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

Abstract

The application provides an enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (DNA sodium) and a preparation method thereof, which is composed of a needle body layer and a backing layer, wherein the needle body layer contains the following components in percentage by mass: PDRN with a molecular weight of 50-300 kDa, content of 5%-15%; and a zwitterionic polymer carrier copolymerized from methacryloyl ethyl sulfobetaine and hyaluronic acid, content of 30%-50%. The patch has the characteristics of good mechanical properties, rapid dissolution, high active retention and high-efficiency penetration promotion, and can be used for treating post-acne pigmentation and repairing skin barrier, and comprehensively improves the skin repair effect and practicality.

Description

Technical Field

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

[0002] With the rapid development of skin repair and aesthetic medicine, concerns about post-acne pigmentation and damaged skin barrier have increased, leading to a significant increase in demand for efficient and safe skin repair technologies. Polynucleotides (PDRNs), derived from salmon testes, possess bioactivities such as promoting cell proliferation, anti-inflammation, and repairing damaged tissue, showing promising application prospects in the field of skin repair.

[0003] Currently, there are three main methods of PDRN administration: topical application is hindered by the stratum corneum of the skin, resulting in low transdermal absorption and poor bioavailability; subcutaneous injection has high absorption efficiency, but it is painful, has low patient compliance, and is prone to infection; enhanced transdermal delivery cosmetic patches can penetrate the stratum corneum through micron-sized needles to deliver active ingredients directly to the epithelial tissue, combining high efficiency and safety, making them an ideal carrier for PDRN delivery.

[0004] However, existing PDRN-enhanced transdermal delivery cosmetic patches still have key problems: First, PDRN has insufficient stability. As a bioactive macromolecule, it is easily degraded and inactivated by environmental factors such as temperature and humidity, affecting the repair effect. Second, the performance of carrier materials is limited. Traditional carriers (such as hyaluronic acid and gelatin) have limited PDRN loading capacity and it is difficult to balance mechanical strength and biocompatibility. Inappropriate dissolution rate can also affect drug release efficiency. Third, the transdermal penetration efficiency needs to be improved. PDRN has insufficient deep penetration ability in skin tissue, and it is difficult to improve bioavailability by simply relying on microneedle physical puncture. Fourth, the function is singular. It only relies on the repair effect of PDRN and lacks synergistic regulation of multiple skin repair needs (such as anti-oxidation, anti-inflammation, and depigmentation), resulting in limited comprehensive effect. Fifth, the preparation process is defective. Traditional processes (such as hot melt casting and UV curing) may cause PDRN inactivation due to high temperature or chemical reagents, and the needle morphology and mechanical strength are difficult to control precisely.

[0005] Therefore, developing enhanced transdermal delivery cosmetic patches that can stably load PDRN, improve its activity retention and transdermal efficiency, and possess both good mechanical properties and synergistic repair functions has become an inevitable trend. This application aims to overcome existing technological bottlenecks and provide a more efficient, stable, and safe solution for skin repair through innovative designs such as optimizing carrier materials, introducing nano-penetration enhancers, and improving the preparation process. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) and its preparation method. The patch possesses excellent mechanical properties, rapid dissolution, high activity retention, and efficient penetration enhancement. It can also be used for the treatment of post-acne pigmentation and skin barrier repair, comprehensively improving skin repair effects and practicality.

[0007] This invention provides an enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA), comprising a needle layer and a backing layer, wherein:

[0008] The needle body layer comprises the following components by mass percentage:

[0009] PDRN with a molecular weight of 50-300kDa, at a content of 5%-15%;

[0010] A zwitterionic polymer carrier composed of methacryloylethyl sulfobetaine and hyaluronic acid, with a content of 30%-50%;

[0011] L-carnosine encapsulated in liposomes with a particle size not exceeding 100 nm is used as a nanoscale penetration enhancer, with a content of 10%-20%.

[0012] A cryoprotectant composed of trehalose and mannitol in a mass ratio of 1:1 to 3:1, with a content of 15% to 25%;

[0013] The backing layer is a block copolymer of biodegradable polycaprolactone and polyethylene glycol, PCL-PEG-PCL, with a molecular weight of 8000-12000 Da.

[0014] Furthermore, the PDRN has a molecular weight of 150-250 kDa and a loading of 8%-12% in the needle layer;

[0015] The grafting rate of the sulfobetaine monomer on the zwitterionic polymer support is 20%-35%;

[0016] The encapsulation efficiency of the liposomes encapsulating L-carnosine is not less than 95%.

[0017] Furthermore, the needle height is 600-900μm, and the single needle load-bearing capacity is not less than 0.35N;

[0018] Complete dissolution time in physiological saline at 35-37℃ should not exceed 3 minutes;

[0019] After an accelerated test at 50℃-60℃ for 48-72 hours, the PDRN activity retention rate was no less than 85%.

[0020] Furthermore, the needle body layer also contains 0.1%-0.3% tranexamic acid and 2%-4% glutathione by weight.

[0021] The backing layer contains 5%-7% by weight of Centella asiatica extract.

[0022] Furthermore, the PDRN is a DNA fragment derived from salmon testes, with a length of 50-500 bp;

[0023] The liposomes contain dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a molar ratio of 6.5:3.5 to 7.5:2.5.

[0024] Furthermore, the thickness ratio of the needle body layer to the backing layer is 3:1-5:1;

[0025] The surface of the backing layer has a micron-scale protrusion structure with a height of 20-50 μm.

[0026] This invention provides a method for preparing an enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) as described in any of the above claims, comprising the following steps:

[0027] (1) Synthesis of zwitterionic polymer support:

[0028] Methacrylethyl sulfobetaine and hyaluronic acid with a molecular weight of 50-100 kDa were dissolved in PBS buffer at a molar ratio of 1:3-1:5. 0.08%-0.12% by mass of ammonium persulfate initiator was added, and the mixture was reacted at 48-52℃ for 6-8 hours, followed by dialysis purification.

[0029] (2) Preparation of nano-penetration enhancer:

[0030] L-carnosine and dipalmitoylphosphatidylcholine (DPPC) were mixed at a mass ratio of 1:2 to 1:4, and liposomes were prepared by thin-film hydration. The liposomes were then sterilized by passing the mixture through a 0.20-0.24 μm filter membrane.

[0031] (3) Preparation of needle solution:

[0032] PDRN, zwitterionic polymer carrier, nano-permeability enhancer, and cryoprotectant were dissolved in deionized water at 4±2℃ and ultrasonically treated with 180-220W power for 4-6 minutes to form a homogeneous solution.

[0033] (4) Low temperature molding:

[0034] The needle solution was injected into a PDMS mold and pre-frozen at -45 to -35°C for 1.5 to 2.5 hours, followed by freeze-drying at a vacuum of 0.08 to 0.12 mPa and a temperature of -30 to -20°C for 22 to 26 hours.

[0035] (5) Backing layer composite:

[0036] A PCL-PEG-PCL chloroform solution with a mass-volume ratio of 18-22% was coated on the back of the needle layer. After the solvent evaporated at 23-27°C, the needle was peeled off and molded.

[0037] Furthermore, the total solids content of the solution in step (3) is 35%-45%;

[0038] The freeze-drying process includes:

[0039] Heat from -45 to -35℃ to -25 to -15℃ at a rate of 1.8-2.2℃ / h, and hold for 3-4 hours;

[0040] Heat from -25 to -15℃ to -5 to 5℃ at a rate of 0.8-1.2℃ / h, and hold for 4-5 hours;

[0041] Heat from -5°C to 5°C at a rate of 0.4-0.6°C / h to 23-27°C, and hold for 2-3 hours.

[0042] This invention provides the application of the enhanced transdermal delivery cosmetic patch in the preparation of skin repair medical devices for the treatment of post-acne pigmentation and for skin barrier repair.

[0043] This invention provides a method for detecting the enhanced transdermal delivery cosmetic patch, comprising:

[0044] PDRN content was determined by high performance liquid chromatography: the chromatographic column was a C18 column, the mobile phase was 0.08-0.12M phosphate buffer at pH 7.2-7.6, the flow rate was 0.8-1.2 mL / min, the detection wavelength was 260 nm, and the injection volume was 10-20 μL.

[0045] The cumulative permeation of L-carnosine was determined using a Franz diffusion cell: the effective permeation area of ​​the diffusion cell was 1.5-2.5 cm², the receiving solution was PBS buffer with pH 7.2-7.4, and the permeation was not less than 75-85 μg / cm² over 24 hours.

[0046] The advantages and positive effects of this invention are:

[0047] 1. Highly efficient transdermal delivery and activity retention: Through the synergistic effect of PDRN with a specific molecular weight (50-300kDa) and zwitterionic polymer carrier (sulfobetaine-hyaluronic acid copolymer), the drug's skin penetration efficiency is significantly improved (24h L-carnosine penetration ≥80μg / cm²), while the cryoprotectant (trehalose / mannitol) ensures the activity stability of PDRN during preparation and storage (activity retention ≥85% after 72h accelerated test at 60℃).

[0048] 2. Precise and controllable delivery system: The dual permeation-enhancing mechanism of nanoscale liposome encapsulation of L-carnosine (particle size ≤100nm, encapsulation rate ≥95%) and microneedle physical penetration breaks through the stratum corneum barrier to achieve targeted delivery of PDRN; the gradient freeze-drying process (stage heating from -40℃ to 25℃) ensures the integrity of the needle structure (single needle load capacity ≥0.35N) and rapid solubility (completely dissolved in 37℃ physiological saline within 3 minutes).

[0049] 3. Multifunctional Repair and Expanded Clinical Applications: The compound formula (containing tranexamic acid, glutathione, and centella asiatica extract) has anti-inflammatory, antioxidant, and repair-promoting functions, and is suitable for indications such as post-acne pigmentation, skin barrier repair, and diabetic ulcers. The biodegradable backing layer (PCL-PEG-PCL) achieves a balance between drug sustained release and mechanical support. Detailed Implementation

[0050] The technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. Based on the embodiments in this disclosure, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this disclosure. Unless otherwise specified, the raw materials, reagents, etc. used in the embodiments are all conventional products that can be obtained commercially; unless otherwise specified, the detection methods are all conventional detection methods in the field of microbial preparations.

[0051] Example 1

[0052] 1.1 Raw material preparation:

[0053] PDRN: DNA fragment derived from salmon testes (prepared by enzymatic digestion). Specific preparation method: Fresh salmon testes were washed with physiological saline, chopped, and homogenized in 0.1 M Tris-HCl buffer (pH 8.0, containing 0.1 M EDTA and 1% SDS). The mixture was incubated at 4°C for 2 hours. Proteinase K (final concentration 50 μg / mL) was added and the mixture was incubated at 37°C for 4 hours. Extraction was performed three times with chloroform-isoamyl alcohol (24:1), and DNA was precipitated with anhydrous ethanol. Enzymatic digestion with DNase I (purchased from Takara, catalog number 2270A) was used: DNA concentration was 1 mg / mL in 50 mM Tris-HCl (pH 7.5) buffer, and DNase I was added at 10 U / mg DNA. The reaction was carried out at 37°C for 2 hours. Fragment length was verified as 200-300 bp by 12% agarose gel electrophoresis. Finally, PDRN with a molecular weight of 200 kDa and a purity ≥98% was obtained by purification using a Sephadex G-50 column (HPLC verification). (Note: 50kDa and 150kDa PDRN were prepared by adjusting the DNaseI hydrolysis time (4h and 1.5h, respectively), and the remaining steps were the same.)

[0054] Hyaluronic acid (HA): Purchased from Bloomage Biotechnology Co., Ltd., product name "medical grade low molecular weight sodium hyaluronate", catalog number HA-MW80k, molecular weight 80kDa (GPC determination, PDI=1.2), purity ≥99%.

[0055] Methacryloxyethyl sulfobetaine (SBMA): purchased from Aladdin Reagent (Shanghai) Co., Ltd., catalog number S105422, purity ≥98%, molecular weight 247.3 Da, water content ≤0.5%.

[0056] L-Carnosine: Purchased from Sigma-Aldrich (USA), product name "L-Carnosine", catalog number C9625, purity ≥99%, optical rotation -12.5°~-14.5°, pharmaceutical grade.

[0057] Dipalmitoylphosphatidylcholine (DPPC): Purchased from AvantiPolarLipids (USA), product name "1,2-Dipalmitoyl-sn-glycero-3-phosphocholine", catalog number 850355P, purity ≥98%, phase transition temperature 41℃, liposome-specific grade.

[0058] Cholesterol: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product name "Cholesterol (Pharmaceutical Grade)", item number C810127, purity ≥99%, water content ≤0.3%.

[0059] Trehalose: Purchased from Shandong Futian Pharmaceutical Co., Ltd., product name "food grade trehalose", item number FT-Tre-01, water content ≤0.2%; Mannitol: Purchased from Roquette (China) Nutritional Food Co., Ltd., product name "food grade mannitol", item number RO-MAN-02, water content ≤0.2%; the two are mixed at a mass ratio of 2:1.

[0060] PCL-PEG-PCL block copolymer: purchased from Jinan Daigang Biotechnology Co., Ltd., product name "degradable medical grade PCL-PEG-PCL", catalog number PCL-PEG-PCL-10k, molecular weight 10000Da (PCL segment: PEG segment = 2:1, determined by GPC), PDI = 1.2.

[0061] Ammonium persulfate (APS): Purchased from Sinopharm Chemical Reagent Co., Ltd., product name "Ammonium persulfate (analytical grade)", item number 10019-60-9, purity ≥98%, stored in a dry place.

[0062] PBS buffer: Prepared in-house. Formula: Mix 0.01M potassium dihydrogen phosphate and disodium hydrogen phosphate, adjust pH to 7.4 with 0.1M NaOH, conductivity 1.5 mS / cm (measured with a DDS-307 conductivity meter).

[0063] Chloroform: Purchased from Aladdin Reagent (Shanghai) Co., Ltd., product name "anhydrous chloroform", item number C105674, purity ≥99.8%, water content ≤0.01%.

[0064] 1.2 Synthesis of zwitterionic polymer supports

[0065] Weigh SBMA (0.01 mol, 2.473 g) and HA (0.04 mol, 3.2 g, converted to 80 kDa molecular weight) at a molar ratio of 1:4, dissolve in 100 mL of PBS buffer (0.01 M, pH 7.4), and magnetically stir for 30 min (stirrer model: IKARCTbasic, speed 200 rpm) until completely dissolved to form a clear solution;

[0066] Add 0.01g APS (0.1% of the total mass of SBMA and HA), purge with high-purity nitrogen (99.999%) to remove oxygen for 30 min (purge rate 10 mL / min), and seal the reaction flask;

[0067] Place in a 50℃ constant temperature water bath (model: HH-S4) and react for 7 hours, during which magnetic stirring (200 rpm) is maintained.

[0068] The reaction product was transferred into a regenerated cellulose dialysis bag with a molecular weight cutoff of 3500 Da (to retain unreacted SBMA monomer with a molecular weight of 247.3 Da), and dialyzed with deionized water for 48 hours (the deionized water was changed every 12 hours, 1 L each time). The solution after dialysis was sterilized by passing it through a 0.22 μm filter membrane.

[0069] After sterilization, the solution was transferred to a freeze-drying bottle and pre-frozen in a -50°C ultra-low temperature freezer (model: ThermoScientificForma900) for 4 hours. Then it was transferred to a freeze dryer (model: ChristAlpha1-4LDplus) and freeze-dried at a vacuum of 0.1 mPa and a temperature of -25°C for 36 hours to obtain a white fluffy powder.

[0070] The grafting rate of sulfobetaine monomers was determined by proton nuclear magnetic resonance spectroscopy (1H-NMR, 400MHz, solvent D2O): with the hydroxyl hydrogen of HA (δ4.5-5.0) as an internal standard and the methyl hydrogen of SBMA (δ3.2-3.3) as a characteristic peak, the grafting rate was calculated to be 28%.

[0071] 1.3 Preparation of nano-penetration enhancer

[0072] Weigh L-carnosine (1.0g) and DPPC (3.0g) in a mass ratio of 1:3, add cholesterol (1.28g, DPPC to cholesterol molar ratio 7:3, based on molecular weight conversion: DPPC 734.0g / mol, cholesterol 386.7g / mol), and dissolve them together in 50mL of chloroform;

[0073] The mixture was transferred to a rotary evaporator (model: RE-52AA), the temperature was set to 35℃ and the rotation speed to 60 rpm, and the mixture was rotary evaporated for 2 hours to form a uniform and transparent lipid film on the inner wall of the flask (without visible cracks).

[0074] Add 20 mL of PBS buffer (0.01 M, pH 7.4) to the flask and hydrate in a constant temperature water bath at 37 °C for 2 h, shaking once every 15 min (amplitude 3 cm) to ensure complete film detachment;

[0075] A probe-type ultrasonic instrument (model: SCIENTZ-IID, power: 300W, frequency: 20kHz) was used to sonicate for 10 minutes (3 seconds working / 5 seconds intermittent) to obtain a milky white liposome suspension.

[0076] The suspension is sterilized by passing it through a 0.22μm polyethersulfone filter membrane (25mm in diameter) to remove large particle agglomerates;

[0077] Characterization and detection:

[0078] Particle size: Measured using a dynamic light scattering instrument (DLS, model: Malvern Zetasizer NanoZS), the average particle size was 85 nm ± 3 nm.

[0079] Encapsulation efficiency: Ultrafiltration centrifugation-HPLC was used (ultrafiltration membrane molecular weight cutoff 10 kDa, centrifugation at 8000 rpm for 30 min). HPLC conditions: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase methanol-water (60:40, v / v), detection wavelength 220 nm, flow rate 1.0 mL / min. The encapsulation efficiency was calculated to be 96.5% ± 1.2%.

[0080] 1.4 Preparation of Needle Solution

[0081] Weigh the components by mass percentage: PDRN 10% (1.0g), zwitterionic polymer carrier 40% (4.0g), nano-penetration enhancer 15% (1.5g), cryoprotectant (trehalose:mannitol = 2:1) 20% (2.0g).

[0082] Add 1.5g of pre-cooled deionized water at 4℃ (15% of the total mass), and stir magnetically for 30 minutes (300 rpm) until initially dissolved;

[0083] The solution was ultrasonically treated for 5 minutes using a probe-type ultrasonic instrument (power 200W, frequency 20kHz) to form a homogeneous and transparent solution (without visible particles).

[0084] Total solids content determination: Take 1 mL of solution into a weighing bottle, dry under reduced pressure at 80℃ (0.09 MPa) to constant weight, and calculate the total solids content as 40% ± 2%.

[0085] 1.5 Low temperature molding

[0086] (1) Slowly inject the needle solution into the PDMS mold (10×10 needle array, 750μm needle height, 50μm needle tip diameter), and gently scrape the mold surface with a polytetrafluoroethylene scraper to avoid generating air bubbles;

[0087] (2) Place the mold in a -40℃ ultra-low temperature freezer for 2 hours to pre-freeze and ensure that the solution is completely frozen;

[0088] (3) Transfer to a freeze dryer and freeze dry according to the following procedure:

[0089] stage Temperature change heating rate Insulation time 1 -40℃→-20℃ 2℃ / h 4h 2 -20℃→0℃ 1℃ / h 6h 3 0℃→25℃ 0.5℃ / h 14h

[0090] (4) The total freeze-drying time is 24 hours. Take out the mold and observe that the needle body layer is intact (no shrinkage or cracking). The height of a single needle is 750μm±20μm.

[0091] 1.6 Backing layer composite

[0092] Preparation of backing layer solution: Weigh 20g of PCL-PEG-PCL, dissolve it in 100mL of chloroform, and stir magnetically for 2h at 200rpm until completely dissolved to obtain a solution with a mass-volume ratio of 20%.

[0093] Coating control: An automatic coater (model: RKPrintCoatKControlCoater) is used to coat the solution onto the back of the needle body layer, with a wet film thickness of 200μm set to ensure uniform coating;

[0094] Thickness ratio control: The thickness of the needle body layer after drying is 750μm, and the thickness of the backing layer after drying is 187.5μm. The ratio of needle body layer to backing layer is 4:1.

[0095] Micron-level protrusion molding: After chloroform has evaporated for 8 hours at 25°C and 45% humidity (solvent residue ≤0.1%), a micro-imprinting mold (protrusion height 35μm, spacing 100μm) is used to imprint on the surface of the backing layer with a pressure of 5N / cm² for 10 minutes.

[0096] Demolding: Gently peel the PDMS mold with tweezers to obtain the complete enhanced transdermal delivery cosmetic patch.

[0097] 1.7 Performance Testing

[0098] To comprehensively verify the mechanical properties, stability, transdermal effect, and clinical application value of the enhanced transdermal delivery cosmetic patch of this invention, the following seven core tests were set up in accordance with the technical requirements of medical device testing specifications and performance limits, skin repair applications, and testing methods. All tests were repeated three times, and the results are expressed as "mean ± standard deviation", as detailed below:

[0099] 1.7.1 Single Needle Bearing Capacity Test

[0100] Test objective: To evaluate the mechanical strength of enhanced transdermal delivery cosmetic patches, ensuring that they can penetrate the stratum corneum of the skin without breaking, and guaranteeing high penetration.

[0101] Test method:

[0102] Equipment and sample preparation: Use a universal testing machine (model: Instron5944) equipped with a 5mm diameter planar probe; take one enhanced transdermal delivery cosmetic patch, cut it into a 1×1cm² microneedle array unit (containing 100 microneedles), fix it on the base of the testing machine, and ensure that the needle tip is facing upward and perpendicular to the probe direction.

[0103] Test parameter settings: Adjust the initial distance between the probe and the needle tip to 0.5cm, set the probe pressing rate to 0.05mm / s, the pressure detection range to 0-1N, and record the pressure-displacement curve in real time.

[0104] Judgment criteria: When the microscope observes obvious deformation of the needle tip (needle tip bending angle > 10°), stop the test and read the pressure value at this time, which is the single needle bearing capacity (total pressure ÷ number of microneedles).

[0105] Test results: The single needle load-bearing capacity is 0.38N±0.02N, proving that the enhanced transdermal delivery cosmetic patch has sufficient mechanical strength and can effectively penetrate the skin barrier.

[0106] 1.7.2 Dissolution time detection

[0107] Detection purpose

[0108] To verify the dissolution rate of the enhanced transdermal delivery cosmetic patch in the skin's physiological environment, ensuring that the active ingredients can be quickly released and absorbed by the skin, avoiding irritation from residual foreign matter.

[0109] Test methods

[0110] Preparation of simulated physiological environment: Measure 50 mL of physiological saline (pH 7.4, temperature controlled by a constant water bath at 37℃, error ±0.5℃), pour it into a 100 mL beaker, place a magnetic stir bar (diameter 8 mm) in the beaker, set the stirring speed to 50 rpm, and ensure that the solution is in a uniform constant temperature state.

[0111] Sample testing: Take one enhanced transdermal delivery cosmetic patch and immerse it completely in physiological saline while starting a timer. Observe the dissolution state of the needle layer every 10 seconds through an optical microscope (magnification 20x) and record the time when "the needle completely disappears and there are no visible solid particles in the solution".

[0112] Test results

[0113] The dissolution time was 2.5 min ± 0.1 min, indicating that the enhanced transdermal delivery cosmetic patch can dissolve rapidly under the skin's physiological environment, ensuring the timely release of active ingredients.

[0114] 1.7.3 Detection of PDRN activity retention rate

[0115] Detection purpose

[0116] To evaluate the storage stability of enhanced transdermal delivery cosmetic patches, with a focus on verifying the activity retention capacity of PDRN under high temperature conditions.

[0117] Test methods

[0118] Accelerated test treatment: Take 10 enhanced transdermal delivery cosmetic patches and divide them into two groups (5 patches in each group). One group serves as a control (stored at 25℃ and 45% humidity), while the other group is placed in a 60℃ constant temperature incubator (model: BinderBD23) for 72 hours, avoiding light and vibration during the period.

[0119] PDRN content determination: The PDRN content of the two groups of samples was determined according to the HPLC method in Section 4.1 (chromatographic column: amino column 250mm×4.6mm, 5μm; mobile phase: 0.1M, pH7.4 phosphate buffer; detection wavelength 260nm; flow rate 1.0mL / min).

[0120] Activity retention rate calculation: Activity retention rate = (PDRN content after accelerated test ÷ PDRN content of control sample) × 100%.

[0121] Test results

[0122] After 72 hours of accelerated testing at 60℃, the PDRN activity retention rate was 88%±1.5%, demonstrating that the enhanced transdermal delivery cosmetic patch effectively improves the stability of PDRN through the synergistic effect of the cryoprotectant (trehalose-mannitol) and the zwitterionic carrier.

[0123] 1.7.4 PDRN content detection

[0124] Purpose of the test: To confirm the actual PDRN loading in the needle body layer and ensure that it is within the concentration range.

[0125] Test methods

[0126] Sample pretreatment: Take one enhanced transdermal delivery cosmetic patch, cut it into pieces and put it into a 50mL centrifuge tube. Add 20mL of 0.1MPBS buffer (pH 7.4) and sonicate it for 30min using a probe sonicator (power 200W, frequency 20kHz) to ensure complete dissolution of the needle. Then filter it through a 0.22μm polyethersulfone filter membrane and take the filtrate as the test solution.

[0127] HPLC detection: Under the same chromatographic conditions as in Section 1.7.3, inject 10 μL of the test solution and record the peak area; plot a standard curve using PDRN standards (concentrations of 10, 20, 50, and 100 μg / mL) and calculate the mass of PDRN in the test sample; calculate the mass percentage of PDRN by combining the total mass of the needle layer (pre-weighed dried needle layer).

[0128] Test results

[0129] The PDRN content was 9.8%±0.2%, which falls within the "8%-12%" loading range, proving that the preparation process can precisely control the amount of PDRN added and ensure consistency between product batches.

[0130] 1.7.5L-Carnosine 24h Cumulative Permeability Detection

[0131] Detection purpose

[0132] To verify the transdermal effect of the nano-penetration enhancer (liposome-encapsulated L-carnosine), ensuring that L-carnosine can effectively penetrate the skin and reach the site of action, and work synergistically with PDRN to exert its repair function.

[0133] Test methods

[0134] Franz diffusion chamber setup: A vertical Franz diffusion chamber (effective transdermal area 1.77 cm²) was used, with a receiving chamber volume of 6.5 mL. 0.01 M PBS buffer (pH 7.4, kept at 37°C in a constant temperature water bath) was added, and the chamber was magnetically stirred (500 rpm). The transdermal barrier was made of fresh pigskin (500 μm thick, dehaired, washed with physiological saline, and disinfected with 0.1% benzalkonium chloride), which was fixed between the drug supply chamber and the receiving chamber of the diffusion chamber to ensure no air bubbles.

[0135] Sample loading and sampling: The enhanced transdermal delivery cosmetic patch was applied to the drug delivery surface of the pigskin and pressed for 30 seconds to ensure adhesion; samples were taken at 1, 4, 8, 12 and 24 hours, 1 mL of receiving fluid was taken each time, and 1 mL of fresh receiving fluid (preheated at 37℃) was added at the same time.

[0136] L-carnosine content determination: The L-carnosine content in the receiving solution was determined by HPLC (chromatographic column: C18 column 250mm×4.6mm, 5μm; mobile phase: methanol-water=60:40, v / v; detection wavelength 220nm; flow rate 1.0mL / min), and the cumulative permeation amount (μg / cm²) over 24h was calculated.

[0137] Test results

[0138] The cumulative permeation of L-carnosine over 24 hours was 85 μg / cm² ± 2.3 μg / cm², demonstrating that liposome encapsulation technology can effectively improve the transdermal efficiency of L-carnosine, providing a guarantee for its synergistic effect with PDRN in repairing the skin.

[0139] 1.7.6 Puncture Rate Detection

[0140] Detection purpose

[0141] Simulating skin with different stratum corneum thicknesses to verify the actual penetration ability of enhanced transdermal delivery cosmetic patches is the core verification indicator for "high penetration".

[0142] Test methods

[0143] Simulated skin preparation: Take 6 3×3cm² silicone films (each with a thickness of 130μm and a hardness close to that of human stratum corneum), stack them on the base of the universal testing machine, with a total thickness of 520μm, to simulate skin with a thicker stratum corneum (such as the T-zone of the face).

[0144] Puncture test: Take an enhanced transdermal delivery cosmetic patch (10×10 array), place it with the needle facing down on the surface of the silicone membrane; start the universal testing machine, apply a constant pressure of 30N (simulating human body pressure), hold for 2 minutes and then remove the patch.

[0145] Puncture rate calculation: Separate the overlapping silicone membranes layer by layer, observe the number of micropores in each membrane using an optical microscope (magnification 50x), and count the number of microneedles with complete micropores (pore diameter ≥ 50μm). Puncture rate = (number of microneedles with micropores ÷ total number of microneedles) × 100%.

[0146] Test results

[0147] The puncture rate of the 520μm silicone membrane was 96%±1.2%, with the first three membranes (total thickness 390μm) achieving a 100% puncture rate and the fourth membrane (520μm) achieving a 96% puncture rate. This demonstrates that the enhanced transdermal delivery cosmetic patch can effectively penetrate the stratum corneum of different thicknesses and has the characteristic of high permeability.

[0148] 1.7.7 Detection of post-acne pigmentation improvement rate

[0149] Detection purpose

[0150] This study validated the effectiveness of enhanced transdermal cosmetic patches in treating post-acne hyperpigmentation through clinical volunteer trials.

[0151] Test methods

[0152] Volunteer selection and grouping: 30 volunteers were selected who met the following criteria: women aged 20-40 with post-acne brown pigmentation on the face (pigmentation area ≥2cm). 2 The baseline ITA value (skin brightness index, the higher the value, the brighter the skin) was 38°±2°, and no other whitening or repair products were used in the past month. The participants were randomly divided into two groups: the experimental group (15 people) used the enhanced transdermal delivery beauty patch of this invention, and the control group (15 people) used a blank enhanced transdermal delivery beauty patch without PDRN (other components were the same).

[0153] Usage and Environmental Control: Both groups used the product twice a week. After cleansing the face, the enhanced transdermal delivery beauty patch was applied to the pigmented area. Press for 1 minute to ensure adhesion, and remove after 4 hours. The trial period was 8 weeks. During this period, all volunteers used only basic moisturizing lotion (commercially available small red box essence lotion) and avoided sun exposure and spicy food. The test environment was kept at a constant temperature of 22±1℃ and humidity of 50±5%. Volunteers were required to sit quietly in this environment for 20 minutes before each test.

[0154] Evaluation of results: The ITA value of the test site was measured using a skin color analyzer (model: MinoltaCM-700d) at the beginning of the experiment (week 0), week 4 and week 8. The increase in ITA value after 8 weeks was calculated to characterize the rate of improvement in pigmentation (the greater the increase in ITA value, the more obvious the improvement in pigmentation).

[0155] Test results

[0156] After 8 weeks, the ITA value in the experimental group increased from 38°±2° to 43.2°±1.8°, with a pigmentation improvement rate of 92%; while the ITA value in the control group only increased from 38°±2° to 39.5°±1.5°, with an improvement rate of less than 5%. The results demonstrate that the enhanced transdermal delivery cosmetic patch of the present invention can significantly improve post-acne pigmentation.

[0157] Example 2: Effect of PDRN molecular weight on the performance of enhanced transdermal delivery cosmetic patches containing highly active PDRN (sodium DNA).

[0158] 2.1 Raw material preparation

[0159] PDRN (50kDa): DNA fragment derived from salmon testes (prepared by enzymatic digestion). Specific preparation method: Fresh salmon testes were taken, washed with physiological saline, cut into small pieces, and homogenized in 0.1 M Tris-HCl buffer (pH 8.0, containing 0.1 M EDTA and 1% SDS). The mixture was then allowed to stand at 4°C for 2 hours. Proteinase K (final concentration 50 μg / mL) was added and the mixture was incubated in a water bath at 37°C for 4 hours. The mixture was extracted three times with chloroform-isoamyl alcohol (24:1), and DNA was precipitated with anhydrous ethanol. DNase I (purchased from Takara, catalog number 2270A) was used for enzymatic digestion: DNA concentration was 1 mg / mL in 50 mM Tris-HCl (pH 7.5) buffer, and DNase I was added at 10 U / mg DNA. The mixture was reacted at 37°C for 4 hours. The fragment length was verified to be 50-100 bp by 12% agarose gel electrophoresis. Finally, the fragments were purified by Sephadex G-50 chromatography column to obtain PDRN with a molecular weight of 50 kDa and a purity of 98.5% (HPLC verification).

[0160] PDRN (150kDa): DNA fragment derived from salmon testes (prepared in-house using enzymatic digestion). Digestion conditions: DNase I reaction at 37℃ for 1.5h, with the remaining steps the same as for 50kDa PDRN preparation. Fragment length 150-200bp, purity 98.2% (verified by HPLC).

[0161] PDRN (250kDa): DNA fragment from salmon testes (prepared in-house using enzymatic digestion). Digestion conditions: DNase I at 37℃ for 2 hours, with the remaining steps the same as for 50kDa PDRN preparation. Fragment length 250-300bp, purity 98.7% (verified by HPLC).

[0162] PDRN (300kDa): DNA fragment from salmon testes (prepared in-house using enzymatic digestion). Digestion conditions: DNase I at 37℃ for 1 hour, with the remaining steps the same as for 50kDa PDRN preparation. Fragment length 300-400bp, purity 98.1% (verified by HPLC).

[0163] 2.2 Other Materials and Equipment

[0164] Completely consistent with Example 1, including raw materials such as methacryloyl ethyl sulfobetaine (SBMA, purity ≥98%), 80kDa hyaluronic acid (HA, purity ≥99%), dipalmitoyl phosphatidylcholine (DPPC, purity ≥98%), and equipment such as a universal testing machine (Instron 5944), a high performance liquid chromatograph (Agilent 1260), and a Franz diffusion cell (effective transdermal area 1.77cm²).

[0165] 2.3 Sample Preparation

[0166] Following the preparation process of Example 1, four groups of enhanced transdermal delivery cosmetic patches with different PDRN molecular weights were prepared:

[0167] 1. Synthesis of zwitterionic polymer supports: SBMA and HA were reacted at a molar ratio of 1:4 at 50℃ for 7 h, with the grafting rate controlled at 28% ± 1%;

[0168] 2. Preparation of nano-penetration enhancers: all were prepared with a DPPC to cholesterol molar ratio of 7:3, an encapsulation efficiency of 96.5% ± 1.2%, and a particle size of 85 nm ± 3 nm.

[0169] 3. Preparation of needle solution: PDRN loading was 10%±0.2%, and total solids content of the solution was 40%±2%;

[0170] 4. Low-temperature molding and backing composite: Both have a needle height of 750μm, a backing layer protrusion of 35μm, and a needle layer to backing layer thickness ratio of 4:1.

[0171] 2.4 Performance Testing Methods and Results

[0172] 2.4.1 Transdermal efficiency testing

[0173] Detection purpose

[0174] PDRN needs to penetrate the stratum corneum of the skin to reach the dermis in order to exert its repair effect. Transdermal efficiency directly determines the clinical effect and is a key indicator for evaluating the impact of molecular weight on product effectiveness.

[0175] Detection methods

[0176] The Franz diffusion cell method was used. The transdermal barrier was fresh pigskin (500 μm thick, sterilized), and the receiving solution was 0.01 M PBS buffer (pH 7.4, constant temperature 37℃, stirring at 500 rpm). Four groups of enhanced transdermal delivery cosmetic patches were applied to the drug delivery surface of the pigskin. Samples were taken after 24 h, and the PDRN content in the receiving solution was determined by HPLC (same as Example 1.7.4). The transdermal efficiency was calculated (transdermal efficiency = (total PDRN in the receiving solution in 24 h ÷ initial total PDRN in the needle body layer) × 100%).

[0177] Test results

[0178] 50kDa group: Transdermal efficiency 72%±2.1% — The small molecular weight makes PDRN easily degraded by skin surface enzymes, and some of it becomes inactive before reaching the dermis.

[0179] 150kDa group: transdermal efficiency 89%±1.5% — moderate molecular weight, which can penetrate the stratum corneum and reduce surface degradation, significantly improving delivery efficiency;

[0180] 250kDa group: Transdermal efficiency 92%±1.2% - the highest transdermal efficiency, at which point the PDRN molecule size matches the skin pores best, and the anti-degradation ability is better than the 50kDa group;

[0181] 300kDa group: transdermal efficiency 85%±1.8% — The large molecular weight leads to increased penetration resistance, and some PDRN remains in the stratum corneum, resulting in a decrease in transdermal efficiency.

[0182] 2.4.2 Detection of PDRN activity retention rate

[0183] Detection purpose

[0184] To verify the stability of PDRNs of different molecular weights during the storage of enhanced transdermal delivery cosmetic patches, ensuring that the product's activity is not lost during its shelf life.

[0185] Detection methods

[0186] Referring to the accelerated test protocol in Example 1.7.3: four groups of samples were placed in a 60℃ constant temperature chamber for 72 hours, and the PDRN content before and after acceleration was determined by HPLC. The activity retention rate was calculated (activity retention rate = content after acceleration ÷ content before acceleration × 100%).

[0187] Test results

[0188] 50kDa group: Activity retention rate 82%±1.3% — Small molecule PDRN has poor structural stability and is prone to chain breakage at high temperatures, resulting in an activity retention rate of less than 85%.

[0189] 150kDa group: Activity retention rate 86%±1.1% — Improved structural stability, reduced risk of chain breakage at high temperature, meeting the activity retention rate requirements;

[0190] 250kDa group: Activity retention rate 87%±1.0% - the highest activity retention rate, stronger resistance to degradation due to macromolecular structure, and no instability of carrier encapsulation due to excessive molecular weight;

[0191] 300kDa group: Activity retention rate 84%±1.4% - Although higher than 50kDa group, due to stronger intermolecular forces, some PDRN aggregated in the carrier, resulting in a slightly lower activity count during detection.

[0192] 2.4.3 Single Needle Bearing Capacity Test

[0193] Detection purpose

[0194] Microneedles need to be sufficiently rigid to penetrate the skin. The single needle's load-bearing capacity directly determines the penetration success rate and is a core indicator for assessing the impact of molecular weight on product usability.

[0195] Detection methods

[0196] The universal testing machine test scheme according to Example 1.7.1 is as follows: probe diameter 5mm, pressing rate 0.05mm / s, record the pressure when the needle is deformed, and calculate the single needle bearing capacity (total pressure ÷ number of microneedles).

[0197] Test results

[0198] 50kDa group: Single needle load capacity 0.32N±0.02N — The interaction between small molecule PDRN and zwitterionic carrier is weak, the needle structure is not compact enough, and the load capacity is less than 0.35N;

[0199] 150kDa group: Single needle load capacity 0.36N±0.02N — PDRN forms stable hydrogen bonds with the carrier, increasing the needle body hardness and meeting the load capacity requirements;

[0200] 250kDa group: Single needle load capacity 0.37N±0.02N — strongest intermolecular interaction, most compact needle structure, and highest load capacity;

[0201] 300kDa group: Single needle load capacity 0.34N±0.02N — Although higher than the 50kDa group, the distribution uniformity of macromolecular PDRN in the carrier is slightly poor, and there are local weak points, resulting in the load capacity not reaching the optimal level.

[0202] 2.4.4 Dissolution time detection

[0203] Detection methods

[0204] Refer to the saline dissolution protocol in Example 1.7.2: saline at 37°C and pH 7.4, stirred at 50 rpm, and record the time for complete dissolution of the needle.

[0205] Test results

[0206] 50kDa group: Dissolution time 2.2min±0.1min — Small molecule PDRN accelerates carrier swelling and dissolution rate, but excessively rapid dissolution may lead to excessively high local concentration of active ingredient;

[0207] 150kDa group: Dissolution time 2.4min ± 0.1min — The dissolution rate is moderate, which can release the components quickly while avoiding excessive local concentration;

[0208] 250kDa group: Dissolution time 2.6min±0.1min — The dissolution rate is slightly slower, but still within 3min, and the release of components is more gradual, with a longer duration of action;

[0209] 300kDa group: Dissolution time 2.8min ± 0.1min — Macromolecular PDRN delays carrier swelling, and the dissolution time is close to the upper limit of 3min. The timeliness of component release needs to be monitored.

[0210] 2.4.5 Experimental Conclusions

[0211] Based on four core performance indicators, the molecular weight of PDRN has a significant impact on the effectiveness, stability, and practicality of enhanced transdermal delivery cosmetic patches.

[0212] The 150-250kDa range is optimal: within this range, the transdermal efficiency (89%-92%), activity retention rate (86%-87%), and single needle carrying capacity (0.36-0.37N) are significantly better than the 50kDa and 300kDa groups, achieving the best balance of "effectiveness-stability-practicability".

[0213] Example 3: Effect of the ratio of DPPC to cholesterol in liposomes on the permeation-enhancing effect of L-carnosine

[0214] 3.1 Raw material preparation (only materials that differ from those in Example 1 are listed; the rest are the same)

[0215] DPPC: Purity ≥98%, pharmaceutical grade, purchased from Sigma-Aldrich, batch number D8503, purity ≥98%, phase transition temperature 41℃, liposome-specific grade;

[0216] Cholesterol: Purity ≥99%, pharmaceutical grade, purchased from Alfa Aesar, batch number A10015, purity ≥99%, water content ≤0.3%;

[0217] Pair DPPC with the following molar ratios:

[0218] 6:4 group: DPPC to cholesterol molar ratio 6:4, total mass of each group 4.28g (corresponding to L-carnosine 1.0g), of which DPPC 3.0g and cholesterol 1.71g;

[0219] 7:3 group (control): Same as Example 1, DPPC 3.0g, cholesterol 1.28g;

[0220] 8:2 group: DPPC to cholesterol molar ratio 8:2, DPPC 3.0g, cholesterol 0.85g.

[0221] 3.2 Testing Equipment

[0222] Dynamic light scattering (DLS): Malvern Zetasizer NanoZS, used to determine liposome particle size;

[0223] High performance liquid chromatography (HPLC): Agilent 1260, equipped with a C18 column (250 mm × 4.6 mm, 5 μm), used to determine L-carnosine content;

[0224] Ultrafiltration centrifuge tubes: 10kDa molecular weight cutoff, Millipore, used for the separation of free L-carnosine;

[0225] Franz diffusion cell: effective transdermal area 1.77 cm², constant temperature 37℃, used for transdermal testing.

[0226] 3.3 Liposome preparation process (unified operation for three groups)

[0227] 3.3.1 Thin film preparation: DPPC and cholesterol were dissolved in 50 mL of chloroform in a certain proportion, transferred to a rotary evaporator (35℃, 60 rpm), and evaporated for 2 h to form a uniform lipid film;

[0228] 3.3.2 Hydration treatment: Add 20 mL of PBS buffer (0.01 M, pH 7.4), hydrate in a constant temperature water bath at 37 °C for 2 h, and shake once every 15 min (amplitude 3 cm).

[0229] 3.3.3 Ultrasonic treatment: Ultrasonic treatment at 300W power for 10 min (3 s working / 5 s intermittent) to obtain liposome suspension;

[0230] 3.3.4 Sterilization treatment: Filter through a 0.22μm filter membrane to remove large particle agglomerates, and store at 4℃ for later use.

[0231] 3.4 Performance Testing Methods and Results

[0232] 3.4.1 Encapsulation efficiency test

[0233] Detection purpose

[0234] Encapsulation efficiency directly reflects the efficiency of liposomes in encapsulating L-carnosine and is a core indicator for measuring the drug loading of penetration enhancers. The higher the encapsulation efficiency, the lower the skin irritation of the free drug and the better the sustained-release effect.

[0235] Detection methods

[0236] The method employed was "ultrafiltration centrifugation-HPLC coupled".

[0237] 1. Take 1 mL of liposome suspension, add it to an ultrafiltration centrifuge tube, centrifuge at 8000 rpm for 30 min, and the supernatant is free L-carnosine;

[0238] 2. Take another 1 mL of suspension, add 1 mL of methanol to break the emulsion (destroy the liposome structure), shake for 10 min, filter through a 0.22 μm filter membrane, and use this as the total drug amount;

[0239] 3. HPLC detection (mobile phase methanol-water = 60:40, detection wavelength 220 nm), calculation of encapsulation efficiency:

[0240] Encapsulation rate = (Total drug amount - Free drug amount) / Total drug amount × 100%.

[0241] Test results

[0242] Group 6:4: Encapsulation rate 92%±1.3% - The high cholesterol ratio (40%) led to excessive fluidity of the liposome bilayer membrane, and some L-carnosine leaked during sonication or centrifugation.

[0243] 7:3 group: Encapsulation rate 96.5%±1.2% - with a moderate cholesterol ratio (30%), it forms a stable "rigid-fluidity balance" structure with DPPC, resulting in the best encapsulation effect;

[0244] 8:2 group: Encapsulation rate 93%±1.5% - The cholesterol ratio was too low (20%), resulting in an overly rigid membrane structure. During hydration, the membrane was difficult to disperse completely, and some L-carnosine was not encapsulated.

[0245] 3.4.2 Average particle size detection

[0246] Detection methods

[0247] DLS assay was performed as follows: 1 mL of liposome suspension was diluted 10 times with PBS (to avoid multiple scattering), injected into a cuvette, and the temperature was set to 25℃. After equilibration for 2 min, the sample was detected. Each sample was repeated 3 times, and the average value was taken.

[0248] Test results

[0249] Group 6:4: Average particle size 95nm±4nm — Excess cholesterol leads to increased membrane fluidity, making liposomes prone to aggregation and resulting in larger particle size;

[0250] Group 7:3: Average particle size 85nm±3nm — smallest particle size and uniform distribution (PDI=0.12), with the best transdermal potential;

[0251] 8:2 group: average particle size 90nm±3nm - insufficient cholesterol leads to increased membrane rigidity, making ultrasonic dispersion more difficult, and some particles are not completely dispersed, with a particle size slightly larger than that of the 7:3 group.

[0252] 3.4.324h cumulative permeability detection (to assess the actual permeability enhancement effect)

[0253] Detection methods

[0254] 1. Transdermal barrier: Fresh pig skin (500 μm thick, dehaired and sterilized) was fixed in a Franz diffusion cell, and the receiving solution was 0.01 M PBS (37 °C, 500 rpm stirring).

[0255] 2. Sample loading: The enhanced transdermal delivery cosmetic patch containing different liposomes (prepared according to the formulation of Example 1) was applied to the surface of the pigskin and pressed for 30 seconds to ensure adhesion;

[0256] 3. Sampling and testing: Samples were taken at 1, 4, 8, 12 and 24 hours, and the L-carnosine content in the receiving solution was determined by HPLC. The cumulative permeation amount (μg / cm²) over 24 hours was calculated.

[0257] Test results

[0258] Group 6:4: 75μg / cm²±2.1μg / cm² — Although the encapsulation efficiency reached 92%, due to the large particle size (95nm) and poor membrane stability, some liposomes ruptured prematurely on the skin surface, resulting in insufficient effective transdermal absorption.

[0259] 7:3 group: 85μg / cm²±2.3μg / cm² — with the smallest particle size (85nm) and stable membrane structure, it can not only penetrate the skin efficiently, but also slowly release L-carnosine in the dermis, resulting in the highest penetration.

[0260] Group 8:2: 78μg / cm²±2.2μg / cm² — Particle size (90nm) is slightly larger than that of Group 7:3, and the membrane rigidity is too strong, which slows down the release rate and the transdermal amount is lower than that of Group 7:3.

[0261] in conclusion

[0262] The ratio of DPPC to cholesterol directly regulates the membrane structure stability, particle size, and drug release behavior of liposomes, thus significantly affecting the transdermal permeation-enhancing effect of L-carnosine, as shown below:

[0263] The optimal ratio of DPPC to cholesterol is 7:3: at this ratio, the encapsulation efficiency of liposomes reaches its highest level (96.5%), which can effectively reduce the leakage of L-carnosine during preparation and storage; at the same time, the average particle size of the formed liposomes is the smallest (85nm), which can pass smoothly through skin pores and reduce penetration resistance; finally, the cumulative penetration of L-carnosine in 24 hours reaches 85μg / cm², which fully proves the scientific validity of the 7:3 ratio of DPPC to cholesterol.

[0264] Liposomes deviating from the 7:3 ratio exhibit significant performance defects: when the DPPC to cholesterol ratio is 6:4, the excessively high cholesterol content leads to overly fluid liposome bilayer membranes, reducing the stability of L-carnosine encapsulation and causing some drugs to be released prematurely at the skin surface, ultimately resulting in a significant decrease in both encapsulation efficiency and transdermal absorption. When the ratio is 8:2, the excessively low cholesterol content results in an overly rigid membrane structure, increasing the difficulty of ultrasonic dispersion (leading to an increase in particle size to 90 nm) and slowing down the release rate of L-carnosine in the dermis, similarly failing to achieve the desired permeation-enhancing effect. The overall performance of liposomes deviating from the 7:3 ratio is far inferior to that of the 7:3 group, further highlighting the necessity of this specific ratio.

[0265] Example 4: Effect of backing layer protrusion height on the fit and drug utilization of enhanced transdermal delivery cosmetic patches containing highly active PDRN (sodium DNA).

[0266] 4.1 Raw material preparation (only materials that differ from those in Example 1 are listed; the rest are the same)

[0267] Backing layer substrate: PCL-PEG-PCL block copolymer, molecular weight 10000Da (PCL segment to PEG segment molar ratio 2:1), medical grade, purity ≥98%, molecular weight distribution coefficient (PDI) of 1.2 as determined by GPC;

[0268] The raised molding mold is a PDMS imprinting mold prepared by photolithography. The raised heights are 0μm (smooth control group), 20μm, 30μm and 50μm respectively. The cross-section of the raised is semi-circular and the spacing is 100μm (to ensure that the raiseds do not overlap and are adapted to the skin texture).

[0269] Skin simulation model: Fresh pigskin (1.0 mm thick, after hair removal, washing with saline solution, and disinfection with 0.1% benzalkonium chloride to simulate the texture and elasticity of human facial skin).

[0270] Other materials, such as chloroform (anhydrous grade, purity ≥99.8%) and PBS buffer (0.01M, pH 7.4), are consistent with the basic example.

[0271] 4.2 Key Testing Equipment

[0272] Imprinting equipment: Small desktop imprinting machine (model: RKPrintCoatK303), controllable pressure range 0-10N / cm², temperature accuracy ±1℃;

[0273] Adhesion testing system: includes a constant temperature and humidity chamber (temperature 37℃±0.5℃, humidity 50%±5%, simulating the human skin environment) and a tension sensor (range 0-5N, accuracy 0.01N, used to monitor the pulling force when the patch falls off).

[0274] High-performance liquid chromatography (HPLC): Model Agilent 1260, equipped with an amino column (250 mm × 4.6 mm, 5 μm) and a C18 column (250 mm × 4.6 mm, 5 μm), used for determining drug content;

[0275] Optical microscope: Model OlympusBX53, magnification 50-200x, used to verify the actual size of the protrusion height (avoiding height deviation caused by mold error).

[0276] 4.3 Sample preparation process

[0277] 4.3.1 Needle body layer preparation (consistent with the basic example)

[0278] 1. Weigh out the following according to the formula: PDRN (salmon testes, molecular weight 200kDa, length 200-300bp), zwitterionic polymer carrier (SBMA-HA copolymer, grafting rate 28%), nano-penetration enhancer (DPPC-cholesterol liposome encapsulating L-carnosine, encapsulation rate 96.5%), and cryoprotectant (trehalose:mannitol = 2:1). Dissolve in deionized water at 4℃ and sonicate at 200W for 5 minutes to form a homogeneous solution (total solids content 40%).

[0279] 2. Inject the needle solution into the PDMS microneedle mold (needle height 750μm), pre-freeze at -40℃ for 2h, and then freeze-dry according to the gradient temperature program (-40℃→-20℃, 2℃ / h; -20℃→0℃, 1℃ / h; 0℃→25℃, 0.5℃ / h) to obtain the molded needle layer (thickness after drying 750μm±20μm).

[0280] 4.3.2 Composite backing layers with different protrusion heights

[0281] 1. Preparation of backing layer solution: Weigh 20g of PCL-PEG-PCL, dissolve it in 100mL of chloroform, and stir magnetically for 2h (200rpm) until completely dissolved to obtain a transparent solution with a mass-volume ratio of 20%;

[0282] 2. Coating and Imprinting:

[0283] The needle body layer is fixed on the stage, and the backing layer solution is uniformly coated on the back of the needle body layer using an automatic coater (wet film thickness 200μm) to ensure that the coating area is completely matched with the needle body layer.

[0284] Immediately cover the wet film surface with the PDMS embossing mold of the corresponding height (with the protrusion facing down), apply a pressure of 5 N / cm² through the embossing machine, and hold for 10 minutes (to ensure that the protrusion is completely replicated).

[0285] Transfer to a clean environment at 25°C and 45% humidity and leave for 8 hours to allow chloroform to evaporate completely (solvent residue ≤0.1%, verified by gas chromatography).

[0286] 3. Thickness ratio control: The thickness of the backing layer after drying is 187.5μm±10μm, and the thickness ratio of the needle layer to the backing layer is 4:1 (750μm:187.5μm), which is within a reasonable range of 3:1 to 5:1;

[0287] 4. Protrusion height verification: Randomly select 10 protrusions using an optical microscope and measure their height (take the average value) to ensure that the deviation between the actual height and the design height is ≤5% (e.g., the actual measured value of a design height of 30μm is 28.5-31.5μm).

[0288] 4.5 Performance Testing Methods and Results

[0289] 4.5.1 Adhesion retention time test (to assess long-term adhesion stability)

[0290] Detection purpose

[0291] The adhesion retention time refers to the longest time that the patch maintains an effective adhesion (without obvious lifting or falling off) on the simulated skin surface, which directly affects the continuous release and absorption of active ingredients—the longer the retention time, the easier it is for the drug to fully penetrate.

[0292] Detection methods

[0293] 1. Preparation of simulated skin: Cut fresh pigskin into 5×5cm² samples, fix them on a constant temperature stage (37℃±0.5℃, humidity 50%±5%), and wipe the surface with physiological saline (simulating the sweat environment of skin).

[0294] 2. Patch Application: Take 10 enhanced transdermal delivery cosmetic patches of varying protrusion heights and apply them to the center of the pigskin. Press firmly with your finger for 30 seconds (approximately 20 N / cm). 2 (simulating the pressure applied during human use);

[0295] 3. Dynamic monitoring: The edge lifting of the patch is monitored in real time through the fit test system. When the edge lifting length of the patch exceeds 1mm, the time is recorded. If there is no lifting within 6 hours, it is recorded as "≥6h".

[0296] 4. Data processing: Take the average value of 10 samples in each group, repeat the experiment 3 times, and calculate the standard deviation.

[0297] Test results

[0298] 0μm (smooth group): Adhesion retention time 2.5h±0.2h — The lack of raised structure results in low friction between the patch and the skin, and it cannot adapt to the fine texture of the skin surface. It is prone to lifting due to simulated sweat evaporation or slight movement.

[0299] 20μm raised group: Adhesion retention time 5.5h±0.3h — The raised structure increases the contact area between the patch and the skin, and the raised part can be embedded in the gaps of the skin texture, which significantly improves the friction and extends the retention time by more than 1 times;

[0300] 30μm raised group: Adhesion retention time 6.8h±0.3h — The height of the raised group matches the depth of the skin texture (about 20-40μm) best, which can firmly embed into the texture without causing local pressure due to excessively high raised groups, thus further extending the retention time;

[0301] 50μm protrusion group: Adhesion retention time 7.2h±0.3h — The protrusion height exceeds the depth of most skin textures. Although the friction is still relatively high, some protrusions are prone to "lifting" effect due to slight skin deformation, so the improvement in retention time is limited (only 0.4h more than the 30μm group).

[0302] 4.5.26h shedding rate test

[0303] Detection purpose

[0304] The detachment rate is a quantitative indicator of fit, reflecting the probability of the patch falling off within a 6-hour usage cycle (which is consistent with the single usage duration for home care). It is directly related to user experience and medication adherence.

[0305] Detection methods

[0306] 1. Sample grouping and bonding: Take 10 patches for each group, apply them to the simulated skin surface according to method 5.1, and place them in a constant temperature and humidity chamber (37℃, 50% humidity).

[0307] 2. Dynamic interference: Apply slight vibrations to the simulated skin every hour (amplitude 5mm, frequency 1Hz, simulating daily human activities such as speaking and facial expression changes).

[0308] 3. Detachment judgment: Observe the patch status after 6 hours. If the detached area of ​​the patch exceeds 50%, it is judged as "detached". Count the number of detached patches in each group and calculate the detachment rate (detachment rate = number of detached patches / total number of patches × 100%).

[0309] 4. Replication verification: Each group of experiments was repeated 3 times, and the mean and standard deviation were taken.

[0310] Test results

[0311] 0μm (smooth group): detachment rate 35%±2.5% - more than 1 / 3 of the patches detached within 6 hours, mainly due to poor bonding stability and inability to withstand slight vibration interference;

[0312] 20μm protrusion group: detachment rate 12%±1.2% — the detachment rate was significantly reduced, with only a few patches detaching due to insufficient edge adhesion;

[0313] 30μm raised group: detachment rate 8%±0.8% - the lowest detachment rate, most patches can remain intact for 6 hours, only a few detached due to uneven raised height during imprinting;

[0314] 50μm protrusion group: detachment rate 5%±0.5% - the detachment rate is slightly lower than that of the 30μm group, but the difference is small (only 3 percentage points). In addition, some samples have excessively high protrusions, which cause local air bubbles to form during bonding, thus increasing the risk of detachment.

[0315] 4.5.3 Drug utilization rate detection

[0316] Detection purpose

[0317] Drug utilization rate refers to the proportion of the effective ingredients (PDRN and L-carnosine) actually absorbed by the skin to the initial drug load of the patch. It is the ultimate functional manifestation of the fit - the better the fit, the less likely the effective ingredients are to be lost due to patch detachment, and the higher the utilization rate.

[0318] Detection methods

[0319] 1. Drug loading determination of patches: Take patches of each protrusion height (3 patches per group), cut them into small pieces, and dissolve them by sonication in 20 mL PBS buffer (0.01 M, pH 7.4) for 30 min. HPLC was used to determine the initial total amount of PDRN (amino column, mobile phase 0.1 M PBS pH 7.4) and L-carnosine (C18 column, mobile phase methanol-water = 60:40).

[0320] 2. Determination of drug residues in the skin: The patch was applied to simulated skin as described in 5.1. After 6 hours, the patch was removed, and the residual drug on the skin surface was washed with physiological saline. The skin was then cut into pieces and 20 mL of PBS buffer was added. The mixture was extracted by sonication for 30 min (300 W power). After filtration through a 0.22 μm filter membrane, the total amount of residual drug in the skin was determined by HPLC.

[0321] 3. Drug utilization rate calculation: Drug utilization rate = (Total drug in skin / Initial drug loading of patch) × 100%;

[0322] 4. Data processing: Take the average of 3 trials for each group and calculate the standard deviation.

[0323] Test results

[0324] 0μm (smooth group): Drug utilization rate 78%±1.8% - Due to the premature detachment of some patches, approximately 22% of the drug was not absorbed by the skin and was lost with the detached patches;

[0325] 20μm protrusion group: drug utilization rate 86%±1.5% - improved adhesion stability reduces drug loss and increases utilization rate by 8 percentage points;

[0326] 30μm protrusion group: drug utilization rate 92%±1.2% - the highest utilization rate, with only 8% of the drug lost due to incomplete penetration into the skin surface, and no drug waste due to patch detachment;

[0327] 50μm protrusion group: drug utilization rate 93%±1.0% - the utilization rate is slightly higher than that of the 30μm group, but the increase is only 1 percentage point. Moreover, due to the excessive protrusion, some needles do not make sufficient contact with the skin, which reduces the local drug penetration efficiency.

[0328] in conclusion

[0329] The height of the backing layer protrusion significantly affects the adhesion stability and drug utilization by altering the contact mode between the patch and the skin (friction, texture compatibility). Specific conclusions are as follows:

[0330] 1. 20-50μm protrusion height can effectively improve adhesion: Compared with no protrusion (0μm), protrusions in this range can extend the adhesion retention time by 2-3 times, reduce the detachment rate by 23-30 percentage points, and increase drug utilization by 8-15 percentage points, proving the key role of micron-level protrusion structure in improving adhesion;

[0331] 2.30μm protrusion is the optimal height: This height matches the skin texture depth best, achieving long-term adhesion for 6.8 hours, a low detachment rate of 8% and a high drug utilization rate of 92%, while avoiding the local pressure and air bubble problems that may be caused by 50μm protrusion. At the same time, the preparation difficulty is relatively low (the precision of the imprinting mold is easy to control, and the yield rate is over 95%), making it the most cost-effective.

[0332] 3. The height of the protrusion is not necessarily better the higher it is: Although the 50μm protrusion is slightly better than the 30μm group in terms of adhesion retention time and detachment rate, the improvement in drug utilization is limited, and the preparation cost increases (the mold precision requirements are higher) and the user comfort decreases, which does not meet the balance principle of "performance-cost-experience".

[0333] In summary, the 20-50μm micron-level protrusions on the surface of the backing layer can effectively solve the problem of poor patch adhesion. Among them, 30μm protrusions are the optimal choice, which can maximize the full penetration of the drug and provide structural support for the actual performance of the product.

[0334] Example 5: Synergistic effect of functional additives (tranexamic acid, glutathione, centella asiatica extract)

[0335] 5.1 Raw material preparation (only materials that differ from those in Example 1 are listed; the rest are the same)

[0336] Tranexamic acid: pharmaceutical grade, purchased from Aladdin Reagent (Shanghai) Co., Ltd., catalog number A100234, purity ≥99%, water solubility ≥50g / 100mL (25℃), no heavy metal residue (≤1ppm); added to the needle body layer according to the following mass percentages: group 10.1%, group 20.2%, group 30.3%, and group with tranexamic acid alone 0.2%.

[0337] Glutathione (reduced): pharmaceutical grade, purchased from Sigma-Aldrich (USA), catalog number G4251, purity ≥98%, optical rotation -21.0°~-23.0°, stability: activity retention ≥95% after 6 months of storage at 4℃; added to the needle body layer in the following mass percentages: group 12%, group 23%, group 34%, and group 3% with glutathione alone.

[0338] Centella Asiatica extract: purchased from Chengdu Kloma Biotechnology Co., Ltd., product name "pharmaceutical grade Centella Asiatica extract", product number KLMA-003, asiaticoside purity ≥40% (HPLC determination), solvent residue ≤0.1%, pH 5.0-7.0; added to the backing layer in the following mass percentages: group 15%, group 26%, group 37%, and group with Centella Asiatica alone 6%.

[0339] The remaining ingredients, including PDRN, hyaluronic acid, SBMA, L-carnosine, DPPC, cholesterol, trehalose, mannitol, PCL-PEG-PCL, APS, PBS buffer, and chloroform, are completely consistent with those in Example 1.

[0340] Testing equipment

[0341] Skin barrier tester: Model Courage+KhazakaMPA580, used to measure TEWL value (transepidermal water loss) and stratum corneum moisture content;

[0342] Enzyme-linked immunosorbent assay (ELISA) analyzer: Model ThermoScientificMultiskanFC, used to detect the level of IL-6 (inflammatory factor) in skin tissue;

[0343] Skin color analyzer: Model MinoltaCM-700d, used to measure ITA value (characterizes skin brightness and assesses improvement in pigmentation).

[0344] Skin irritation assessment tool: Standard skin irritation rating card (0-5 points, 0 points no reaction, 5 points severe redness, swelling and oozing).

[0345] 5.2 Sample preparation process

[0346] 5.2.1 Basic Process (Parts consistent with the basic embodiment)

[0347] Synthesis of zwitterionic polymer support: SBMA and HA were reacted at a molar ratio of 1:4 at 50°C for 7 h, purified by dialysis, and then lyophilized.

[0348] Preparation of nano-permeability enhancer: Liposomes were prepared by thin-film hydration method using L-carnosine and DPPC in a mass ratio of 1:3, and sterilized by 0.22μm filter membrane;

[0349] Low-temperature molding: Pre-freeze the needle solution at -40℃ for 2 hours, then freeze-dry with gradient temperature increase (-40℃→-20℃→0℃→25℃).

[0350] Backing layer composite: coated with 20% PCL-PEG-PCL chloroform solution, solvent evaporated at 25℃, thickness ratio 4:1.

[0351] 5.2.2 Steps for adding functional additives (differentiated part)

[0352] Group Tranexamic acid (needle layer) Glutathione (needle layer) Centella Asiatica extract (backing layer) Add method Group 1 0.1% 2% 5% 1. When preparing the needle solution, first dissolve tranexamic acid and glutathione, then add other components; 2. Add 5% Centella asiatica extract to the backing layer solution and stir for 3 hours until homogeneous. Group 2 0.2% 3% 6% In the same group 1, only the concentration was adjusted. Group 3 0.3% 4% 7% In the same group 1, only the concentration was adjusted. single tranexamic acid group 0.2% 0% 0% Only 0.2% tranexamic acid was added to the needle body layer; nothing else was added. Glutathione-only group 0% 3% 0% Only 3% glutathione was added to the needle body layer, and nothing else. Single Centella Asiatica group 0% 0% 6% Only 6% Centella Asiatica extract is added to the backing layer; none is added otherwise.

[0353] 5.2.3 Sample Quality Control

[0354] Total solids content of the needle solution: 40% ± 2% (determined by vacuum drying method);

[0355] Centella asiatica extract dispersibility: The backing solution was sonicated for 10 min (200 W) to ensure no agglomeration;

[0356] Additive activity retention: After preparation, storage at 4℃, HPLC analysis showed that the activity retention of tranexamic acid and glutathione was ≥98% within 3 days.

[0357] 5.3 Performance Testing Methods and Results

[0358] 5.3.1 Improvement rate of post-acne hyperpigmentation (assessing the synergistic effect of whitening and anti-inflammatory)

[0359] Detection purpose

[0360] Improving post-acne pigmentation requires a synergistic effect of "anti-inflammatory (reducing post-inflammatory pigmentation) + whitening (decomposing existing melanin)," and the effect is quantified by changes in ITA value (the higher the value, the brighter the skin tone).

[0361] Detection methods

[0362] Volunteer selection: 36 female volunteers aged 20-40 with post-acne brown pigmentation on their faces (area ≥2cm). 2 (Baseline ITA value 38°±2°), no whitening / anti-inflammatory products used in the past month, and randomly divided into 6 groups (6 people in each group, corresponding to 6 sample groups);

[0363] Usage instructions: Use twice a week. After cleansing the face, apply the patch to the pigmented area and remove it after 4 hours. The cycle is 8 weeks. The test environment is a constant temperature of 22±1℃ and humidity of 50±5%. Volunteers should sit quietly for 20 minutes before each test.

[0364] ITA value determination: ITA values ​​of the test sites were measured using a skin color analyzer at weeks 0 (baseline), 4, and 8. The improvement rate at 8 weeks was calculated (Improvement rate = (8-week ITA value - baseline ITA value) / baseline ITA value × 100%).

[0365] Test results

[0366] Group 8-week improvement rate of pigmentation after acne treatment Synergistic effect analysis Group 1 85%±1.5% The concentration is low, resulting in insufficient anti-inflammatory and whitening effects, and a slightly lower improvement rate. Group 2 92%±1.2% The three components are at optimal concentrations: tranexamic acid inhibits inflammatory factors and reduces pigment production, glutathione breaks down melanin, and centella asiatica repairs the skin barrier and promotes absorption, resulting in the best synergistic effect. Group 3 93%±1.0% It has the highest concentration and slightly better whitening and anti-inflammatory effects, but its irritation level is higher (see 5.4), making it less cost-effective. single tranexamic acid group 62%±2.0% It can only reduce the formation of new pigments, but cannot break down existing melanin, and its improvement rate is significantly lower than that of the compound group. Glutathione-only group 58%±1.8% It can only break down melanin, but uncontrolled inflammation leads to the continuous generation of new pigment, resulting in a low improvement rate. Single addition of Centella asiatica group 45%±2.2% It can only repair the skin barrier and has no direct anti-inflammatory or whitening effect, resulting in the lowest improvement rate.

[0367] 5.3.2 TEWL value decrease rate (assessing the skin barrier repair effect)

[0368] Detection purpose

[0369] The TEWL value (transepidermal water loss) reflects the integrity of the skin barrier. The lower the value, the better the barrier function. Centella asiatica extract is the core repair ingredient, while tranexamic acid has anti-inflammatory properties that can reduce barrier damage. The two work together to improve repair efficiency.

[0370] Detection methods

[0371] Test subjects: 24 volunteers with impaired skin barrier (TEWL value ≥ 20g / ( (The individuals with stratum corneum moisture content ≤20 AU) were randomly divided into 6 groups (4 people in each group).

[0372] Testing steps: Before using the sample (week 0) and after 4 weeks of use, measure the TEWL value of the test site with a skin barrier analyzer and calculate the reduction rate (reduction rate = (TEWL value at week 0 - TEWL value at week 4) / TEWL value at week 0 × 100%).

[0373] Environmental control: Before the test, volunteers sat quietly for 30 minutes in a constant temperature and humidity environment (22℃, 50% humidity) to avoid interference from sweat.

[0374] Test results

[0375] Group 4-week TEWL value decrease rate Synergistic effect analysis Group 1 38%±1.8% The lower the concentration of Centella asiatica, the slower the barrier repair speed. Group 2 42%±1.5% Centella asiatica extract promotes collagen production, while tranexamic acid reduces the ongoing damage to the skin barrier caused by inflammation. The synergistic effect of these two ingredients maximizes the efficiency of barrier repair. Group 3 45%±1.2% The highest concentration of Centella asiatica has a slightly better repair effect, but high concentrations may cause mild local irritation. single tranexamic acid group 20%±1.5% It only has anti-inflammatory properties and no direct barrier repair effect, resulting in a low reduction rate. Glutathione-only group 15%±1.2% It has no anti-inflammatory or repair function and has no significant effect on TEWL value. Single addition of Centella asiatica group 35%±1.8% It can only repair the barrier, but the lack of control over inflammation causes the repair effect to be negated, resulting in a lower rate of decline compared to the compound group.

[0376] 5.3.3 IL-6 reduction rate (assessing anti-inflammatory effect)

[0377] Detection purpose

[0378] IL-6 is a core inflammatory factor, and its content increases when there is acne or skin barrier damage. Tranexamic acid can inhibit the release of IL-6, and Centella asiatica extract can reduce the infiltration of inflammatory cells. The two work synergistically to enhance the anti-inflammatory effect.

[0379] Detection methods

[0380] Sample processing: Each group of enhanced transdermal delivery cosmetic patches was applied to the back skin of mice (3 mice per group, to establish an inflammation model: 1% capsaicin was applied to induce inflammation), and skin tissue was collected 24 hours later;

[0381] IL-6 detection: Skin tissue was minced and homogenized with PBS buffer, centrifuged and the supernatant was collected. The IL-6 content was measured using an ELISA kit, and the reduction rate was calculated (reduction rate = (IL-6 content in model group - IL-6 content in sample group) / IL-6 content in model group × 100%).

[0382] Test results

[0383] Group IL-6 reduction rate Synergistic effect analysis Group 1 52%±1.5% The concentration of tranexamic acid is low, resulting in insufficient anti-inflammatory efficacy. Group 2 58%±1.2% Tranexamic acid inhibits IL-6 release, and Centella asiatica extract reduces the aggregation of inflammatory cells; the synergistic effect of the two results in optimal anti-inflammatory efficacy. Group 3 60%±1.0% Tranexamic acid had the highest concentration and slightly better anti-inflammatory effect, but its irritant properties increased. single tranexamic acid group 45%±1.8% The anti-inflammatory effect of tranexamic acid alone, without the synergistic effect of Centella asiatica, is lower than that of the compound group. Glutathione-only group 10%±0.8% It has no anti-inflammatory function and no significant effect on IL-6. Single addition of Centella asiatica group 38%±1.5% The anti-inflammatory effect of Centella asiatica alone, without the synergistic effect of tranexamic acid, is lower than that of the combined group.

[0384] 5.3.4 Skin irritation score (to assess safety)

[0385] Detection purpose

[0386] High concentrations of additives may cause skin irritation (e.g., excessively high concentrations of tranexamic acid may cause dryness, and excessively high concentrations of centella asiatica extract may cause allergies), so it is necessary to balance efficacy and safety.

[0387] Detection methods

[0388] Test subjects: 24 healthy volunteers (skin types I-IV), randomly divided into 6 groups (4 people in each group);

[0389] Irritation test: Apply the sample to the inside of the volunteer's forearm (3×3cm) 2 Remove the mask after 24 hours and observe the skin reaction. Score the skin reaction using a standard scoring card (0 points: no reaction; 1 point: slight redness; 2 points: obvious redness; 3 points: redness and swelling; 4 points: redness and swelling + papules; 5 points: redness and swelling + oozing).

[0390] Scoring statistics: The average score of 4 people in each group was taken, and the experiment was repeated 3 times.

[0391] Test results

[0392] Group Skin irritation score Security Analysis Group 1 1.0±0.2 Low concentration results in minimal irritation, but insufficient effectiveness. Group 2 1.1±0.2 It causes only slight redness, with no other discomfort, and offers the best balance between safety and effectiveness. Group 3 1.5±0.2 Some volunteers experienced noticeable redness and increased irritation. Although this did not meet the "discomfort" standard, the safety level was lower than that of group 2. single tranexamic acid group 0.8±0.1 It has low irritant properties, but no synergistic effect. Glutathione-only group 0.9±0.1 It has low irritant properties, but no synergistic effect. Single addition of Centella asiatica group 1.0±0.1 It has low irritant properties, but no synergistic effect.

[0393] in conclusion

[0394] The functional additives exhibited a significant synergistic effect: the improvement rate of post-acne pigmentation (85%-93%), the decrease rate of TEWL value (38%-45%), and the reduction rate of IL-6 (52%-60%) in the compound additive groups (groups 1-3) were all much higher than those in the single additive groups (improvement rate 45%-62%, decrease rate 15%-35%, and reduction rate 10%-45%). This demonstrates that the anti-inflammatory effect of tranexamic acid, the whitening effect of glutathione, and the barrier repair effect of Centella asiatica extract can form a functional closed loop of "anti-inflammatory-whitening-repair", with a significant synergistic effect of 1+1+1>3.

[0395] Group 2 (0.2% tranexamic acid + 3% glutathione + 6% centella asiatica extract) is the optimal ratio: this group achieves a 92% improvement rate in post-acne pigmentation, a 42% reduction rate in TEWL value, and a 58% reduction rate in IL-6, while only showing a slight irritation score of 1.1, fully meeting the "effectiveness-safety" balance requirement for skin repair.

[0396] Both excessively high and excessively low concentrations have drawbacks: the low concentration in Group 1 results in insufficient efficacy, while the high concentration in Group 3, although slightly better, increases irritation and production costs (e.g., 7% Centella asiatica extract costs 15% more than 6%), which does not meet the cost-effectiveness principle of industrial production.

[0397] In summary, the combination of adding 0.2% tranexamic acid and 3% glutathione to the needle body layer and 6% Centella asiatica extract to the backing layer can maximize the synergistic effect of the three, providing core support for the product's treatment of post-acne pigmentation and skin barrier repair function.

[0398] Comparative Example 1: Performance Comparison between Traditional Hyaluronic Acid Carriers and Zwitterionic Polymer Carriers

[0399] Hyaluronic acid (HA) is a commonly used carrier material for enhanced transdermal delivery cosmetic patches. However, unmodified HA suffers from drawbacks such as insufficient stability, limited transdermal efficiency, and low mechanical strength. This comparative example uses unmodified HA to replace the zwitterionic polymer carrier (methacryloyl ethyl sulfobetaine-hyaluronic acid copolymer, SBMA-HA) in Example 1. Under identical conditions, the effects of the two carriers on PDRN activity retention, transdermal efficiency, and mechanical properties are systematically compared. This demonstrates the breakthrough improvement of carrier function brought about by zwitterionic modification and verifies its irreplaceable role in this application.

[0400] This comparative example only replaces the carrier material with unmodified hyaluronic acid (80kDa) with the same molecular weight as the HA fragment in Example 1. All other parameters (including PDRN specifications, liposome formulation, cryoprotectant ratio, preparation process, detection method, etc.) are completely consistent with Example 1, ensuring that the difference in detection results is only caused by the structural difference of the carrier material (whether it has been modified with zwitterions).

[0401] Experimental materials and equipment

[0402] 1.1 Comparison of carrier materials

[0403] Material parameters Comparative Example 1 (Unmodified HA) Example 1 (SBMA-HA copolymer) Source of raw materials Prepared by microbial fermentation, pharmaceutical grade, purchased from Bloomage Biotechnology Co., Ltd., product number HA-MW80k Same as left, modified by SBMA grafting molecular weight 80 kDa (GPC measured, PDI=1.3) 80kDa (HA backbone, total molecular weight after SBMA grafting is approximately 95kDa) Chemical structure Pure HA chain (repeating units: D-glucuronic acid and N-acetylglucosamine) HA-linked SBMA (grafting rate 28%, sulfobetaine group) Water-soluble 20g / 100mL (25℃) 35g / 100mL (25℃, zwitterionic groups enhance hydrophilicity) isoelectric point Approximately 3.0 (easily aggregates under acidic conditions) Approximately 7.0 (close to physiological pH, making it less prone to aggregation)

[0404] 1.2 Other raw materials: PDRN, SBMA (only used in Example 1 for synthesizing the vector, Comparative Example 1 did not have the SBMA grafting step), L-carnosine, DPPC, cholesterol, trehalose, mannitol, PCL-PEG-PCL, APS, PBS buffer, chloroform, etc., are all completely consistent with Example 1 (Note: Comparative Example 1 did not have the SBMA grafting step, so SBMA was not actually added, and only other raw materials consistent with Example 1 were retained).

[0405] Testing equipment: Same as in Example 1, including HPLC (to measure PDRN content), Franz diffusion cell (to measure transdermal absorption), universal testing machine (to measure single needle load capacity), and constant temperature accelerated test chamber (to measure activity retention rate), etc.

[0406] 1.3 Sample preparation process

[0407] 1.3.1 Comparative Example 1 (Unmodified HA Carrier) Preparation Steps

[0408] Carrier solution preparation: Weigh 40g of unmodified HA (80kDa), dissolve it in 100mL of 4℃ deionized water, and stir magnetically for 2h until completely dissolved to form a 40% (w / w) transparent solution;

[0409] Needle body solution preparation: According to the formulation in Example 1, PDRN (10%), unmodified HA carrier (40%), nano-penetration enhancer (15%), and cryoprotectant (trehalose:mannitol = 2:1, 25%) were added to deionized water in sequence, and the solution was sonicated at 200W for 5 minutes to form a homogeneous solution (total solid content 40%).

[0410] Low-temperature molding: completely consistent with Example 1 - injection into a PDMS mold (needle height 750μm), pre-freezing at -40℃ for 2h, and freeze-drying by gradient temperature increase (-40℃→-20℃→0℃→25℃).

[0411] Backing layer composite: Same as Example 1—coating with 20% PCL-PEG-PCL chloroform solution, solvent evaporated at 25°C, needle layer to backing layer thickness ratio 4:1.

[0412] 1.3.2 Preparation of Control Sample for Example 1 (SBMA-HA Vector)

[0413] Prepared according to the standard procedure of Example 1, ensuring that all steps and parameters are completely consistent with Comparative Example 1 except for the carrier material, as a benchmark for performance comparison.

[0414] 1.4 Performance Testing Methods and Results

[0415] 1.3.1 Detection of PDRN activity retention rate

[0416] PDRN is a bioactive polypeptide that is susceptible to degradation due to environmental factors such as temperature and pH. The carrier must maintain its structural stability through spatial encapsulation or charge interactions. This assay uses accelerated testing to evaluate the protective effects of two carriers on PDRN activity.

[0417] Detection methods

[0418] Accelerated test conditions: The two samples were placed in a 60℃ constant temperature chamber (simulating long-term storage) and samples were taken at 0 days and 3 days (72h).

[0419] Activity assay: The content of active PDRN in the sample was determined by ELISA (PDRN-specific antibody), and the retention rate was calculated (retention rate = 72h active PDRN content / initial content × 100%).

[0420] Parallel validation: Each group was tested three times, and the mean and standard deviation were taken.

[0421] Test results

[0422] Comparative Example 1 (Unmodified HA): Activity retention rate 62%±2.3% — The carboxyl group of unmodified HA is negatively charged under neutral conditions, forming a weak electrostatic repulsion with the amino group of PDRN, and cannot effectively encapsulate PDRN; in addition, hydrogen bonds are easily formed between HA chains, leading to aggregation, which causes some PDRN to degrade when exposed to high temperature environment.

[0423] Example 1 (SBMA-HA): Activity retention rate 88%±1.5% - The sulfobetaine group (zwitterion) of SBMA forms a "charge neutralization" effect with PDRN. At the same time, the HA main chain and the SBMA side chain form a three-dimensional network structure, which stably encapsulates PDRN inside, significantly reducing degradation caused by high temperature.

[0424] 1.3.2 24h transdermal dose detection

[0425] The carrier not only needs to load the drug, but also needs to work synergistically with nano-penetration enhancers to improve transdermal efficiency. Unmodified HA is too hydrophilic and may hinder liposomes from penetrating the skin, while the amphiphilicity of zwitterionic carriers makes it easier for them to interact with the stratum corneum of the skin.

[0426] Detection methods

[0427] Transdermal model: A Franz diffusion cell was used with fresh pigskin (500 μm thick) as a barrier and 37℃ PBS (0.01 M, pH 7.4) as the receiving solution.

[0428] Sample loading: Two types of enhanced transdermal delivery cosmetic patches were applied to the surface of pigskin and pressed for 30 seconds to ensure needle penetration;

[0429] Sampling and testing: After 24 hours, the receiving solution was collected and the PDRN content was determined by HPLC (amino column, mobile phase 0.1M PBS pH 7.4), and the transdermal transdermal dose per unit area (μg / cm²) was calculated.

[0430] Test results

[0431] Comparative Example 1 (Unmodified HA): Transdermal absorption rate 45 μg / cm² ± 2.1 μg / cm² — Unmodified HA easily forms a viscous hydration layer on the skin surface, which hinders the penetration of liposomes (nano-penetration enhancers) into the stratum corneum; and the interaction between HA chains and the stratum corneum is weak, making it impossible to open skin channels.

[0432] Example 1 (SBMA-HA): Transdermal absorption rate 85 μg / cm² ± 2.3 μg / cm² — The zwitterionic groups of SBMA can form hydrogen bonds and hydrophobic interactions with the amino acid residues of keratinocytes, temporarily opening skin channels; at the same time, the compatibility between the carrier and liposomes is better, promoting the efficient transdermal delivery of PDRN by the nano-penetration enhancer.

[0433] 1.3.3 Single Needle Bearing Capacity Test

[0434] Microneedles need to have sufficient mechanical strength to penetrate the skin (single needle load capacity ≥ 0.35N). Unmodified HA has weak interchain forces, which may lead to needle breakage, while zwitterionic modification can enhance structural stability through side chain interactions.

[0435] Detection methods

[0436] Testing equipment: Universal testing machine (model Instron5944), equipped with a 5N pressure sensor, loading rate 1mm / min;

[0437] Test procedure: The enhanced transdermal delivery cosmetic patch was fixed on the stage with the needle tip pointing vertically upward. A flat indenter was used to slowly contact a single needle body, and the maximum pressure (i.e., single needle load-bearing capacity) when the needle body broke was recorded.

[0438] Statistical method: 10 needles were tested in each group, and the mean and standard deviation were taken.

[0439] Test results

[0440] Comparative Example 1 (Unmodified HA): Single needle load capacity 0.28N±0.02N — Unmodified HA is mainly connected by hydrogen bonds, with weak inter-chain forces. Under pressure, it is prone to plastic deformation, which leads to premature needle breakage and failure to effectively penetrate the skin.

[0441] Example 1 (SBMA-HA): Single needle load capacity 0.42N±0.03N — The sulfonyl and betaine groups of the SBMA side chain form intramolecular / intermolecular ionic bonds, which work together with the hydrogen bonds of the HA main chain to form a “rigid and flexible” network structure, significantly improving mechanical strength and meeting the needs of skin insertion.

[0442] in conclusion

[0443] Amphoteric modification is key to improving the stability of the carrier: SBMA-HA improves the PDRN activity retention rate from 62% to 88% through the charge interaction between zwitterions and PDRN and the three-dimensional network encapsulation, thus solving the core defect of traditional HA carriers that cannot protect bioactive components.

[0444] The zwitterionic structure significantly enhances the transdermal synergistic effect: The interaction between the amphiphilicity of SBMA and the stratum corneum of the skin, combined with liposome nano-permeability enhancers, increases the transdermal amount from 45 μg / cm² to 85 μg / cm², breaking through the transdermal barrier caused by the excessive hydrophilicity of traditional HA.

[0445] Ionic bonds and hydrogen bonds enhance mechanical properties: The ionic bonds of the SBMA side chain and the hydrogen bonds of the HA main chain form multiple forces, which increases the single needle load capacity from 0.28N to 0.42N, meeting the mechanical requirements for skin insertion and avoiding the needle breakage problem of traditional HA carriers.

[0446] In summary, compared with traditional unmodified HA, zwitterionic polymer carriers have shown breakthrough advantages in three aspects: PDRN stability protection, improved transdermal efficiency, and enhanced mechanical properties. They are the core materials for achieving the product's efficient skin repair function, and their performance improvement effect cannot be achieved by traditional HA carriers.

[0447] Comparative Example 2: Performance Comparison of Free L-Carnosine and Liposome-Encapsulated L-Carnosine

[0448] L-Carnosine, a key active ingredient in skin repair, directly impacts product efficacy due to its transdermal efficiency and irritation. Free L-carnosine is easily blocked by the skin barrier and may cause local irritation, while liposomes, as nanocarriers, can enhance transdermal absorption and reduce irritation through encapsulation. This comparative study systematically compared the transdermal absorption and skin irritation differences between the two formulations by replacing liposome-encapsulated L-carnosine with free L-carnosine in the same formulation system. This clarified the optimizing effect of liposome encapsulation on L-carnosine function and validated the necessity of nanoscale penetration enhancers.

[0449] Strictly following the "single variable control method": only the form of L-carnosine (free state vs. liposome-encapsulated state) was changed, and all other parameters (including PDRN specifications, zwitterionic carrier grafting rate, cryoprotectant ratio, preparation process, detection conditions, etc.) were completely consistent with Example 1, ensuring that the difference in detection results was only caused by whether L-carnosine was encapsulated in liposomes.

[0450] 2. Experimental Materials and Equipment

[0451] 2.1 Comparison of L-carnosine forms

[0452] Material parameters Comparative Example 2 (Free L-Carnosine) Example 1 (L-carnosine encapsulated in liposomes) Raw material specifications Purity ≥99%, pharmaceutical grade, water solubility ≥20g / 100mL, optical rotation -12.5°~-14.5° Same as left, encapsulated by DPPC-cholesterol liposomes Form of existence Free small molecules (molecular weight 226.24 Da) Nanoliposome encapsulation (particle size 85nm±3nm, PDI=0.12) Encapsulated state Unencapsulated, directly exposed to the solution The encapsulation efficiency was 96.5% ± 1.2%, and L-carnosine was encapsulated by a liposome bilayer membrane. stability It is easily oxidized (75% activity retention rate after 30 days of storage at 4℃). Liposome membrane protection (92% activity retention after 30 days of storage at 4°C)

[0453] Among them, free L-carnosine was purchased from Sigma-Aldrich (USA), product name "L-Carnosine", catalog number C9625.

[0454] 2.2 Other Materials and Equipment

[0455] The remaining ingredients, including PDRN, hyaluronic acid, SBMA, DPPC, cholesterol (Comparative Example 2 did not have a liposome preparation step, so DPPC and cholesterol were not actually used to encapsulate L-carnosine), trehalose, mannitol, PCL-PEG-PCL, APS, PBS buffer, chloroform, etc., were all completely consistent with those in Example 1.

[0456] Testing equipment: Franz diffusion cell (for transdermal measurement), skin irritation rating scale, HPLC (for L-carnosine content), dynamic light scattering instrument (for verifying liposome particle size), etc.

[0457] 3. Sample preparation process

[0458] 3.1 Preparation steps of Comparative Example 2 (Free L-Carnosine)

[0459] Preparation of free L-carnosine solution: Weigh out an amount of free L-carnosine equal to the amount of L-carnosine encapsulated in liposomes in Example 1 (to ensure that the absolute content of active L-carnosine is consistent), dissolve in deionized water at 4°C, and stir magnetically for 10 min until completely dissolved;

[0460] Needle body solution preparation: According to the formulation in Example 1, PDRN (10%), zwitterionic carrier (40%), free L-carnosine (15%, based on pure L-carnosine) and cryoprotectant (25%) were mixed sequentially and sonicated at 200W for 5 minutes to form a homogeneous solution (total solid content 40%).

[0461] Low-temperature molding and backing layer lamination: exactly the same as in Example 1 - injection into a PDMS mold, freeze-drying, and lamination with a PCL-PEG-PCL backing layer to ensure consistent parameters such as needle height and thickness ratio.

[0462] 3.2 Preparation of control sample for Example 1 (liposome-encapsulated L-carnosine)

[0463] The liposomes were prepared according to the standard procedure of Example 1, wherein the liposomes were prepared by membrane hydration (DPPC:cholesterol = 7:3, L-carnosine to DPPC mass ratio 1:3), ensuring that all steps were completely consistent with Comparative Example 2 except for the form in which L-carnosine was present.

[0464] 4. Performance Testing Methods and Results

[0465] 4.124h transdermal dose detection

[0466] The stratum corneum of the skin acts as a natural barrier against free small molecules, while nanoliposomes can enhance drug delivery efficiency by penetrating through hair follicles, sweat gland channels, or directly across the interstitial spaces of the stratum corneum. This study compares the transdermal transdermal capabilities of two forms of L-carnosine.

[0467] Detection methods

[0468] Transdermal model: Franz diffusion cell (effective transdermal area 1.77 cm²), with fresh pigskin (500 μm thick, dehaired and sterilized) as a barrier, and PBS (0.01 M, pH 7.4, magnetic stirring 500 rpm) at 37℃ as the receiving solution.

[0469] Sample loading: Two micro-patterns are applied to two different skin surfaces respectively, and the needle is inserted into the skin after pressing for 30 seconds;

[0470] Detection and calculation: After 24 hours, the receiving solution was collected and the L-carnosine content was determined by HPLC (C18 column, mobile phase methanol-water = 60:40, detection wavelength 220nm), and the transdermal transdermal dose per unit area (μg / cm²) was calculated.

[0471] Test results

[0472] Comparative Example 2 (Free L-Carnosine): Transdermal absorption 38 μg / cm² ± 1.8 μg / cm² — Free L-Carnosine is a water-soluble small molecule that is difficult to pass through the hydrophobic lipid barrier of the stratum corneum. Most of it remains on the skin surface or only penetrates to the superficial layer of the stratum corneum and cannot effectively reach the dermis.

[0473] Example 1 (liposome encapsulation): Transdermal capacity 85 μg / cm² ± 2.3 μg / cm² — The phospholipid bilayer of liposomes has a similar lipid structure to that of the stratum corneum, and can open skin channels through the "fusion-penetration" mechanism; at the same time, the 85 nm nanoparticle size can easily pass through skin appendages (hair follicles, sweat glands) into the dermis, significantly improving transdermal efficiency.

[0474] 4.2 Skin Irritation Score

[0475] When free L-carnosine comes into direct contact with the skin, it may stimulate keratinocytes due to excessively high local concentrations. The sustained-release effect of liposomes can reduce the local instantaneous concentration and reduce irritation.

[0476] Detection methods

[0477] Volunteer testing: 12 healthy volunteers (skin types I-IV) were randomly divided into 2 groups (6 people in each group) to test the samples of Comparative Example 2 and Example 1 respectively;

[0478] Test procedure: Apply the sample to the inner forearm (3×3cm²), remove it after 4 hours, and observe the skin reaction at 0h and 24h. Score according to the standard scoring card (0 points: no reaction; 1 point: slight redness; 2 points: obvious redness; 3 points: redness and swelling; 4 points: redness and swelling + papules; 5 points: redness and swelling + oozing).

[0479] Data statistics: The average and standard deviation of the 24-hour scores are taken.

[0480] Test results

[0481] Comparative Example 2 (Free L-Carnosine): Irritation score 3.2±0.3 — Free L-Carnosine is rapidly released on the skin surface, and the excessively high local concentration leads to dehydration of stratum corneum cells. Among the 6 volunteers, 4 showed obvious redness and swelling (3 points), and 2 showed redness and swelling + papules (4 points).

[0482] Example 1 (liposome encapsulation): Irritation score 1.1±0.2 - The liposome membrane slowly ruptures to release L-carnosine, avoiding a sudden increase in local concentration, resulting in only slight redness (1 point), with no obvious discomfort, and significantly improved safety.

[0483] in conclusion

[0484] Liposome encapsulation significantly enhances the transdermal efficiency of L-carnosine: Through fusion with lipids in the stratum corneum and the advantage of nanoscale size, liposomes increase the transdermal transdermal capacity of L-carnosine from 38 μg / cm² to 85 μg / cm², solving the problem that free L-carnosine is difficult to penetrate the skin barrier and ensuring that it plays a repairing role in the dermis.

[0485] Liposome encapsulation effectively reduces skin irritation: The sustained-release properties of liposomes avoid the local high concentration of free L-carnosine, reducing the skin irritation score from 3.2 to 1.1, achieving a balance of "high efficiency and low irritation";

[0486] The dual functions of nanoliposomes are irreplaceable: pure free L-carnosine cannot simultaneously meet the requirements of high transdermal penetration and low irritation, while liposomes optimize both key indicators through encapsulation, proving their necessity as a nanoscale penetration enhancer.

[0487] In summary, liposome encapsulation is a core technology for enhancing the penetration and safety of L-carnosine. Its functional advantages cannot be achieved by replacing free L-carnosine, thus providing a key guarantee for the skin repair effect and user comfort of the product.

[0488] Comparative Example 3: Performance Comparison between No Cryoprotectant and Cryoprotectant (Trehalose-Mannitol)

[0489] Cryoprotectants (trehalose and mannitol) play a triple role in the preparation of enhanced transdermal delivery cosmetic patches, providing "activity protection, structural support, and mechanical enhancement": they inhibit ice crystal formation and maintain the structural stability of active ingredients during freeze-drying, and enhance the strength of the needle skeleton after molding. This comparative example, by completely removing trehalose and mannitol from the formulation (with other conditions consistent with Example 1), systematically compares the effects of the presence or absence of cryoprotectants on PDRN activity retention, needle molding integrity, and mechanical properties, clarifying the supporting role of cryoprotectants in the core performance of the product and verifying their necessity in the formulation.

[0490] Strictly following the "single variable control method": only the cryoprotectant (trehalose and mannitol, originally 25% of the formula) in the needle layer was removed, and the total mass was made up by increasing the amount of 4°C deionized water (ensuring that the total solid content of the needle solution is still 40%, consistent with Example 1); all other parameters (PDRN specifications, zwitterionic carrier grafting rate, liposome formula, freeze-drying process, detection method, etc.) were exactly the same as in Example 1, ensuring that the difference in detection results was only caused by the "presence or absence of cryoprotectant".

[0491] 3.1 Experimental Materials and Equipment

[0492] 3.1.1 Is there a comparison of cryogenic protectants?

[0493] Material parameters Comparative Example 3 (without cryoprotectant) Example 1 (containing cryoprotectant) Core differences No trehalose, no mannitol, and the mass is made up with deionized water. Trehalose:mannitol = 2:1 (mass ratio), accounting for 25% Low temperature protectant function No antifreeze, shaping, or moisturizing effects. Trehalose: Inhibits ice crystal formation and protects PDRN structure; Mannitol: Enhances needle rigidity. Needle solution properties The viscosity is low before freeze drying (120 mPa·s at 25°C). The viscosity before freeze-drying is moderate (viscosity at 25℃: 350 mPa·s). Structural stability after freeze-drying Easily shrinks and breaks Intact structure, no shrinkage

[0494] Specifically, deionized water: pre-cooled at 4°C, used to replace the cryoprotectant (trehalose + mannitol) in Example 1, to make up the total mass (to ensure that the total solid content of the needle solution is still 40%) (Example 1 contains trehalose and mannitol, while Comparative Example 3 does not contain these two raw materials, which is the core difference).

[0495] 3.1.2 Other materials and equipment

[0496] The remaining ingredients, including PDRN, hyaluronic acid, SBMA, L-carnosine, DPPC, cholesterol, PCL-PEG-PCL, APS, PBS buffer, and chloroform, are completely consistent with those in Example 1 (Note: Comparative Example 3 does not contain trehalose and mannitol, so these two ingredients are not added).

[0497] Testing equipment: constant temperature accelerated test chamber (to measure PDRN activity), optical microscope (to count needle fragmentation rate), universal testing machine (to measure single needle load-bearing capacity), viscometer (to measure the viscosity of the needle solution).

[0498] 3.2 Sample preparation process

[0499] 3.2.1 Comparative Example 3 (without cryoprotectant) Preparation Steps

[0500] Preparation of needle solution:

[0501] According to the formulation ratio of Example 1, weigh out PDRN (10%, 1.0g), zwitterionic carrier (40%, 4.0g), and nano-penetration enhancer (15%, 1.5g).

[0502] The original formula's 25% cryoprotectant (2.5g) was replaced with 4℃ deionized water, while the total solution mass remained 10g. The solution was then magnetically stirred for 30 minutes until initially dissolved.

[0503] A homogeneous solution was formed by ultrasonication at 200W for 5 minutes. The viscosity at 25°C was measured to be 120 mPa·s (350 mPa·s in Example 1). The total solid content was measured to be 40% by vacuum drying (consistent with Example 1).

[0504] Low temperature molding:

[0505] Completely consistent with Example 1: The needle solution was injected into the PDMS mold (needle height 750 μm) and pre-frozen at -40°C for 2 hours;

[0506] Freeze-drying was performed using a gradient temperature program (-40℃→-20℃, 2℃ / h; -20℃→0℃, 1℃ / h; 0℃→25℃, 0.5℃ / h), with a total freeze-drying time of 24 hours.

[0507] Backing layer composite:

[0508] Same as Example 1: Coated with 20% PCL-PEG-PCL chloroform solution, solvent evaporated at 25°C for 8 hours, needle body layer to backing layer thickness ratio 4:1.

[0509] 3.2.2 Preparation of the control sample of Example 1 (containing cryoprotectant)

[0510] Prepared according to the standard procedure of Example 1, ensuring accurate addition of the cryoprotectant (trehalose 1.67g + mannitol 0.83g) as a performance comparison benchmark.

[0511] 3.3 Performance Testing Methods and Results

[0512] 3.3.1 Detection of PDRN activity retention rate

[0513] Detection purpose

[0514] Without a cryoprotectant during freeze-drying, ice crystals can easily damage the DNA strand structure of PDRN; at the same time, PDRN is easily oxidized and degraded during storage, and cryoprotectants can maintain its activity through a "coating-moisturizing" effect.

[0515] Detection methods

[0516] Accelerated test conditions: The two samples were placed in a 60℃ constant temperature chamber (simulating a 12-month storage environment) and samples were taken at 0 days (initial) and 3 days (72h);

[0517] Activity assay: The content of active PDRN was determined by HPLC (amino column, mobile phase 0.1M, pH 7.4 phosphate buffer, detection wavelength 260nm), and the retention rate was calculated (retention rate = 72h active content / initial active content × 100%).

[0518] Parallel validation: Each group was tested three times, and the mean and standard deviation were taken.

[0519] Test results

[0520] Comparative Example 3 (without cryoprotectant): Activity retention rate 58% ± 2.0% — During freeze-drying, without trehalose to inhibit ice crystal formation, the DNA strand of PDRN was physically damaged by ice crystals; and without the antioxidant effect of mannitol, PDRN was further oxidized and degraded under accelerated conditions at 60℃, resulting in a loss of nearly 42% of activity;

[0521] Example 1 (containing cryoprotectant): Activity retention rate 88%±1.5% - Trehalose binds to PDRN through a "water substitution" mechanism to avoid ice crystal damage; mannitol can scavenge free radicals and reduce oxidative degradation, with an activity loss of only 12%.

[0522] 3.3.2 Needle fragmentation rate test

[0523] Detection purpose

[0524] Low-temperature protectants are the "skeleton aids" for the freeze-drying molding of needles: trehalose can form a glassy structure to maintain the shape of the needle, and mannitol can enhance the density of the structure. Without protectants, the needle is prone to breakage due to freeze-drying shrinkage or mechanical contact.

[0525] Detection methods

[0526] Sampling and observation: 10 pieces of each of the two types of samples were taken, and a 10×10 microneedle array (100 needles in total) was selected from each piece. The integrity of the needles was observed through an optical microscope (50x magnification).

[0527] Criteria for determining breakage: The following conditions are considered as breakage: "fracture (separation of needle tip from needle body)," "chipping (missing area of ​​needle body edge ≥10%)", and "shrinkage deformation (needle body height shortened ≥20%)".

[0528] Fragmentation rate calculation: Fragmentation rate = (total number of fragmented needles / total number of needles) × 100%, with 3 repeated tests per group.

[0529] Test results

[0530] Comparative Example 3 (without cryoprotectant): Fragmentation rate 42%±3.5% - The solution viscosity was low (120mPa・s) during freeze-drying. After the water sublimated, no protectant was formed to form a skeleton, and the needles shrank and deformed. Moreover, the structure was loose, and it broke with slight touch when demolding. An average of 42 needles broke in 10 samples.

[0531] Example 1 (containing cryoprotectant): 0% breakage rate - The glassy structure formed by trehalose works synergistically with the compactness of mannitol, resulting in intact needle morphology after freeze-drying (height 750μm±20μm), with no breakage during demolding and subsequent operations.

[0532] 3.3.3 Single needle load-bearing capacity test (to evaluate the mechanical reinforcing effect of the cryoprotectant)

[0533] Detection purpose

[0534] The needle body must have sufficient load-bearing capacity (≥0.35N) to penetrate the skin. The cryoprotectant can enhance the structural rigidity by forming "hydrogen bond-hydrophobic interaction" with the carrier. Without the protectant, the mechanical properties of the needle body will be significantly reduced.

[0535] Detection methods

[0536] Testing equipment: Universal testing machine (Instron 5944), equipped with a 5mm diameter flat probe, loading rate 0.05mm / s, pressure range 0-1N;

[0537] Test procedure: Fix the sample to the base with the needle tip facing upwards, and press the probe vertically downwards until the needle body shows obvious deformation (needle tip bending angle > 10°). Record the pressure value at this time and calculate the single needle bearing capacity (total pressure ÷ number of needles).

[0538] Statistical method: 10 complete needles were tested in each group, and the mean and standard deviation were taken.

[0539] Test results

[0540] Comparative Example 3 (without cryoprotectant): Single needle load capacity 0.22N±0.02N - without mannitol to enhance rigidity, and the needle structure is loose (density after freeze-drying is 1.05g / cm³, compared to 1.32g / cm³ in Example 1), it is prone to plastic deformation under pressure, and the load capacity is far lower than the requirement for skin insertion.

[0541] Example 1 (containing cryoprotectant): Single needle load capacity 0.42N±0.03N - Mannitol forms a hydrophobic interaction with the SBMA side chain of the zwitterionic carrier, and trehalose forms hydrogen bonds with the HA main chain, which synergistically enhances the rigidity of the needle body, and the load capacity meets the insertion requirements.

[0542] in conclusion

[0543] Low-temperature protectants are the core guarantee for PDRN activity: the anti-ice crystal effect of trehalose and the antioxidant effect of mannitol work synergistically to increase the PDRN activity retention rate from 58% to 88%, avoiding activity loss during freeze-drying and storage, and ensuring that the active ingredients can perform their repair function.

[0544] The cryoprotectant determines the integrity of the needle body: the glassy skeleton formed by trehalose and the density of mannitol reduce the needle body breakage rate from 42% to 0%, solving the problem of needle body shrinkage and breakage without the protectant, and ensuring the manufacturability and stability of the product.

[0545] The cryoprotectant significantly enhances mechanical properties: through multiple interactions with the carrier, the cryoprotectant increases the single needle load-bearing capacity from 0.22N to 0.42N, meeting the mechanical requirements for skin insertion and avoiding the defect of "needle collapse and inability to penetrate the stratum corneum" without the protectant.

[0546] In summary, the cryoprotectant composed of trehalose and mannitol is not simply a "freeze-drying aid," but a key component that simultaneously ensures PDRN activity, needle molding integrity, and mechanical properties. Its function cannot be replaced by other ingredients and is a necessary condition for achieving the core performance of the product.

[0547] 3.4 Validation of the detection method

[0548] The accuracy, repeatability, and applicability of detection methods are the core of ensuring controllable product quality. The following supplements key operational details, condition screening basis, and complete methodological validation for the determination of PDRN content (HPLC method) and the determination of L-carnosine cumulative permeation (Franz diffusion cell method) to ensure that the test results are reliable and reproducible.

[0549] 3.4.1 Determination of PDRN content (HPLC method)

[0550] Method Principles

[0551] PDRN is a DNA fragment (a negatively charged molecule) derived from salmon testes. Using an amino column can effectively retain PDRN through "polar interaction". Using 0.1M, pH 7.4 phosphate buffer as the mobile phase can avoid irreversible adsorption of PDRN on the chromatographic column, while ensuring its response stability at the detection wavelength of 260nm (the characteristic absorption peak of nucleic acid substances). Finally, the content is calculated by the external standard method.

[0552] Reagent and instrument preparation

[0553] (1) Reagent preparation

[0554] 0.1M PBS buffer (pH 7.4): Accurately weigh 0.68g of potassium dihydrogen phosphate (KH2PO4) and 1.42g of dipotassium hydrogen phosphate (K2HPO4·3H2O), dissolve in 1000mL of ultrapure water (resistivity ≥18.2MΩ·cm), calibrate the pH to 7.4±0.02 with 0.1M sodium hydroxide (NaOH) or 0.1M hydrochloric acid (HCl), filter through a 0.22μm aqueous filter membrane, and degas by sonication for 15min (300W) before use;

[0555] PDRN standard stock solution (1000 μg / mL): Weigh 10 mg of PDRN standard (purity ≥99%, molecular weight 200 kDa) accurately, dissolve in 0.1 M PBS buffer and bring the volume up to 10 mL. Store at 4°C protected from light. Shelf life is 7 days.

[0556] PDRN standard working solutions: The stock solution is serially diluted with 0.1M PBS buffer to prepare a series of working solutions of 10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL and 200μg / mL, and should be prepared and used immediately.

[0557] (2) Specifications of instruments and consumables

[0558] High performance liquid chromatograph: equipped with a UV detector (model Agilent 1260).

[0559] Chromatographic column: Amino-bonded silica column (250mm×4.6mm, 5μm, brand: WatersXBridgeAmide);

[0560] Ultrasonic instrument: 200W power, 20kHz frequency (model: SCIENTZ-IID);

[0561] Filter membrane: 0.22μm aqueous polyethersulfone filter membrane (diameter 25mm).

[0562] Sample pretreatment

[0563] Sample taking: Take one enhanced transdermal delivery cosmetic patch, cut it into 1mm×1mm fine particles with sterile scissors (to ensure complete dissolution), and accurately weigh the mass (recorded as m, unit g).

[0564] Extraction and dissolution: Transfer the powder to a 50mL centrifuge tube, add 10mL of 0.1MPBS buffer (pH 7.4), place it in an ultrasonic instrument, and set the parameters as follows: power 200W, frequency 20kHz, working time 3s / interval 5s, total ultrasonic time 30min (preliminary experimental verification: the dissolution rate was only 85% after 20min, and reached 99.5% after 30min, so 30min was determined).

[0565] Purification and filtration: After sonication, centrifuge (5000 rpm, 10 min), take the supernatant and filter it through a 0.22 μm aqueous filter membrane, discard 1 mL of the initial filtrate, and take the subsequent filtrate as the test solution;

[0566] Blank control: Take a blank enhanced transdermal delivery cosmetic patch without PDRN (other components are the same as the sample) and prepare a blank control solution according to the above steps to eliminate matrix interference.

[0567] Chromatographic conditions

[0568] Chromatographic parameters Setting value Screening criteria chromatographic column Amino column (250mm × 4.6mm, 5μm) Compared to the C18 column (which shows weak PDRN retention and peak tailing), the amino column shows strong retention of polar PDRN and symmetrical peak shape (tailing factor 1.05-1.15). mobile phase 0.1M PBS (pH 7.4) Compared to methanol-PBS mixed phases (resolution ≤ 1.2), pure PBS buffer can completely separate PDRN from impurities (resolution ≥ 1.5). Flow rate 1.0 mL / min At a flow rate of 0.8 mL / min, the retention time was too long (15 min); at 1.2 mL / min, the peak shape broadened; and at 1.0 mL / min, both efficiency and peak shape were considered. Column temperature 30℃ At room temperature (25℃), the retention time fluctuated by ±0.5 min; a constant temperature of 30℃ resulted in a retention time RSD ≤ 0.3%. Detection wavelength 260nm Scanning the 200-400 nm UV spectrum, PDRN showed maximum absorption at 260 nm, and the blank matrix did not interfere. Injection volume 10μL The response value is low with an injection volume of 5 μL, easily overloaded with 20 μL, and the peak area has the best linear relationship with concentration with an injection volume of 10 μL.

[0569] Methodological Validation

[0570] (1) System suitability test: 100 μg / mL PDRN standard working solution was injected and the chromatogram was recorded: theoretical plate number (N): ≥3000 (actual measurement 3850); resolution (R): resolution between PDRN peak and adjacent impurity peak ≥1.5 (actual measurement 2.2); tailing factor (T): 0.95-1.15 (actual measurement 1.08); retention time (tR): stable at 8.5±0.2 min (actual measurement 8.45 min), which meets the system suitability requirements.

[0571] (2) Linearity: A series of standard working solutions of 10-200 μg / mL were injected. The PDRN concentration (C, μg / mL) was used as the abscissa and the peak area (A) was used as the ordinate. Linear regression was performed: Regression equation: A=12563C+452 (n=5); Correlation coefficient (R²): 0.9998 (R²>0.999, which meets the linearity requirement); Linear range: 10-200 μg / mL, covering the expected concentration of PDRN in the sample (8-12 μg / mL, which falls within the linear range after dilution).

[0572] (3) Precision and repeatability: The same test solution (PDRN concentration 9.8 μg / mL) was injected 6 times consecutively, and the peak area was measured: peak area RSD = 0.8% (RSD < 2%, good repeatability); intermediate precision: the same sample was measured by 2 experimenters on different days (Day 1, Day 2) using different instruments (Agilent 1260, Waterse 2695): the results of the two measurements RSD = 1.0% (RSD < 2%, good intermediate precision).

[0573] (4) Accuracy (Recovery Test) The “spiking recovery method” was adopted. A sample with known content (PDRN content 9.8%) was taken, and PDRN standards at three levels of low, medium and high were added respectively (the amount of spiked PDRN was 80%, 100% and 120% of the amount of PDRN in the sample). The sample was prepared and measured according to the sample pretreatment steps: PDRN amount in the spiked sample (μg) PDRN amount added (μg) Total PDRN amount measured (μg) Recovery rate (%) Average recovery rate (%) RSD (%) Low 98.0 78.4 175.2 98.5 99.8 1.2 Medium 98.0 98.0 195.8 99.8 High 98.0 117.6 214.5 101.2 Average recovery rate 99.8%, RSD=1.2% (recovery rate 95%-105%, RSD<2%, accuracy meets the requirements).

[0574] (5) Stability of the test solution: Take the test solution and inject it at 0h, 4h, 8h, 12h and 24h respectively, and measure the peak area:

[0575] The peak area RSD within 24 hours was 1.1% (RSD < 2%, the test solution was stable for 24 hours under light-protected conditions at 4℃); the stability of the standard solution was determined by taking 100 μg / mL of the standard working solution, storing it at 4℃ under light-protected conditions for 7 days, and determining the content: the content RSD within 7 days was 1.3% (RSD < 2%, the standard solution was stable within 7 days).

[0576] (6) Specificity: Blank control solution, test solution, and PDRN standard solution were injected separately: the blank control solution showed no chromatographic peak at the PDRN retention time (no matrix interference); the PDRN peak and impurity peak in the test solution were completely separated (resolution 2.2), indicating good specificity. 4.2 Determination of cumulative L-carnosine permeation (Franz diffusion cell method) 4.2.1 Method principle: The Franz diffusion cell simulates the transdermal process of L-carnosine in human skin through the structure of "drug supply chamber-skin barrier-receiving chamber"; fresh pig skin (the stratum corneum structure is close to that of human skin) is used as a barrier, and the temperature is kept constant at 37℃ to simulate the physiological temperature of the skin. Magnetic stirring is used to ensure uniform concentration of the receiving solution; the content of L-carnosine in the receiving solution at different times is determined by HPLC, the cumulative permeation amount over 24 hours is calculated, and the permeation-enhancing effect is evaluated.

[0577] Reagent and instrument preparation

[0578] (1) Reagent preparation: 0.01M PBS buffer (pH 7.4): Weigh 0.068g of potassium dihydrogen phosphate and 0.142g of dipotassium hydrogen phosphate, dissolve in 1000mL of ultrapure water, calibrate the pH to 7.4±0.02, filter through a 0.22μm filter membrane, degas and use as receiving solution; L-carnosine standard stock solution (1000μg / mL): Take 10mg of L-carnosine standard (purity ≥99%), dissolve in 0.01M PBS buffer and make up to 10mL, store at 4℃ protected from light; L-carnosine standard working solution: serially dilute to 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL and 100μg / mL, prepare fresh and use immediately.

[0579] (2) Instruments and Consumables: Franz diffusion cell: vertical, effective transdermal area 1.77 cm², receiving cell volume 6.5 mL (model: TK-6A); constant temperature water bath: temperature control accuracy ±0.5℃ (model: HH-S4); magnetic stirrer: speed accuracy ±10 rpm (model: IKARCTbasic); skin barrier: fresh pig skin (taken from pig abdomen, thickness 500±50 μm, hair removed and disinfected, stored at 4℃, used within 24 hours); HPLC instrument: equipped with C18 column (model: Agilent ZORBAXSB-C18, 250 mm × 4.6 mm, 5 μm).

[0580] 4.2.3 Sample pretreatment and diffusion cell setup

[0581] (1) Pig skin treatment (key step, avoid barrier differences) Hair removal: Use an electric hair remover to remove hair from the surface of the pig skin to avoid damaging the stratum corneum; Cleaning and disinfection: Rinse 3 times with physiological saline (0.9% NaCl) to remove surface dirt; Wipe the surface with 0.1% benzalkonium chloride solution, disinfect for 10 minutes, and then rinse 3 times with physiological saline to remove disinfectant residue; Thickness control: Use a skin thickness gauge (accuracy 0.01mm) to randomly measure 5 points to ensure the thickness is 500±50μm. If the thickness is too thick, use a scalpel to gently thin it (only remove the subcutaneous tissue and retain the intact stratum corneum).

[0582] (2) Assembly and filling of the receiving chamber of the diffusion cell: Inject 6.5 mL of 0.01 M PBS buffer (preheated at 37 °C) into the receiving chamber of the Franz diffusion cell, and place an 8 mm magnetic stir bar (500 rpm) to ensure no air bubbles; Skin fixation: Fix the treated porcine keratin layer upwards between the drug supply chamber and the receiving chamber, and clamp it with a clamp to ensure that there is no gap between the skin and the pool wall (to avoid leakage of the receiving liquid); Sample addition to the drug supply chamber: Take one enhanced transdermal delivery cosmetic patch, remove the backing, and place it with the needle layer downwards on the surface of the porcine keratin layer. Press gently for 30 seconds (approximately 20 N / cm², simulating the pressure when using a human body) to ensure that the needle penetrates the skin; Seal the drug supply chamber with parafilm to prevent moisture evaporation.

[0583] Sampling and Testing

[0584] Sampling time points: 1h, 4h, 8h, 12h, and 24h were set as sampling times (preliminary experiments showed that L-carnosine began to permeate within 1h, the permeation rate tended to stabilize after 8h, and osmotic equilibrium was reached at 24h, so this time point was selected).

[0585] Sampling procedure: Each time, accurately pipette 1 mL of the receiving solution (recorded as V sampling), and immediately add 1 mL of fresh 0.01 M PBS buffer preheated at 37°C (recorded as V replenishment) to ensure that the volume of the receiving chamber remains constant;

[0586] HPLC detection: After filtering the sample solution through a 0.22 μm aqueous filter membrane, the L-carnosine content was determined under the following chromatographic conditions: Column: Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: methanol-0.01 M PBS (pH 7.4) = 60:40 (v / v); Flow rate: 1.0 mL / min; Column temperature: 30 ℃; Detection wavelength: 220 nm; Injection volume: 10 μL.

[0587] Cumulative permeability calculation

[0588] Since fresh receiving solution is added after each sampling, the cumulative permeability needs to be corrected, as shown in the following formula:

[0589] Q t : Cumulative permeation at time point t (μg / cm²);

[0590] C t : L-carnosine concentration in the receiving solution at time point t (μg / mL);

[0591] V0: Initial volume of the receiving chamber (6.5 mL);

[0592] C i: L-carnosine concentration in the receiving solution at time point t-1 (μg / mL);

[0593] V s : Sample volume per time (1 mL);

[0594] A: Effective transdermal area (1.77cm²).

[0595] Methodological Validation

[0596] (1) System suitability test: 50 μg / mL L-carnosine standard working solution was injected: theoretical plate number ≥3000 (actual measurement 4200); resolution ≥1.5 (actual measurement 2.5); tailing factor 0.95-1.15 (actual measurement 1.05); retention time stable at 6.8±0.2 min (actual measurement 6.78 min), system suitability is qualified.

[0597] (2) Linearity: The working solution of the series of standard solutions with concentrations of 5-100 μg / mL was injected. The linear regression equation was: A=8972C+321 (n=5); R²=0.9997 (R²>0.999, good linearity); the linear range covered the expected concentration of L-carnosine in the receiving solution (5-8 μg / mL).

[0598] (3) Precision repeatability: The same receiving solution sample (L-carnosine concentration 7.5 μg / mL) was injected 6 times consecutively, and the peak area RSD was 1.1% (RSD < 2%). Intermediate precision: The results were measured by different experimenters and different instruments, and the RSD was 1.3% (RSD < 2%).

[0599] (4) Accuracy (Recovery Test): Add low, medium, and high levels of L-carnosine standards (5 μg / mL, 10 μg / mL, and 20 μg / mL) to the blank receiving solution and determine the accuracy according to the detection procedure.

[0600] spiking level Amount added (μg) Measured amount (μg) Recovery rate (%) Average recovery rate (%) RSD (%) Low 5.0 4.89 97.8 99.9 1.5 middle 10.0 9.98 99.8 / / high 20.0 20.46 102.3 / / The average recovery rate was 99.9%, and the RSD was 1.5% (meeting the requirements of 95%-105%, indicating good accuracy). / / / / /

[0601] (5) Skin integrity verification

[0602] After the experiment, the pigskin barrier was removed, and its surface was coated with 0.1% methylene blue solution. Observation was performed after 10 minutes.

[0603] No blue penetration into the skin (indicating an intact skin barrier and no leakage);

[0604] Tiny blue spots are visible at the microneedle puncture site (indicating that the needle was effectively inserted and the transdermal pathway was normal).

[0605] in conclusion

[0606] PDRN content determination (HPLC method): It exhibits good linearity (R²=0.9998) in the range of 10-200 μg / mL, and meets the requirements for precision (RSD≤1.0%), accuracy (recovery rate 99.8%), and stability (24hRSD=1.1%). It can accurately determine the PDRN content in enhanced transdermal delivery cosmetic patches.

[0607] L-carnosine cumulative permeation assay (Franz diffusion cell method): linear range 5-100 μg / mL (R²=0.9997), precision and accuracy meet the standards, porcine skin barrier integrity is controllable, and the transdermal permeation enhancement effect of L-carnosine can be reliably evaluated.

[0608] Both testing methods have clear operational details and are reproducible, providing a scientific basis for product quality control and performance evaluation.

[0609] This invention focuses on the core objective of "highly efficient delivery of active PDRN + enhanced skin repair function." Through component screening, structural optimization, and process innovation, it achieves a comprehensive breakthrough in the effectiveness, stability, and practicality of enhanced transdermal delivery cosmetic patches, as detailed below:

[0610] 1. Synergistic Component Composition to Ensure Core Functions: The optimal combination of components was selected—PDRN derived from salmon testes with a molecular weight of 150-250kDa is the core active ingredient. SBMA-HA zwitterion carrier with a grafting rate of 28% enhances its stability (88% activity retention rate after accelerated treatment at 60℃). Liposomes with a 7:3 ratio of DPPC to cholesterol enhance L-carnosine penetration (85μg / cm² transdermal absorption in 24 hours). Trehalose and mannitol in a 2:1 ratio act as a cryoprotectant to prevent needle breakage (0% breakage rate). Combined with 0.2% tranexamic acid, 3% glutathione, and 6% Centella asiatica extract, a closed loop of "anti-inflammatory-whitening-barrier repair" is formed, resulting in a 92% improvement rate in post-acne pigmentation after 8 weeks.

[0611] 2. Optimized structure for enhanced user experience: The design features a needle height of 750μm and a needle layer to backing layer thickness ratio of 4:1, combined with micron-level protrusions in the backing layer with a height of 35μm. This extends the adhesion retention time to 6.8h, reduces the detachment rate to 8%, and increases drug utilization to 92%. At the same time, the single needle has a load-bearing capacity of 0.38N and a dissolution time of 2.5min in 37℃ saline, meeting the requirements for skin penetration and rapid release of ingredients.

[0612] 3. Controllable process ensures stable quality: Through the standardized process of "SBMA-HA copolymerization (50℃ reaction for 7h) - liposome film hydration (35℃ rotary evaporation) - gradient freeze drying (pre-freezing at -40℃ followed by stepwise heating) - backing layer coating and imprinting", the parameters of each step are controllable, ensuring consistent performance between product batches, and solvent residue ≤0.1%, meeting safety requirements.

[0613] 4. Performance Breakthrough, Highlighting Creativity: Compared with traditional solutions, this invention replaces unmodified HA with zwitterionic carriers, increasing the PDRN activity retention rate from 62% to 88%; encapsulates free L-carnosine with liposomes, increasing the transdermal absorption rate from 38 μg / cm² to 85% and reducing irritation to 1.1 points; and by adding a cryoprotectant, the single needle load-bearing capacity is increased from 0.22N to 0.38N, solving the pain points of traditional microneedles such as "low activity, poor transdermal absorption, and fragility".

[0614] All experimental data are based on three parallel experiments with an error range of ≤2%. The operational details of each step are clearly defined and can be fully reproduced by those skilled in the art, meeting the "fully disclosed" requirements of the patent. This provides an efficient and safe new solution for the treatment of post-acne pigmentation and skin barrier repair.

[0615] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. An enhanced transdermal delivery cosmetic patch containing highly effective active PDRN (sodium DNA), characterized in that: It consists of a needle body layer and a backing layer, wherein: The needle body layer comprises the following components by mass percentage: PDRN with a molecular weight of 50-300kDa, at a content of 5%-15%; A zwitterionic polymer carrier composed of methacryloylethyl sulfobetaine and hyaluronic acid, with a content of 30%-50%; L-carnosine encapsulated in liposomes with a particle size not exceeding 100 nm is used as a nanoscale penetration enhancer, with a content of 10%-20%. A cryoprotectant composed of trehalose and mannitol in a mass ratio of 1:1 to 3:1, with a content of 15% to 25%; The backing layer is a block copolymer of biodegradable polycaprolactone and polyethylene glycol, PCL-PEG-PCL, with a molecular weight of 8000-12000 Da.

2. The enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) according to claim 1, characterized in that: The PDRN has a molecular weight of 150-250 kDa and a loading of 8%-12% in the needle layer; The grafting rate of the sulfobetaine monomer on the zwitterionic polymer support is 20%-35%; The encapsulation efficiency of the liposomes encapsulating L-carnosine is not less than 95%.

3. The enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) according to claim 1, characterized in that: The needle height is 600-900μm, and the single needle load-bearing capacity is not less than 0.35N; Complete dissolution time in physiological saline at 35-37℃ should not exceed 3 minutes; After an accelerated test at 50℃-60℃ for 48-72 hours, the PDRN activity retention rate was no less than 85%.

4. The enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) according to claim 1, characterized in that: The needle body layer also contains 0.1%-0.3% tranexamic acid and 2%-4% glutathione by weight; The backing layer contains 5%-7% by weight of Centella asiatica extract.

5. The enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) according to claim 1, characterized in that: The PDRN is a DNA fragment derived from salmon testes, with a length of 50-500 bp; The liposomes contain dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a molar ratio of 6.5:3.5 to 7.5:2.

5.

6. The enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) according to claim 1, characterized in that: The thickness ratio of the needle body layer to the backing layer is 3:1-5:1; The surface of the backing layer has a micron-scale protrusion structure with a height of 20-50 μm.

7. The method for preparing the enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Synthesis of zwitterionic polymer support: Methacrylethyl sulfobetaine and hyaluronic acid with a molecular weight of 50-100 kDa were dissolved in PBS buffer at a molar ratio of 1:3-1:

5. 0.08%-0.12% by mass of ammonium persulfate initiator was added, and the mixture was reacted at 48-52℃ for 6-8 hours, followed by dialysis purification. (2) Preparation of nano-penetration enhancer: L-carnosine and dipalmitoylphosphatidylcholine (DPPC) were mixed at a mass ratio of 1:2 to 1:4, and liposomes were prepared by thin-film hydration. The liposomes were then sterilized by passing the mixture through a 0.20-0.24 μm filter membrane. (3) Preparation of needle solution: PDRN, zwitterionic polymer carrier, nano-permeability enhancer, and cryoprotectant were dissolved in deionized water at 4±2℃ and ultrasonically treated with 180-220W power for 4-6 minutes to form a homogeneous solution. (4) Low temperature molding: The needle solution was injected into a PDMS mold and pre-frozen at -45 to -35°C for 1.5 to 2.5 hours, followed by freeze-drying at a vacuum of 0.08 to 0.12 mPa and a temperature of -30 to -20°C for 22 to 26 hours. (5) Backing layer composite: A PCL-PEG-PCL chloroform solution with a mass-volume ratio of 18-22% was coated on the back of the needle layer. After the solvent evaporated at 23-27°C, the needle was peeled off and molded.

8. The method for preparing the enhanced transdermal delivery cosmetic patch containing highly active PDRN (sodium DNA) as described in claim 7, characterized in that: In step (3), the total solids content of the solution is 35%-45%; The freeze-drying process includes: Heat from -45 to -35℃ to -25 to -15℃ at a rate of 1.8-2.2℃ / h, and hold for 3-4 hours; Heat from -25 to -15℃ to -5 to 5℃ at a rate of 0.8-1.2℃ / h, and hold for 4-5 hours; Heat from -5°C to 5°C at a rate of 0.4-0.6°C / h to 23-27°C, and hold for 2-3 hours.

9. The application of the enhanced transdermal delivery cosmetic patch according to claim 1 in the preparation of skin repair medical devices, characterized in that: Used for the treatment of post-acne hyperpigmentation and for skin barrier repair.

10. A method for detecting the enhanced transdermal delivery cosmetic patch as described in any one of claims 1-6, characterized in that... include: PDRN content was determined by high performance liquid chromatography: the chromatographic column was a C18 column, the mobile phase was 0.08-0.12M phosphate buffer at pH 7.2-7.6, the flow rate was 0.8-1.2 mL / min, the detection wavelength was 260 nm, and the injection volume was 10-20 μL. The cumulative permeation of L-carnosine was determined using a Franz diffusion cell: the effective permeation area of ​​the diffusion cell was 1.5-2.5 cm², the receiving solution was PBS buffer with pH 7.2-7.4, and the permeation was not less than 75-85 μg / cm² over 24 hours.

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