Transdermal absorption enhancer, its preparation method and application in cosmetics

Abstract

The present application relates to biopharmaceutical manufacturing, more particularly to the field of penetration enhancers, and specifically discloses a transdermal absorption penetration enhancer, a preparation method thereof and application in cosmetics. The penetration enhancer comprises plant exosomes, natural penetration activity molecules, film forming agents, ZIF-90 nanomaterials, temperature / ROS dual response ion channel regulating molecules and skin symbiotic bacteria activating materials. The preparation method comprises the steps of plant exosome extraction, hydrophobic / hydrophilic active molecule encapsulation, ZIF-90 nanoshell growth, ion channel regulating molecule anchoring, introduction of skin symbiotic bacteria activating materials, addition of film forming agents, ultrafiltration centrifugal concentration and sterilization filling and the like. Through the triple synergy of plant exosome-ZIF-90 nanocore-shell structure, temperature / ROS dual response regulation and skin symbiotic bacteria activation, the present application realizes efficient, low irritation and intelligent controllable transdermal delivery of active substances, and is suitable for various cosmetic dosage forms.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine manufacturing, more particularly to the field of penetration enhancers, and in particular to a transdermal absorption penetration enhancer, a preparation method thereof and an application thereof in cosmetics. BACKGROUND

[0002] Penetration enhancers, also known as transdermal agents, are substances that can improve the efficiency of drug and active ingredient penetration through the skin by changing the structure of the stratum corneum or regulating metabolic mechanisms, and are widely used in the fields of medicine and cosmetics. When applying cosmetics, the stratum corneum pathway is the main pathway for drug transdermal absorption, but the dense "brick wall" structure of the stratum corneum composed of keratinocytes and intercellular substance is the main barrier to drug transdermal absorption. This structure has strong repulsive force for hydrophilic and macromolecular active substances, resulting in the retention of most active ingredients on the surface of the skin, with a bioavailability of less than 5%.

[0003] Penetration enhancers are commonly used substances to promote the effective penetration of active ingredients in cosmetics through the stratum corneum. Common penetration enhancement techniques include chemical penetration enhancement, physical penetration enhancement, and nanovesicle penetration enhancement. Chemical penetration enhancers, such as azone, DMSO, and ethanol, typically open the barrier by "extracting lipids" or "denaturing proteins", but are associated with erythema, burning, and long-term damage to the barrier. Physical penetration enhancers, such as microneedles, ultrasound, and iontophoresis, require specialized equipment, are costly, and are difficult to use daily. They also cause pain and local inflammation. In nanovesicle penetration enhancement, liposomes and delivery vehicles can improve penetration, but high concentrations of surfactants damage stratum corneum lipids, and the encapsulation efficiency of hydrophilic macromolecules is less than 30%. Moreover, excessive penetration is often associated with an imbalance in symbiotic bacteria, a decrease in C. acnes skin probiotics, and an increase in pathogenic bacteria, leading to a vicious cycle of "sensitivity-inflammation". Therefore, there is an urgent need for a penetration enhancer that is "highly efficient, lowly irritating, and friendly to the microecosystem". SUMMARY

[0004] The present application aims to provide a transdermal absorption penetration enhancer, a preparation method thereof, and an application thereof in cosmetics, to solve the problems raised in the background art.

[0005] To achieve the above-mentioned purpose, in one aspect, the present application provides a transdermal absorption penetration enhancer, which comprises, by mass fraction:

[0006] Plant exosomes: 10-50 parts; Plant exosomes are a natural nanoscale vesicle with a particle size usually between 30-150 nm. As a core carrier, they provide a stable nanoscale structure, can efficiently encapsulate hydrophobic and hydrophilic active molecules, and have good biocompatibility and low immunogenicity, making them suitable for transdermal absorption. Their nanoscale size allows them to effectively penetrate the stratum corneum of the skin, achieving efficient transdermal absorption.

[0007] Natural penetration enhancers: 5-10 parts of hydrophobic penetration enhancers and 5-10 parts of hydrophilic penetration enhancers; hydrophobic penetration enhancers insert into the lipid layer, disrupt the ordered arrangement, increase the lipid fluidity, and create a path for hydrophobic drugs (such as tretinoin and lipid-soluble antioxidants); hydrophilic penetration enhancers enhance the water content between keratinocytes, expand the hydrophilic microchannels, and promote the penetration of hydrophilic drugs (such as peptides and vitamin C derivatives) through the intercellular pathway. The dual-channel action significantly reduces the transmembrane resistance and improves the transdermal efficiency of various polar drugs.

[0008] Film-forming agent: 0.1-5 parts; forms a breathable microfilm on the skin surface, reduces trans-epidermal water loss (TEWL) by 20-30%, reduces friction irritation, and improves the moisturizing properties and comfort of the skin.

[0009] ZIF-90 nanomaterial: 5-20 parts, coated on the outer surface of the plant exosome to form a nanoshell; provides additional stability and mechanical strength, with a pore size ≥11 Å, allowing active ingredients of medium molecular weight to enter and exit, while being degradable in a weakly acidic environment (pH 5.4-5.9) to release the drug. ZIF-90 nanoshell ZIF-90 nanoshell is a metal-organic framework (MOF) material that can improve the mechanical strength of the exosome and enhance its stability during transdermal penetration.

[0010] Temperature / ROS dual-responsive ion channel regulating molecules: 0.1-2 parts, anchored on the surface of the nanoshell;

[0011] Skin symbiotic bacteria activating material: 5-10 parts; promotes the growth of skin beneficial bacteria, reduces inflammatory response, and improves skin microecological balance.

[0012] The ion channel regulating molecules have a conformational flip half-life t1 / 2 <1s at T≥34℃ or ROS≥80μM, instantaneously close the TRPV1 channel, reduce skin irritation, and ensure a mild and non-irritating use process.

[0013] TRPV1 (full name: Transient Receptor Potential Vanilloid 1) is a non-selective cation channel belonging to the vanilloid subtype of the transient receptor potential (TRP) channel family, mainly expressed on the membranes of skin keratinocytes, sensory neurons (C-fibers) and various non-neural cells. Activation of TRPV1 in keratinocytes can promote intercellular lipid secretion, but excessive activation can damage the barrier integrity. After the channel opens, a large amount of Ca²⁺ flows in, triggering the release of neuropeptides (such as substance P, CGRP), leading to burning pain, erythema, and vasodilation. After the introduction of ion channel modulating molecules in the present application, when T≥34℃ or ROS≥80μM, TRPV1 is transiently closed, Ca²⁺ influx is blocked, and in turn, the side effects such as erythema are reduced. For inflamed skin (pH≈5.5), the ZIF-90 nanoshell degrades at this time, releasing the entrapped active molecules. In a weakly acidic environment, the degradation rate of ZIF-90 is moderate, which can effectively control the release amount of Zn²⁺, avoiding toxicity to skin cells.

[0014] Preferably, the plant exosome is derived from plant tissues of grape, ginseng, cactus, rose, or citrus.

[0015] Preferably, the hydrophobic penetration-promoting active molecule includes any one of β-sitosterol, geraniol, menthol, and the hydrophilic penetration-promoting active molecule includes hesperidin or rutin.

[0016] Preferably, the film-forming agent is selected from one of mannan, trimethylpentanediol / adipic acid / glycerin crosspolymer.

[0017] Preferably, the temperature / ROS dual-responsive ion channel modulating molecule is Capsazepine linked by an azobenzene bridge, which is a pure antagonistic type of photoswitch. When T≥34℃, the conformational flip half-life t1 / 2=0.7±0.2 s, and when ROS≥80μM, the conformational flip half-life t1 / 2=0.6±0.1 s.

[0018] Preferably, the skin symbiotic bacteria activating material is one of fructooligosaccharides, seaweed extract, arabinoxylan oligosaccharides, fructo-oligosaccharides, lactobacillus fermentation product, cold-pressed flaxseed oil, and purple onion extract.

[0019] Fructooligosaccharides are prebiotics that promote the proliferation of skin beneficial bacteria, such as Staphylococcus epidermidis, and inhibit the growth of harmful bacteria. Arabinoxylan oligosaccharides are natural prebiotics that stimulate the growth of the commensal strain Staphylococcus epidermidis. Fructo-oligosaccharides are prebiotics obtained by cold-pressing Polymnia sonchifolia roots, which promote skin microbiota balance. Lactobacillus fermentate (such as Microbiome ME-2) has the effect of reducing pigment deposition, increasing skin moisture content, and improving skin elasticity. Seaweed extract (such as EPS SEAPUR®) is a mixture extracted from brown kelp, green microalgae Chlorella, marine exopolysaccharide solution, and Earth seawater, which can reverse stress-related microbiota imbalance and enhance microbial diversity. Alpha-linolenic acid in cold-pressed flaxseed oil can promote the synthesis of antimicrobial peptide LL-37 by microorganisms. Purple onion extract contains quercetin-3-glucoside, which can inhibit the quorum sensing system of pathogenic bacteria.

[0020] In another aspect, the application discloses a preparation method of the transdermal absorption enhancer, comprising the following steps:

[0021] S1: After homogenizing the plant tissue, differential centrifugation is performed, the precipitate is resuspended to obtain plant exosomes; ensure high purity and integrity of the exosomes to provide a good foundation for subsequent encapsulation.

[0022] S2: Dissolve the hydrophobic permeation active molecule in an ethanol-water mixed solvent, mix with the plant exosomes, and use low-temperature microjet technology for encapsulation at a pressure of 800-1200 bar for 2-5 cycles;

[0023] S3: Form a ZIF-90 nanoshell layer on the surface of the plant exosomes by in-situ growth; form a uniform nanoshell layer on the surface of the plant exosomes to enhance stability and functionality.

[0024] S4: Dissolve the hydrophilic permeation active molecule in a buffer solution with a pH of 7.0-8.0, mix with the product of step S3, and incubate at room temperature for 10-60 min;

[0025] S5: Dissolve the channel regulating molecule in ethanol to prepare a solution of 1-5 mg / mL, and add the solution to the product of step S4, and stir in the dark at room temperature for 10-24 h;

[0026] S6: Replace the original buffer with a filtrate with a conductivity of ≤50 μS / cm.

[0027] Due to the difference in pH value between the buffer of the hydrophilic penetration-promoting active molecule and the buffer of the film-forming agent, the ultrafiltration replacement avoids the precipitation of the hydrophilic penetration-promoting active molecule when the pH value changes suddenly, removes free metal ions (Zn2+), organic solvents and fragment impurities, reduces irritation, and protects the structural integrity by gradient osmotic pressure regulation (290±10 mOsm / kg) to avoid the rupture of the exosome-ZIF-90 complex due to the sudden change in osmotic pressure (integrity rate >95%).

[0028] S7: Dissolve the film-forming agent in a buffer with a pH of 5.8-6.0, stir at a speed of 300-800 rpm until completely transparent, add the skin symbiotic bacteria activating material, homogenize at 550-600 rpm for 10-15 min, and then add dropwise to the product of step S6;

[0029] S8: Concentrate by centrifugation using an ultrafiltration membrane with a molecular weight cutoff of 300-500 kDa;

[0030] S9: Sterilization filtration, nitrogen filling and packaging.

[0031] Preferably, the homogenate comprises:

[0032] Mix the plant tissue with the buffer at a mass-volume ratio of 1:(5-8), homogenize intermittently at 3-5℃ and 15000-23000 rpm for 2-5 times, homogenize for 10-15 s, and pause for 4-5 s.

[0033] Preferably, the differential centrifugation comprises:

[0034] Centrifuge at 4-6℃ and 2000-10000g for 10-30 min to obtain the supernatant, then centrifuge at 5000-20000g for 10-60 min, filter the supernatant with a 0.2-0.3μm microfiltration membrane, take the filtrate, and perform ultracentrifugation at 100000-200000g for 30-120 min.

[0035] In another aspect, the application also discloses the use of the transdermal absorption penetration enhancer in cosmetics.

[0036] The application has the following advantages:

[0037] The transdermal absorption enhancer of the present application realizes multiple advantages such as high efficiency, low irritation, micro-ecological friendliness and process scalability by innovatively combining plant exosomes, ZIF-90 nanoshell, ion channel regulating molecules and skin symbiotic bacteria activating materials. The enhancer uses the natural nanovesicle structure of plant exosomes and the large-pore nanoshell of ZIF-90 to significantly improve the encapsulation efficiency and skin permeability of active ingredients. At the same time, the ion channel regulating molecules can instantaneously close the TRPV1 channel when the temperature is above 34℃ or ROS is greater than or equal to 80μM, effectively reducing skin irritation, with a redness score less than 0.5, ensuring a gentle and non-irritating use. In addition, skin symbiotic bacteria activating materials such as fructooligosaccharides and seaweed extracts significantly increase the abundance of skin beneficial bacteria, reduce inflammatory response and improve skin microecological balance. The entire preparation process is controlled at a temperature below 35℃, suitable for industrial production, easy to scale up and quality control, providing an efficient, safe and multifunctional transdermal absorption solution for the cosmetics and pharmaceutical fields.

[0038] The preparation method of the present application ensures efficient preparation and stability of the enhancer by optimizing the steps of plant exosome extraction, hydrophobic / hydrophilic active molecule encapsulation, ZIF-90 nanoshell growth, ion channel regulating molecule anchoring, introduction of skin symbiotic bacteria activating materials, addition of film forming agent, ultrafiltration centrifugal concentration and sterilization filling. The method first extracts plant exosomes through low-temperature homogenization and differential centrifugation technology to ensure high purity and integrity. Then, the low-temperature microjet technology is used to efficiently encapsulate hydrophobic active molecules inside the exosomes, and the electrostatic adsorption is used to combine hydrophilic active molecules on the surface of the exosomes, forming a double-layer encapsulation structure. The in-situ growth of ZIF-90 nanoshell further enhances the mechanical strength and encapsulation capacity of the exosomes, while imparting pH responsiveness and realizing intelligent release. The anchoring of ion channel regulating molecules provides the enhancer with temperature and ROS responsive intelligent control function, effectively reducing skin irritation. In addition, the introduction of materials such as fructooligosaccharides and seaweed extracts significantly improves the skin microecology. The entire preparation process is parameterized, and the operation steps are detailed, ensuring the repeatability and stability of the product, suitable for large-scale industrial production, providing an efficient, safe and multifunctional transdermal absorption enhancer preparation scheme for the cosmetics and pharmaceutical fields.

[0039] Other features and advantages of the present application will be described in detail in the following specific embodiments.

[0040] It should be noted that all reagents and raw materials in the present application are commercially available, and the purity of the reagents is analytical pure. DETAILED DESCRIPTION

[0041] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present application, and are not intended to limit the present application.

[0042] Preparation of Capsazepine-azobenzene photoswitch:

[0043] 1. According to parts by weight, p-aminoazobenzoic acid 1.0 parts and glutaric anhydride 0.48 parts, 4-dimethylaminopyridine 0.05 parts are dissolved in N,N-dimethylformamide 20 parts, stirred at room temperature for 2 hours to obtain azobenzene glutaric acid monoamide; then directly add NHS 0.53 parts and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride 0.88 parts, and stir for 1 hour to form azobenzene-NHS ester.

[0044] 2. Add Capsazepine 1.7 parts and N,N-diisopropylethylamine 0.7 parts to the mixture in 1, stir at room temperature for 2 hours to complete the amide coupling to obtain a mixture.

[0045] 3. Pour the mixture in 2 into 150 parts of ice water, precipitate red solid, filter and pass through a silica gel column (dichloromethane:methanol=20:1) to obtain the finished product Capsazepine-azobenzene photoswitch.

[0046] Example 1 (grape exosome)

[0047] A transdermal absorption penetration enhancer, comprising, in parts by mass:

[0048] Plant exosome (grape): 10 parts;

[0049] β-sitosterol (hydrophobic): 5 parts;

[0050] Hesperidin (hydrophilic): 5 parts;

[0051] β-1,4-mannan (film-forming agent): 0.1 parts;

[0052] ZIF-90: 20 parts;

[0053] Capsazepine-azobenzene photoswitch: 0.1 parts;

[0054] Raftinose (symbiotic bacteria activation): 5 parts.

[0055] Preparation method:

[0056] S1: Plant exosome extraction

[0057] Homogenate: Take fresh grape pulp at 3℃ and remove unusable parts (such as seeds, rotten areas);

[0058] The treated fresh grape pulp was mixed with PBS buffer solution with a concentration of 50 mM at pH 7.4 at a ratio of 1:5 (w / v, g / mL), and homogenized intermittently at 15000 rpm for 3 times, 10 s each time, with a pause of 4 s;

[0059] Differential centrifugation: the homogenized product was centrifuged at 2000 g for 30 min at 4°C to obtain the supernatant, and then centrifuged at 5000 g for 60 min to obtain the supernatant, which was filtered through a 0.2 μm microfiltration membrane, and then ultracentrifuged at 100000 g for 120 min to obtain the plant exosome;

[0060] S2: Hydrophobic penetration-promoting active molecule encapsulation

[0061] β-sitosterol was mixed with an ethanol-water (v / v: 70 / 30) mixed solvent at a ratio of 8:1 (w / v, mg / mL), and then mixed with the plant exosome. Low-temperature microfluidization technology was used for encapsulation, with a pressure of 800 bar and 2 cycles;

[0062] S3: In-situ growth of ZIF-90 nanoshell

[0063] Zn(NO3)2 (10 mM) and 2-methylimidazole (80 mM) were added to the product of S2, and the mixture was reacted at 25°C for 60 min to grow the ZIF-90 nanoshell in situ;

[0064] S4: Hydrophilic penetration-promoting active molecule encapsulation

[0065] Hesperidin was mixed with 50 mM PBS buffer solution at pH 7.4 at a ratio of 10:1 (w / v, mg / mL), and then mixed with the product of step S3. The mixture was incubated at room temperature for 30 min;

[0066] S5: Channel-regulating molecule anchoring

[0067] Capsazepine-azobenzene photoswitch was dissolved in ethanol to prepare a 1 mg / mL solution, which was added dropwise to the product of step S4. The mixture was stirred at room temperature in the dark for 12 h;

[0068] S6: Ultrafiltration to replace the original buffer with a filtrate with a conductivity of 40 μS / cm;

[0069] The product prepared in S5 was centrifuged at 4000 g at 4°C using a 300 kDa ultrafiltration membrane, and 10 times the volume of HEPES at a concentration of 5 mM at pH 6.0 was used for replacement, to obtain a filtrate with a conductivity of 40 μS / cm.

[0070] S7: β-1,4-mannan was mixed with acetic acid buffer at pH 6.0 at 15:1 (w / v, mg / mL), stirred at 500 rpm until completely transparent, added raffinose, and homogenized at 600 rpm for 15 min, then added dropwise to the product of step S6;

[0071] S8: The product in S7 was concentrated by centrifugation using an ultrafiltration membrane with a molecular weight cut-off of 300 kDa;

[0072] S9: The product in S8 was sterilized by filtration using a 0.22 μm sterilization grade microporous filter, and filled with nitrogen.

[0073] Example 2 (Panax ginseng exosome)

[0074] A transdermal absorption penetration enhancer includes, by mass fraction:

[0075] Plant exosome (Panax ginseng): 50 parts;

[0076] Geraniol (hydrophobic): 10 parts;

[0077] Rutin (hydrophilic): 10 parts;

[0078] Trimethylpentanediol / adipic acid / glycerin crosspolymer: 5 parts;

[0079] ZIF-90: 5 parts;

[0080] Capsazepine-azobenzene photoswitch: 2 parts;

[0081] Fucoidan (molecular weight 50 kDa): 10 parts.

[0082] Preparation method:

[0083] S1: Plant exosome extraction

[0084] Homogenization: Fresh Panax ginseng was pre-cooled at 5°C, and unusable parts (such as seeds, rotten areas) were removed;

[0085] The treated fresh Panax ginseng was mixed with Tris-sodium acetate buffer at pH 7.0 and a concentration of 35 mM at 1:8 (w / v, g / mL), and homogenized at 22000 rpm for 5 times, 15 s each time, with 5 s pauses;

[0086] Differential centrifugation: The homogenized product was centrifuged at 5°C, 10000 g for 10 min, and the supernatant was taken, then centrifuged at 20000 g for 10 min, and the supernatant was filtered with a 0.22 μm microfiltration membrane, then the filtrate was taken and ultracentrifuged at 200000 g for 30 min, and the precipitate was resuspended to obtain plant exosomes;

[0087] S2: Hydrophobic penetration enhancer active molecule encapsulation

[0088] Geraniol was mixed with ethanol-water (v / v: 60 / 40) mixed solvent at 8:1 (w / v, mg / mL), and then mixed with plant exosome. Low-temperature microjet technology was used for loading, with a pressure of 1200 bar and 5 cycles;

[0089] S3: In-situ growth of ZIF-90 nanoshell

[0090] Zn(NO3)2 (10 mM) and 2-methylimidazole (80 mM) were added to the product of S2, and ZIF-90 nanoshell was grown in situ at 25°C for 60 min;

[0091] S4: Hydrophilic active molecule loading

[0092] Rutin was mixed with 50 mM Tris-HCl buffer at pH 7.0 at 15:1 (w / v, mg / mL), and then mixed with the product of step S3, and incubated at room temperature for 60 min;

[0093] S5: Channel regulating molecule anchoring

[0094] Capsazepine-azobenzene photoswitch was dissolved in ethanol to prepare a 5 mg / mL solution, which was added dropwise to the product of step S4, and stirred at room temperature in the dark for 24 h;

[0095] S6: Ultrafiltration to replace the original buffer with a filtrate conductivity of 45 μS / cm;

[0096] The product in S5 was replaced with 10 times the volume of pH 6.0 5 mM HEPES using a 300 kDa ultrafiltration membrane at 4°C and 4000g centrifugation, to a conductivity of 45 μS / cm.

[0097] S7: Trimethylpentanediol / adipic acid / glycerol cross-linked polymer was mixed with acetic acid buffer at pH 5.9 at 15:1 (w / v, mg / mL), and stirred at 800 rpm until completely transparent. Skin symbiotic bacteria activating material was added, and homogenized at 550 rpm for 10 min, then added dropwise to the product of step S6;

[0098] S8: The product in S7 was concentrated by centrifugation using an ultrafiltration membrane with a molecular weight cutoff of 500 kDa;

[0099] S9: The product in S8 was sterilized by filtration using a 0.22 μm sterilization grade microporous filter, and filled with nitrogen.

[0100] Example 3 (cactus exosome)

[0101] A transdermal absorption enhancer, comprising, by mass fraction:

[0102] Plant exosome (cactus): 30 parts;

[0103] Menthol (hydrophobic): 7.5 parts;

[0104] Hesperidin (hydrophilic): 7.5 parts;

[0105] β-1,4-mannan: 2.5 parts;

[0106] ZIF-90: 12 parts;

[0107] Capsazepine-azobenzene photoswitch: 1.0 part

[0108] Arabinoxylo-oligosaccharide AXOS-3A: 7.5 parts.

[0109] Preparation method:

[0110] S1: Plant exosome extraction

[0111] Homogenization: Fresh cactus was pre-cooled at 4℃, and unusable parts (such as seeds, rotten areas) were removed;

[0112] The treated fresh cactus was mixed with sodium citrate buffer solution with a pH of 7.2 and a concentration of 50 mM at a ratio of 1:7 (w / v, g / mL), and was intermittently homogenized at 20000 rpm for 5 times, 14 s each time, with a pause of 5 s;

[0113] Differential centrifugation: The homogenized product was centrifuged at 4℃, 5000g for 20 min to obtain the supernatant, and then centrifuged at 15000g for 30 min to obtain the supernatant. The supernatant was filtered with a 0.3μm microfiltration membrane, and the filtrate was obtained. The precipitate was resuspended by ultracentrifugation at 150000g for 60 min, and plant exosomes were obtained;

[0114] S2: Hydrophobic penetration-promoting active molecule encapsulation

[0115] Menthol was mixed with ethanol-water (v / v: 55 / 45) mixed solvent at a ratio of 8:1 (w / v, mg / mL), and then mixed with plant exosomes. Low-temperature microjet technology was used for encapsulation, with a pressure of 1000 bar and 3 cycles;

[0116] S3: In-situ growth of ZIF-90 nanoshell

[0117] Zn(NO3)2 (10mM) and 2-methylimidazole (80mM) were added to the product of S2, and ZIF-90 nanoshell was grown in situ at 25℃ for 60 min;

[0118] S4: Hydrophilic penetration-promoting active molecule encapsulation

[0119] Mix hesperidin with 50 mM PBS buffer at pH 7.0 at 10:1 (w / v, mg / mL), and then mix with the product of step S3, and incubate at room temperature for 60 min;

[0120] S5: Channel regulation molecule anchoring

[0121] Dissolve Capsazepine-azobenzene photoswitch in ethanol to prepare a 2 mg / mL solution, and then add the solution dropwise to the product of step S4, and stir in the dark at room temperature for 10 h;

[0122] S6: Ultrafiltration to replace the original buffer to a conductivity of 50 μS / cm in the filtrate;

[0123] Centrifuge the product in S5 at 4℃ and 4000g using a 300 kDa ultrafiltration membrane, and replace with 10 volumes of HEPES at pH 6.0 and a concentration of 5 mM, to a conductivity of 50 μS / cm.

[0124] S7: Mix β-1,4-mannan with acetic acid buffer at pH 5.8 at 15:1 (w / v, g / mL), and stir at a speed of 500 rpm until completely transparent, add arabinoxylo-oligosaccharide AXOS-3A, and homogenize at 580 rpm for 12 min, and then add dropwise to the product of step S6;

[0125] S8: Concentrate the product in S7 by centrifugation using an ultrafiltration membrane with a molecular weight cut-off of 400 kDa;

[0126] S9: Sterilize the product in S8 by sterilizing filtration using a 0.22 μm microporous filter, and fill with nitrogen.

[0127] Example 4 (Rose Exosome)

[0128] A transdermal absorption enhancer includes, by mass fraction:

[0129] Plant exosome (rose): 20 parts;

[0130] β-sitosterol (hydrophobic): 8 parts;

[0131] Rutin (hydrophilic): 6 parts;

[0132] Trimethylpentanediol / adipic acid / glycerin crosspolymer: 1.5 parts;

[0133] ZIF-90: 15 parts;

[0134] Capsazepine-azobenzene photoswitch: 0.5 parts;

[0135] Inulin-type fructan: 8 parts.

[0136] Preparation method:

[0137] S1: Plant exosome extraction

[0138] Homogenate: Fresh rose was pre-cooled at 4℃, and the unusable parts (such as seeds, rotten areas) were removed;

[0139] The treated fresh rose was mixed with sodium citrate buffer solution with a pH of 7.2 and a concentration of 50 mM at a ratio of 1:7 (w / v, g / mL), and was intermittently homogenized at 15000 rpm for 3 times, 13 s each time, with a pause of 5 s;

[0140] Differential centrifugation: The homogenized product was centrifuged at 6000 g for 30 min at 4℃, and the supernatant was taken. Then, it was centrifuged at 18000 g for 30 min, and the supernatant was filtered with a 0.22 μm microfiltration membrane. The filtrate was obtained by ultracentrifugation at 180000 g for 60 min, and the precipitate was resuspended to obtain plant exosomes;

[0141] S2: Hydrophobic penetration-promoting active molecule encapsulation

[0142] β-sitosterol was mixed with an ethanol-water (v / v: 70 / 30) mixed solvent at a ratio of 8:1 (w / v, mg / mL), and then mixed with plant exosomes. Low-temperature microjet technology was used for encapsulation, with a pressure of 1000 bar and 3 cycles;

[0143] S3: In-situ growth of ZIF-90 nanoshell

[0144] Zn(NO3)2 (10 mM) and 2-methylimidazole (80 mM) were added to the product of S2, and ZIF-90 nanoshell was grown in situ at 25℃ for 60 min;

[0145] S4: Hydrophilic penetration-promoting active molecule encapsulation

[0146] Rutin was mixed with Tris-HCl buffer solution with a pH of 7.0 and a concentration of 50 mM at a ratio of 8:1 (w / v, mg / mL), and then mixed with the product of step S3. Incubation was carried out at room temperature for 60 min;

[0147] S5: Channel-regulating molecule anchoring

[0148] Capsazepine-azobenzene photoswitch was dissolved in ethanol to prepare a 2 mg / mL solution, which was added dropwise to the product of step S4. Stirring was carried out at room temperature in the dark for 12 h;

[0149] S6: Ultrafiltration to replace the original buffer with a filtrate conductivity of 48 μS / cm;

[0150] The product in S5 was concentrated by centrifugation at 4℃, 4000g, using a 300 kDa ultrafiltration membrane, and was replaced with 10 volumes of HEPES at pH 6.0 and a concentration of 5 mM, to a conductivity of 48 μS / cm.

[0151] S7: The trimethylpentanediol / adipic acid / glycerol cross-linked polymer was mixed with acetic acid buffer at pH 6.0 at a ratio of 15:1 (w / v, g / mL), stirred at a speed of 800 rpm until completely transparent, inositol-type fructooligosaccharides were added, and homogenized at 600 rpm for 15 min, then added dropwise to the product of step S6;

[0152] S8: The product in S7 was concentrated by centrifugation using an ultrafiltration membrane with a molecular weight cut-off of 500 kDa;

[0153] S9: The product in S8 was sterilized by filtration using a 0.22 μm sterilization-grade microporous filter, and filled with nitrogen.

[0154] Comparative Example 1

[0155] The ZIF-90 nanoshell was not included in the penetration enhancer component, and the other steps in the preparation procedure and the raw material components were the same as in Example 1.

[0156] Comparative Example 2

[0157] The channel modulating molecule was not included in the penetration enhancer component, and the other steps in the preparation procedure and the raw material components were the same as in Example 1.

[0158] Comparative Example 3

[0159] The non-symbiont-activating material was not included in the penetration enhancer component, and the preparation procedure was as follows:

[0160] S1: Plant exosome extraction

[0161] Homogenization: Fresh cactus was pre-cooled at 4℃, and unusable parts (such as seeds, rotten areas) were removed;

[0162] The treated fresh cactus was mixed with sodium citrate buffer at pH 7.2 and a concentration of 50 mM at a ratio of 1:7 (w / v, g / mL), and was intermittently homogenized at 20000 rpm for 5 times, with a homogenization time of 14 s and a pause time of 5 s;

[0163] Differential centrifugation: The homogenized product was centrifuged at 4℃, 5000g for 20 min to obtain the supernatant, and then centrifuged at 15000g for 30 min to obtain the supernatant, which was filtered through a 0.3 μm microfiltration membrane, and the filtrate was obtained and subjected to ultracentrifugation at 150000g for 60 min, and the precipitate was resuspended to obtain the plant exosomes;

[0164] S2: Hydrophobic penetration enhancer active molecule encapsulation

[0165] The menthol was mixed with an ethanol-water (v / v: 55 / 45) mixed solvent at 8:1 (w / v, mg / mL), and then mixed with the plant exosome. The low-temperature microjet technology was used for loading, the pressure was 1000 bar, and the cycle was 3 times.

[0166] S3: In-situ growth of ZIF-90 nanoshell

[0167] Zn(NO3)2 (10 mM) and 2-methylimidazole (80 mM) were added to the product of S2, and the ZIF-90 nanoshell was grown in situ at 25°C for 60 min;

[0168] S4: Hydrophilic penetration enhancer active molecule loading

[0169] The hesperidin was mixed with 50 mM PBS buffer at pH 7.0 at 10:1 (w / v, mg / mL), and then mixed with the product of step S3, and incubated at room temperature for 60 min;

[0170] S5: Channel regulating molecule anchoring

[0171] Capsazepine-azobenzene photoswitch was dissolved in ethanol to prepare a 2 mg / mL solution, which was added dropwise to the product of step S4, and stirred at room temperature in the dark for 10 h;

[0172] S6: Ultrafiltration to replace the original buffer to the conductivity of the filtrate of 50 μS / cm;

[0173] The product in S5 was centrifuged at 4°C, 4000g, with a 300 kDa ultrafiltration membrane, and replaced with 10 volumes of pH 6.0 HEPES at a concentration of 5 mM, to a conductivity of 50 μS / cm.

[0174] S7: β-1, 4-mannan was mixed with acetic acid buffer at pH 5.8 at 15:1 (w / v, g / mL), and stirred at a speed of 500 rpm until completely transparent, and then added dropwise to the product of step S6;

[0175] S8: The product in S7 was concentrated by centrifugation using an ultrafiltration membrane with a molecular weight cutoff of 400 kDa;

[0176] S9: The product in S8 was sterilized by 0.22 μm sterilization grade microporous filter, and filled with nitrogen.

[0177] Experimental Example 1

[0178] The penetration enhancers obtained in Examples 1-4 and Comparative Examples 1-3 were subjected to in vitro transdermal penetration experiments (Franz diffusion cell method), and the diffusion cell parameters were as follows: effective diffusion area 1.77 m 2, receiving liquid pH 7.4 PBS + 0.02% NaN3+20% PEG400, 37±0.5℃, stirring 600rpm; test the 24h cumulative penetration rate of each penetration enhancer and the total leakage rate of active substances (including hydrophobic penetration active molecules and hydrophilic penetration active molecules) after 3 freeze-thaw cycles (freeze-thaw conditions: place the sample in a -80 ℃ ultra-low temperature freezer for 2h, take out the frozen sample and place it in a 25 ℃ constant temperature water bath for 30min, cycle 3 times); test results as shown in Table 1 (all numerical values are "mean ± standard deviation").

[0179] Table 1 in vitro transdermal penetration performance table

[0180] No. 24h cumulative permeation rate (%) Active leakage rate after 3 freeze-thaw cycles (%) Example 1 78.4±4.2 4.1±0.8 Example 2 75.9±3.8 5.0±1.1 Example 3 74.2±4.0 3.7±0.9 Example 4 73.5±3.6 4.5±1.0 Comparative Example 1 46.2±3.5 41.3±5.2 Comparative Example 2 70.1±4.1 5.2±1.3 Comparative Example 3 71.8±3.9 4.8±1.2

[0181] As can be seen from Table 1, the 24h cumulative penetration rate of the penetration enhancers of Examples 1-4 is more than 70%, and the leakage rate of active substances after 3 freeze-thaw cycles is less than 6%. The main reason is that the components (plant exosomes, ZIF-90 shell, film-forming agent, regulatory molecules, and symbiotic bacteria activating material) synergize with each other to form a complete penetration system. Plant exosomes provide biocompatibility and encapsulation capacity, ZIF-90 shell provides physical protection and stability, film-forming agent enhances penetration efficiency, regulatory molecules achieve intelligent response, and symbiotic bacteria activating material maintains microecological balance. This synergistic effect allows active ingredients to efficiently penetrate the skin surface while reducing leakage.

[0182] In Comparative Example 1, there is no ZIF-90 nanoshell, resulting in a significant decrease in penetration rate (only about 59% of Example 1) and a very high active substance leakage rate (41.3%), indicating that the ZIF-90 nanoshell is crucial for stability and penetration performance. Comparative Example 2 has no regulatory molecules, and the penetration rate is slightly lower than that of the examples, but the leakage rate is similar to that of the examples, indicating that the regulatory molecules have some effect on the penetration performance, but the main effect is reflected in the subsequent temperature / ROS responsiveness. Comparative Example 3 has no symbiotic bacteria activating material, and the penetration rate and leakage rate are similar to those of the examples, indicating that the symbiotic bacteria activating material has little effect on the penetration performance, but it may have potential effects on the microecological balance.

[0183] Experimental Example 2 (temperature / ROS dual-responsive verification)

[0184] TRPV1-HEK293 cell calcium imaging experiments were performed on the penetration enhancers prepared in Examples 1 and 2 and Comparative Example 2, and the experimental conditions were as follows:

[0185] Temperature group: Tyrode solution was preheated to 34℃±0.2℃, and the sample was added quickly;

[0186] ROS group: H2O2 final concentration 100μM (≈ROS 80-120μM), real-time record 180s.

[0187] Confocal 488nm / 520nm, frame rate 10Hz

[0188] The time required for the real-time fluorescence intensity F to decay to 50% of the initial fluorescence intensity F0 is t1 / 2.

[0189] TRPV1 off rate = (1-Fmax / F0) x 100%, wherein Fmax is the maximum fluorescence intensity.

[0190] The experimental results are shown in Table 2.

[0191] Table 2 Results of cell calcium imaging experiment

[0192] No. fluorescence decay t1 / 2(s) at 34°C stimulation H2O2 100 μM stimulation t1 / 2 (s) TRPV1 off rate (%) Example 1 0.7 0.5 90 Example 2 0.7 0.6 88 Comparative Example 2 > 5 s (no significant decay) >5 s 15

[0193] As can be seen from Table 2, Comparative Example 2 has no regulatory molecules, and cannot achieve rapid temperature / ROS response, and the TRPV1 off rate is extremely low (only 15%), indicating that the regulatory molecules are the key to achieving intelligent response.

[0194] Experimental Example 3 (human patch test)

[0195] The skin penetration enhancers prepared in Example 1 and Example 2 and Comparative Example 2 are made into skin penetration enhancer masks. The components and preparation method of the mask can be selected from the salvia miltiorrhiza mask and its preparation method disclosed in Example 1 of Chinese invention patent (CN201110139220.9). The skin penetration enhancer in the mask is replaced with an equal amount of the skin penetration enhancer of Example 1 and Example 2 and Comparative Example 2. Thirty-two sensitive skin subjects are selected, and half of the face is used for the skin penetration enhancer mask of Example 1 and Example 2 and Comparative Example 2, and the other half of the face is used for the blank mask (the mask disclosed in Example 1 of CN201110139220.9 without the skin penetration enhancer of the present application) for patch testing.

[0196] The subjects' pricking sensation is recorded on a 0-4 scale, and the number of people at level 0 is counted to evaluate the irritability of the product.

[0197] Scale levels:

[0198] 0 level: no pricking sensation.

[0199] 1 level: slight pricking, almost imperceptible.

[0200] 2 level: mild pricking, can be clearly felt.

[0201] 3 level: moderate pricking, more obvious and may cause discomfort.

[0202] 4 level: severe pricking, very strong and may be unbearable.

[0203] Skin transepidermal water loss was measured using Tewameter TM300 to evaluate skin barrier function.

[0204] Skin erythema index was measured using Mexameter MX18 to evaluate erythema improvement by a* value drop.

[0205] The experimental results are shown in Table 3.

[0206] Table 3 Human patch test results table

[0207] No. 0-grade stinging sensation ratio (%) TEWL reduction rate (%) Redness score reduction Example 1 93 18 1.9 Example 2 90 17 1.7 Comparative Example 2 60 9 1.0 Blank mask 45 4 0.5

[0208] As shown in Table 3, the penetration-promoting masks of Example 1 and Example 2 showed extremely low irritation in sensitive skin subjects, and more than 90% of the subjects did not feel tingling. Comparative Example 2 lacked temperature / ROS dual-responsive ion channel modulating molecules, and the irritation was significantly higher than that of the examples, and only 60% of the subjects did not feel tingling.

[0209] The penetration-promoting masks of Example 1 and Example 2 significantly improved skin barrier function, with TEWL reduction rates of 18% and 17%, respectively. Comparative Example 2 lacked channel modulating molecules, and the improvement effect on skin barrier function was limited, with a TEWL reduction rate of only 9%.

[0210] The penetration-promoting masks of Example 1 and Example 2 significantly reduced skin erythema, with erythema scores decreasing by 1.9 and 1.7, respectively. Comparative Example 2 lacked channel modulating molecules, and the anti-inflammatory effect was poor, with an erythema score decrease of only 1.0.

[0211] Experimental Example 4 (Skin symbiotic bacteria diversity detection)

[0212] After using the penetration enhancers of Example 3, Example 4, and Comparative Example 3 to prepare penetration-promoting masks according to the method of Experimental Example 3 for 28 days, samples were collected from the face or other designated skin sites (before use and after 28 days of use), and 16S rRNA gene sequencing technology was used to detect the Shannon index, Propionibacterium relative abundance, and Staphylococcus epidermidis of the skin microbial community. The data before and after 28 days of use were compared, and the Shannon index increase, Propionibacterium relative abundance increase, and Staphylococcus epidermidis stability were calculated. The detection results are as follows:

[0213] Table 4 Skin symbiotic bacteria diversity detection table

[0214] No. Shannon index improvement (%) Propionibacterium relative abundance increase (%) Staphylococcus epidermidis stability Example 3 38 29 Maintained Example 4 32 26 Maintained Comparative Example 3 7 5 Slightly decreased

[0215] As can be seen from Table 4, the penetration enhancer of Example 3 and Example 4 significantly improves the diversity of skin microbial community (the Shannon index is increased by 38% and 32% respectively), indicating that the penetration enhancer can effectively activate skin symbiotic bacteria and promote the richness and evenness of microbial community. The Shannon index of Comparative Example 3 (without symbiotic bacteria activating material) is only increased by 7%, indicating that the symbiotic bacteria activating material plays a key role in improving the diversity of microbial community.

[0216] Propionibacterium is a beneficial skin symbiotic bacteria that can maintain the acidic environment of the skin and inhibit the growth of harmful bacteria, so the penetration enhancer has a positive impact on skin health. The penetration enhancer of Example 3 and Example 4 significantly increases the relative abundance of propionibacterium (increased by 29% and 26% respectively), indicating that the penetration enhancer can effectively promote the growth of propionibacterium. The relative abundance of propionibacterium of Comparative Example 3 is only increased by 5%, indicating that the symbiotic bacteria activating material also plays a key role in increasing the relative abundance of propionibacterium.

[0217] Staphylococcus epidermidis is a common skin symbiotic bacteria that plays an important role in maintaining skin barrier function. The penetration enhancer of Example 3 and Example 4 has no significant effect on the abundance of Staphylococcus epidermidis, indicating that the penetration enhancer promotes the growth of beneficial bacteria while not disrupting the balance of skin microbial community. Comparative Example 3 has a slight effect on the abundance of Staphylococcus epidermidis, indicating that the symbiotic bacteria activating material also has a certain effect on maintaining skin barrier function.

[0218] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application. In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

[0219] In addition, various different embodiments of the present application can also be combined in any manner, as long as they do not deviate from the idea of the present application, and they should also be considered as disclosed by the present application.

Claims

1. A transdermal absorption enhancer, characterized in that, The product comprises, by weight parts: 10-50 parts plant exosomes; natural permeation-enhancing active molecules, including 5-10 parts hydrophobic and 5-10 parts hydrophilic permeation-enhancing active molecules; 0.1-5 parts film-forming agent; 5-20 parts ZIF-90 nanomaterials, coated on the outer surface of the plant exosomes to form a nanoshell; 0.1-2 parts temperature / ROS dual-response ion channel regulating molecules, anchored on the surface of the nanoshell; and 5-10 parts skin symbiotic bacteria activating material. Hydrophobic permeation-enhancing active molecules include any one of β-sitosterol, geraniol, and menthol, while hydrophilic permeation-enhancing active molecules include hesperidin or rutin. The skin symbiotic bacteria activating material is one of the following: fructooligosaccharides, seaweed extract, arabinoxylan oligosaccharides, fructooligosaccharides, lactobacillus fermentation products, cold-pressed flaxseed oil, and purple onion extract; When the ion channel regulating molecule is at T≥34℃ or ROS≥80μM, the conformational inversion half-life t1 / 2<1s, and the TRPV1 channel is instantaneously shut down. The ion channel regulating molecule is a purely antagonistic photoswitch of Capsazepine bridged by azobenzene. At T≥34℃, the conformational inversion half-life t1 / 2 = 0.7±0.2 s; at ROS 80≥μM, the conformational inversion half-life t1 / 2 = 0.6±0.1 s. The preparation method is as follows: (1) Dissolve 1.0 part of p-aminoazobenzoic acid, 0.48 part of glutaric anhydride, and 0.05 part of 4-dimethylaminopyridine in 20 parts of N,N-dimethylformamide according to the weight ratio, and stir at room temperature for 2 hours to obtain azobenzoic glutaric acid monoamide; then directly add 0.53 parts of NHS and 0.88 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir for 1 hour to generate azobenzoic-NHS ester, and obtain a mixture; (2) Add 1.7 parts of Capsazepine and 0.7 parts of N,N-diisopropylethylamine to the mixture in (1), stir at room temperature for 2 hours to complete the amide coupling, and obtain a mixture; (3) Pour the mixture in (2) into 150 parts of ice water, precipitate a red solid, filter and pass through a silica gel column, the eluent is 20:1 dichloromethane and methanol, and the product is obtained; Methods for preparing transdermal absorption enhancers include: S1: Homogenize plant tissue, centrifuge at differential speed, resuspend the precipitate to obtain plant exosomes; S2: Dissolve hydrophobic permeability-enhancing molecules in a mixed solvent of ethanol and water, mix with plant exosomes, and encapsulate using low-temperature microfluidic technology at a pressure of 800-1200 bar for 2-5 cycles; S3: Form a ZIF-90 nanoshell on the surface of plant exosomes using an in-situ growth method; S4: Dissolve hydrophilic permeability-enhancing molecules in a buffer solution of pH 7.0-8.0, mix with the product from S3, and incubate at room temperature for 10-60 min; S5: Dissolve ion channel regulating molecules in ethanol to prepare 1-5 A solution of mg / mL was added dropwise to the product of step S4, and stirred at room temperature in the dark for 10-24 hours; S6: Ultrafiltration was used to replace the original buffer solution until the conductivity of the filtrate was ≤50μS / cm; S7: The film-forming agent was dissolved in a buffer solution with a pH of 5.8-6.0 and stirred at 300-800 rpm until completely transparent. Skin symbiotic bacteria activating material was added and homogenized at 550-600 rpm for 10-15 minutes, and then added dropwise to the product of step S6; S8: The product was concentrated by centrifugation using an ultrafiltration membrane with a molecular weight cutoff of 300-500 kDa; S9: The product was sterilely filtered and filled with nitrogen.

2. The transdermal absorption enhancer according to claim 1, characterized in that, The plant exosomes are derived from plant tissues of grapes, ginseng, cactus, roses, or citrus.

3. The transdermal absorption enhancer according to claim 1, characterized in that, The film-forming agent is selected from mannan and trimethylpentane / adipic acid / glycerol cross-linked polymer.

4. A method for preparing the transdermal absorption enhancer according to any one of claims 1-3, characterized in that, Includes the following steps: S1: After homogenizing the plant tissue, centrifuge at a differential speed, resuspend the precipitate, and obtain plant exosomes; S2: Dissolve the hydrophobic permeability-enhancing active molecule in an ethanol-water mixed solvent, mix it with plant exosomes, and encapsulate it using low-temperature microfluidic technology at a pressure of 800-1200 bar for 2-5 cycles. S3: A ZIF-90 nanoshell is formed on the surface of plant exosomes via in-situ growth. S4: Dissolve the hydrophilic permeation-enhancing active molecule in a buffer solution with a pH of 7.0-8.0, mix it with the product from step S3, and incubate at room temperature for 10-60 min; S5: Dissolve the channel-regulating molecule in ethanol to prepare a solution of 1-5 mg / mL, add the solution dropwise to the product of step S4, and stir at room temperature in the dark for 10-24 h. S6: Ultrafiltration replaces the original buffer solution until the conductivity of the filtrate is ≤50μS / cm; S7: Dissolve the film-forming agent in a buffer solution with a pH of 5.8-6.0, stir at 300-800 rpm until completely transparent, add the skin symbiotic bacteria activating material, homogenize at 550-600 rpm for 10-15 min, and then dropwise add it to the product of step S6; S8: Concentrate by centrifugation using an ultrafiltration membrane with a molecular weight cutoff of 300-500 kDa; S9: Sterilization filtration, nitrogen filling.

5. The method for preparing the transdermal absorption enhancer according to claim 4, characterized in that: The homogenate includes: Mix the plant tissue with the buffer solution at a mass-to-volume ratio of 1:(5-8), and homogenize intermittently 2-5 times at 3-5℃ and 15000-23000rpm, homogenizing for 10-15s and pausing for 4-5s.

6. The method for preparing the transdermal absorption enhancer according to claim 5, characterized in that, The differential centrifugation includes: Centrifuge at 2000-10000g for 10-30 min at 4-6℃, collect the supernatant, then centrifuge at 5000-20000g for 10-60 min. Filter the supernatant through a 0.2-0.3μm microfiltration membrane, collect the filtrate, and then ultracentrifuge at 100000-200000g for 30-120 min.

7. The use of a transdermal absorption enhancer according to any one of claims 1-3 in the preparation of cosmetics.