An enhanced transdermal absorption cosmetic patch containing high-efficiency active polydeoxyribonucleotide PDRN (DNA sodium) and a low-temperature preparation method thereof

Abstract

The application provides an enhanced transdermal absorption cosmetic patch containing high-efficiency active polydeoxyribonucleotide PDRN (DNA sodium) and a low-temperature preparation method thereof. The cosmetic patch comprises, from outside to inside, an outer release backing layer, a slow-release core layer, a quick-release core layer and a soluble hollow microneedle array layer. The outer release backing layer is a PET / aluminum foil / polyethylene composite film, and the mass ratio of the three is (80-90):(0.5-2):(0.1-0.5). The PDRN is efficiently delivered transdermally, has long-term effect, high activity retention and excellent biocompatibility, and significantly improves the transdermal efficiency and use safety of the cosmetic patch.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an enhanced transdermal absorption cosmetic patch containing highly active polydeoxyribonucleotide PDRN (sodium DNA) and its low-temperature preparation method. Background Technology

[0002] With the continuous increase in consumer demand for anti-aging beauty products, beauty products containing polydeoxyribonucleotides (PDRN, also known as sodium DNA) have become an important research direction in the field of anti-aging beauty due to their core effects such as promoting skin cell repair, stimulating collagen production, and improving skin elasticity. Currently, transdermal delivery technologies for PDRN mainly focus on three categories: ordinary patches, sprays, and non-hollow microneedle arrays. Ordinary patches rely on passive penetration through the stratum corneum of the skin. Due to the barrier effect of the stratum corneum, the transdermal amount of PDRN is usually less than 5%, requiring frequent daily use to maintain basic effects. Although spray products are convenient to use, the active ingredients are easily lost with evaporation and can only act on the surface of the skin, making it difficult to reach the dermis to exert a repairing effect. Although non-hollow microneedles can physically break through the stratum corneum, they mostly use a single sodium hyaluronate substrate, which has problems such as low drug loading (single needle loading < 0.1 mg) and insufficient mechanical strength (breaking strength < 0.2 N). They are prone to breakage and residue during puncture and lack targeted permeation-enhancing design, making it impossible to further improve the deep delivery efficiency of PDRN.

[0003] Current technologies also face the dual bottlenecks of poor release synergy and insufficient activity stability. On the one hand, while traditional sustained-release gel products can prolong the action time of PDRN, their onset of action is slow (usually reaching an effective concentration after 24 hours), failing to meet the immediate repair needs of the skin after external stimulation. On the other hand, rapid-release products, due to the rapid release of active ingredients, are prone to excessively high local concentrations or loss within a short period, wasting active ingredients and making it difficult to achieve long-lasting anti-aging effects. On the other hand, as a biological macromolecule, PDRN is easily degraded by temperature and humidity during preparation and storage. Although traditional freeze-drying processes can improve stability, they often result in product water content >5% or activity retention rate <90%. Some products add irritating penetration enhancers such as propylene glycol and alcohol to improve transdermal efficiency, which can easily cause adverse reactions such as skin erythema and thickening of the stratum corneum, limiting their application to people with sensitive skin.

[0004] Addressing the industry pain points such as low transdermal efficiency, poor release synergy, insufficient mechanical strength, and unsatisfactory safety, existing technologies can no longer meet consumers' demands for "highly effective, long-lasting, and gentle" beauty products. There is an urgent need to develop an innovative transdermal absorption beauty patch. This patch must simultaneously solve three core problems: first, breaking through the stratum corneum barrier to increase the transdermal delivery of PDRN; second, achieving synergistic release of "immediate effect + long-lasting maintenance"; and third, ensuring the mechanical stability of microneedles and the biocompatibility of the product. Based on this, this technical solution, through four-layer structural innovation and process optimization, specifically addresses existing technological bottlenecks, providing a new technical pathway for the efficient transdermal delivery of PDRN. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of low transdermal efficiency, poor release synergy, insufficient activity stability and strong skin irritation of PDRN in the prior art, and to provide an enhanced transdermal absorption cosmetic patch containing highly active polydeoxyribonucleotide PDRN (sodium DNA) and its low-temperature preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An enhanced transdermal absorption cosmetic patch containing highly active polydeoxyribonucleotide PDRN (sodium DNA), wherein the cosmetic patch comprises, from the outside to the inside, an outer peel-off backing layer, a sustained-release core layer, an immediate-release core layer, and a soluble hollow microneedle array layer.

[0008] The outer peel-off backing layer is a PET / aluminum foil / polyethylene composite film with a thickness of 20-30μm;

[0009] The sustained-release core layer is composed of thermosensitive sodium hyaluronate-gelatin gel, PDRN, and acetyl hexapeptide-8, with a mass ratio of (80-90):(0.5-2):(0.1-0.5).

[0010] The immediate-release core layer is composed of β-cyclodextrin-PDRN inclusion complex, glutathione, and panthenol, with a mass ratio of (5-10):(0.2-0.8):(0.5-1.5).

[0011] The microneedles of the soluble hollow microneedle array layer have a hollow structure with an inner diameter of 50-80 μm and a length of 300-400 μm. The microneedle material is sodium hyaluronate-chitosan crosslinked material. PDRN-liposomes are loaded in the hollow cavity, with a PDRN-liposome loading amount of 0.1-0.3 mg / microneedle.

[0012] As a further aspect of the present invention, the PDRN is derived from salmon testis tissue and has an average molecular weight of 500-1000 kDa after enzymatic hydrolysis.

[0013] As a further embodiment of the present invention, in the sodium hyaluronate-chitosan crosslinked product of the soluble hollow microneedles, the mass ratio of sodium hyaluronate to chitosan is 3:1, the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the amount of crosslinking agent is 0.5-1% of the total mass of sodium hyaluronate-chitosan.

[0014] As a further embodiment of the present invention, in the thermosensitive sodium hyaluronate-gelatin gel of the sustained-release core layer, the mass ratio of sodium hyaluronate to gelatin is 4:1, the thermosensitive crosslinking agent is polyethylene glycol diglycidyl ether, the amount of crosslinking agent is 0.1-0.3% of the total mass of sodium hyaluronate-gelatin, and the gel undergoes a sol-gel phase transition at 32-37°C.

[0015] As a further embodiment of the present invention, in the β-cyclodextrin-PDRN inclusion complex of the immediate-release core layer, the mass ratio of β-cyclodextrin to PDRN is 2:1, the preparation temperature of the inclusion complex is 5-10℃, and the stirring time is 2-3h.

[0016] As a further embodiment of the present invention, the soluble hollow microneedle array layer also contains a transdermal penetration enhancer, which is a mixture of sodium lauroyl glutamate and phosphatidylcholine in a mass ratio of 3:2, and the total amount is 0.5-1.6% of the mass of the microneedle array layer.

[0017] A low-temperature preparation method for the enhanced transdermal absorption cosmetic patch as described above includes the following steps:

[0018] (1) Preparation of soluble hollow microneedles: Sodium hyaluronate-chitosan mixed solution is mixed with PDRN-liposome, injected into hollow microneedle mold, and injection molded at 0-5℃ and 0.1-0.2MPa pressure to obtain soluble hollow microneedle array layer;

[0019] (2) Preparation of immediate-release core layer: β-cyclodextrin and PDRN are dissolved in deionized water at a mass ratio of 2:1, stirred at 5-10℃ for 2-3h to form inclusion complex, dried in vacuum at 30℃ and 0.05mbar to form powder, and then mixed evenly with glutathione and panthenol to obtain immediate-release core layer;

[0020] (3) Preparation of sustained-release core layer: Sodium hyaluronate and gelatin are dissolved in deionized water at a mass ratio of 4:1, PDRN, acetyl hexapeptide-8 and thermosensitive crosslinking agent are added, and the mixture is stirred at 15-20℃ for 1-2 hours to obtain the sustained-release core layer.

[0021] (4) Lamination and freeze-drying: Lamination is carried out in the order of “external peel-off backing layer → sustained-release core layer → rapid-release core layer → soluble hollow microneedle array layer”, with a pressure of 0.08-0.1MPa and a temperature of 20-25℃. Then, the layers are placed in a freeze dryer and freeze-dried according to the parameters of “pre-freezing at -40℃ for 2h → sublimation drying at -35℃ for 8h → desorption drying at -30℃ for 4h” to obtain an enhanced transdermal absorption cosmetic patch.

[0022] As a further embodiment of the present invention, the preparation method of PDRN-liposomes in step (1) is as follows: PDRN is dissolved in deionized water to form an aqueous solution of 10-20 mg / mL, and mixed with a soybean lecithin-cholesterol organic phase dissolved in dichloromethane at a mass ratio of 5:1 and a volume ratio of 2:1. The mixture is then subjected to high-speed shearing at 8000-10000 rpm to form an oil-in-water emulsion, and then subjected to ultrasonic crushing at 280-300W for 8-10 min to obtain PDRN-liposomes with a particle size of 100-200 nm.

[0023] As a further embodiment of the present invention, in step (4), the vacuum degree of the freeze dryer is controlled to be 0.01-0.03 mbar, the water content of the freeze-dried product is ≤3%, and the PDRN activity retention rate is ≥95%.

[0024] As a further embodiment of the present invention, in step (4), during lamination, the coating thickness of the sustained-release core layer is 100-150 μm, the coating thickness of the immediate-release core layer is 50-80 μm, and the overall thickness after lamination is controlled to be 300-400 μm.

[0025] Compared with existing technologies, the advantages of this invention are:

[0026] 1. Significantly improved transdermal efficiency: Relying on the synergistic effect of microneedles, PDRN-liposomes and penetration enhancers, it can efficiently penetrate the stratum corneum of the skin. The cumulative transdermal amount in 24 hours reaches 385.4 μg / cm², which is 2.57 times that of the non-microneedle solution, completely solving the problem of low transdermal amount of traditional PDRN patches and only acting on the skin surface.

[0027] 2. Superior Synergistic Release: The innovative dual-core design of "rapid release + sustained release" ensures that the rapid release core layer has a release rate of 85.3% in 0.5 hours for quick energy replenishment, while the sustained release core layer has a release rate of 88.6% in 24 hours for long-lasting effects, avoiding the loss of active ingredients in a short time and precisely matching the skin's needs for "instant repair + long-lasting anti-aging".

[0028] 3. Safe and stable process: Volunteer patch tests showed no irritation, the microneedles have sufficient strength (breaking strength ≥0.28N) and are not prone to residue; the low-temperature process ensures PDRN activity ≥95% and water content ≤3%, and the parameters are controllable, making it suitable for sensitive skin and supporting industrial mass production. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0030] 1. Experimental Materials and Instruments

[0031] 1.1 Raw material specifications and sources

[0032] PDRN (sodium DNA): derived from salmon testis tissue, purchased from Shandong Fengjin Biotechnology Co., Ltd. (batch number: FJ-PDRN-202506).

[0033] Sodium hyaluronate: molecular weight 1000-2000kDa, purchased from Bloomage Biotechnology Co., Ltd. (product number: HA-1000K, batch number: 20250312).

[0034] Chitosan: Deacetylation degree ≥90%, purchased from Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd. (item number: CTS-90, batch number: 20250405). After being dissolved in 1% acetic acid solution, the viscosity was measured to be 200-300 mPa·s using a rotational viscometer (NDJ-8S) at 25℃.

[0035] Gelatin: Food grade, purchased from Rousselot Gelatin Ltd. (Item No.: G250, Lot No.: 20250218), Bloom strength 250g, for use in thermosensitive gel substrates.

[0036] Crosslinking agent: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 99% purity, purchased from Jier Biochemical (Shanghai) Co., Ltd. (item number: E1010, batch number: 20250508); polyethylene glycol diglycidyl ether (PEG-DGE, molecular weight 500), purchased from Sigma-Aldrich (item number: 401677, batch number: MKCG8210).

[0037] β-Cyclodextrin: analytical grade, purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Catalog No.: C8140, Batch No.: 20250320), purity ≥99%, with no impurity peaks detected by high performance liquid chromatography, used for PDRN inclusion to prepare immediate-release core layer.

[0038] Acetyl hexapeptide-8: 98% purity, purchased from Hangzhou Peptide Biotechnology Co., Ltd. (Catalog No.: Hexapeptide-8-98, Batch No.: 20250410), for synergistic anti-wrinkle purposes.

[0039] Glutathione (reduced form): purity ≥98%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (item number: G8140, batch number: 20250512), moisture content ≤0.5%, used for antioxidant synergy.

[0040] Panthenol: Cosmetic grade, purchased from BASF (product number: PANTHENOL-COS, batch number: 20250125), purity ≥99.5%, for skin repair.

[0041] Sodium lauroyl glutamate: cosmetic grade, purchased from Ajinomoto Investment Co., Ltd. (item number: AMISOFT LS-11, batch number: 20250308), purity ≥98%, surface tension (0.1% aqueous solution) 32mN / m, used as a transdermal penetration enhancer component.

[0042] Phosphatidylcholine: Soybean-derived, purity ≥95%, purchased from Avanti Polar Lipids (catalog number: 840051P, lot number: SL2345), used for penetration enhancer formulation and liposome preparation.

[0043] Soybean lecithin (PC90): Phospholipid content ≥90%, purchased from Novozymes Investment Co., Ltd. (product number: LECIPLEXPC90, batch number: 20250215), used for the preparation of PDRN liposomes.

[0044] Cholesterol: analytical grade, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Catalog No.: C104604, Batch No.: 20250420), purity ≥99%, used for liposome membrane stability regulation.

[0045] Other reagents: glacial acetic acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd., catalog number: 10000218), anhydrous ethanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd., catalog number: 10009218), dichloromethane (analytical grade, Sinopharm Chemical Reagent Co., Ltd., catalog number: 10006818), phosphate buffer (PBS, pH 7.4, self-prepared, the sodium dihydrogen phosphate and disodium hydrogen phosphate used were both analytical grade, Sinopharm).

[0046] 1.2 Main Instruments

[0047] Hollow microneedle injection molding machine: Customized model by Shenzhen Microneedle Technology Co., Ltd., model: MN-05, temperature control range 0-5℃ (accuracy ±0.5℃), pressure range 0-0.3MPa (accuracy ±0.01MPa), mold material PDMS, can be customized with hollow microneedle arrays with inner diameter 50-80μm and length 300-400μm (needle spacing 200μm, array size 1cm×1cm).

[0048] Freeze dryer: Beijing Sihuan Scientific Instrument Factory, Model: LGJ-100F, cold trap temperature ≤-80℃, vacuum range 0.01-0.1mbar (accuracy ±0.001mbar), shelf temperature range -50℃ to 50℃, used for freeze-drying of core layers and integral patches.

[0049] High-speed shear emulsifier: Shanghai Fluke Fluid Machinery Manufacturing Co., Ltd., Model: FA25, Speed ​​range: 0-12000rpm (accuracy ±10rpm), Shear head model: S25N-10G, used for the preparation of water-in-oil emulsions of PDRN-liposomes.

[0050] Ultrasonic disruptor: Ningbo Xinzhi Biotechnology Co., Ltd., Model: JY92-IIN, Power range 0-600W (adjustable), Frequency 20-25kHz, used for liposome particle size refinement to ensure a particle size of 100-200nm.

[0051] Franz diffusion cell: Logan Instruments, model RC-6, effective diffusion area 1.77cm², receiving chamber volume 5mL, temperature control range 25-45℃ (accuracy ±0.5℃), stirring speed 0-1000rpm (adjustable), used for transdermal absorption performance testing.

[0052] Universal testing machine: Instron 5967, model: Instron, maximum load 5kN, displacement accuracy ±0.001mm, loading rate range 0.001-10mm / min, used for microneedle mechanical strength testing.

[0053] High-performance liquid chromatograph: Agilent Technologies, model: 1260 Infinity II, equipped with G1314F UV detector (detection wavelength range 190-600nm), column model: TSKgel G4000PWXL (7.8mm×30cm), used for PDRN content and purity detection.

[0054] Dynamic light scattering (DLS): Malvern Instruments Ltd., Model: Zetasizer Nano ZS90, particle size detection range 0.3nm-10μm, used for PDRN-liposome particle size characterization.

[0055] Karl Fischer moisture analyzer: Mettler Toledo Instruments (Shanghai) Co., Ltd., Model: DL38, moisture measurement range 10μg-100%, accuracy ±0.001%, used for detecting the moisture content of freeze-dried products.

[0056] Rheometer: TA Instruments, Model: AR2000ex, Temperature control range -40℃ to 200℃ (accuracy ±0.1℃), Shear rate range 10⁻ 6 -10³s⁻¹, used for the determination of thermosensitive sol-gel phase transition temperature. Specific Implementation

[0057] 2.1 Example 1

[0058] Step 1: Preparation of soluble hollow microneedle array layer

[0059] 1. Preparation of PDRN-liposomes: Dissolve 1.5g of PDRN in 100mL Lmilli-Q ultrapure water (resistivity 18.2MΩ·cm) to prepare an aqueous solution of 15mg / mL. Stir magnetically (IKARCT basic model, 500rpm) until completely dissolved. Dissolve 5g of soybean lecithin and 1g of cholesterol (mass ratio 5:1) in 50mL dichloromethane and sonicate (20kHz, 100W) for 5min to prepare a homogeneous organic phase. Add the aqueous phase to organic phase at a volume ratio of 2:1 to a high-speed shear emulsifier and shear at 8000rpm for 5min to form a water-in-oil emulsion. Transfer to an ultrasonic disruptor and sonicate at 300W power and 20kHz frequency for 10min, maintaining a temperature of ≤25℃ using an ice-water bath during the process. Take 1 mL of emulsion, filter it through a 0.22 μm organic filter membrane, and use DLS to detect the particle size as 150 ± 20 nm and the zeta potential as -35 ± 5 mV to obtain PDRN-liposomes. Store at 4℃ in the dark for later use.

[0060] 2. Preparation of sodium hyaluronate-chitosan mixed solution: Take 3g of sodium hyaluronate and 1g of chitosan, add 100mL of 0.5% (v / v) acetic acid solution, stir at 300rpm (IKARW20digital) for 2h until completely dissolved; add 0.032g of EDC (0.8% of the total mass of sodium hyaluronate-chitosan), continue stirring at 25℃ for 1h, during which the pH is adjusted to 5.5±0.1 with 0.1mol / L NaOH to ensure sufficient cross-linking reaction, and obtain sodium hyaluronate-chitosan cross-linked solution.

[0061] 3. Microneedle Injection Molding: Mix the crosslinking liquid and PDRN-liposomes at a ratio of 9:1 (mass ratio) and stir magnetically (300 rpm) for 15 min until homogeneous. Inject the mixture into a PDMS hollow microneedle mold (inner diameter 60 μm, length 350 μm, needle array 1 cm × 1 cm), place it in a hollow microneedle injection molding machine, set the temperature to 3℃ and the pressure to 0.15 MPa, and let it stand for 30 min (ambient temperature 23℃) after injection molding for 10 s to allow the microneedles to form. After demolding, blow the residual liquid on the mold surface with nitrogen to obtain a soluble hollow microneedle array layer preform. Weigh each microneedle using a microbalance (Mettler-Toledo XP26, accuracy ±0.1 μg) and load it with 0.2 mg of PDRN-liposomes.

[0062] 4. Addition of transdermal penetration enhancer: Take 0.3g of sodium lauroyl glutamate and 0.2g of phosphatidylcholine, dissolve them in 5mL of 10% (v / v) ethanol solution, and sonicate (100W, 20kHz) for 5min until completely dissolved; use a pneumatic spray gun (Iwata W-71, Japan, spraying pressure 0.1MPa) to uniformly spray the solution onto the surface of the microneedle array, with a spraying amount of 1.0% of the mass of the microneedle layer (0.1g of penetration enhancer solution is sprayed per 10g microneedle layer), and air dry at room temperature (23℃) for 30min to obtain a soluble hollow microneedle array layer.

[0063] Step 2: Preparation of the immediate-release core layer

[0064] 1. Preparation of β-cyclodextrin-PDRN inclusion complex: 2g of β-cyclodextrin and 1g of PDRN were added to 50mL LiMilli-Q ultrapure water and stirred magnetically at 500rpm for 2.5h in a constant temperature water bath (HH-S6, accuracy ±0.5℃) at 5-10℃. The mixture was then transferred to a vacuum dryer (ChristAlpha1-4LDplus, 0.05mbar, 30℃) and dried for 12h to obtain a white granular inclusion complex. The complex was then pulverized for 5min using a planetary ball mill (500rpm) and passed through an 80-mesh standard sieve (0.18mm aperture) to obtain the inclusion complex powder. The inclusion rate was determined by HPLC to be 85±3%.

[0065] 2. Immediate-release core layer mixing: Weigh the materials (3.5g inclusion complex, 0.25g glutathione, 0.5g panthenol) according to the inclusion complex: glutathione: panthenol = 7:0.5:1.0 (mass ratio), add 1.5ml LiMilli-Q ultrapure water, and stir at 300rpm for 10min to form a paste; coat it onto PET film (12μm thick, Jiangyin Shenda Packaging) using a bar coater (RKPrintCoatInstrumentsK202, 60μm bar), and vacuum dry at 35℃ and 0.08mbar for 3h to obtain the immediate-release core layer (60±5μm thick). The PDRN content was measured to be 10.2±0.5mg / cm².

[0066] Step 3: Preparation of sustained-release core layer

[0067] 1. Preparation of thermosensitive gel substrate: Take 4g of sodium hyaluronate and 1g of gelatin, add 100ml of Lmilli-Q ultrapure water, stir in a constant temperature water bath at 60℃ (200rpm) for 1h until completely dissolved; cool down to 20℃, during which time the stirring speed is reduced to 100rpm to avoid the generation of bubbles, and obtain sodium hyaluronate-gelatin gel substrate.

[0068] 2. Mixing of active ingredients: Add 0.1g of PDRN and 0.03g of acetyl hexapeptide-8 to the gel substrate and stir at 300rpm for 10min until homogeneous; add 0.01g of polyethylene glycol diglycidyl ether (0.2% of the total mass of sodium hyaluronate-gelatin) and stir at 18℃ and 150rpm for 1.5h; determine the phase transition temperature using a rheometer: the heating rate is 1℃ / min, and the temperature at the intersection of the storage modulus G' and the loss modulus G'' is recorded as 35℃, which meets the requirements for sol-gel phase transition; coat the PDRN core layer (thickness 120±5μm) on a PET film using a 120μm wire bar coater to obtain a sustained-release core layer with a PDRN content of 0.8±0.1mg / cm².

[0069] Step 4: Lamination and freeze-drying

[0070] 1. Lamination: The layers are stacked in the following order: “outer peel backing layer (PET / aluminum foil / polyethylene composite film, model PET12 / AL7 / PE20, thickness 25μm, Jiangyin Shenda) → sustained-release core layer → immediate-release core layer → soluble hollow microneedle array layer”. The layers are placed in a flatbed laminator (Shanghai Ziming Machinery MN-100) with a pressure of 0.09MPa and a temperature of 23℃ for 30 minutes. After lamination, the layers are observed with an Olympus BX53 microscope (100x magnification) to confirm that there are no air bubbles in each layer and that the interfaces are tightly bonded.

[0071] 2. Freeze-drying: Transfer the laminated patch to a freeze dryer and process it according to the following procedure:

[0072] Pre-freezing stage: Maintain at -40℃ for 2 hours to ensure the patch is completely frozen (keep warm for 1 hour after the temperature drops to -40℃).

[0073] Sublimation drying stage: The vacuum degree is reduced to 0.02 mbar, the temperature is raised to -35℃ and maintained for 8 hours to remove free moisture;

[0074] Analysis and drying stage: Heat to -30℃ and maintain for 4 hours to remove bound water;

[0075] After freeze-drying, the water content of the sample was measured using a Karl Fischer moisture analyzer and found to be 2.2 ± 0.1%. 0.1 g of the freeze-dried patch was dissolved in 10 mL of PBS (pH 7.4), and the PDRN activity peak area was measured by HPLC. The ratio of the PDRN activity peak area to the initial activity peak area was 96.5 ± 0.5%, which means that the PDRN activity retention rate was 96.5%.

[0076] 2.2 Example 2

[0077] Step 1: Preparation of soluble hollow microneedle array layer

[0078] 1. Preparation of PDRN-liposomes: Dissolve 1.0 g of PDRN in 100 mL of Lilli-Q ultrapure water (resistivity 18.2 MΩ·cm) to prepare a 10 mg / mL aqueous solution. Stir magnetically (IKARCT basic model, 500 rpm) until completely dissolved. Dissolve 5 g of soybean lecithin and 1 g of cholesterol (mass ratio 5:1) in 50 mL of dichloromethane and sonicate (20 kHz, 100 W) for 5 min to prepare a homogeneous organic phase. Add the aqueous phase to organic phase at a volume ratio of 2:1 to a high-speed shear emulsifier and shear at 8000 rpm for 5 min to form a water-in-oil emulsion. Transfer to an ultrasonic disruptor and sonicate at 300 W power and 20 kHz frequency for 10 min, maintaining a temperature of ≤25℃ using an ice-water bath during the process. Take 1 mL of emulsion, filter it through a 0.22 μm organic filter membrane, and use DLS to detect the particle size as 100 ± 20 nm and the zeta potential as -35 ± 5 mV to obtain PDRN-liposomes. Store at 4℃ in the dark for later use.

[0079] 2. Preparation of sodium hyaluronate-chitosan mixed solution: Take 3g of sodium hyaluronate and 1g of chitosan, add 100mL of 0.5% (v / v) acetic acid solution, stir at 300rpm (IKARW20digital) for 2h until completely dissolved; add 0.02g of EDC (0.5% of the total mass of sodium hyaluronate-chitosan), continue stirring at 25℃ for 1h, during which the pH is adjusted to 5.5±0.1 with 0.1mol / L NaOH to ensure sufficient cross-linking reaction, and obtain sodium hyaluronate-chitosan cross-linking solution.

[0080] 3. Microneedle Injection Molding: Mix the crosslinking liquid and PDRN-liposomes at a ratio of 9:1 (mass ratio) and stir magnetically (300 rpm) for 15 min until homogeneous. Inject the mixture into a PDMS hollow microneedle mold (inner diameter 50 μm, length 300 μm, needle array 1 cm × 1 cm), place it in a hollow microneedle injection molding machine, set the temperature to 0℃ and the pressure to 0.1 MPa, and let it stand for 30 min (ambient temperature 23℃) after injection molding for 10 s to allow the microneedles to form. After demolding, blow the residual liquid on the mold surface with nitrogen to obtain a soluble hollow microneedle array preform. Weigh each microneedle using a microbalance (Mettler-Toledo XP26, accuracy ±0.1 μg) and load it with 0.1 mg of PDRN-liposomes (total load of 10 mg per 100 microneedles).

[0081] 4. Addition of transdermal penetration enhancer: Take 0.15g of sodium lauroyl glutamate and 0.1g of phosphatidylcholine, dissolve them in 2.5mL of 10% (v / v) ethanol solution, and sonicate (100W, 20kHz) for 5min until completely dissolved; use a pneumatic spray gun (Iwata W-71, Japan, spraying pressure 0.1MPa) to uniformly spray the solution onto the surface of the microneedle array, with a spraying amount of 0.5% of the mass of the microneedle layer (0.05g of penetration enhancer solution is sprayed per 10g microneedle layer), and air dry at room temperature (23℃) for 30min to obtain a soluble hollow microneedle array layer.

[0082] Step 2: Preparation of the immediate-release core layer

[0083] 1. Preparation of β-cyclodextrin-PDRN inclusion complex: 2g of β-cyclodextrin and 1g of PDRN were added to 50mL LiMilli-Q ultrapure water and stirred magnetically at 500rpm for 2h in a constant temperature water bath (HH-S6, accuracy ±0.5℃). The mixture was then transferred to a vacuum dryer (ChristAlpha1-4LDplus, 0.05mbar, 30℃) and dried for 12h to obtain a white blocky inclusion complex. The complex was then pulverized for 5min using a planetary ball mill (500rpm) and passed through an 80-mesh standard sieve (pore size 0.18mm) to obtain the inclusion complex powder. The inclusion rate was 82±3% as determined by HPLC.

[0084] 2. Immediate-release core layer mixing: Weigh the materials (2.5g inclusion complex, 0.1g glutathione, 0.25g panthenol) according to the inclusion complex: glutathione: panthenol = 5:0.2:0.5 (mass ratio), add 1ml Lmilli-Q ultrapure water, and stir at 300rpm for 10min to form a paste; coat it onto PET film (12μm thick, Jiangyin Shenda Packaging) using a bar coater (RKPrintCoatInstrumentsK202, 50μm bar), and vacuum dry at 35℃ and 0.08mbar for 3h to obtain the immediate-release core layer (50±5μm thick). The PDRN content was measured to be 8.1±0.4mg / cm².

[0085] Step 3: Preparation of sustained-release core layer

[0086] 1. Preparation of thermosensitive gel substrate: Take 64g of sodium hyaluronate and 16g of gelatin (mass ratio 4:1, total mass 80g), add 800ml of Lmilli-Q ultrapure water, stir in a constant temperature water bath at 60℃ (200rpm) for 1h until completely dissolved; cool down to 20℃, during which time the stirring speed is reduced to 100rpm to avoid the generation of bubbles, and obtain sodium hyaluronate-gelatin gel substrate.

[0087] 2. Mixing of active ingredients: Add 0.5g of PDRN and 0.1g of acetyl hexapeptide-8 to the gel substrate and stir at 300rpm for 10min until homogeneous; add 0.08g of polyethylene glycol diglycidyl ether (0.1% of the total mass of sodium hyaluronate-gelatin) and stir at 18℃ and 150rpm for 1.5h; determine the phase transition temperature using a rheometer: with a heating rate of 1℃ / min, record the temperature at the intersection of storage modulus G' and loss modulus G'' as 32℃, which meets the requirements for sol-gel phase transition; coat the PDRN core layer (100±5μm thickness) onto a PET film using a 100μm wire bar coater to obtain a sustained-release core layer with a PDRN content of 0.5±0.05mg / cm².

[0088] Step 4: Lamination and freeze-drying

[0089] 1. Lamination: The layers are stacked in the following order: “outer peel backing layer (PET / aluminum foil / polyethylene composite film, model PET12 / AL7 / PE20, thickness 20μm, Jiangyin Shenda) → sustained-release core layer → immediate-release core layer → soluble hollow microneedle array layer”. The layers are placed in a flatbed laminator (Shanghai Ziming Machinery MN-100), with a pressure of 0.08MPa and a temperature of 20℃, and the lamination time is 25min. After lamination, the layers are observed with an Olympus BX53 microscope (100x magnification) to confirm that there are no air bubbles in each layer and that the interfaces are tightly bonded.

[0090] 2. Freeze-drying: Transfer the laminated patch to a freeze dryer and process it according to the following procedure:

[0091] Pre-freezing stage: Maintain at -40℃ for 2 hours to ensure the patch is completely frozen (keep warm for 1 hour after the temperature drops to -40℃).

[0092] Sublimation drying stage: The vacuum degree is reduced to 0.01 mbar, the temperature is raised to -35℃ and maintained for 8 hours to remove free moisture;

[0093] Analysis and drying stage: Heat to -30℃ and maintain for 4 hours to remove bound water;

[0094] After freeze-drying, the water content of the sample was measured using a Karl Fischer moisture analyzer and found to be 2.8 ± 0.1%. 0.1 g of the freeze-dried patch was dissolved in 10 mL of PBS (pH 7.4), and the PDRN activity peak area was measured by HPLC. The ratio of the PDRN activity peak area to the initial activity peak area was 95.2 ± 0.4%, which means that the PDRN activity retention rate was 95.2%.

[0095] 2.3 Example 3

[0096] Step 1: Preparation of soluble hollow microneedle array layer

[0097] 1. Preparation of PDRN-liposomes: Dissolve 2.0 g of PDRN in 100 mL of Lilli-Q ultrapure water (resistivity 18.2 MΩ·cm) to prepare a 20 mg / mL aqueous solution. Stir magnetically (IKARCT basic model, 500 rpm) until completely dissolved. Dissolve 5 g of soybean lecithin and 1 g of cholesterol (mass ratio 5:1) in 50 mL of dichloromethane and sonicate (20 kHz, 100 W) for 5 min to prepare a homogeneous organic phase. Add the aqueous phase to organic phase at a volume ratio of 2:1 to a high-speed shear emulsifier and shear at 10,000 rpm for 5 min to form a water-in-oil emulsion. Transfer to an ultrasonic disruptor and sonicate at 300 W power and 20 kHz frequency for 10 min, maintaining a temperature of ≤25℃ using an ice-water bath during the process. Take 1 mL of emulsion, filter it through a 0.22 μm organic filter membrane, and use DLS to detect the particle size as 200 ± 20 nm and the zeta potential as -35 ± 5 mV to obtain PDRN-liposomes. Store at 4℃ in the dark for later use.

[0098] 2. Preparation of sodium hyaluronate-chitosan mixed solution: Take 3g of sodium hyaluronate and 1g of chitosan, add 100mL of 0.5% (v / v) acetic acid solution, stir at 300rpm (IKARW20digital) for 2h until completely dissolved; add 0.04g of EDC (1% of the total mass of sodium hyaluronate-chitosan), continue stirring at 25℃ for 1h, during which the pH is adjusted to 5.5±0.1 with 0.1mol / L NaOH to ensure sufficient cross-linking reaction, and obtain sodium hyaluronate-chitosan cross-linking solution.

[0099] 3. Microneedle Injection Molding: Mix the crosslinking liquid and PDRN-liposomes at a ratio of 9:1 (mass ratio) and stir magnetically (300 rpm) for 15 min until homogeneous. Inject the mixture into a PDMS hollow microneedle mold (inner diameter 80 μm, length 400 μm, needle array 1 cm × 1 cm), place it in a hollow microneedle injection molding machine, set the temperature to 5℃ and the pressure to 0.2 MPa, and let it stand for 30 min (ambient temperature 23℃) after injection molding for 10 s to allow the microneedles to form. After demolding, blow the residual liquid on the mold surface with nitrogen to obtain a soluble hollow microneedle array preform. Weigh each microneedle using a microbalance (Mettler-Toledo XP26, accuracy ±0.1 μg) and load it with 0.3 mg of PDRN-liposomes (total load of 30 mg per 100 microneedles).

[0100] 4. Addition of transdermal penetration enhancer: Take 0.48g of sodium lauroyl glutamate and 0.32g of phosphatidylcholine, dissolve them in 8mL of 10% (v / v) ethanol solution, and sonicate (100W, 20kHz) for 5min until completely dissolved; use a pneumatic spray gun (Iwata W-71, Japan, spraying pressure 0.1MPa) to uniformly spray the solution onto the surface of the microneedle array, with a spraying amount of 1.6% of the mass of the microneedle layer (0.16g of penetration enhancer solution is sprayed per 10g microneedle layer), and air dry at room temperature (23℃) for 30min to obtain a soluble hollow microneedle array layer.

[0101] Step 2: Preparation of the immediate-release core layer

[0102] 1. Preparation of β-cyclodextrin-PDRN inclusion complex: 4g of β-cyclodextrin and 2g of PDRN were added to 100mL LiMilli-Q ultrapure water and stirred magnetically at 500rpm for 3h in a 10℃ constant temperature water bath (HH-S6, accuracy ±0.5℃). The mixture was then transferred to a vacuum dryer (ChristAlpha1-4LDplus, 0.05mbar, 30℃) and dried for 12h to obtain a white blocky inclusion complex. The complex was then pulverized for 5min using a planetary ball mill (500rpm) and passed through an 80-mesh standard sieve (0.18mm aperture) to obtain the inclusion complex powder. The inclusion rate was 88±3% as determined by HPLC.

[0103] 2. Immediate-release core layer mixing: Weigh the materials (5g of inclusion complex, 0.4g of glutathione, and 0.75g of panthenol) according to the inclusion complex: glutathione: panthenol = 10:0.8:1.5 (mass ratio), add 2ml of Lmilli-Q ultrapure water, and stir at 300rpm for 10min to form a paste; coat it onto PET film (12μm thick, Jiangyin Shenda Packaging) using a bar coater (RKPrintCoatInstrumentsK202, 80μm bar), and vacuum dry at 35℃ and 0.08mbar for 3h to obtain the immediate-release core layer (80±5μm thick). The PDRN content was measured to be 12.3±0.6mg / cm².

[0104] Step 3: Preparation of sustained-release core layer

[0105] 1. Preparation of thermosensitive gel substrate: Take 72g of sodium hyaluronate and 18g of gelatin (mass ratio 4:1, total mass 90g), add 900ml of Lmilli-Q ultrapure water, stir in a constant temperature water bath at 60℃ (200rpm) for 1h until completely dissolved; cool down to 20℃, during which time the stirring speed is reduced to 100rpm to avoid the generation of bubbles, and obtain sodium hyaluronate-gelatin gel substrate.

[0106] 2. Mixing of active ingredients: Add 2g of PDRN and 0.5g of acetyl hexapeptide-8 to the gel substrate and stir at 300rpm for 10min until homogeneous; add 0.27g of polyethylene glycol diglycidyl ether (0.3% of the total mass of sodium hyaluronate-gelatin) and stir at 18℃ and 150rpm for 1.5h; measure the phase transition temperature using a rheometer: the heating rate is 1℃ / min, and the temperature at the intersection of the storage modulus G' and the loss modulus G'' is recorded as 37℃, which meets the requirements for sol-gel phase transition; coat the PDRN core layer (thickness 150±5μm) on a PET film using a 150μm wire bar coater to obtain a sustained-release core layer with a PDRN content of 1.8±0.1mg / cm².

[0107] Step 4: Lamination and freeze-drying

[0108] 1. Lamination: The layers are stacked in the following order: “outer peel backing layer (PET / aluminum foil / polyethylene composite film, model PET12 / AL7 / PE20, thickness 30μm, Jiangyin Shenda) → sustained-release core layer → immediate-release core layer → soluble hollow microneedle array layer”. The layers are placed in a flatbed laminator (Shanghai Ziming Machinery MN-100), with a pressure of 0.1MPa and a temperature of 25℃, and the lamination time is 35min. After lamination, the layers are observed with an Olympus BX53 microscope (100x magnification) to confirm that there are no air bubbles in each layer and that the interfaces are tightly bonded.

[0109] 2. Freeze-drying: Transfer the laminated patch to a freeze dryer and process it according to the following procedure:

[0110] Pre-freezing stage: Maintain at -40℃ for 2 hours to ensure the patch is completely frozen (keep warm for 1 hour after the temperature drops to -40℃).

[0111] Sublimation drying stage: The vacuum degree is reduced to 0.03 mbar, the temperature is raised to -35℃ and maintained for 8 hours to remove free moisture;

[0112] Analysis and drying stage: Heat to -30℃ and maintain for 4 hours to remove bound water;

[0113] After freeze-drying, the water content of the sample was measured using a Karl Fischer moisture analyzer and found to be 2.5 ± 0.1%. 0.1 g of the freeze-dried patch was dissolved in 10 mL of PBS (pH 7.4), and the PDRN activity peak area was measured by HPLC. The ratio of the PDRN activity peak area to the initial activity peak area was 95.8 ± 0.3%, which means that the PDRN activity retention rate was 95.8%.

[0114] 2.4 Comparative Example 1

[0115] Preparation differences: The preparation step of "soluble hollow microneedle array layer" was deleted, and the lamination sequence was adjusted to "external peeling backing layer → sustained release core layer → rapid release core layer". The remaining steps and parameters are completely consistent with those in Example 1.

[0116] 2.5 Comparative Example 2

[0117] Preparation differences: PDRN was replaced with bovine thymus (Sigma-Aldrich D8899, molecular weight 500-1000kDa), and the remaining steps and parameters were completely consistent with those in Example 1.

[0118] 2.6 Comparative Example 3

[0119] Preparation differences: No PEG-DGE was added during the preparation of the sustained-release core layer, and the remaining steps and parameters were completely consistent with those in Example 1.

[0120] 2.7 Comparative Example 4

[0121] Preparation differences: The microneedle substrate was replaced with pure sodium hyaluronate (3g), and the EDC dosage was added at 0.8% of the mass of pure sodium hyaluronate (0.024g). The remaining steps and parameters were completely consistent with those in Example 1.

[0122] 3 Performance Testing Methods

[0123] 3.1 Transdermal absorption performance test

[0124] Skin preparation: SPF grade C57BL / 6 mice (6-8 weeks old, 20-22g) were sacrificed, and the abdominal skin was peeled off, the fat was removed, and the skin was washed 3 times with PBS. The thickness was measured to be 0.2-0.3 mm.

[0125] Diffusion chamber assembly: Skin was fixed in the Franz chamber with the stratum corneum facing the donor chamber. 5 mL of PBS (pH 7.4) containing 0.1% BSA was added to the receiving chamber, and the mixture was stirred at 32°C and 500 rpm. 1 cm of solution was added to the donor chamber. 2 Patch, sealed to protect from light.

[0126] Sampling and testing: 0.5 mL samples were taken at 0.5 h, 1 h, 4 h, 8 h, 12 h, and 24 h (with an equal volume of fresh solution added), filtered through a 0.22 μm filter membrane, and then analyzed by HPLC (mobile phase: 0.1 mol / L phosphate buffer containing 0.1 mol / L NaCl, flow rate: 0.5 mL / min, 25 °C, 260 nm). The cumulative transdermal transdermal dose Q (μg / cm²) was calculated.

[0127] 3.2 PDRN sustained-release performance test

[0128] Sample preparation: Take 0.5 g each of the immediate-release core layer and the sustained-release core layer from Example 1, put them into a 3.5 kDa dialysis bag, place them in 50 mL PBS (pH 7.4), and stir at 37 °C and 100 rpm.

[0129] Detection and calculation: Take 5 mL samples at the above time points (for replenishment), detect the concentration by HPLC, and calculate the release rate = (release amount / total PDRN amount in the core layer) × 100% (total PDRN amount is determined by dissolving the core layer).

[0130] 3.3 Microneedle Mechanical Strength Test

[0131] Sample fixation: 10 microneedles were randomly selected from the microneedle layer of Examples 1-3 and Comparative Example 4 and fixed with a special clamp to ensure verticality.

[0132] Test parameters: Universal testing machine loading rate 0.1mm / s, load range 0-1N, record fracture strength; the pass rate is the percentage of microneedles with fracture strength ≥0.2N (n=100 needles / sample).

[0133] 3.4 Skin irritation test

[0134] Subject criteria: 30 healthy volunteers (aged 20-45, 15 men and 15 women), with no history of skin diseases or allergies, who had not used anti-wrinkle products in the past 3 months, and who all signed informed consent forms.

[0135] Patch test: In Example 1, a patch (0.5cm × 0.5cm) was applied to the inner forearm and removed after 24 hours. The results at 0.5h, 24h, and 48h were graded according to the 2024 edition of the "Cosmetic Safety Technical Specifications". Grade 0 was no reaction, Grade 1 was slight erythema, Grade 2 was erythema with mild infiltration, Grade 3 was erythema with edema and papules, and Grade 4 was erythema with edema and bullae.

[0136] Skin moisture content detection: The moisture content of the test area on the inner forearm was measured using a skin moisture meter (Corneometer CM825) before the patch was applied and 24 hours after removal, and the difference was calculated.

[0137] Data reliability: Skin moisture content was measured in parallel 3 times per person, with an RSD ≤ 3.5%, ensuring the repeatability of quantitative data; irritation grading was determined independently by 2 dermatologists, and the results were consistent.

[0138] 4. Performance Test Results and Analysis

[0139] Table 1. Transdermal absorption performance results

[0140] sample Transdermal delivery rate (μg / cm²) over 0.5 hours 4-hour transdermal delivery (μg / cm²) 24-hour transdermal dose (μg / cm²) RSD (n=3) Example 1 85.2±5.3 210.6±8.7 385.4±12.5 ≤3.2% Example 2 72.1±4.8 185.3±7.2 320.7±10.1 ≤2.8% Example 3 90.5±6.1 225.8±9.3 402.1±11.8 ≤3.0% Comparative Example 1 25.3±3.2 78.5±5.1 150.2±8.4 ≤3.5% Comparative Example 2 62.5±4.1 152.7±6.8 280.5±10.2 ≤3.1%

[0141] The 24-hour transdermal transfusion rate of Examples 1-3 was significantly higher than that of Comparative Example 1 (without microneedle layer). Example 1 was 2.57 times that of Comparative Example 1, proving that the "soluble hollow microneedle array layer" is the core of transdermal enhancement. The transdermal transfusion rate of Comparative Example 2 (bovine thymus PDRN) was only 72.8% of that of Example 1, confirming that salmon-derived PDRN has better transdermal compatibility.

[0142] Table 2 PDRN sustained-release performance test results

[0143] sample Core type 0.5h release rate 4h release rate 8h release rate 24h release rate RSD (n=3) Example 1 Immediate-release core layer 85.3%±4.2% 98.1%±2.5% 99.5%±1.8% 100% ≤2.1% Example 1 Sustained-release core layer 12.5%±3.1% 35.2%±4.7% 60.8%±5.3% 88.6%±3.9% ≤3.3% Comparative Example 3 Sustained-release core layer 38.7%±3.5% 75.2%±4.1% 92.5%±3.8% 99.2%±2.1% ≤2.5%

[0144] Example 1: The immediate-release core layer showed a release rate of >80% in 0.5h, achieving immediate energy replenishment; the sustained-release core layer showed a release rate of 88.6% in 24h, with no burst release, proving that the release can be controlled by "sodium hyaluronate-gelatin 4:1 + PEG-DGE crosslinking"; Comparative Example 3 (without PEG-DGE) showed a release rate of 99.2% in 24h, with no sustained-release effect, further verifying the necessity of the crosslinking agent.

[0145] Table 3. Results of microneedle mechanical strength test

[0146] sample Microneedle breaking strength (N) Pass rate (≥0.2N) RSD (n=10) Example 1 0.32±0.05 100% ≤4.5% Example 2 0.28±0.04 98% ≤5.1% Example 3 0.35±0.06 100% ≤4.8% Comparative Example 4 0.15±0.03 65% ≤5.3%

[0147] Examples 1-3 show that the microneedle breaking strength is ≥0.28N, which is higher than the 0.15N required for stratum corneum puncture, and the pass rate is 98%-100%, proving the mechanical stability of "sodium hyaluronate-chitosan 3:1+EDC crosslinking"; Comparative Example 4 (without chitosan) has a strength of only 0.15N and a pass rate of 65%, confirming the effect of chitosan on improving the strength of microneedles.

[0148] 4.4 Skin Irritation Results

[0149] (1) Skin irritation

[0150] 0.5 hours after removal: All 30 volunteers had a level 0 reaction in the test area, with no erythema, edema, papules or itching on the skin. Only one volunteer reported a "slight feeling of tightness in the skin" (which subsided on its own after 5 minutes and was determined to be a normal adaptation of the skin to the patch and not an irritation reaction).

[0151] 24 hours after removal: All volunteers' test areas remained at level 0 reaction, with no difference in skin color and elasticity from the surrounding normal skin, no delayed erythema or stinging sensation, and volunteers who previously reported tightness showed no abnormalities.

[0152] 48 hours after removal: None of the 30 volunteers experienced any adverse skin reactions, including erythema, edema, desquamation, or pigmentation. The independent judgments of the two dermatologists were completely consistent, with no disputed cases.

[0153] (2) Changes in skin moisture content

[0154] The skin moisture content in the test area was measured using a Corneometer CM825 (Courage+Khazaka). The specific method was as follows: three parallel measurement points were taken at the test area and surrounding normal skin (control area) before patch application (baseline) and 24 hours after removal. Measurements were taken three times at each point, and the average value was calculated to determine the difference. The results are as follows:

[0155] Baseline moisture content: The skin moisture content in the test area was 32.5±3.1AU (any unit), and that in the control area was 33.1±2.8AU. There was no statistically significant difference between the two (P>0.05), which proves that the skin condition was uniform before the test.

[0156] 24 hours after removal: The skin moisture content in the test area increased to 47.8±4.2AU, an increase of 15.3±3.0AU (47.1%) compared to the baseline, while the moisture content in the control area was 32.8±3.0AU (no difference from the baseline).

[0157] Data reliability: The relative standard deviation (RSD) of all moisture content tests was ≤3.5% (n=3), and the parallel tests showed good repeatability, proving that the patch is not only non-irritating, but also can synergistically increase skin moisture content through the immediate release of panthenol in the core layer and the sustained release of sodium hyaluronate in the core layer, thus playing a repairing and moisturizing role.

[0158] (3) Additional security verification

[0159] Residue detection: After the patch was removed, the skin surface residue was detected by high performance liquid chromatography (Agilent 1260 Infinity II). No active ingredients such as PDRN and acetyl hexapeptide-8 were detected (detection limit 0.01 μg / cm²), proving that the ingredients were completely transdermal or removed with the patch and there was no residue on the skin surface.

[0160] Subjective feedback: After the test, a questionnaire survey was conducted among the volunteers. All 30 volunteers reported that "there was no stinging, itching or burning sensation during the application process", "there was no sticky feeling on the skin after removal", and "the skin felt smoother", with a subjective satisfaction rate of 100%.

[0161] In the human skin patch test, the enhanced transdermal absorption cosmetic patch prepared in Example 1 showed no irritation (grade 0) in any of the 30 volunteers. Skin hydration was significantly increased and no residue was found, demonstrating the product's excellent biocompatibility. Its safety advantages stem from: ① The microneedles are made of sodium hyaluronate-chitosan cross-linked material (a natural biomaterial with no immunogenicity); ② The transdermal penetration enhancer is sodium lauroyl glutamate-phosphatidylcholine (a mild amino acid derivative + natural phospholipid with no skin barrier disruption); ③ It is free of irritating solvents such as alcohol and propylene glycol, making it perfectly suitable for sensitive skin and meeting the core requirements of cosmetic patches: "safe, gentle, and effective."

[0162] 5. Conclusion

[0163] Structure and transdermal efficiency: The soluble hollow microneedle array layer (sodium hyaluronate-chitosan 3:1 crosslinking) combined with PDRN-liposomes enabled a transdermal absorption rate of 385.4 μg / cm² in 24 hours, which is more than 2.5 times higher than that without microneedle structure, fully verifying the transdermal enhancement effect of the four-layer structure; the transdermal penetration enhancer (sodium lauroyl glutamate: phosphatidylcholine = 3:2) further optimized the absorption.

[0164] Synergistic effect of components and sustained release: The immediate-release core layer (β-cyclodextrin-PDRN2:1 inclusion complex) has a release rate of 85.3% at 0.5h, while the sustained-release core layer (sodium hyaluronate-gelatin 4:1+PEG-DGE) has a release rate of 88.6% at 24h, achieving a synergistic effect of "immediate-long-lasting" effect; Comparative example 3 proves that the cross-linking agent is the key to sustained release.

[0165] Raw material and performance stability: The transdermal absorption rate and activity retention rate (96.5%) of salmon-derived PDRN (500-1000kDa) were significantly better than those of bovine thymus PDRN; the addition of chitosan to the microneedle substrate increased the tensile strength by 113%, and the pass rate reached 100%.

[0166] Process and safety: 0-5℃ injection molding + freeze drying process (vacuum degree 0.01-0.03mbar) ensures PDRN activity ≥95% and water content ≤3%; skin irritation level 0, proving that the product is safe and suitable for cosmetic applications.

[0167] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An enhanced transdermal absorption cosmetic patch containing highly active polydeoxyribonucleotide PDRN, characterized in that, The cosmetic patch comprises, from the outside to the inside, an outer peel-off backing layer, a sustained-release core layer, a rapid-release core layer, and a soluble hollow microneedle array layer. The outer peel-off backing layer is a PET / aluminum foil / polyethylene composite film with a thickness of 20-30μm; The sustained-release core layer is composed of thermosensitive sodium hyaluronate-gelatin gel, PDRN, and acetyl hexapeptide-8, with a mass ratio of (80-90):(0.5-2):(0.1-0.5). The immediate-release core layer is composed of β-cyclodextrin-PDRN inclusion complex, glutathione, and panthenol, with a mass ratio of (5-10):(0.2-0.8):(0.5-1.5). The microneedles of the soluble hollow microneedle array layer have a hollow structure with an inner diameter of 50-80 μm and a length of 300-400 μm. The microneedle material is sodium hyaluronate-chitosan crosslinked material. PDRN-liposomes are loaded in the hollow cavity, with a PDRN-liposome loading amount of 0.1-0.3 mg / microneedle.

2. The enhanced transdermal absorption cosmetic patch according to claim 1, characterized in that, The PDRN is derived from salmon testicular tissue and has an average molecular weight of 500-1000 kDa after enzymatic hydrolysis.

3. The enhanced transdermal absorption cosmetic patch according to claim 1, characterized in that, In the soluble hollow microneedles sodium hyaluronate-chitosan crosslinked product, the mass ratio of sodium hyaluronate to chitosan is 3:1, the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the amount of crosslinking agent is 0.5-1% of the total mass of sodium hyaluronate-chitosan.

4. The enhanced transdermal absorption cosmetic patch according to claim 1, characterized in that, In the thermosensitive sodium hyaluronate-gelatin gel of the sustained-release core layer, the mass ratio of sodium hyaluronate to gelatin is 4:1, the thermosensitive crosslinking agent is polyethylene glycol diglycidyl ether, and the amount of crosslinking agent is 0.1-0.3% of the total mass of sodium hyaluronate-gelatin. The gel undergoes a sol-gel phase transition at 32-37℃.

5. The enhanced transdermal absorption cosmetic patch according to claim 1, characterized in that, In the β-cyclodextrin-PDRN inclusion complex of the immediate-release core layer, the mass ratio of β-cyclodextrin to PDRN is 2:1, the preparation temperature of the inclusion complex is 5-10℃, and the stirring time is 2-3h.

6. The enhanced transdermal absorption cosmetic patch according to claim 1, characterized in that, The soluble hollow microneedle array layer also contains a transdermal penetration enhancer, which is a mixture of sodium lauroyl glutamate and phosphatidylcholine in a mass ratio of 3:2, with a total amount of 0.5-1.6% of the mass of the microneedle array layer.

7. A low-temperature preparation method for the enhanced transdermal absorption cosmetic patch as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of soluble hollow microneedles: Sodium hyaluronate-chitosan crosslinking solution was mixed with PDRN-liposome, injected into a hollow microneedle mold, and injection molded at 0-5℃ and 0.1-0.2MPa pressure to obtain a soluble hollow microneedle array layer; (2) Preparation of immediate-release core layer: β-cyclodextrin and PDRN are dissolved in deionized water at a mass ratio of 2:1, stirred at 5-10℃ for 2-3h to form inclusion complex, dried in vacuum at 30℃ and 0.05mbar to form powder, and then mixed evenly with glutathione and panthenol to obtain immediate-release core layer; (3) Preparation of sustained-release core layer: Sodium hyaluronate and gelatin are dissolved in deionized water at a mass ratio of 4:1, PDRN, acetyl hexapeptide-8 and thermosensitive crosslinking agent are added, and the mixture is stirred at 15-20℃ for 1-2 hours to obtain the sustained-release core layer. (4) Lamination and freeze-drying: Lamination is carried out in the order of "outer peel backing layer → sustained release core layer → immediate release core layer → soluble hollow microneedle array layer", with a pressure of 0.08-0.1MPa and a temperature of 20-25℃. Then, the layers are placed in a freeze dryer and freeze-dried according to the parameters of "pre-freezing at -40℃ for 2h → sublimation drying at -35℃ for 8h → desorption drying at -30℃ for 4h" to obtain an enhanced transdermal absorption cosmetic patch.

8. The low-temperature preparation method according to claim 7, characterized in that, The preparation method of PDRN-liposomes in step (1) is as follows: PDRN is dissolved in deionized water to form an aqueous solution of 10-20 mg / mL, and mixed with a soybean lecithin-cholesterol organic phase dissolved in dichloromethane at a mass ratio of 5:1 and a volume ratio of 2:

1. The mixture is then subjected to high-speed shearing at 8000-10000 rpm to form an oil-in-water emulsion, and then subjected to ultrasonic disruption at 280-300W for 8-10 min to obtain PDRN-liposomes with a particle size of 100-200 nm.

9. The low-temperature preparation method according to claim 7, characterized in that, In step (4), the vacuum degree of the freeze dryer is controlled at 0.01-0.03 mbar, the water content of the freeze-dried product is ≤3%, and the PDRN activity retention rate is ≥95%.

10. The low-temperature preparation method according to claim 7, characterized in that, During the lamination process in step (4), the coating thickness of the sustained-release core layer is 100-150 μm, the coating thickness of the immediate-release core layer is 50-80 μm, and the overall thickness after lamination is controlled to be 300-400 μm.