation of a skin health product, characterized in that the skin health product is a cosmetic.  20. Use of a plant extract mediated drug transdermal delivery system according to claim 19 for the preparation of a skin health product, characterized in that the cosmetic is a cosmetic having whitening and freckle function.  The use of a plant extract-mediated drug transdermal delivery system according to claim 20 for the preparation of a skin health product, characterized in that the cosmetic is a whitening and freckle cosmetic containing siRNA regulating melanin-related gene expression. .  22. Use of a plant extract mediated drug transdermal method according to any one of claims 15 to 17 for the preparation of a skin health product.  23. Use of a plant extract mediated drug transdermal method according to claim 22 for the preparation of a skin health product, characterized in that the skin health product is a cosmetic.  24. The use of a plant extract mediated drug transdermal method according to claim 23 for the preparation of a skin health product, characterized in that the cosmetic is a cosmetic having whitening and freckle function.  The use of a plant extract-mediated drug transdermal method according to claim 24 for the preparation of a skin health product, characterized in that the cosmetic is a whitening and freckle cosmetic containing siRNA which regulates expression of a melanin-related gene.