CN116602884A - A multi-effect anti-aging co-delivery nano composition suitable for microneedles and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of nano-compositions, and particularly relates to a multi-effect anti-aging co-delivery nano-composition suitable for microneedles, and a preparation method and application thereof. The invention provides a multi-effect anti-aging co-delivery nano composition suitable for microneedles, which comprises a composite active factor, an emulsifying agent, a co-emulsifying agent, a preservative and water, wherein the composite active factor comprises polydeoxyribonucleotide, acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydro oat alkaloid and ergothioneine. The combination and collocation of the composite active factors can realize excellent skin anti-aging effect through multi-path and multi-target synergism.

Description

A multi-effect anti-aging co-delivery nanocomposite suitable for microneedles and its preparation method and application

Technical Field

The present invention belongs to the technical field of nanocomposites, and in particular relates to a multi-effect anti-aging co-delivery nanocomposites suitable for microneedles, and a preparation method and application thereof.

Background Art

With the increase in the average life expectancy of the population and the aging of society, modern consumers are gradually becoming more aware of skin health management. Therefore, in-depth exploration of the mechanism of skin aging and the development of corresponding means to delay skin aging have become one of the research hotspots in the medical beauty industry. According to the product ingredients, the common anti-aging products on the current market have a single mechanism of action, and the anti-aging effect is often unsatisfactory. Based on the main action pathways involved in the skin aging process, it is of great significance to select different functional ingredients of natural origin, high safety, and excellent effects for scientific combination to develop new, synergistic, and efficient anti-aging products.

On the other hand, the application of functional skin care products begins on the surface of the skin, and needs to complete the transdermal absorption process, eventually enter the deep tissue of the epidermis, and accumulate to an effective concentration in the target cells at the target site to successfully play the target role. For various functional ingredients, the specific physiological structure of the skin protects the body from external damage while also limiting its transdermal delivery efficiency. Therefore, reducing the resistance of the stratum corneum, increasing skin permeability, allowing anti-aging functional ingredients to smoothly enter the epidermis, dermis and subcutaneous tissue involved in the skin aging process, and exerting anti-aging effects at multiple levels and targets is the key to solving the problem.

At present, the relatively mature microneedle technology can solve the problem of transdermal penetration of some effective ingredients. A certain number of microneedles are arranged in an array on a carrier, which can break through the barrier of the skin stratum corneum to form recoverable instant microchannels, so that the effective ingredients can be directly penetrated into the epidermis or dermis without causing pain or skin damage. However, the anti-aging products currently on the market generally use a single anti-aging ingredient. Due to the complexity of different anti-aging ingredients in terms of molecular size, solubility, stability, etc., different anti-aging ingredients interfere or react with each other, and it is impossible to realize the compound application of multiple anti-aging ingredients in microneedle technology.

Summary of the invention

The purpose of the present invention is to provide a multi-effect anti-aging co-delivery nanocomposition suitable for microneedles and its preparation method and application. The anti-aging nanocomposition provided by the present invention has multi-effect anti-aging functions and has efficient anti-aging performance.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides a multi-effect anti-aging co-delivery nanocomposition suitable for microneedles, comprising the following components in percentage by weight:

1-10% of composite active factor, 2-10% of emulsifier, 2-12% of auxiliary emulsifier, 0.1-0.5% of preservative and the balance of water;

The composite active factors include polydeoxyribonucleotide, acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydroavenous alkaloids and ergothioneine.

Preferably, taking the mass of the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles as 100%, the composite active factor includes the following components in percentage by mass: polydeoxyribonucleotide 0.1-2%, acetyl hexapeptide-8 0.001-0.01%, palmitoyl pentapeptide-4 0.001-0.1%, dihydrooat alkaloid 1-5% and ergothioneine 0.1-2%.

Preferably, the particle size of the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles is 10 to 500 nm.

Preferably, the emulsifier comprises one or more of lecithin and/or polyoxyethylene hydrogenated castor oil.

Preferably, the co-emulsifier includes one or more of butylene glycol, hexylene glycol and propylene glycol.

Preferably, the preservative comprises p-hydroxyacetophenone.

The present invention provides a method for preparing a multi-effect anti-aging co-delivery nanocomposition suitable for microneedles as described in the above technical solution, comprising the following steps:

The composite active factor, emulsifier, co-emulsifier, preservative and water are uniformly mixed to obtain a mixture;

The mixed material is subjected to micronization treatment to obtain a micron-sized dispersion;

The micron-scale dispersion is subjected to nano-processing to obtain the anti-aging nano-composition.

Preferably, the micronization treatment is shear mixing, the rotation speed of the shear mixing is 4000-30000 rpm, and the time is 1-20 min.

Preferably, the nano-treatment is a high-pressure homogenization treatment or a high-pressure microfluidization treatment;

The pressure of the high-pressure homogenization treatment is 300-1600 bar, the temperature is 20-70° C., and the number of cycles is 1-10 times;

The pressure of the high-pressure microfluidic treatment is 3000-16000 psi, the temperature is 20-70° C., and the number of cycles is 1-10 times.

The present invention provides the use of the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles described in the above technical scheme or the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles obtained by the preparation method described in the above technical scheme in the preparation of anti-skin aging cosmetics.

The present invention provides a multi-effect anti-aging co-delivery nanocomposite suitable for microneedles, comprising the following components in percentage by mass: 1-10% of composite active factors, 2-10% of emulsifiers, 2-12% of co-emulsifiers, 0.1-0.5% of preservatives and the remainder of water; the composite active factors include polydeoxyribonucleotides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydrooat alkaloids and ergothioneine. Among them, polydeoxyribonucleotides can induce the regeneration of skin tissues and cells damaged by physical, chemical stimulation and ultraviolet rays, replenish the elastic fiber structure reduced by aging, and restore the healthy elasticity of the skin; dihydrooat alkaloids are active ingredients derived from oats, which can block the occurrence of cell inflammation, reduce the occurrence of inflammatory immune reactions and skin neurodermatitis reactions, and can play multiple functions such as anti-histamine, anti-oxidation, anti-irritation, anti-inflammatory, and improvement of erythema; ergothioneine is an amino acid derivative with a strong antioxidant effect, which can remove active oxygen free radicals, protect Protect mitochondrial oxidative damage, thereby delaying cell aging; acetyl hexapeptide-8 is a neurotransmitter inhibitory peptide that competes for SNAP-25 sites in the melt bubble complex, making it impossible for muscles to effectively receive neurotransmitters, weakening muscle contraction, relaxing muscles, and preventing the formation of wrinkles; palmitoyl pentapeptide-4 acts on dermal fibroblasts, stimulating the synthesis of skin elastin, fibronectin, glycosaminoglycans, and collagen to supplement the extracellular matrix, solving the problem of improving the appearance of skin aging from a deep level, smoothing wrinkles, and enhancing skin firmness and glossiness. The present invention combines active factors such as polydeoxyribonucleotides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydrooat alkaloids, and ergothioneine to achieve excellent skin anti-aging effects through multi-pathway and multi-target synergy.

At the same time, thanks to the structural characteristics of nanoparticles, the product can be applied with microneedle introduction technology, which promotes the efficient penetration of effective ingredients through the stratum corneum of the skin. The multi-effect anti-aging nano-composition has good skin permeability and retention properties, which effectively promotes the further diffusion of effective ingredients after passing through the stratum corneum, reaching various target sites, and enriching them at high concentrations, retaining them for a long time, and sustained release, and finally being taken up by tissue cells, thereby improving their bioavailability and overall skin anti-aging effects and prolonging the duration of action.

The present invention provides a method for preparing a multi-effect anti-aging co-delivery nanocomposition suitable for microneedles, comprising the following steps: mixing a composite active factor, an emulsifier, an emulsifier co-emulsifier, a preservative and water to obtain a mixture; subjecting the mixture to micronization treatment to obtain a micron-level dispersion; subjecting the micron-level dispersion to nanoization treatment to obtain the anti-aging nanocomposition. The preparation method of the present invention combines micronization treatment and nanoization treatment, and encapsulates a variety of scientifically matched anti-aging functional ingredients with emulsifiers, emulsifier co-emulsifiers and water to obtain a nano-particle-sized multi-effect anti-aging nanocomposition, which has good water dispersibility, improves the solubility of some water-insoluble functional ingredients, and the irritation of some functional substances, so that each functional ingredient can reach a sufficient concentration in the product to smoothly exert the corresponding functional effect.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

FIG1 is the cell safety evaluation result of Test Example 2;

FIG2 is the irritation evaluation result of Test Example 3;

FIG3 is the results of the measurement of the cumulative permeation and skin retention in the free composition, co-delivered nanocomposition and microneedle delivery group of Test Example 5;

FIG4 is the result of skin penetration behavior observed by laser confocal microscopy in Test Example 6;

FIG5 is a study result of the cell proliferation promoting effect of Test Example 7;

FIG6 is a study result of the cell antioxidant effect of Test Example 8;

FIG. 7 is the results of the cell anti-aging effect test in Test Example 9;

FIG8 is the results of the study on the cell anti-aging factors in Test Example 10.

DETAILED DESCRIPTION

The present invention provides a multi-effect anti-aging co-delivery nanocomposition suitable for microneedles, comprising the following components in percentage by weight:

1-10% of composite active factor, 2-10% of emulsifier, 2-12% of auxiliary emulsifier, 0.1-0.5% of preservative and the balance of water;

The composite active factor comprises polydeoxyribonucleotide (PDRN), acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydroavenous alkaloid and ergothioneine.

In the present invention, unless otherwise specified, the required raw materials for preparation are all commercially available products well known to those skilled in the art.

In terms of mass percentage, the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention includes 1 to 10% of the composite active factor, preferably 2 to 7%. In the present invention, the mass percentage of the composite active factor cannot be too high or too low; when the content of the composite active factor component is too low, its overall anti-aging effect is not significant; when the content of the composite active factor component increases, its effect is improved to a certain extent, but when the content of the effective component exceeds 10%, it will cause the particle size of the anti-aging nanocomposition to increase significantly, which is not conducive to the long-term stability of the product.

In the present invention, the composite active factors include polydeoxyribonucleotides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydrooat alkaloids and ergothioneine. Taking the mass of the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles as 100%, the composite active factors preferably include: 0.1-2% polydeoxyribonucleotides, 0.001-0.01% acetyl hexapeptide-8, 0.001-0.1% palmitoyl pentapeptide-4, 1-5% dihydrooat alkaloids and 0.1-2% ergothioneine.

The multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention includes 0.1-2% of polydeoxyribonucleotides, preferably 1-2%, in terms of mass percentage. In the present invention, polydeoxyribonucleotides, as repair and reconstruction factors, can induce the regeneration of skin tissues and cells damaged by physical and chemical stimulation and ultraviolet rays, replenish the elastic fiber structure reduced by aging, and restore the healthy elasticity of the skin. Its mechanism of action includes selectively stimulating receptors, shortening the time for cells to transition to the proliferation phase; promoting the secretion of cell growth factors to stimulate cell growth; rapidly generating capillaries, providing nutrition and oxygen, and providing nucleotide raw materials for tissue regeneration.

The multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention comprises 0.001-0.01% of acetyl hexapeptide-8, preferably 0.005-0.01%, by mass percentage. In the present invention, acetyl hexapeptide-8, as a signal anti-wrinkle factor, participates in the competition for the site of SNAP-25 in the melt bubble complex, so that the muscle cannot effectively receive neurotransmitters, the muscle contraction is weakened, the muscle is relaxed, and the formation of wrinkles is prevented.

The multi-effect anti-aging co-delivery nanocomposition suitable for microneedle provided by the present invention comprises 0.001-0.1% palmitoyl pentapeptide-4, preferably 0.005-0.01%, by mass percentage. In the present invention, palmitoyl pentapeptide-4 acts on dermal fibroblasts as a regenerative wrinkle-relieving factor, and can stimulate and promote the synthesis of skin elastin, fibronectin, glycosaminoglycans, and collagen to supplement the extracellular matrix, thereby improving the appearance of skin aging, smoothing wrinkles, and enhancing skin firmness and glossiness from a deep level.

The multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention comprises 1-5% of dihydrooat alkaloids, preferably 2-5%, in terms of mass percentage. In the present invention, dihydrooat alkaloids are derived from the active ingredients of oats (Avena Sativa), and as anti-allergic and anti-inflammatory factors, they have the effects of inhibiting the degradation of keratinocyte nuclear factor NF-κB-α associated with inflammation, preventing the phosphorylation of p65 protein subunit on nuclear factor NF-κB, thereby blocking the occurrence of cell inflammation; reducing the occurrence of inflammatory immune response and skin neurodermatitis reaction, and can play multiple functions such as anti-histamine, anti-oxidation, anti-irritation, anti-inflammatory, and improving erythema.

In terms of mass percentage, the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention includes 0.1-2% ergothioneine, preferably 1-2%. In the present invention, ergothioneine, as a light protection factor, has a strong antioxidant effect, can remove reactive oxygen free radicals, and protect mitochondrial oxidative damage, thereby achieving the effect of delaying cell aging. As a super antioxidant, it is the only antioxidant that can repair mitochondria with a clear mechanism. At the same time, it has been proven that it can inhibit the activation and generation of inflammatory cytokines caused by UVB irradiation, and has a fibroblast protection effect.

According to the development process and mechanism of skin aging, the present invention combines polydeoxyribonucleotides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydrooat alkaloids and ergothioneine active factors to stimulate the formation of collagen and elastic fibers, promote the construction of extracellular matrix, resist oxidative inflammatory stimulation, protect against ultraviolet damage, and regulate skin neurotransmitters. It can achieve multi-pathway and multi-target effectiveness, thereby achieving excellent skin anti-aging effects.

The multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention comprises 2 to 10% of an emulsifier, preferably 2 to 8%, by mass percentage. In the present invention, the emulsifier preferably comprises lecithin and/or polyoxyethylene hydrogenated castor oil, more preferably lecithin and polyoxyethylene hydrogenated castor oil, and the mass ratio of the lecithin to the polyoxyethylene hydrogenated castor oil is preferably 1:(1 to 3), more preferably 1:1.

In terms of mass percentage, the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention includes 5 to 12% of an emulsifier, preferably 5 to 10%. In the present invention, the emulsifier preferably includes one or more of butanediol, hexanediol and propylene glycol, more preferably butanediol, 1,2-hexanediol and 1,3-propylene glycol. In the present invention, the mass ratio of butanediol, 1,2-hexanediol and 1,3-propylene glycol is preferably (1 to 3): (2.5 to 5): (2.5 to 5), more preferably 1:2.5:2.5. The present invention uses a mixed combination of butanediol, 1,2-hexanediol and 1,3-propylene glycol, which is beneficial to the dispersion of the composition and improves the solubility of the composite active factor.

In the present invention, when the types and contents of the emulsifier and the co-emulsifier in the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles are within the above preferred ranges, the prepared anti-aging nanocomposition has a smaller particle size, a uniform size distribution, and high stability.

The multi-effect anti-aging co-delivery nanocomposition suitable for microneedles provided by the present invention comprises 0.1 to 0.5% of a preservative, measured in percentage by mass. In the present invention, the preservative preferably comprises p-hydroxyacetophenone.

Measured by mass percentage, the multi-effect anti-aging co-delivery nanocomposition suitable for microneedle provided by the present invention includes a balance of water. In the present invention, the water is preferably distilled water or purified water.

In the present invention, the particle size of the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles is preferably 10 to 500 nm, more preferably 10 to 200 nm. The multi-effect anti-aging co-delivery nanocomposition within this particle size range has higher stability.

The present invention provides a method for preparing a multi-effect anti-aging co-delivery nanocomposition suitable for microneedles as described in the above technical solution, comprising the following steps:

The composite active factor, the emulsifier, the co-emulsifier, the preservative and water are mixed to obtain a mixture;

The mixed material is subjected to micronization treatment to obtain a micron-sized dispersion;

The micron-scale dispersion is nano-processed to obtain the multi-effect anti-aging co-delivery nano-composition suitable for microneedles.

The invention mixes the composite active factor, the emulsifier, the auxiliary emulsifier, the preservative and water to obtain the mixture.

In the present invention, the mixing temperature is preferably 30 to 50° C., more preferably 45° C.; the mixing preferably includes the following steps:

(1) heating the polydeoxyribonucleotide, part of the co-emulsifier and part of the water for a first mixing to obtain phase A, wherein the part of the co-emulsifier is preferably butanediol; the temperature of the first mixing is preferably 45° C.; and the heating is preferably water bath heating;

(2) heating acetyl hexapeptide-8, palmitoyl pentapeptide-4, another part of the co-emulsifier and another part of water for a second mixing to obtain phase B; the other part of the co-emulsifier is preferably propylene glycol; the temperature of the second mixing is preferably 45° C.; and the heating is preferably water bath heating;

(3) heating the dihydroavenate alkaloids, the emulsifier, the remaining co-emulsifier and the remaining water for a third mixing to obtain phase C; the remaining co-emulsifier is preferably hexylene glycol; the temperature of the third mixing is preferably 45° C.; and the heating is preferably water bath heating;

(4) heating ergothioneine and the preservative for a fourth mixing to obtain phase D; the temperature of the fourth mixing is preferably 45° C.; and the heating is preferably water bath heating;

(5) The phase B and the phase C are mixed for the fifth time, and the obtained mixed phase is mixed for the sixth time with the phase A and the phase D. The temperature of the fifth mixing is preferably 45° C., and the fifth mixing is preferably performed under stirring, and the stirring speed is preferably 500 rpm.

After obtaining the mixed material, the present invention performs micronization treatment on the mixed material to obtain a micron-grade dispersion.

In the present invention, the micronization treatment is preferably shear mixing. The rotation speed of the shear mixing is preferably 4000-30000 rpm, more preferably 10000-30000 rpm, and the time is preferably 1-20 min, more preferably 5-15 min.

After obtaining the micron-sized dispersion, the present invention performs nano-processing on the micron-sized dispersion to obtain the multi-effect anti-aging co-delivery nano-composition suitable for microneedles. In the present invention, the nano-processing is preferably a high-pressure homogenization process or a high-pressure microfluidization process; the pressure of the high-pressure homogenization process is preferably 300-1600 bar, more preferably 500-1500 bar, the temperature is preferably 20-70°C, more preferably 40-60°C, and the number of cycles is preferably 1-10 times, more preferably 2-6 times; the pressure of the high-pressure microfluidization process is preferably 3000-16000 psi, more preferably 5000-15000 psi, the temperature is preferably 20-70°C, more preferably 40-60°C, and the number of cycles is preferably 1-10 times, more preferably 2-6 times.

The present invention uses a method combining micronization and nanoization to package and deliver a variety of scientifically matched functional ingredients using emulsifiers and co-emulsifiers. Based on the structure and characteristics of nanocarriers, the multi-effect anti-aging nanocomposition obtained by package nanoization has good water dispersibility, improves the solubility of some water-insoluble functional ingredients, and the irritation of some functional substances, so that each functional ingredient can reach a sufficient concentration in the product to smoothly exert the corresponding functional effect. Thanks to the structural characteristics of the nanocarrier, the product can be applied to the microneedle introduction technology, promote the efficient penetration of the functional ingredients through the stratum corneum of the skin, and the multi-effect anti-aging nanocomposition has good skin permeability and retention performance, effectively promotes the functional ingredients to further diffuse after penetrating the stratum corneum, reach various target sites, and enrich at high concentration, retain for a long time, slow release and controlled release, and finally be taken up by tissue cells, improve its bioavailability and overall skin anti-aging effect, and prolong the action time.

The present invention provides the use of the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles described in the above technical solution or the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles obtained by the preparation method described in the above technical solution in the preparation of anti-skin aging cosmetics. The multi-effect anti-aging co-delivery nanocomposition suitable for microneedles described in the present invention can be used to prepare cosmetics with anti-aging, anti-wrinkle, repair, whitening and other effects. The form of the cosmetics includes but is not limited to toner, cream, lotion, essence and gel. The multi-effect anti-aging co-delivery nanocomposition suitable for microneedles described in the present invention is easily soluble in water and can be used in combination with microneedle introduction technology.

In the present invention, the anti-aging cosmetic preferably comprises the multi-effect anti-aging co-delivery nanocomposition suitable for microneedles as described in the above technical solution and a blank essence. The mass percentage of the anti-aging nanocomposition added to the cosmetic is preferably 0.1-30%.

In the present invention, the blank essence preferably comprises the following components in percentage by weight: 1-3% glycerol, 0.1-0.3% carbomer, 0.1-0.2% xanthan gum, 0.1-0.5% phenoxyethanol and the balance water _; more preferably 2% glycerol, 0.3% carbomer, 0.1% xanthan gum, 0.5% phenoxyethanol and the balance water.

In the present invention, the anti-aging nanocomposition can be used in combination with microneedle introduction technology. Microneedles are a new type of physical penetration-promoting technology. The microneedle device arranges hundreds of microneedles with a height of 10-2000 μm and a width of 10-50 μm on the base. The length, size and shape of the microneedles can be precisely regulated and individually designed according to needs. The microneedles can pass through the stratum corneum in a directional manner without touching the pain nerves, forming an instant delivery channel on the surface of the skin, so that the efficacy substance reaches the specified depth of the skin and is directly placed in the epidermis or upper dermis. The channel can be automatically restored within 20-30 minutes after the microneedle action. Microneedle technology can play a role in safely and effectively promoting the transdermal penetration of efficacy ingredients, and provides a new research idea for improving the efficacy of cosmetics. The present invention encapsulates multiple efficacy ingredients with different anti-aging mechanisms in a nanocarrier, and the integrity and biological activity of the ingredient structure are still maintained after encapsulation. At the same time, the light and thermal stability of the efficacy ingredients can be improved, the solubility and water dispersibility of the insoluble active ingredients can be improved, and the irritation of some active ingredients can be reduced. Thanks to the characteristics and advantages of nano-carriers, the combination of multi-effect anti-aging nano-compositions and microneedle introduction technology provides a basis for the rapid penetration of active ingredients through the stratum corneum of the skin and directly reaching the target site. In addition, nano-carriers have small particle sizes and large specific surface areas, and exhibit sustained-release properties and high bioadhesion in transdermal delivery, which can effectively promote the penetration and diffusion of high-content active ingredients in the target site, retain the effects, prolong the duration of action, improve their bioavailability, and enhance the overall anti-aging effect of the skin.

In order to further illustrate the present invention, an anti-aging nanocomposition provided by the present invention and its preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

Example 1

By mass percentage, 1% PDRN, 1% butanediol and 30% water were mixed and dissolved in a 45°C water bath to obtain phase A;

Mix 0.005% acetyl hexapeptide-8, 0.005% palmitoyl pentapeptide-4, 2.5% 1,3-propylene glycol and 23.99% water, heat and dissolve in a 45°C water bath to obtain phase B;

Mix 2.5% dihydroavenate alkaloids, 2.5% lecithin, 2.5% PEG-40 hydrogenated castor oil, 2.5% 1,2-hexanediol and 30% water, and heat in a water bath at 45°C to dissolve to obtain phase C;

Mix 1% ergothioneine extract with 0.5% p-hydroxyacetophenone, heat in a 45°C water bath to dissolve, and obtain phase D;

Under stirring conditions of 45°C and 500 rpm, the above phase B and phase C are mixed, and then phase A and phase D are added in sequence, mixed, and then subjected to high-speed shear treatment at a speed of 10,000 rpm for 8 minutes to obtain a micron-sized dispersion;

The micron-sized dispersion was subjected to high-pressure homogenization treatment at a temperature of 45° C. and a pressure of 700 bar, and the process was repeated twice. The mixture was cooled to room temperature to obtain a multi-effect anti-aging co-transport nanocomposite.

Example 2

The formula is the same as that of Example 1, except that after obtaining the micron-sized dispersion according to the steps of Example 1, high-pressure microfluidization treatment is carried out under the conditions of a pressure of 8000 psi and a temperature of 45°C, and the process is repeated 3 times, and the mixture is cooled to room temperature to obtain a multi-effect anti-aging co-delivery nanocomposite.

Example 3-15

The formula is shown in Table 1, and the preparation method is the same as that of Example 1, except that some ingredients and contents in the formula are slightly changed.

Comparative Examples 1-15

The formula is shown in Table 2. The preparation method is the same as that of Example 1, except that some ingredients and contents in the formula are greatly changed.

Comparative Example 16

All the effective ingredients in the formula of Example 1 and 2% dimethyl sulfoxide were added to the remaining amount of purified water, and ultrasonic dissolution was performed at 45° C. to obtain a free multi-effect anti-aging composition.

Comparative Example 17

The preparation method is the same as that of Example 1, except that the effective ingredient in the formula is only 2.5% dihydroavenate alkaloids, and a single-package nanocarrier containing 2.5% dihydroavenate alkaloids is obtained.

Comparative Example 18

The preparation method is the same as that in Example 1, except that the effective ingredients in the formula are 1% PDRN, 1% ergothioneine, 0.005% acetyl hexapeptide-8 and 0.005% palmitoyl pentapeptide-4 raw materials, and a nanocomposite containing 1% PDRN, 1% ergothioneine, 0.005% acetyl hexapeptide-8 and 0.005% palmitoyl pentapeptide-4 is obtained.

Table 1 Types and mass percentages of raw materials used in each embodiment

Table 2 Types and mass percentages of raw materials used in each comparative example

Application Example 1

Preparation of sample serum for efficacy testing

2.0% glycerol, 0.3% carbomer, 0.1% xanthan gum, 0.5% phenoxyethanol, and 97.2% purified water were stirred and dissolved at room temperature to obtain a blank essence solution. Each group of test samples was prepared according to the table below.

Table 3 Test essence composition

Control group 1 Blank Essence Control group 2 Blank essence + 20% sample obtained in comparative example 16 Sample set Blank essence + 20% nanocarrier obtained in Example 1 Microneedle Kit Blank essence + 20% nanocarrier obtained in Example 1

Test Example 1 Stability Test

The multi-effect anti-aging co-delivery nanocompositions prepared in Examples 1-15 and Comparative Examples 1-10 were placed in a sealed container and stored at room temperature and 45°C for 3 months respectively, and freeze-thaw experiments were carried out. The appearance of the samples under various conditions before and after storage was checked, the particle size of the nanocompositions under various conditions before and after storage was tested, and the stability of the nanocompositions was comprehensively evaluated.

Freeze-thaw experiment: Place the sample in a sealed container, keep it at -20℃ for 48 hours, then place it at 45℃ for 48 hours, repeat twice, and observe the changes in the sample.

The particle size of the nanocomposite is measured by ZetasizerNano-ZS90 laser particle size analyzer. Take an appropriate amount of the nanocomposite and dilute it 50 times with ultrapure water so that the average light intensity of the sample liquid is 200-300. The particle size measurement angle of the laser particle size analyzer is 90° and the test temperature is 25°C.

Table 4 Anti-aging nanocomposite stability results

It can be seen from the data in Table 4 that the anti-aging nanocompositions (Examples 1-15) prepared under the conditions of the functional substances, emulsifiers, co-emulsifiers and corresponding contents of the present invention have uniform appearance, without stratification or precipitation. On the contrary, the nanocompositions cannot be formed (Comparative Examples 2, 4, 5-9), or the stability is extremely poor, and stratification, precipitation and the like are prone to occur (Comparative Examples 1, 3, 10). With the preferred types and content conditions of functional substances, emulsifiers, and co-emulsifiers, the particle size of the multi-effect nanocomposition obtained is lower, and the particle size of the nanocomposition prepared under non-preferred conditions is relatively larger, but the particle size is maintained within the range of 10 to 500 nm. After being observed at room temperature and 45°C for 3 months, as well as after freeze-thaw experiments, the multi-effect anti-aging co-delivery nanocompositions of each embodiment are still stable and meet the requirements of practical applications.

Test Example 2 Cell Safety Evaluation

Test method: CCK-8 method was used for testing. HSF cells (human skin fibroblasts) and HaCaT cells (human keratinocytes) were inoculated into 96-well plates respectively. After culturing for 24 h at 5% CO ₂ and 37° C., 100 μL of DMEM complete medium (PDRN concentrations of 50, 100, 200, 400 and 800 μg/mL) containing the multi-effect anti-aging nanocomposition described in Example 1 and the free anti-aging composition described in Comparative Example 16 were added to the 96-well plates of the two cells respectively. After further culturing for 24 h, the cell survival rate was measured by CCK-8 method. The control group was treated with only 100 μL of DMEM complete medium.

As shown in Figure 1 (A), within the concentration range of 50-400 μg/mL, the HaCaT cell activity of the free composition and the co-delivered nanocomposition was above 80%, with no cytotoxicity. As shown in Figure 1 (B), within the concentration range of 50-800 μg/mL, the HSF cell activity of the free composition and the co-delivered nanocomposition was above 80%, with no cytotoxicity. The above results show that the multi-effect anti-aging co-delivered nanocomposition is safe.

Test Example 3 Evaluation of the irritation of chicken embryo chorioallantoic membrane

The multi-effect anti-aging nanocomposition described in Examples 1-15 was diluted 10 times with physiological saline, 0.2 mL of the sample was dripped on the surface of the chorioallantoic membrane, and the changes in the blood vessels were observed within 5 minutes, and the initial time of congestion, bleeding and coagulation of the chorioallantoic membrane blood vessels was recorded, and the stimulation score IS was calculated.

IS＝[(301-secH)×5+(301-secL)×7+(301-secC)×9]/300

Wherein, secH is the initial time of surface congestion, s; secL is the initial time of surface bleeding, s; secC is the initial time of surface coagulation, s.

The irritation was graded according to the size of the mean value, where 0-0.9, 1.0-4.9, 5.0-8.9 and 9-21.0 were classified as no irritation, slight irritation, moderate irritation and severe irritation, respectively.

The final results of Examples 1-15 of the present invention are similar, and the irritation evaluation results of the multi-effect anti-aging co-delivery nanocomposition of Example 1 are shown in Figure 2. When the nanocomposition diluted 10 times was in contact with the chicken embryo chorioallantoic membrane for 300s, there was no bleeding, vascular melting, or coagulation in the capillaries, and the reaction integral was 0.7, indicating that the multi-effect anti-aging co-delivery nanocomposition diluted 10 times was safe and non-irritating.

Test Example 4 Patch Test

45 subjects were selected in the experiment and their reactions were recorded. 10% of the multi-effect anti-aging nanocomposite filter paper described in Examples 1 to 15 was placed in a spot tester, and the normal hole was used as a blank control. The sample and the blank control were both attached to the curved side of the subject's forearm, and gently pressed with the palm to evenly apply it to the skin for 24 hours. After removing the spot tester, wait for 30 minutes and observe the skin reaction after the indentation disappears. Observe the skin reaction again 24 hours and 48 hours after removing the spot tester.

The results showed that none of the 45 subjects developed light red spots, erythema, edematous erythema, significant redness and swelling, infiltration or papules, and papules or blisters, etc., indicating that the multi-effect anti-aging nano-composition in this trial is non-irritating to human skin.

Test Example 5: Determination of Cumulative Transmission Amount and Skin Retention Amount

The vertical Franz diffusion cell method was used to conduct an in vitro pig skin transdermal experiment. Take 0.5 g of each of the 5% multi-effect anti-aging nanocomposite essence in the sample group of Application Example 1 and the 5% free multi-effect anti-aging essence in the control group 2 in the supply chamber, and use a microneedle vertical surface to continuously inject 0.5 g of the essence in Application Example 1. PBS (pH 7.4) was used as the receiving liquid, and HPLC analysis was used to calculate the cumulative permeation amount of specific drugs per unit area at different times. After 24 hours, the skin homogenate supernatant was taken for HPLC analysis to calculate the skin retention amount per unit area of specific functional ingredients. The cumulative skin permeation amount of functional ingredients at different sampling times was calculated according to the following formula.

Where Qn is the cumulative drug permeation, Cn is the drug concentration measured at the nth time, Ci is the drug concentration measured at the ith point, V0 is the volume of the diffusion cell, i.e. the amount of release medium added, and Vi is the amount of each sampling. The cumulative permeation per unit area Q = Qn/S, where S is the area of the diffusion cell, 2.27 cm ² .

As shown in Figure 3, the 24h cumulative permeation of dihydroavenate alkaloids per unit area of skin in the free composition, co-delivery nanocomposition and microneedle delivery group were 8.65μg/ ^cm2 , 29.42μg/ ^cm2 and 82.37μg/ ^cm2 , respectively, and the skin retention was 24.36μg/ ^cm2 , 75.26μg/ ^cm2 and 95.34μg/ ^cm2 , respectively. Compared with the free group, the cumulative permeation of dihydroavenate alkaloids per unit area of skin in the co-delivery group and microneedle group increased by 240.1% and 852.3%, respectively, and the skin retention of dihydroavenate alkaloids increased by 208.9% and 291.4%, respectively. This shows that the active ingredients can effectively promote the transdermal absorption and retention of the active ingredients in the skin after being encapsulated by nanocarriers, improve their skin bioavailability, and are more conducive to transdermal absorption after microneedle treatment.

Test Example 6 Observation of skin penetration behavior using laser confocal microscopy

The test experimental device is the same as the above transdermal experimental device. The 5% multi-effect anti-aging co-delivered nanocomposite essence in the sample group of Application Example 1 labeled with Rhodamine B (RhoB) and the 5% free anti-aging composition essence in the control group 2 are placed in the supply chamber. At the same time, the essence in Application Example 1 is continuously injected vertically on the microneedle surface. PBS is used as the receiving liquid. Stir and diffuse at 37°C. After 2h and 4h, the residual sample on the skin is gently wiped off, the skin in the target area is removed, and the skin is rinsed again. After thorough cleaning, the residual moisture is wiped off. The sample is frozen and sectioned, and the section is observed by laser confocal microscopy.

As can be seen from Figure 4, the fluorescence penetration depth of the skin increases with time. The results show that in the same period of time, the fluorescence intensity of the RhoB-labeled multi-effect anti-aging co-delivery nanocomposite essence in the skin is significantly stronger than that of the free form, and the multi-effect anti-aging co-delivery nanocomposite essence can enter the active epidermis and dermis. After microneedle delivery, the fluorescence intensity and penetration depth of the skin are further enhanced, reaching the deep dermis, indicating that microneedles combined with nanocarriers can promote the rapid penetration of the encapsulated ingredients into the skin, thereby exerting a better anti-aging effect.

Test Example 7 Study on the Cell Proliferation Effect

HaCaT and HSF cells were inoculated in 96-well plates, 100 μL per well, and cultured for 24 h at 5% CO ₂ and 37° C. 100 μL of DMEM complete medium containing the multi-effect anti-aging nanocomposition of Example 1 and the free multi-effect anti-aging composition of Comparative Example 16 (at concentrations of 100, 200 and 400 μg/mL) was added to each well, and only 100 μL of DMEM complete medium was added to the control group. After continued culture for 48 h, the cell proliferation rate was measured by CCK-8 method.

As shown in (A) of Figure 5, compared with the control group, the multi-effect anti-aging nanocomposition has a proliferation-promoting effect on HaCaT cells at a concentration of 200 and 400 μg/mL (P<0.01 or P<0.05). Compared with the free composition, the multi-effect anti-aging nanocomposition has a proliferation-promoting effect on HaCaT cells at a concentration of 400 μg/mL (P<0.01). As shown in (B) of Figure 5, compared with the control group and the free composition of the same concentration, the multi-effect anti-aging nanocomposition has a proliferation-promoting effect on HSF cells at a concentration of 400 μg/mL (P<0.01 or P<0.05). The above data prove that the multi-effect anti-aging nanocomposition can effectively combat skin aging by promoting the proliferation and renewal of HaCaT and HSF cells, and the effect of the anti-aging nanocomposition obtained by nanocarrier technology on promoting cell proliferation is further improved. Among them, the legend of "100μg/mL" in Figure 5 corresponds to the bar chart filled with "black dots", the legend of "200μg/mL" corresponds to the bar chart filled with "grids", and the legend of "400μg/mL" corresponds to the bar chart filled with "twill" (the same as in the figure below).

Test Example 8 Study on Cellular Antioxidant Effect

HSF cells were inoculated in a 24-well plate, 500 μL per well, and cultured for 24 h at 5% CO ₂ and 37°C. They were divided into a control group, a model group, and a drug-treated group. The control group was added with only DMEM complete medium, the model group was added with DMEM complete medium containing 0.8 mmol/L H ₂ O ₂ , and the drug-treated group was added with DMEM complete medium containing 0.8 mmol/L H ₂ O ₂ and the multi-effect anti-aging co-delivery nanocomposition of Example 1, and DMEM complete medium of free multi-effect anti-aging composition of Comparative Example 16 with different concentrations (concentrations of 100, 200, and 400 μg/mL), and cultured together for 24 h. Cell activity was determined by CCK8, and SOD activity, GSH content, and MDA content in the cell supernatant were tested by a kit.

As shown in Figure 6 (A), the cell survival rate of HSF cells in the model group decreased to 40.95% after injury by _0.8mmol / _LH2O2 , and the cell injury effect was obvious, indicating that _{the H2O2} oxidative damage HaCaT cell model was successfully established. Compared with the model group, the anti-aging co-delivery nanocomposition at a concentration of 200 and 400μg/mL can significantly increase the cell activity of oxidative damage (P<0.01). Compared with the free anti-aging composition, the anti-aging co-delivery nanocomposition at a concentration of 200 and 400μg/mL can significantly increase the cell activity of oxidative damage (P<0.05 or P<0.01).

As shown in Figure 6 (B), the SOD activity of HSF cells in the model group after injury by _0.8mmol / _LH2O2 was 19.54U/mgprot, which was significantly lower than that in the control group. Compared with the model group, the anti-aging co-delivery nanocomposition significantly increased the SOD activity (P<0.01). Compared with the free anti-aging composition, the anti-aging co-delivery nanocomposition significantly increased the SOD activity of oxidatively damaged cells (P<0.05 or P<0.01).

As shown in Figure 6 (C), the GSH content of HSF cells in the model group after injury by _0.8mmol / _LH2O2 was 7.37mg/Lt, which was significantly lower than that in the control group. Compared with the model group, the GSH content of the free anti-aging composition group and the anti-aging co-delivery nanocomposition group increased significantly (P<0.05 or P<0.01). Compared with the free anti-aging composition, the anti-aging co-delivery nanocomposition at a concentration of 400μg/mL can significantly increase the GSH content of oxidatively damaged cells (P<0.01).

As shown in Figure 6 (D), the MDA content of HSF cells in the model group increased to 10.98nmol/mL after injury by 0.8mmol/LH ₂ O ₂ , which was significantly higher than that in the control group. Compared with the model group, the anti-aging co-delivery nanocomposition at a concentration of 200 and 400μg/mL significantly reduced the MDA content (P<0.01).

The above experimental results show that the anti-aging co-delivery nanocomposition can increase the SOD activity and GSH content, and can effectively reduce cell oxidative damage. Compared with the same dose of free anti-aging composition, the anti-aging co-delivery nanocomposition has a better skin antioxidant effect.

Test Example 9 Cell Anti-aging Effect Detection

HSF cells were inoculated in 24-well plates, 500 μL per well, and cultured for 24 h at 5% CO ₂ and 37° C. The experiment was divided into a blank control group, a model group, and a sample group (Example 1, Comparative Example 16, Comparative Example 17, Comparative Example 18). The blank control group was added with complete culture medium, the model group was added with 10 mg/mL D-galactose to induce the construction of an aging model, the sample group was added with the culture medium dilution of the composition prepared in Example 1, Comparative Example 16, Comparative Example 17 and Comparative Example 18 diluted 1000 times, and then added with 10 mg/mL D-galactose, placed in a _CO2 incubator (37°C, 5% _CO2 ) and incubated for 48 hours, according to the instructions of the kit, each group of cells was washed 3 times with 0.01 mol/L PBS, 1 mL of β-galactosidase staining fixative was added to each well, fixed at room temperature for 15 minutes, washed 3 times with PBS, 1 mL of β-galactosidase staining working solution was added, incubated at 37°C overnight, and the staining results were observed under a microscope. Three fields of view were randomly selected for each group, the number of positive cells in 100 cells was counted, and the positive cell rate was calculated. The results are shown in Figure 7.

As shown in Figure 7, after HSF cells were modeled with 10 mg/mL D-galactose, the positive cell rate increased significantly compared with the blank control group. Compared with the model group, the positive cell rates of Comparative Example 16, Comparative Example 18 and Example 1 were significantly reduced (P<0.01), while the positive cell rate of Comparative Example 17 had no significant change (P>0.05). Compared with Comparative Example 16, the positive cell rate of Example 1 was significantly reduced (P<0.01), and compared with Comparative Example 18, the positive cell rate of Example 1 was significantly reduced (P<0.01). The results show that the single-package nanocarrier (Comparative Example 17) with only dihydroavenous alkaloids has no anti-aging effect, the positive cell rate of the nanocomposite (Comparative Example 18) with four raw materials of polydeoxyribonucleotides, acetyl hexapeptide-8, palmitoyl pentapeptide-4 and ergothioneine is 41%, and the positive cell rate of the nanocomposite (Example 1) with five raw materials of polydeoxyribonucleotides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, ergothioneine and dihydroavenous alkaloids is 17%, and the positive cell rate of Example 1 is reduced by 141% compared with Comparative Example 18. The free composition (Comparative Example 16) with five raw materials has a positive cell rate of 45%, and the positive cell rate of Example 1 is reduced by 165% compared with Comparative Example 16. It is proven that the combination of dihydrooat alkaloids with polydeoxyribonucleotides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, and ergothioneine can promote the effects of anti-aging ingredients, and the anti-aging effect of the encapsulated nanocomposite is greatly enhanced compared to the free ingredients.

Test Example 10: Study on Cellular Anti-aging Factors

HSF cells were seeded in a 24-well plate, 500 μL per well, and cultured for 24 h at 5% CO ₂ and 37°C. The control group was only added with DMEM complete medium, the model group was irradiated with 20 J/cm ² of UVA, and the drug-treated group was added with DMEM complete medium containing the multi-effect anti-aging co-delivery nanocomposition of Example 1 and DMEM complete medium containing different concentrations of free multi-effect anti-aging composition of Comparative Example 16 (concentrations of 100, 200 and 400 μg/mL), respectively, and the cells were irradiated with 20 J/cm ² of UVA after pretreatment for 1 h, placed in a CO ₂ incubator for incubation for 24 h, and the supernatant was taken to test the content of type I collagen (ColⅠ), type III collagen (ColⅢ) and hyaluronic acid (HA) using an Elisa kit.

As shown in Figure 8, after HSF was irradiated with UVA at 20J/ ^cm2 , the secretion of ColⅠ, ColⅢ, and HA by cells was significantly reduced. As shown in Figure 8A, compared with the model group, the free anti-aging composition and the anti-aging co-delivery nanocomposition can significantly increase the secretion of ColⅠ (P<0.05 or P<0.01). Compared with the free anti-aging composition, the anti-aging co-delivery nanocomposition can significantly increase the secretion of ColⅠ when the concentration is 200 and 400μg/mL (P<0.05 or P<0.01). As shown in Figure 8 (B), compared with the model group, the anti-aging co-delivery nanocomposition can significantly increase the secretion of ColⅢ when the concentration is 100, 200, and 400μg/mL (P<0.01). Compared with the free anti-aging composition, the anti-aging co-delivery nanocomposition can significantly increase the secretion of ColⅢ (P<0.05 or P<0.01). As shown in Figure 8C, compared with the model group, the free component concentration and the anti-aging co-delivery nanocomposition can significantly increase the HA content (P<0.05 or P<0.01). Compared with the free anti-aging composition, the anti-aging co-delivery nanocomposition concentration of 200 and 400 μg/mL can significantly increase the HA secretion (P<0.01). The above experimental results show that the anti-aging co-delivery nanocomposition can effectively counteract the reduction of collagen and hyaluronic acid caused by UVA irradiation, and has a significant anti-skin aging effect. Compared with the same dose of free anti-aging composition, the anti-aging co-delivery nanocomposition has better skin anti-aging effect.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (10)

1. A multi-effect anti-aging co-delivery nano composition suitable for microneedles, characterized in that it comprises the following components in mass percentage: Compound active factor 1-10%, emulsifier 2-10%, co-emulsifier 2-12%, preservative 0.1-0.5% and the rest water; The composite active factor includes polydeoxyribonucleotide, acetyl hexapeptide-8, palmitoyl pentapeptide-4, dihydroavenous alkaloids and ergothioneine. 2. The multi-effect anti-aging co-transport nano composition suitable for microneedles according to claim 1, wherein the mass of the multi-effect anti-aging co-transport nano composition suitable for microneedles is 100% In total, the composite active factor includes: polydeoxyribonucleotide 0.1-2%, acetyl hexapeptide-80.001-0.01%, palmitoyl pentapeptide-40.001-0.1%, dihydroavenous alkaloid 1-5% and ergothioneine 0.1 to 2%. 3. The multi-effect anti-aging co-delivery nano composition suitable for microneedles according to claim 1, characterized in that the particle size of the multi-effect anti-aging co-transport nano composition suitable for microneedles is 10- 500nm. 4. The multi-effect anti-aging co-delivery nano composition suitable for microneedles according to claim 1, wherein the emulsifier comprises lecithin and/or polyoxyethylene hydrogenated castor oil. 5. The multi-effect anti-aging co-delivery nano composition suitable for microneedles according to claim 1 or 4, wherein the co-emulsifier comprises one or more of butanediol, hexanediol and propylene glycol kind. 6. The multi-effect anti-aging co-delivery nano composition suitable for microneedles according to claim 1, wherein the preservative comprises p-hydroxyacetophenone. 7. The preparation method of the multi-effect anti-aging co-delivery nano composition suitable for microneedles according to any one of claims 1 to 6, characterized in that it comprises the following steps: Mix the complex active factor, emulsifier, co-emulsifier, preservative and water to obtain a mixture; Micronizing the mixture to obtain a micron-scale dispersion; The micron-scale dispersion is subjected to nano-processing to obtain the multi-effect anti-aging co-delivery nano composition suitable for microneedles. 8 . The preparation method according to claim 7 , wherein the micronization treatment is shear mixing, the rotation speed of the shear mixing is 4000-30000 rpm, and the time is 1-20 min. 9. The preparation method according to claim 7, characterized in that, the nano-processing is high-pressure homogeneous treatment or high-pressure micro-jet treatment; The pressure of the high-pressure homogenization treatment is 300-1600 bar, the temperature is 20-70°C, and the number of cycles is 1-10 times; The pressure of the high-pressure micro-jet treatment is 3000-16000 psi, the temperature is 20-70° C., and the number of cycles is 1-10 times. 10. The multi-effect anti-aging co-delivery nano composition suitable for microneedles according to any one of claims 1-6 or the multi-effect anti-aging co-delivery nanocomposite suitable for microneedles obtained by the preparation method described in any one of claims 7-9. Application of anti-aging co-delivery nanocomposition in the preparation of anti-aging skin cosmetics.