Enhanced transdermal delivery beauty patch containing efficient active PDRN (DNA sodium) and preparation method of enhanced transdermal delivery beauty patch

Abstract

The invention provides an enhanced transdermal delivery beauty patch containing efficient active PDRN (DNA sodium) and a preparation method thereof, the enhanced transdermal delivery beauty patch is composed of a needle body layer and a backing layer, the needle body layer comprises the following components by mass: 5-15% of PDRN with a molecular weight of 50-300 kDa, 10-20% of a polymer with a molecular weight of 50-300 kDa, 5-15% of a polymer with a molecular weight of 50-300 kDa, 5-15% of a polymer with a molecular weight The content of the zwitterionic polymer carrier is 30%-50%, and the zwitterionic polymer carrier is formed by copolymerizing methacryloylethyl sulfobetaine and hyaluronic acid. The composition has the characteristics of good mechanical properties, rapid dissolution, high activity retention and efficient permeation promotion, can be used for treatment of pigmentation after acne and skin barrier repair, and comprehensively improves the skin repair effect and practicability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (DNA sodium) and a preparation method thereof. BACKGROUND

[0002] With the rapid development of skin repair and medical aesthetics, the problems such as post-acne pigmentation and damaged skin barrier have attracted more attention, and the demand for high-efficiency and safe skin repair technology has increased significantly. Polynucleotide (PDRN) is derived from salmon testis, and has biological activities such as promoting cell proliferation, anti-inflammatory and repairing damaged tissues, and has a good application prospect in the field of skin repair.

[0003] At present, there are mainly three ways to administer PDRN: topical application is hindered by the stratum corneum, and the transdermal absorption rate is low and the bioavailability is poor; subcutaneous injection has high absorption efficiency, but is obviously painful, has low patient compliance and is easy to cause infection; the enhanced transdermal delivery cosmetic patch can break through the stratum corneum through micron-sized needles and deliver active ingredients directly to the epithelial tissue, and has high efficiency and safety, and is an ideal carrier for PDRN delivery.

[0004] However, the existing PDRN enhanced transdermal delivery cosmetic patch still has some key problems: first, the stability of PDRN is insufficient, as a biological macromolecule, it is easily degraded and inactivated by environmental factors such as temperature and humidity, affecting the repair effect; second, the performance of the carrier material is limited, the traditional carrier (such as hyaluronic acid and gelatin) has limited loading capacity for PDRN, and it is difficult to balance the mechanical strength and biocompatibility, and the improper dissolution rate will also affect the drug release efficiency; third, the transdermal penetration efficiency needs to be improved, PDRN has insufficient penetration ability in the deep layer of the skin tissue, and it is difficult to improve the bioavailability simply by relying on physical puncture of microneedles; fourth, the function is single, only relying on the repair effect of PDRN, lacking the synergistic regulation of multiple demands of skin repair (such as antioxidant, anti-inflammatory and depigmentation), and the comprehensive effect is limited; fifth, the preparation process has defects, the traditional process (such as hot melt pouring and ultraviolet curing) may cause PDRN to be inactivated due to high temperature or chemical reagents, and the shape and mechanical strength of the needle body are difficult to control accurately.

[0005] Therefore, it is an inevitable trend to develop an enhanced transdermal delivery cosmetic patch that can stably load PDRN, improve the activity retention rate and transdermal efficiency, and has good mechanical properties and synergistic repair function. The present application optimizes the carrier material, introduces a nano penetration enhancer, and improves the preparation process through innovative design, so as to overcome the technical bottlenecks of the prior art and provide a more efficient, stable and safe solution for skin repair. SUMMARY

[0006] To solve the above technical problems, the application provides an enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA) and a preparation method thereof, which has the characteristics of good mechanical properties, rapid dissolution, high active retention and high-efficiency penetration promotion, and can be used for post-acne pigmentation treatment and skin barrier repair, and comprehensively improves the skin repair effect and practicability.

[0007] The application provides an enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA), which is composed of a needle body layer and a backing layer, wherein: The needle body layer contains the following components by mass percentage: PDRN with a molecular weight of 50-300 kDa, content of 5%-15%; Amphoteric ion polymer carrier copolymerized from methacryl ethyl sulfobetaine and hyaluronic acid, content of 30%-50%; L-muscle peptide wrapped by liposome with a particle size of not more than 100 nm as a nanoscale penetration enhancer, content of 10%-20%; Cryoprotective agent composed of trehalose and mannitol in a mass ratio of 1:1-3:1, content of 15%-25%; The backing layer is a block copolymer PCL-PEG-PCL of degradable polycaprolactone with a molecular weight of 8000-12000 Da and polyethylene glycol.

[0008] Further, the molecular weight of the PDRN is 150-250 kDa, and the loading amount in the needle body layer is 8%-12%; The grafting rate of the sulfobetaine monomer of the amphoteric ion polymer carrier is 20%-35%; The encapsulation rate of the liposome-wrapped L-muscle peptide is not less than 95%.

[0009] Further, the needle body height is 600-900 mu m, and the single needle bearing capacity is not less than 0.35 N; The complete dissolution time in physiological saline at 35-37 DEG C is not more than 3 min; After 48-72 h of accelerated test at 50 DEG C-60 DEG C, the PDRN active retention rate is not less than 85%.

[0010] Further, the needle body layer further contains 0.1%-0.3% of tranexamic acid and 2%-4% of glutathione by mass percentage; The backing layer adds 5%-7% of centella asiatica extract by mass percentage.

[0011] Further, the PDRN is a DNA fragment from salmon sperm nest, and the length is 50-500 bp; The liposome comprises dipalmitoyl phosphatidylcholine DPPC and cholesterol, and the molar ratio is 6.5:3.5-7.5:2.5.

[0012] Further, 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-level convex structure with a height of 20-50 μm.

[0013] The application provides a preparation method of the enhanced transdermal delivery cosmetic patch containing the high-efficiency active PDRN (DNA sodium) as described in any one of the above, comprising the following steps: (1) Synthesis of zwitterionic polymer carrier: Methacryl ethyl sulfobetaine and hyaluronic acid with a molecular weight of 50-100 kDa are dissolved in PBS buffer at a molar ratio of 1:3-1:5, 0.08%-0.12% ammonium persulfate initiator is added, and the reaction is carried out at 48-52°C for 6-8h, and then the solution is purified by dialysis. (2) Preparation of nano-penetration enhancer: L-carnosine and dipalmitoyl phosphatidylcholine DPPC are mixed at a mass ratio of 1:2-1:4, liposomes are prepared by the film hydration method, and bacteria are removed by a 0.20-0.24 μm filter. (3) Preparation of needle body solution: PDRN, zwitterionic polymer carrier, nano-penetration enhancer, and cryoprotectant are dissolved in 4±2°C deionized water, and the solution is ultrasonically treated at a power of 180-220W for 4-6min to form a homogeneous solution. (4) Cryogenic forming: The needle body solution is injected into a PDMS mold, pre-frozen at -45 to -35°C for 1.5-2.5h, and then freeze-dried at a vacuum degree of 0.08-0.12mPa and a temperature of -30 to -20°C for 22-26h. (5) Backing layer compounding: A PCL-PEG-PCL chloroform solution with a mass-volume ratio of 18-22% is coated on the back of the needle body layer, and then the solvent is volatilized at 23-27°C to form a patch.

[0014] Further, the total solid content of the solution in step (3) is 35%-45%. The freeze-drying procedure comprises: From -45 to -35°C, the temperature is raised to -25 to -15°C at a rate of 1.8-2.2°C / h, and the temperature is maintained for 3-4h; From -25 to -15°C, the temperature is raised to -5 to 5°C at a rate of 0.8-1.2°C / h, and the temperature is maintained for 4-5h; From -5 to 5°C, the temperature is raised to 23-27°C at a rate of 0.4-0.6°C / h, and the temperature is maintained for 2-3h.

[0015] The application provides application of the enhanced transdermal delivery cosmetic patch in preparation of a skin repair medical device for post-acne hyperpigmentation treatment and for skin barrier repair.

[0016] The application provides a detection method of the enhanced transdermal delivery cosmetic patch, comprising: The PDRN content is determined by high performance liquid chromatography, a C18 column is used as the chromatographic column, a phosphate buffer with a concentration of 0.08-0.12 M and a pH of 7.2-7.6 is used as the mobile phase, the flow rate is 0.8-1.2 mL / min, the detection wavelength is 260 nm, and the injection volume is 10-20 μL; The L-carnosine cumulative penetration amount is determined by a Franz diffusion cell, the effective penetration area of the diffusion cell is 1.5-2.5 cm², the receiving liquid is a PBS buffer with a pH of 7.2-7.4, and the 24 h penetration amount is not less than 75-85 μg / cm².

[0017] The application has the advantages and positive effects that: 1. Efficient transdermal delivery and activity maintenance: through synergistic effect of PDRN with a specific molecular weight (50-300 kDa) and a zwitterionic polymer carrier (sulfobetaine-hyaluronic acid copolymer), the skin penetration efficiency of the drug (24 h L-carnosine penetration amount ≥ 80 μg / cm²) is significantly improved, and a low-temperature protective agent (trehalose / mannitol) ensures the activity stability of PDRN in preparation and storage (activity retention rate ≥ 85% after 72 h of 60°C accelerated test).

[0018] 2. Precise and controllable delivery system: the dual penetration enhancement mechanism of nanoscale liposome-encapsulated L-carnosine (particle size ≤ 100 nm, encapsulation efficiency ≥ 95%) and microneedle physical penetration breaks through the stratum corneum barrier and realizes targeted delivery of PDRN; a gradient freeze-drying process (-40℃-25℃ staged temperature rise) guarantees the structural integrity (single needle bearing capacity ≥ 0.35 N) and rapid solubility (completely dissolved in 37℃ physiological saline within 3 min) of the needle body.

[0019] 3. Multifunctional repair and clinical application expansion: the composite formula (containing tranexamic acid, glutathione and asiaticoside extract) has anti-inflammatory, antioxidant and pro-repair functions, is suitable for post-acne hyperpigmentation, skin barrier repair and diabetic ulcers and other indications, and realizes the balance between drug sustained release and mechanical support through a degradable backing layer (PCL-PEG-PCL). DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present disclosure. The raw materials, reagents and the like used in the embodiments, if not specifically stated, are all conventional products that can be obtained by purchase; the detection methods, if not specifically stated, are all conventional detection methods in the field of microbial preparations.

[0021] Embodiment 1 1.1 Preparation of raw materials: PDRN: DNA fragments from salmon sperm (prepared by enzyme digestion). Specific preparation method: take fresh salmon sperm, wash with physiological saline, cut into pieces, homogenate with 0.1M Tris-HCl buffer (pH 8.0 containing 0.1M EDTA, 1% SDS), 4°C for 2h; add proteinase K (final concentration 50μg / mL) 37°C water bath for 4h, chloroform-isoamyl alcohol (24:1) extraction for 3 times, DNA precipitation with anhydrous ethanol; DNase I (purchased from Takara, product number 2270A) enzyme digestion: in 50mM Tris-HCl (pH 7.5) buffer, DNA concentration 1mg / mL, DNase I addition amount 10U / mg DNA, 37°C reaction for 2h, verified by 12% agarose gel electrophoresis that the fragment length is 200-300bp, finally purified by Sephadex G-50 column to obtain PDRN with molecular weight of 200kDa, purity ≥98% (verified by HPLC). (Note: 50kDa and 150kDa PDRN are prepared by adjusting the enzyme digestion time of DNase I (4h and 1.5h respectively), and the rest of the steps are the same).

[0022] Hyaluronic acid (HA): purchased from Huaxi Biotechnology Co., Ltd., product name "medical grade low molecular weight sodium hyaluronate", product number HA-MW80k, molecular weight 80kDa (determined by GPC, PDI=1.2), purity ≥99%.

[0023] Methacryloyl ethyl sulfobetaine (SBMA): purchased from Aldrich Reagent (Shanghai) Co., Ltd., product number S105422, purity ≥98%, molecular weight 247.3Da, water content ≤0.5%.

[0024] L-carnosine: purchased from Sigma-Aldrich (USA), product name "L-Carnosine", product number C9625, purity ≥99%, optical rotation -12.5°~ -14.5°, pharmaceutical grade.

[0025] Dipalmitoylphosphatidylcholine (DPPC): purchased from Avanti Polar Lipids (USA), product name "1,2-Dipalmitoyl-sn-glycero-3-phosphocholine", product code 850355P, purity ≥98%, phase transition temperature 41℃, special grade for liposome.

[0026] Cholesterol: purchased from Shanghai Macklin Biochemical Technology Co., Ltd., product name "Cholesterol (pharmaceutical grade)", product code C810127, purity ≥99%, water content ≤0.3%.

[0027] Trehalose: purchased from Shandong Futian Pharmaceutical Co., Ltd., product name "Food grade trehalose", product code FT-Tre-01, water content ≤0.2%; Mannitol: purchased from Rongguang (China) Nutritional Food Co., Ltd., product name "Food grade mannitol", product code RO-MAN-02, water content ≤0.2%; both are used in a mass ratio of 2:1.

[0028] PCL-PEG-PCL block copolymer: purchased from Jinan Daigang Biological Technology Co., Ltd., product name "Degradable medical grade PCL-PEG-PCL", product code PCL-PEG-PCL-10k, molecular weight 10000 Da (PCL segment: PEG segment = 2:1, determined by GPC), PDI = 1.2.

[0029] Ammonium persulfate (APS): purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., product name "Ammonium persulfate (analytical pure)", product code 10019-60-9, purity ≥98%, stored dry.

[0030] PBS buffer: prepared by ourselves, formula: 0.01M potassium dihydrogen phosphate and sodium phosphate mixed, pH adjusted to 7.4 with 0.1M NaOH, conductivity 1.5mS / cm (measured by DDS-307 conductivity meter).

[0031] Chloroform: purchased from Aladdin Reagent (Shanghai) Co., Ltd., product name "Anhydrous chloroform", product code C105674, purity ≥99.8%, water content ≤0.01%.

[0032] 1.2 Synthesis of zwitterionic polymer carrier SBMA (0.01 mol, 2.473 g) and HA (0.04 mol, 3.2 g, calculated according to 80kDa molecular weight) were weighed according to the molar ratio of 1:4, dissolved in 100 mL of PBS buffer (0.01M, pH 7.4), and magnetically stirred for 30 min (stirrer model: IKARCT basic, speed 200 rpm) until completely dissolved, forming a transparent solution; Add 0.01 g of APS (0.1% of the total mass of SBMA and HA), pass high-purity nitrogen (99.999%) to remove oxygen for 30 min (air flow rate 10 mL / min), seal the reaction bottle; Place in a 50°C constant temperature water bath (model: HH-S4), react for 7 h, and keep magnetic stirring (200 rpm) during the reaction; Transfer the reaction product into a regenerated cellulose dialysis bag with a molecular weight cutoff of 3500 Da (cutoff unreacted SBMA monomer, molecular weight 247.3 Da), dialyze with deionized water for 48 h (replace deionized water every 12 h, 1 L each time), and sterilize the solution after dialysis through a 0.22 μm filter membrane; Transfer the sterilized solution to a freeze-drying bottle, pre-freeze in a -50°C ultra-low temperature refrigerator (model: Thermo Scientific Forma 900) for 4 h, then transfer to a freeze dryer (model: Christ Alpha 1-4 LD plus), and freeze dry under the conditions of vacuum degree 0.1 mPa and temperature -25°C for 36 h to obtain white fluffy powder; Determine the grafting rate of sulfobetaine monomer by nuclear magnetic resonance hydrogen spectrum (1H-NMR, 400 MHz, solvent D2O): take the hydroxyl hydrogen of HA (δ4.5-5.0) as the internal standard, and the methyl hydrogen of SBMA (δ3.2-3.3) as the characteristic peak, and calculate that the grafting rate is 28%.

[0033] 1.3 Preparation of nano-penetration enhancer Take L-carnosine (1.0 g) and DPPC (3.0 g) in a mass ratio of 1:3, add cholesterol (1.28 g, molar ratio of DPPC to cholesterol 7:3, converted according to molecular weight: DPPC 734.0 g / mol, cholesterol 386.7 g / mol), and dissolve them in 50 mL of chloroform; Transfer the mixed solution to a rotary evaporator (model: RE-52AA), set the temperature to 35°C and the rotation speed to 60 rpm, and rotary evaporate for 2 h to form a uniform transparent lipid film on the inner wall of the flask (no visible cracks); Add 20 mL of PBS buffer (0.01 M, pH 7.4) to the flask, hydrate in a 37°C constant temperature water bath for 2 h, and shake every 15 min during the period (amplitude 3 cm) to ensure that the film is completely detached; Use a probe-type ultrasonic instrument (model: SCIENTZ-IID, power 300 W, frequency 20 kHz) to ultrasonically treat for 10 min (work for 3 s / interval for 5 s) to obtain a milky white liposome suspension; Sterilize the suspension through a 0.22 μm polyether sulfone filter membrane (diameter 25 mm) to remove large-particle agglomerates; Characterization and detection: Particle size: measured by dynamic light scattering instrument (DLS, model: Malvern Zetasizer Nano ZS), average particle size 85 nm ± 3 nm; Encapsulation efficiency: ultrafiltration centrifugation-HPLC combined method (ultrafiltration membrane molecular weight cut-off 10 kDa, 8000 rpm centrifugation for 30 min), HPLC conditions: C18 column (250 mm x 4.6 mm, 5 μm), mobile phase methanol-water (60:40, v / v), detection wavelength 220 nm, flow rate 1.0 mL / min, calculated encapsulation efficiency 96.5% ± 1.2%.

[0034] 1.4 Needle body solution preparation The components were weighed according to the mass percentage: PDRN 10% (1.0 g), zwitterionic polymer carrier 40% (4.0 g), nano-penetration enhancer 15% (1.5 g), cryoprotectant (trehalose: mannitol = 2:1) 20% (2.0 g); Add 1.5 g of deionized water pre-cooled at 4°C (15% of the total mass), magnetically stir for 30 min (speed 300 rpm) until initial dissolution; Use a probe-type ultrasonic instrument (power 200 W, frequency 20 kHz) for ultrasonic treatment for 5 min to form a homogeneous transparent solution (no visible particles with the naked eye); Total solid content determination: take 1 mL of the solution in a weighing bottle, dry at 80°C under reduced pressure (0.09 MPa) to constant weight, and calculate the total solid content to be 40% ± 2%.

[0035] 1.5 Low-temperature molding (1) Slowly inject the needle body solution into the PDMS mold (needle body array 10 x 10, needle body height 750 μm, needle tip diameter 50 μm), use a polytetrafluoroethylene scraper to gently scrape the mold surface to avoid air bubbles; (2) Place the mold in a -40°C ultra-low temperature refrigerator for pre-freezing for 2 h to ensure that the solution is completely frozen; (3) Transfer to a freeze dryer and follow the program below for freeze-drying: Stage Temperature change Temperature rising rate Soaking time 1 -40℃→-20℃ 2℃ / h 4h 2 -20℃→0℃ 1℃ / h 6h 3 0℃→25℃ 0.5℃ / h 14h (4) Total freeze-drying time 24 h, remove the mold, observe that the needle layer is formed completely (no shrinkage, cracking), and the single needle height is 750 μm ± 20 μm.

[0036] 1.6 Backing layer compounding Prepare the backing layer solution: weigh 20 g of PCL-PEG-PCL and dissolve in 100 mL of chloroform, magnetically stir for 2 h at a speed of 200 rpm until completely dissolved, to obtain a solution with a mass volume ratio of 20%; Coating control: The solution was coated on the back of the needle layer using an automatic coater (model: RKPrintCoat K Control Coater), with a wet film thickness of 200 μm, ensuring uniform coating; Thickness ratio control: The thickness of the needle layer was 750 μm after drying, and the thickness of the backing layer was 187.5 μm after drying, with a needle layer: backing layer ratio of 4:1; Micrometer-level protrusion molding: After the chloroform was volatilized for 8 h in an environment of 25°C and 45% humidity (solvent residue ≤0.1%), a micro-imprinting mold (protrusion height 35 μm, spacing 100 μm) was used to imprint the surface of the backing layer, with a pressure of 5 N / cm² and a holding time of 10 min; Demolding: The PDMS mold was gently peeled off with tweezers, and the complete enhanced transdermal delivery cosmetic patch was obtained.

[0037] 1.7 Performance testing To comprehensively verify the mechanical properties, stability, transdermal effect, and clinical application value of the enhanced transdermal delivery cosmetic patch of the present application, the following 7 core tests were set up in accordance with the technical requirements of the medical device detection specification and performance limitation, skin repair application, and detection method. All tests were repeated 3 times, and the results were expressed as "average value ± standard deviation", as follows: 1.7.1 Single needle bearing capacity test Test purpose: To evaluate the mechanical strength of the enhanced transdermal delivery cosmetic patch, ensuring that it can penetrate the stratum corneum without breaking, and ensuring high penetration.

[0038] Test method: Equipment and sample preparation: A universal material testing machine (model: Instron 5944) was used, equipped with a 5 mm diameter flat probe; an enhanced transdermal delivery cosmetic patch was taken, cut into a 1×1 cm² microneedle array unit (containing 100 microneedles), and fixed on the testing machine base, ensuring that the needle head was upward and perpendicular to the probe direction.

[0039] Test parameter setting: The initial distance between the probe and the needle tip was adjusted to 0.5 cm, the probe down pressure rate was set to 0.05 mm / s, the pressure detection range was 0-1 N, and the real-time pressure-displacement curve was recorded.

[0040] Judgment standard: When the needle head was observed to have obvious deformation (needle tip bending angle >10°) under a microscope, the test was stopped, and the pressure value at that time was read, which was the single needle bearing capacity (total pressure ÷ number of microneedles).

[0041] Test results: The single needle bearing capacity was 0.38 N±0.02 N, proving that the enhanced transdermal delivery cosmetic patch had sufficient mechanical strength and could effectively penetrate the skin barrier.

[0042] 1.7.2 Dissolution time test Detection purpose Verify the dissolution rate of the enhanced transdermal delivery cosmetic patch in the physiological environment of the skin, ensure that the active ingredients are released quickly and absorbed by the skin, and avoid irritation caused by residual foreign objects.

[0043] Test method Preparation of simulated physiological environment: 50 mL of physiological saline (pH 7.4, 37°C constant temperature water bath control, error ±0.5°C) was measured and poured into a 100 mL beaker, a magnetic stirrer (diameter 8 mm) was placed, the stirring rate was set to 50 rpm, and the solution was ensured to be in a uniform and constant temperature state.

[0044] Sample testing: Take one enhanced transdermal delivery cosmetic patch and completely immerse it in physiological saline, while starting the timer; every 10 seconds, observe the dissolution state of the needle body layer through an optical microscope (magnification 20 times) and record the time when the needle body completely disappears and the solution has no visible solid particles.

[0045] Test results The dissolution time is 2.5 min ± 0.1 min, indicating that the enhanced transdermal delivery cosmetic patch can be quickly dissolved in the physiological environment of the skin, ensuring the timely release of active ingredients.

[0046] 1.7.3 PDRN activity retention rate detection Detection purpose Evaluate the storage stability of the enhanced transdermal delivery cosmetic patch, focusing on verifying the activity retention ability of PDRN under high temperature conditions.

[0047] Test method Accelerated test processing: Take 10 enhanced transdermal delivery cosmetic patches and divide them into two groups (5 pieces each), one group as a control (25°C, humidity 45% storage), and the other group placed in a 60°C constant temperature oven (model: Binder BD23) for continuous 72 hours, avoiding light and vibration during the period.

[0048] PDRN content determination: Refer to the HPLC method in Section 4.1 (chromatographic column: amino column 250 mm x 4.6 mm, 5 μm; mobile phase: 0.1 M, pH 7.4 phosphate buffer; detection wavelength 260 nm; flow rate 1.0 mL / min), respectively. Determine the PDRN content of the two groups of samples.

[0049] Activity retention rate calculation: Activity retention rate = (PDRN content after accelerated test ÷ PDRN content of control sample) x 100%.

[0050] Test results After 72h of 60°C accelerated test, the PDRN activity retention rate was 88% ± 1.5%, which proved that the enhanced transdermal delivery cosmetic patch effectively improved the stability of PDRN through the synergistic effect of cryoprotectant (trehalose-mannitol) and zwitterionic carrier.

[0051] 1.7.4 PDRN content detection Detection purpose: confirm the actual load of PDRN in the needle body layer to ensure that it meets the concentration range.

[0052] Test method Sample pretreatment: take one piece of enhanced transdermal delivery cosmetic patch, cut it into pieces and put it into a 50mL centrifuge tube, add 20mL of 0.1M PBS buffer (pH 7.4), and use a probe ultrasonic instrument (power 200W, frequency 20kHz) to ultrasonically treat for 30min to ensure that the needle body is completely dissolved; then filter with a 0.22μm polyether sulfone filter membrane, and take the filtrate as the test sample solution.

[0053] HPLC detection: same as the chromatographic conditions in section 1.7.3, take 10μL of the test sample solution for injection, record the peak area; draw a standard curve by PDRN standard (concentration 10, 20, 50, 100μg / mL), calculate the mass of PDRN in the test sample; combine the total mass of the needle body layer (previously weigh the dry needle body layer mass) to calculate the mass percentage of PDRN.

[0054] Detection results The PDRN content was 9.8% ± 0.2%, within the load range of "8%-12%", which proved that the preparation process could precisely control the addition of PDRN, ensuring the consistency between product batches.

[0055] 1.7.5 L-carnosine 24h cumulative permeation amount detection Detection purpose Verify the transdermal effect of the nano-penetration enhancer (liposome-encapsulated L-carnosine) to ensure that L-carnosine can effectively penetrate the skin and reach the action site, and synergize with PDRN to play a repair function.

[0056] Test method Franz diffusion cell setup: use a vertical Franz diffusion cell (effective transdermal area 1.77cm²), the receiving cell volume is 6.5mL, add 0.01M PBS buffer (pH 7.4, 37°C constant temperature water bath incubation), magnetic stirring (speed 500rpm); the transdermal barrier is fresh pig skin (thickness 500μm, after depilation, physiological saline washing, 0.1% new jie'er disinfection treatment), fixed between the diffusion cell drug supply chamber and the receiving chamber to ensure no air bubbles.

[0057] Sample loading and sampling: The enhanced transdermal delivery cosmetic patch was attached to the drug application surface of the pig skin, and pressed for 30 seconds to ensure adhesion; 1, 4, 8, 12, and 24 hours of sampling, each time taking 1 mL of receiving liquid, while adding 1 mL of fresh receiving liquid (37°C preheating).

[0058] L-carnosine content determination: HPLC method was used to determine the L-carnosine content in the receiving liquid (chromatographic column: C18 column 250 mm x 4.6 mm, 5 μm; mobile phase: methanol-water = 60:40, v / v; detection wavelength 220 nm; flow rate 1.0 mL / min), and the 24h cumulative penetration (μg / cm²) was calculated.

[0059] Test results The 24h cumulative penetration of L-carnosine was 85 μg / cm²±2.3 μg / cm², which proved that the liposome encapsulation technology could effectively improve the transdermal efficiency of L-carnosine, providing a guarantee for its synergy with PDRN to repair the skin.

[0060] 1.7.6 Puncture rate detection Detection purpose Simulating skin with different thickness of stratum corneum, verifying the actual penetration ability of the enhanced transdermal delivery cosmetic patch, is the core verification index of "high penetration".

[0061] Test method Preparation of simulated skin: Take 6 pieces of 3x3cm² silicone membrane (each thickness 130 μm, hardness close to human stratum corneum), overlap and place on the base of the universal material testing machine, total thickness 520 μm, simulate the skin with thicker stratum corneum (such as the T zone of the face).

[0062] Puncture test: Take the enhanced transdermal delivery cosmetic patch (10x10 array), with the needle facing down, attach it to the surface of the silicone membrane; start the universal material testing machine, apply a constant pressure of 30N (simulate human pressing force), and remove the patch after 2 minutes.

[0063] Puncture rate calculation: Separate the overlapped silicone membranes layer by layer, observe the number of micropores on each membrane by optical microscope (magnification 50x), count the number of microneedles with complete micropores (pore diameter ≥ 50 μm), and the puncture rate = (number of microneedles with micropores ÷ total number of microneedles) x 100%.

[0064] Test results The puncture rate of 520 μm silicone membrane was 96%±1.2%, of which the first 3 layers of membrane (total thickness 390 μm) had a puncture rate of 100%, and the 4th layer of membrane (520 μm) had a puncture rate of 96%, proving that the enhanced transdermal delivery cosmetic patch could effectively penetrate stratum corneum of different thickness, with the characteristics of high penetration.

[0065] 1.7.7 Detection of improvement rate of post-acne hyperpigmentation Purpose of detection Through clinical volunteer test, the actual effect of the enhanced transdermal delivery cosmetic patch in the treatment of post-acne hyperpigmentation is verified.

[0066] Test method Volunteer screening and grouping: 30 volunteers who meet the following conditions are screened: 20-40 year-old women, with post-acne brown hyperpigmentation on the face (hyperpigmentation area ≥2 cm 2 ), baseline ITA value (skin brightness index, the higher the value, the brighter the skin color) 38°±2°, no other whitening or repair products used in the past 1 month; randomly divided into two groups, the test group (15 people) uses the enhanced transdermal delivery cosmetic patch of the application, and the control group (15 people) uses a blank enhanced transdermal delivery cosmetic patch without PDRN (other components are the same).

[0067] Method of use and environmental control: both groups use it twice a week, after cleaning the face, the enhanced transdermal delivery cosmetic patch is attached to the hyperpigmented area, pressed for 1 min to ensure adhesion, and removed after 4 h; the test period is 8 weeks, during which all volunteers only use basic moisturizing water and cream (commercially available small red box essence water and cream), avoid sun exposure and spicy diet; the test environment is kept at a constant temperature of 22±1℃ and humidity of 50±5%, and volunteers need to sit in this environment for 20 min before each test.

[0068] Effect evaluation: the skin color tester (model: Minolta CM-700d) is used to measure the ITA value of the test site before the test (0 weeks), at 4 weeks and at 8 weeks, and the ITA value improvement rate after 8 weeks is calculated to represent the improvement rate of hyperpigmentation (the greater the ITA value improvement rate, the more obvious the improvement of hyperpigmentation).

[0069] Test results The ITA value of the test group after 8 weeks is improved from 38°±2° to 43.2°±1.8°, and the improvement rate of hyperpigmentation is 92%; the ITA value of the control group is only improved from 38°±2° to 39.5°±1.5°, and the improvement rate is less than 5%. The results prove that the enhanced transdermal delivery cosmetic patch of the application can significantly improve post-acne hyperpigmentation.

[0070] Example 2: Effect of PDRN molecular weight on the performance of the enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (DNA sodium) 2.1 Preparation of raw materials PDRN (50 kDa): DNA fragments from salmon testis (self-prepared by enzymatic method). Preparation method: Fresh salmon testis was washed with normal saline and cut into pieces, then homogenized in 0.1 M Tris-HCl buffer (pH 8.0, containing 0.1 M EDTA and 1% SDS), and placed at 4°C for 2 h. Proteinase K (final concentration 50 μg / mL) was added and the mixture was incubated at 37°C for 4 h. DNA was extracted with chloroform-isoamyl alcohol (24:1) for 3 times and precipitated with anhydrous ethanol. DNase I (purchased from Takara, product number 2270A) was used for enzymatic digestion: in 50 mM Tris-HCl (pH 7.5) buffer, the DNA concentration was 1 mg / mL, and the amount of DNase I added was 10 U / mg DNA. The reaction was carried out at 37°C for 4 h. The fragment length was verified by 12% agarose gel electrophoresis to be 50-100 bp. Finally, the PDRN with a molecular weight of 50 kDa was purified by Sephadex G-50 column, and the purity was 98.5% (verified by HPLC).

[0071] PDRN (150 kDa): DNA fragments from salmon testis (self-prepared by enzymatic method). Enzymatic digestion conditions: DNase I was incubated at 37°C for 1.5 h. The rest of the steps were the same as those for the preparation of 50 kDa PDRN. The fragment length was 150-200 bp, and the purity was 98.2% (verified by HPLC).

[0072] PDRN (250 kDa): DNA fragments from salmon testis (self-prepared by enzymatic method). Enzymatic digestion conditions: DNase I was incubated at 37°C for 2 h. The rest of the steps were the same as those for the preparation of 50 kDa PDRN. The fragment length was 250-300 bp, and the purity was 98.7% (verified by HPLC).

[0073] PDRN (300 kDa): DNA fragments from salmon testis (self-prepared by enzymatic method). Enzymatic digestion conditions: DNase I was incubated at 37°C for 1 h. The rest of the steps were the same as those for the preparation of 50 kDa PDRN. The fragment length was 300-400 bp, and the purity was 98.1% (verified by HPLC).

[0074] 2.2 Other materials and equipment The materials and equipment were the same as those in Example 1, including methacryloyl ethyl sulfobetaine (SBMA, purity ≥98%), 80 kDa hyaluronic acid (HA, purity ≥99%), dipalmitoyl phosphatidylcholine (DPPC, purity ≥98%), and universal material testing machine (Instron 5944), high-performance liquid chromatograph (Agilent 1260), Franz diffusion cell (effective transdermal area 1.77 cm²), etc.

[0075] 2.3 Sample preparation Following the preparation process of Example 1, four groups of enhanced transdermal delivery cosmetic patches with different PDRN molecular weights were prepared: 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%; 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. 3. Preparation of needle solution: PDRN loading was 10%±0.2%, and total solids content of the solution was 40%±2%; 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.

[0076] 2.4 Performance Testing Methods and Results 2.4.1 Transdermal efficiency testing Detection purpose 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.

[0077] Detection methods 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%).

[0078] Test results 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. 150kDa group: transdermal efficiency 89%±1.5% — moderate molecular weight, which can penetrate the stratum corneum and reduce surface degradation, significantly improving delivery efficiency; 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; 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.

[0079] 2.4.2 PDRN activity retention rate detection Detection purpose To verify the stability of PDRN with different molecular weights during the storage of the enhanced transdermal delivery cosmetic patch, and ensure that the activity does not decrease during the shelf life of the product.

[0080] Detection method Refer to the accelerated test scheme of Example 1.7.3: Place 4 groups of samples in a 60°C constant temperature box for 72h, and measure the PDRN content before and after acceleration by HPLC method, and calculate the activity retention rate (activity retention rate = content after acceleration ÷ content before acceleration × 100%).

[0081] Detection results 50kDa group: activity retention rate 82% ± 1.3% - small molecule PDRN has poor structural stability, and chain breakage easily occurs at high temperature, with an activity retention rate of less than 85%; 150kDa group: activity retention rate 86% ± 1.1% - structural stability is improved, and the risk of chain breakage at high temperature is reduced, meeting the activity retention rate requirement; 250kDa group: activity retention rate 87% ± 1.0% - the highest activity retention rate, the large molecule structure has stronger anti-degradation ability, and does not cause instability of the carrier due to excessive molecular weight; 300kDa group: activity retention rate 84% ± 1.4% - although higher than the 50kDa group, due to the strong intermolecular force, some PDRN aggregates in the carrier, resulting in a slightly lower activity count during detection.

[0082] 2.4.3 Single needle bearing capacity detection Detection purpose The microneedle needs to have sufficient hardness to penetrate the skin, and the single needle bearing capacity directly determines the penetration success rate, which is a core indicator for evaluating the impact of molecular weight on product practicability.

[0083] Detection method Refer to the universal material testing machine test scheme of Example 1.7.1: probe diameter 5mm, down pressure rate 0.05mm / s, record the pressure when the needle is deformed, calculate the single needle bearing capacity (total pressure ÷ number of microneedles).

[0084] Detection results 50kDa group: single needle bearing capacity 0.32N ± 0.02N - the interaction between small molecule PDRN and zwitterionic carrier is weak, and the needle body structure is not tight enough, with a bearing capacity of less than 0.35N; 150kDa group: single needle bearing capacity 0.36N ± 0.02N - PDRN forms stable hydrogen bond with the carrier, and the needle body hardness is improved, meeting the bearing capacity requirement; 250kDa group: single needle bearing capacity 0.37N ± 0.02N - strongest intermolecular interaction, tightest needle body structure, highest bearing capacity; 300kDa group: single needle bearing capacity 0.34N ± 0.02N - although higher than the 50kDa group, the uniformity of the distribution of macromolecular PDRN in the carrier is slightly poor, and there are weak points locally, resulting in suboptimal bearing capacity.

[0085] 2.4.4 Dissolution time detection Detection method Reference to the physiological saline dissolution scheme of Example 1.7.2: 37℃, pH 7.4 physiological saline, 50rpm stirring, record the complete dissolution time of the needle body.

[0086] Detection results 50kDa group: dissolution time 2.2min ± 0.1min - small molecule PDRN accelerates carrier swelling, fastest dissolution speed, but too fast dissolution may lead to local high concentration of active ingredients; 150kDa group: dissolution time 2.4min ± 0.1min - moderate dissolution speed, both rapid release of ingredients and avoidance of local high concentration; 250kDa group: dissolution time 2.6min ± 0.1min - slightly slower dissolution speed, but still within 3min, and the release of ingredients is more gentle, with longer duration; 300kDa group: dissolution time 2.8min ± 0.1min - macromolecular PDRN delays carrier swelling, dissolution time close to the 3min upper limit, need to pay attention to the timeliness of ingredient release.

[0087] 2.4.5 Test conclusion In summary, the molecular weight of PDRN has a significant impact on the effectiveness, stability, and practicality of the enhanced transdermal delivery cosmetic patch: 150-250kDa interval is optimal: the transdermal efficiency (89%-92%), active retention rate (86%-87%), and single needle bearing capacity (0.36-0.37N) in this interval are significantly better than those of the 50kDa and 300kDa groups, achieving the best balance of "effectiveness-stability-practicality".

[0088] Example 3: Effect of DPPC to cholesterol ratio in liposomes on L-carnosine penetration 3.1 Raw material preparation (only materials different from Example 1 are listed, the rest are the same) DPPC: purity ≥98%, pharmaceutical grade, purchased from Sigma-Aldrich, batch number D8503, purity ≥98%, phase transition temperature 41℃, liposome special grade; Cholesterol: purity > 99%, pharmaceutical grade, purchased from Alfa Aesar, batch number A10015, purity > 99%, water content < 0.3%; Formulated with DPPC in the following molar ratios: 6:4 group: DPPC and cholesterol molar ratio 6:4, total mass 4.28 g per group (corresponding to L-carnosine 1.0 g), of which DPPC 3.0 g, cholesterol 1.71 g; 7:3 group (control): same as Example 1, DPPC 3.0 g, cholesterol 1.28 g; 8:2 group: DPPC and cholesterol molar ratio 8:2, DPPC 3.0 g, cholesterol 0.85 g.

[0089] 3.2 Detection equipment Dynamic light scattering instrument (DLS): Malvern Zetasizer Nano ZS, used to determine the particle size of liposomes; High-performance liquid chromatograph (HPLC): Agilent 1260, equipped with a C18 column (250 mm x 4.6 mm, 5 μm), used to determine the content of L-carnosine; Ultrafiltration centrifuge tube: molecular weight cutoff 10 kDa, Millipore, used to separate free L-carnosine; Franz diffusion cell: effective transdermal area 1.77 cm², constant temperature 37°C, used for transdermal test.

[0090] 3.3 Liposome preparation process (three groups are operated uniformly) 3.3.1 Thin film preparation: DPPC and cholesterol were dissolved in 50 mL chloroform in the proportion, transferred to a rotary evaporator (35°C, 60 rpm), and evaporated for 2 h to form a uniform lipid film; 3.3.2 Hydration treatment: add 20 mL PBS buffer (0.01 M, pH 7.4), constant temperature water bath at 37°C for 2 h, oscillate once every 15 min (amplitude 3 cm); 3.3.3 Ultrasonic treatment: 300 W power ultrasonic for 10 min (work 3 s / interval 5 s), get liposome suspension; 3.3.4 Sterilization treatment: 0.22 μm filter membrane filtration, remove large particle size agglomerates, 4°C cold storage for standby.

[0091] 3.4 Performance detection method and result 3.4.1 Encapsulation efficiency detection Detection purpose Encapsulation efficiency directly reflects the wrapping efficiency of liposomes for L-carnosine, and is the core indicator for measuring the drug loading of penetration enhancer. The higher the encapsulation efficiency, the lower the irritation of free drug to the skin, and the better the sustained release effect.

[0092] Detection method Using "ultrafiltration centrifugation-HPLC combined method": 1. Take 1 mL of liposome suspension, add an ultrafiltration centrifuge tube, centrifuge at 8000 rpm for 30 min, and the supernatant is free L-carnosine; 2. Take another 1 mL of the suspension, add 1 mL of methanol to break the liposome structure, shake for 10 min, and filter through a 0.22 μm filter membrane as the total drug amount; 3. HPLC detection (mobile phase methanol-water=60:40, detection wavelength 220 nm), calculate the encapsulation efficiency: Encapsulation efficiency = (total drug amount-free drug amount) / total drug amount x 100%.

[0093] Detection results 6:4 group: encapsulation efficiency 92% ± 1.3% - too high cholesterol ratio (40%) leads to too strong liposome bilayer membrane fluidity, part of L-carnosine leaks during ultrasonic or centrifugation; 7:3 group: encapsulation efficiency 96.5% ± 1.2% - moderate cholesterol ratio (30%), forms a stable "rigidity-fluidity balance" structure with DPPC, best wrapping effect; 8:2 group: encapsulation efficiency 93% ± 1.5% - too low cholesterol ratio (20%) leads to too rigid membrane structure, it is difficult for the thin film to completely disperse during hydration, part of L-carnosine is not wrapped.

[0094] 3.4.2 Average particle size detection Detection method Using DLS measurement: take 1 mL of liposome suspension, dilute 10 times with PBS (to avoid multiple scattering), inject into a cuvette, set the temperature to 25°C, equilibrate for 2 min, then detect, repeat 3 times for each sample, and take the average value.

[0095] Detection results 6:4 group: average particle size 95nm ± 4nm - too much cholesterol leads to increased membrane fluidity, liposomes are prone to aggregation, and particle size is large; 7:3 group: average particle size 85nm ± 3nm - smallest particle size and uniform distribution (PDI=0.12), best transdermal potential; 8:2 group: average particle size 90nm ± 3nm - insufficient cholesterol leads to increased membrane rigidity, ultrasonic dispersion is difficult, part of the particles are not completely dispersed, and the particle size is slightly larger than that of the 7:3 group.

[0096] 3.4.324h cumulative penetration amount detection (evaluate the actual penetration effect) Detection method 1. Transdermal barrier: Fresh pig skin (thickness 500 μm, depilation and disinfection), fixed in Franz diffusion cell, receiving liquid is 0.01M PBS (37°C, 500 rpm stirring); 2. Sample loading: The enhanced transdermal delivery cosmetic patch containing different liposomes (prepared according to the formulation of Example 1) was attached to the surface of the pig skin, and pressed for 30 s to ensure adhesion; 3. Sampling detection: Sampling was performed at 1, 4, 8, 12, and 24 h, respectively, and the L-carnosine content in the receiving liquid was determined by HPLC, and the 24 h cumulative penetration amount (μg / cm²) was calculated.

[0097] Test results 6:4 group: 75 μg / cm²±2.1 μg / cm² - although the encapsulation rate reached 92%, but due to the large particle size (95 nm) and poor membrane stability, part of the liposomes ruptured on the skin surface in advance, and the effective transdermal amount was insufficient; 7:3 group: 85 μg / cm²±2.3 μg / cm² - the smallest particle size (85 nm) and stable membrane structure, not only can efficiently penetrate the skin, but also slowly release L-carnosine in the dermis, with the highest penetration amount; 8:2 group: 78 μg / cm²±2.2 μg / cm² - the particle size (90 nm) is slightly larger than the 7:3 group, and the release rate is slowed down due to the excessive rigidity of the membrane, resulting in a lower transdermal amount than the 7:3 group.

[0098] Conclusion The ratio of DPPC to cholesterol has a direct regulatory effect on the membrane structure stability, particle size, and drug release behavior of liposomes, which in turn significantly affects the transdermal penetration effect of L-carnosine, which is specifically manifested as follows: DPPC and cholesterol 7:3 is the optimal ratio: under this ratio, the encapsulation rate of liposomes reaches the highest (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 (85 nm), which can smoothly pass through the skin pores and reduce the penetration resistance; finally, the 24 h cumulative penetration amount of L-carnosine reaches 85 μg / cm², fully proving the scientificity of DPPC and cholesterol in a ratio of 7:3.

[0099] Liposomes deviated from the 7:3 ratio have obvious performance defects: when the ratio of DPPC to cholesterol is 6:4, the proportion of cholesterol is too high, which makes the liposome bilayer membrane too fluid, reducing the stability of L-carnosine encapsulation and causing part of the drug to be released in the skin surface layer, ultimately significantly reducing the encapsulation rate and transdermal amount; when the ratio is 8:2, the proportion of cholesterol is too low, which makes the membrane structure too rigid, not only increasing the difficulty of ultrasonic dispersion (resulting in particle size increasing to 90 nm), but also slowing down the release speed of L-carnosine in the dermis layer, which also cannot achieve the ideal penetration effect. The comprehensive performance of the two kinds of liposomes deviated from the ratio is far inferior to that of the 7:3 group, further highlighting the necessity of the specific ratio of 7:3.

[0100] Example 4: Effect of backing layer protrusion height on the adhesion and drug utilization of enhanced transdermal delivery cosmetic patches containing high-efficiency active PDRN (DNA sodium) 4.1 Raw material preparation (only the different materials from Example 1 are listed, the rest are the same) Back layer substrate: PCL-PEG-PCL block copolymer, molecular weight 10000 Da (molar ratio of PCL segment to PEG segment 2:1), medical grade, purity ≥98%, GPC measured molecular weight distribution coefficient (PDI) is 1.2; Protrusion forming mold: PDMS imprinting mold prepared by photolithography technology, protrusion height is 0 μm (smooth control group), 20 μm, 30 μm, 50 μm respectively, the protrusion cross section is semicircular, the interval is 100 μm (to ensure that the protrusions do not overlap and are compatible with skin texture); Skin simulation model: fresh pig skin (thickness 1.0 mm, after depilation, physiological saline washing, 0.1% new jieer disinfection, simulating human facial skin texture and elasticity); Other materials: chloroform (anhydrous grade, purity ≥99.8%), PBS buffer (0.01M, pH7.4) and the like, all consistent with the basic example.

[0101] 4.2 Key detection equipment Imprinting forming equipment: small desktop imprinting machine (model: RKPrintCoatK303), controllable pressure range 0-10 N / cm², temperature accuracy ±1℃; Adhesion test system: contains constant temperature and humidity chamber (temperature 37℃±0.5℃, humidity 50%±5%, simulating human skin environment), tension sensor (range 0-5N, accuracy 0.01N, used to monitor the pulling force when the patch falls off); High performance liquid chromatograph (HPLC): model Agilent1260, equipped with amino column (250mm×4.6mm, 5μm) and C18 column (250mm×4.6mm, 5μm), used to determine the drug content; Optical microscope: Model Olympus BX53, magnification 50-200x, used to verify the actual size of the protrusion height (avoiding height deviation caused by mold error).

[0102] 4.3 Sample preparation procedure 4.3.1 Preparation of needle body layer (consistent with basic embodiment) 1. According to the formula, PDRN (from salmon sperm nest, molecular weight 200 kDa, length 200-300 bp), zwitterionic polymer carrier (SBMA-HA copolymer, grafting rate 28%), nano-penetration enhancer (DPPC-cholesterol liposome-encapsulated L-carnosine, encapsulation efficiency 96.5%), cryoprotectant (trehalose: mannitol = 2:1) were dissolved in deionized water at 4°C, and a homogeneous solution was formed by ultrasonic treatment at 200W for 5min (total solid content 40%); 2. The needle body solution was injected into the PDMS microneedle mold (needle body height 750μm), pre-frozen at -40°C for 2h, and freeze-dried according to the gradient warming program (-40°C→-20°C, 2°C / h; -20°C→0°C, 1°C / h; 0°C→25°C, 0.5°C / h), to obtain a shaped needle body layer (thickness 750μm±20μm after drying).

[0103] 4.3.2 Compound of backing layer with different protrusion heights 1. Preparation of backing layer solution: 20g of PCL-PEG-PCL was dissolved in 100mL of chloroform, and magnetic stirring was carried out at 200rpm for 2h until complete dissolution, to obtain a transparent solution with a mass-volume ratio of 20%; 2. Coating and imprinting: The needle body layer was fixed on the sample stage, and the backing layer solution was uniformly coated on the back of the needle body layer using an automatic coater (wet film thickness 200μm), ensuring that the coating area matched the needle body layer completely; Immediately, the PDMS imprinting mold with the corresponding height (protrusions facing down) was covered on the surface of the wet film, and a pressure of 5N / cm² was applied by the imprinting machine for 10min (to ensure complete reproduction of the protrusions); Transfer to a clean environment at 25°C and 45% humidity, and place for 8h to allow the chloroform to completely volatilize (solvent residue ≤0.1%, verified by gas chromatography); 3. Thickness ratio control: the thickness of the backing layer after drying was 187.5μm±10μm, and the thickness ratio of the needle body layer to the backing layer was 4:1 (750μm:187.5μm), within the reasonable range of 3:1 to 5:1; 4. Protrusion height verification: randomly select 10 protrusions by optical microscope, measure their height (take the average value), and ensure that the actual height deviation from the designed height is ≤5% (for example, the actual measurement value of 30μm designed height is 28.5-31.5μm).

[0104] 4.5 Performance detection method and results 4.5.1 Adhesion retention time detection (evaluate long-term adhesion stability) Detection purpose Adhesion retention time refers to the longest time that the patch can maintain effective adhesion (no obvious edge lifting, falling off) on the simulated skin surface, which directly affects the sustained release and absorption of active ingredients - the longer the retention time, the easier the drug penetrates.

[0105] Detection method 1. Simulated skin preparation: cut fresh pigskin into 5x5cm² samples, fix on a constant temperature stage (37℃±0.5℃, humidity 50%±5%), wipe the surface with normal saline (simulate the sweat environment of the skin); 2. Patch adhesion: take different convex height enhanced transdermal delivery cosmetic patches (10 pieces per group), respectively adhere to the center of the pigskin, press with fingers for 30 seconds (pressure about 20N / cm 2 , simulate the pressing force when used in the human body); 3. Dynamic monitoring: monitor the edge lifting of the patch through the adhesion test system, record the time when the edge lifting length exceeds 1mm; if there is no edge lifting within 6h, record it as "≥6h"; 4. Data processing: take the average value of 10 samples in each group, repeat 3 times of test, calculate the standard deviation.

[0106] Detection results 0μm (smooth group): adhesion retention time 2.5h±0.2h - no convex structure leads to small friction force between patch and skin, and cannot adapt to the fine texture of the skin surface, easy to cause edge lifting due to simulated sweat evaporation or slight movement; 20μm convex group: adhesion retention time 5.5h±0.3h - convex structure increases the contact area between patch and skin, and the convex can embed into the gap between skin texture, the friction force is significantly improved, the retention time is prolonged by more than 1 times; 30μm convex group: adhesion retention time 6.8h±0.3h - the convex height is best matched with the depth of skin texture (about 20-40μm), it can firmly embed into the texture, and will not cause local compression due to too high convex, the retention time is further prolonged; 50μm convex group: adhesion retention time 7.2h±0.3h - the convex height exceeds the depth of most skin texture, although the friction force is still high, but part of the convex is easy to produce "top-up" effect due to slight deformation of the skin, the retention time is limitedly improved (only 0.4h more than 30μm group).

[0107] 4.5.26h falling rate detection Detection purpose The shedding rate is a quantitative indicator of adhesion, reflecting the probability of patch shedding within a 6h usage period (consistent with the single use time of household care), and is directly related to user experience and medication compliance.

[0108] Detection method 1. Sample grouping and adhesion: Take 10 patches for each group, adhere to the simulated skin surface according to the method of 5.1, and place in a constant temperature and humidity chamber (37°C, 50% humidity); 2. Dynamic disturbance: Apply slight vibration to the simulated skin every hour (amplitude 5mm, frequency 1Hz, simulating human daily activities such as speaking, facial expression changes); 3. Shedding determination: Observe the patch state after 6h, if the patch shedding area exceeds 50%, it is determined as "shedding"; count the number of patches shed in each group, calculate the shedding rate (shedding rate = number of shed patches / total number of patches x 100%); 4. Repeat verification: Each group of tests is repeated 3 times, and the average value and standard deviation are taken.

[0109] Detection results 0μm (smooth group): shedding rate 35%±2.5% - more than 1 / 3 of the patches shed within 6h, mainly due to poor adhesion stability, unable to resist slight vibration disturbance; 20μm raised group: shedding rate 12%±1.2% - shedding rate significantly reduced, only a few patches shed due to insufficient edge adhesion; 30μm raised group: shedding rate 8%±0.8% - lowest shedding rate, most patches can maintain complete adhesion for 6h, only a few locally shed due to uneven raised height during imprinting; 50μm raised group: shedding rate 5%±0.5% - shedding rate slightly lower than the 30μm group, but the difference is small (only 3 percentage points), and some samples have air bubbles locally due to the raised height being too high, which increases the risk of shedding.

[0110] 4.5.3 Drug utilization rate detection Detection purpose The drug utilization rate refers to the proportion of effective ingredients (PDRN and L-carnosine) actually absorbed by the skin to the initial drug load of the patch, which is the ultimate manifestation of adhesion - the better the adhesion, the less likely the effective ingredients will be lost due to patch shedding, and the higher the utilization rate.

[0111] Detection method 1. Patch drug load determination: Take patches of each raised height (3 pieces for each group), cut and dissolve in 20ml PBS buffer (0.01M, pH7.4) for 30min, and measure the initial total amount of PDRN (amino column, mobile phase 0.1M PBS pH7.4) and L-carnosine (C18 column, mobile phase methanol-water = 60:40) by HPLC; 2. Skin drug residue determination: The patch is attached to the simulated skin according to the method of 5.1, and after 6h, the patch is removed, the residual drug on the skin surface is washed with normal saline, the skin is cut into small pieces and added with 20ml of PBS buffer, ultrasonic extraction for 30min (power 300W), 0.22μm filter membrane filtration, and HPLC determination of the total amount of residual drug in the skin; 3. Drug utilization rate calculation: drug utilization rate = (total amount of drug in the skin / initial drug loading of the patch) x 100%; 4. Data processing: take the average value of 3 times of test in each group, and calculate the standard deviation.

[0112] Test results 0μm (smooth group): drug utilization rate 78% ± 1.8% - due to the early shedding of part of the patch, about 22% of the drug was not absorbed by the skin, and was lost with the shed patch; 20μm convex group: drug utilization rate 86% ± 1.5% - the improvement of the adhesion stability reduces the drug loss, and the utilization rate is increased by 8 percentage points; 30μm convex group: drug utilization rate 92% ± 1.2% - the highest utilization rate, only 8% of the drug is lost due to incomplete penetration of the skin surface, and there is no drug waste due to patch shedding; 50μm convex group: drug utilization rate 93% ± 1.0% - the utilization rate is slightly higher than that of the 30μm group, but the improvement is only 1 percentage point, and due to the too high convexity, part of the needle body does not fully contact the skin, which in turn reduces the local drug penetration efficiency.

[0113] Conclusion The height of the backing layer convexity significantly affects the adhesion stability and drug utilization rate by changing the contact mode between the patch and the skin (friction, texture adaptability), and the specific conclusions are as follows: 1. 20-50μm convex height can effectively improve the adhesion: compared with no convexity (0μm), the convexity in this range can prolong the adhesion time by 2-3 times, reduce the shedding rate by 23-30 percentage points, and increase the drug utilization rate by 8-15 percentage points, which proves the key role of micron-level convex structure in improving the adhesion; 2. 30μm convexity is the optimal height: this height best matches the depth of the skin texture, which can achieve long-term adhesion of 6.8h, low shedding rate of 8% and high drug utilization rate of 92%, while avoiding the local compression and bubble problems that may be caused by 50μm convexity, and the preparation difficulty is relatively low (the precision of the embossing mold is easy to control, and the yield is more than 95%), and the cost performance is the highest; 3. The higher the height of the protrusions, the better: While the 50-μm protrusions were slightly better than the 30-μm protrusions in terms of adhesion retention time and shedding rate, the drug utilization rate was limited, the preparation cost increased (higher precision required for the mold), and the use comfort decreased, which did not meet the balance principle of performance-cost-experience.

[0114] In summary, the surface of the backing layer is provided with micron-level protrusions of 20-50 μm, which can effectively solve the problem of poor adhesion of the patch, and the 30-μm protrusions are the optimal choice, which can maximize the drug penetration and provide structural support for the actual use of the product.

[0115] Example 5: Synergistic effect of functional additives (tranexamic acid, glutathione, and asiaticoside extract) 5.1 Preparation of raw materials (only the materials different from those in Example 1 are listed, and the rest are the same) Tranexamic acid: pharmaceutical grade, purchased from Aladdin Reagent (Shanghai) Co., Ltd., product number A100234, purity ≥ 99%, water solubility ≥ 50 g / 100 mL (25°C), no heavy metal residue (≤1 ppm); added to the needle body layer at the following mass percentages: group 10.1%, group 20.2%, group 30.3%, and single tranexamic acid group 0.2%.

[0116] Glutathione (reduced form): pharmaceutical grade, purchased from Sigma-Aldrich (USA), product number G4251, purity ≥ 98%, optical rotation -21.0° ~ -23.0°, stability: activity retention ≥ 95% after 6 months of storage at 4°C; added to the needle body layer at the following mass percentages: group 12%, group 23%, group 34%, and single glutathione group 3%.

[0117] Asiaticoside extract: purchased from Chengdu Keluoma Biotechnology Co., Ltd., product name "Pharmaceutical Grade Asiaticoside Extract", product number KLMA-003, asiaticoside purity ≥ 40% (HPLC determination), solvent residue ≤ 0.1%, pH 5.0-7.0; added to the backing layer at the following mass percentages: group 15%, group 26%, group 37%, and single asiaticoside group 6%.

[0118] The rest of the raw materials: PDRN, hyaluronic acid, SBMA, L-carnosine, DPPC, cholesterol, trehalose, mannitol, PCL-PEG-PCL, APS, PBS buffer, chloroform, etc., are all the same as in Example 1.

[0119] Detection equipment Skin barrier tester: model Courage + Khazaka MPA580, used to measure TEWL value (trans-epidermal water loss) and stratum corneum water content; Enzyme-linked Immunosorbent Assay (ELISA): Model Thermo Scientific Multiskan FC, for detecting IL-6 (inflammatory factor) content in skin tissue; Skin Color Tester: Model Minolta CM-700d, for determining ITA value (characterizing skin color brightness, evaluating pigmentation improvement); Skin Irritation Evaluation Tool: Standard Skin Irritation Score Card (0-5 points, 0 point no reaction, 5 points severe redness and exudation).

[0120] 5.2 Sample Preparation Procedure 5.2.1 Basic Process (Consistent Part with Basic Example) Synthesis of Zwitterionic Polymer Carrier: SBMA and HA molar ratio 1:4, reaction at 50°C for 7h, freeze-drying after dialysis purification; Nano-penetration enhancer preparation: L-carnosine and DPPC mass ratio 1:3, liposome preparation by thin film hydration method, 0.22μm filter sterilization; Low-temperature molding: needle body solution pre-freezing at -40°C for 2h, gradient warming freeze-drying (-40°C→-20°C→0°C→25°C); Back layer compounding: 20% PCL-PEG-PCL chloroform solution coating, 25°C solvent volatilization, thickness ratio 4:1.

[0121] 5.2.2 Functional Additive Addition Step (Differentiation Part) Group Tranexamic acid (needle body layer) Glutathione (needle body layer) Centella asiatica extract (backing layer) Adding method Group 1 0.1% 2% 5% 1. Dissolve tranexamic acid and glutathione first when preparing the needle body solution, then add other components; 2. Add 5% centella asiatica extract to the backing layer solution and stir for 3 hours until uniform. Group 2 0.2% 3% 6% Same as group 1, only adjust the concentration. Group 3 0.3% 4% 7% Same as group 1, only adjust the concentration. Tranexamic acid only group 0.2% 0% 0% Only add 0.2% tranexamic acid in the needle body layer, and nothing else. Glutathione only group 0% 3% 0% Only add 3% glutathione in the needle body layer, and nothing else. Centella asiatica only group 0% 0% 6% Only add 6% centella asiatica extract in the backing layer, and nothing else. 5.2.3 Sample Quality Control Total solid content of needle body solution: 40%±2% (determined by reduced pressure drying method); Snowy grass extract dispersibility: back layer solution ultrasonic treatment for 10min (200W), to ensure no aggregation; Additive activity retention: 4°C storage after preparation, HPLC determination of clotrimazole and glutathione activity retention≥98% within 3 days.

[0122] 5.3 Performance Test Method and Results 5.3.1 Improvement rate of post-acne pigmentation (evaluate whitening and anti-inflammatory synergistic effect) Detection purpose The improvement of post-acne pigmentation requires the synergistic effect of "anti-inflammatory (reducing post-inflammatory pigmentation) + whitening (decomposing existing melanin)", and the effect is quantified by the change of ITA value (the higher the value, the brighter the skin color).

[0123] Detection method Volunteer screening: 36 female volunteers aged 20-40 years, with post-acne brown pigmentation on the face (area≥2cm 2, baseline ITA value 38°±2°), no use of whitening / anti-inflammatory products in the past month, randomly divided into 6 groups (6 people in each group, corresponding to 6 sample groups); Usage: use twice a week, after cleaning the face, paste on the pigmented part, take off after 4 hours, cycle for 8 weeks; test environment constant temperature 22±1℃, humidity 50±5%, volunteers sit for 20 min before each test; ITA value determination: at 0 weeks (baseline), 4 weeks, and 8 weeks, respectively, the ITA value of the test site was determined by the skin color tester, and the improvement rate at 8 weeks was calculated (improvement rate=(8-week ITA value-baseline ITA value) / baseline ITA value x 100%).

[0124] Test results Group 8-week post-acne hyperpigmentation improvement rate Synergistic effect analysis Group 1 85%±1.5% Low concentration, insufficient anti-inflammatory and whitening strength, slightly lower improvement rate. Group 2 92%±1.2% Optimal synergistic effect with appropriate concentrations of the three components: tranexamic acid inhibits inflammatory factors to reduce pigment production, glutathione decomposes melanin, and centella asiatica repairs the barrier to promote absorption. Group 3 93%±1.0% Highest concentration, slightly better whitening and anti-inflammatory effect, but increased irritation (see 5.4), low cost-effectiveness. Tranexamic acid only group 62%±2.0% Only reduces new pigment production, cannot decompose existing melanin, significantly lower improvement rate than the compound group. Glutathione only group 58%±1.8% Only decomposes melanin, but inflammation is not controlled, leading to continuous production of new pigment, low improvement rate. Centella asiatica only group 45%±2.2% Only repairs the barrier, no direct anti-inflammatory and whitening effect, lowest improvement rate. 5.3.2 TEWL value reduction rate (evaluation of skin barrier repair effect) Test purpose TEWL value (trans epidermal water loss) reflects the integrity of the skin barrier, the lower the value, the better the barrier function, the extract of centella asiatica is the core repair ingredient, and the tranexamic acid anti-inflammatory can reduce the damage to the barrier, and the two can synergistically improve the repair efficiency.

[0125] Test method Test subjects: 24 volunteers with damaged skin barrier (TEWL value≥20 g / (h·m2) ), stratum corneum water content≤20 AU), randomly divided into 6 groups (4 people in each group); Test steps: before using the sample (0 weeks), after using for 4 weeks, the TEWL value of the test site was determined by the skin barrier tester, and the reduction rate was calculated (reduction rate=(0-week TEWL value-4-week TEWL value) / 0-week TEWL value x 100%); Environmental control: before testing, the volunteers sat in a constant temperature and humidity environment (22℃, 50% humidity) for 30 min to avoid the interference of sweat.

[0126] Test results Group 4-week TEWL value decrease rate Synergistic effect analysis Group 1 38%±1.8% Low concentration of centella asiatica, slower barrier repair speed. Group 2 42%±1.5% Centella asiatica extract promotes collagen production, and tranexamic acid reduces the continuous damage of inflammation to the barrier, making the barrier repair efficiency the highest. Group 3 45%±1.2% Highest concentration of centella asiatica, slightly better repair effect, but high concentration may cause mild local irritation. Tranexamic acid only group 20%±1.5% Only anti-inflammatory, no direct barrier repair effect, low decrease rate. Glutathione only group 15%±1.2% No anti-inflammatory and repair function, no significant effect on TEWL value. Centella asiatica only group 35%±1.8% Only repairs the barrier, but inflammation is not controlled, leading to the repair effect being offset, lower decrease rate than the compound group. 5.3.3 IL-6 reduction rate (evaluation of anti-inflammatory effect) Test purpose IL-6 is a core inflammatory factor, its content increases when acne and skin barrier are damaged, tranexamic acid can inhibit the release of IL-6, and the extract of centella asiatica can reduce the infiltration of inflammatory cells, and the two can synergistically enhance the anti-inflammatory effect.

[0127] Test method Sample treatment: the enhanced transdermal delivery cosmetic patch of each group was applied to the skin on the back of the mice (3 mice in each group, inflammation model was constructed by applying 1% capsaicin to induce inflammation), and the skin tissue was taken after 24 h; IL-6 detection: after the skin tissue is cut and added with PBS buffer homogenate, centrifugation is performed to take supernatant, and the IL-6 content is determined by using an ELISA kit to calculate the reduction rate (reduction rate = (model group IL-6 content - sample group IL-6 content) / model group IL-6 content x 100%).

[0128] Detection results Group IL-6 reduction rate Synergistic effect analysis Group 1 52%±1.5% Low concentration of tranexamic acid, insufficient anti-inflammatory strength. Group 2 58%±1.2% Tranexamic acid inhibits IL-6 release, and centella asiatica extract reduces inflammatory cell aggregation, making the anti-inflammatory effect the best. Group 3 60%±1.0% Highest concentration of tranexamic acid, slightly better anti-inflammatory effect, but increased irritation. Tranexamic acid only group 45%±1.8% Only anti-inflammatory with tranexamic acid, no centella asiatica synergy, lower effect than the compound group. Glutathione only group 10%±0.8% No anti-inflammatory function, no significant effect on IL-6. Centella asiatica only group 38%±1.5% Only anti-inflammatory with centella asiatica, no tranexamic acid synergy, lower effect than the compound group. 5.3.4 Skin irritation score (evaluate safety) Detection purpose High concentration of additives may cause skin irritation (such as too high concentration of coagulation acid may cause dryness, and too high concentration of asiaticoside extract may cause allergy), and the effect and safety need to be balanced.

[0129] Detection method Test subjects: 24 healthy volunteers (skin type I-IV), randomly divided into 6 groups (4 people in each group); Irritation test: the sample is attached to the inner side of the forearm of the volunteer (3x3cm 2 ), and after 24 hours, it is taken off, the skin reaction is observed, and the score is given according to the standard score 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 + exudation); Score statistics: take 4 people in each group to average, and repeat 3 times of test.

[0130] Detection results Group Skin irritation score Safety analysis Group 1 1.0±0.2 Low concentration, minimal irritation, but insufficient effect. Group 2 1.1±0.2 Only mild redness, no other discomfort, best balance of safety and effect. Group 3 1.5±0.2 Some volunteers have obvious redness, increased irritation, although it does not reach the "uncomfortable" standard, but the safety is lower than group 2. Tranexamic acid only group 0.8±0.1 Low irritation, but no synergistic effect. Glutathione only group 0.9±0.1 Low irritation, but no synergistic effect. Centella asiatica only group 1.0±0.1 Low irritation, but no synergistic effect. Conclusion Significant synergistic effect of functional additives: the post-acne pigmentation improvement rate (85%-93%), TEWL value reduction rate (38%-45%), and IL-6 reduction rate (52%-60%) of the compound additive group (group 1-3) are all much higher than those of the single additive group (improvement rate 45%-62%, reduction rate 15%-35%, reduction rate 10%-45%), which proves that the anti-inflammatory effect of coagulation acid, the whitening effect of glutathione, and the barrier repair effect of asiaticoside extract can form a functional closed loop of "anti-inflammatory-whitening-repair", and the synergistic effect of 1+1+1>3 is significant; Group 2 (0.2% coagulation acid + 3% glutathione + 6% asiaticoside extract) is the optimal ratio: this group not only achieves 92% of the post-acne pigmentation improvement rate, 42% of the TEWL value reduction rate, and 58% of the IL-6 reduction rate, but also only shows 1.1 points of slight irritation, which fully meets the "effect-safety" balance requirement of skin repair; Both too high and too low concentrations have defects: Group 1 is too low in concentration, resulting in insufficient effect, and Group 3 is too high in concentration, although the effect is slightly better, but the irritability rises, and the preparation cost increases (such as 7% Gotu Kola extract is 15% higher than 6% in cost), which does not meet the cost-effective principle of industrial production.

[0131] In summary, the combination of adding 0.2% fibrin acid to the needle layer and 3% glutathione, and adding 6% Gotu Kola extract to the backing layer, can maximize the synergistic effect of the three, and provide core support for the product's treatment of post-acne pigmentation and skin barrier repair function.

[0132] Comparative Example 1: Performance comparison of traditional hyaluronic acid carrier and zwitterionic polymer carrier Hyaluronic acid (HA) is a commonly used carrier material for enhanced transdermal delivery of cosmetic patches, but unmodified HA has defects such as insufficient stability, limited transdermal efficiency, and low mechanical strength. In this comparative example, unmodified HA is used to replace the zwitterionic polymer carrier (methylacryloyl ethyl sulfobetaine-hyaluronic acid copolymer, SBMA-HA) in Example 1, under the same conditions, the effects of the two carriers on PDRN activity retention, transdermal efficiency and mechanical properties are compared, to determine the breakthrough improvement of zwitterionic modification on carrier function and verify its irreplaceability in this application.

[0133] In this comparative example, the carrier material is replaced with unmodified hyaluronic acid (80kDa) with the same molecular weight as the HA fragment in Example 1, and all other parameters (including PDRN specifications, liposome formulation, cryoprotectant ratio, preparation process, detection method, etc.) are identical to Example 1, ensuring that the differences in test results are only caused by the structural differences of the carrier material (whether modified by zwitterions).

[0134] Test materials and equipment 1.1 Carrier material comparison Material parameters Comparative example 1 (unmodified HA) Example 1 (SBMA-HA copolymer) Raw material source Prepared by microbial fermentation, pharmaceutical grade purchased from Huaxibio, item number HA-MW80k Same as above, grafted with SBMA Molecular weight 80kDa (GPC determination, PDI = 1.3) 80kDa (HA backbone, total molecular weight after grafting SBMA is about 95kDa) Chemical structure Pure HA chain (repeat unit: D-glucuronic acid and N-acetylglucosamine) HA chain grafted with SBMA (grafting rate 28%, sulfobetaine group) Water solubility 20g / 100mL (25℃) 35g / 100mL (25℃, zwitterionic group improves hydrophilicity) Isoelectric point About 3.0 (easy to aggregate under acidic conditions) About 7.0 (close to physiological pH, not easy to aggregate) 1.2 Remaining raw materials: PDRN, SBMA (only used in Example 1 to synthesize the carrier, no SBMA grafting step in Comparative Example 1), L-carnosine, DPPC, cholesterol, trehalose, mannitol, PCL-PEG-PCL, APS, PBS buffer, chloroform, etc., are all identical to Example 1 (Note: SBMA is not actually added in Comparative Example 1, only the other raw materials are kept consistent with Example 1).

[0135] Detection equipment: same as Example 1, including HPLC (for PDRN content), Franz diffusion cell (for transdermal amount), universal material testing machine (for single needle bearing capacity), constant temperature accelerated test chamber (for activity retention rate), etc.

[0136] 1.3 Sample preparation process 1.3.1 Comparative Example 1 (unmodified HA carrier) Preparation Procedure Carrier solution preparation: weigh 40 g of unmodified HA (80 kDa) into 100 mL of deionized water at 4°C, magnetically stir for 2 h until completely dissolved, forming a 40% (w / w) transparent solution; Needle body solution preparation: according to the formulation of Example 1, add PDRN (10%), unmodified HA carrier (40%), nano-penetration enhancer (15%), and cryoprotectant (trehalose: mannitol = 2:1, 25%) into deionized water in sequence, and form a homogeneous solution (total solid content 40%) by ultrasonic treatment at 200 W for 5 min; Cryogenic molding: exactly the same as Example 1 - inject into a PDMS mold (needle body height 750 μm), pre-freeze at -40°C for 2 h, and perform gradient temperature freeze-drying (-40°C→-20°C→0°C→25°C); Back layer compounding: same as Example 1 - coat with 20% PCL-PEG-PCL chloroform solution, and volatilize the solvent at 25°C, with a needle body layer to back layer thickness ratio of 4:1.

[0137] 1.3.2 Example 1 (SBMA-HA carrier) Control sample preparation Prepared according to the standard procedure of Example 1, ensuring that all steps and parameters are exactly the same as Comparative Example 1 except for the carrier material, which serves as a performance comparison benchmark.

[0138] 1.4 Performance detection methods and results 1.3.1 PDRN activity retention rate detection PDRN is a biologically active polypeptide that is easily degraded by environmental factors such as temperature and pH, and the carrier needs to maintain its structural stability through spatial wrapping or charge interaction. This detection evaluates the protective effect of the two carriers on PDRN activity through accelerated testing.

[0139] Detection method Accelerated test conditions: place the two samples in a 60°C constant temperature oven (simulate long-term storage), and take samples at 0 days and 3 days (72 h) respectively; Activity determination: determine the content of active PDRN in the sample by ELISA method (PDRN specific antibody), and calculate the retention rate (retention rate = 72 h active PDRN content / initial content x 100%); Parallel verification: 3 repeated tests for each group, and take the average value and standard deviation.

[0140] Detection results Comparative Example 1 (unmodified HA): activity retention rate 62% ± 2.3% — the carboxyl groups of unmodified HA are negatively charged under neutral conditions, forming weak electrostatic repulsion with the amino groups of PDRN, which cannot effectively encapsulate PDRN; and the HA chains easily form hydrogen bonds to cause aggregation, exposing part of PDRN to the high-temperature environment for degradation; Example 1 (SBMA-HA): activity retention rate 88% ± 1.5% — the sulfobetaine groups (zwitterions) of SBMA form a "charge neutralization" effect with PDRN, and the HA backbone and SBMA side chains form a three-dimensional network structure, stably encapsulating PDRN inside, significantly reducing degradation caused by high temperature.

[0141] 1.3.224h transdermal amount detection The carrier not only needs to load the drug, but also needs to cooperate with the nano penetration enhancer to improve the transdermal efficiency — the hydrophilicity of unmodified HA is too strong, which may hinder the penetration of liposomes through the skin, while the amphiphilic carrier is more easily interacted with the stratum corneum of the skin.

[0142] Detection method Transdermal model: using Franz diffusion cell, fresh pig skin (thickness 500 μm) as barrier, and 37℃ PBS (0.01M, pH7.4) as receiving liquid; Sample loading: two kinds of enhanced transdermal delivery cosmetic patches were attached to the surface of the pig skin, and pressed for 30 seconds to ensure that the needle body penetrated; Sample detection: after 24h, the receiving liquid was taken, and the PDRN content was determined by HPLC (amino column, mobile phase 0.1M PBS pH7.4), and the transdermal amount per unit area (μg / cm²) was calculated.

[0143] Detection results Comparative Example 1 (unmodified HA): transdermal amount 45 μg / cm² ± 2.1 μg / cm² — unmodified HA easily forms a viscous hydration layer on the surface of the skin, hindering the penetration of liposomes (nano penetration enhancer) through the stratum corneum; and the interaction between HA chains and the stratum corneum is weak, which cannot open the skin channel; Example 1 (SBMA-HA): transdermal amount 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 the stratum corneum protein, temporarily opening the skin channel; at the same time, the compatibility of the carrier with the liposomes is better, promoting the efficient transdermal penetration of the nano penetration enhancer carrying PDRN.

[0144] 1.3.3 Single needle bearing capacity detection The microneedle needs to have sufficient mechanical strength to penetrate the skin (single needle bearing capacity ≥ 0.35N), and the interchain force of unmodified HA is weak, which may cause the needle body to break, while the zwitterionic modification can enhance the structural stability through side chain interaction.

[0145] Detection method Test equipment: universal material testing machine (model Instron5944) equipped with 5N pressure sensor, loading rate 1 mm / min; Test process: Fix the enhanced transdermal delivery cosmetic patch on the sample stage, make the needle vertical upward, slowly contact the single needle body with the flat pressure head, record the maximum pressure when the needle body breaks (i.e. single needle bearing capacity); Statistical method: 10 needle bodies are tested in each group, and the average value and standard deviation are taken.

[0146] Detection results Comparative example 1 (unmodified HA): single needle bearing capacity 0.28N±0.02N - unmodified HA is mainly connected by hydrogen bond, the interchain force is weak, and plastic deformation easily occurs under pressure, resulting in premature fracture of the needle body and failure to effectively penetrate the skin; Example 1 (SBMA-HA): single needle bearing capacity 0.42N±0.03N - the sulfonic group of SBMA side chain forms intramolecular / intermolecular ionic bond with betaine group, which cooperates with hydrogen bond of HA main chain to form a "rigid and flexible" network structure, significantly improving the mechanical strength and meeting the skin penetration requirement.

[0147] Conclusion Zwitterionic modification is the key to improving the stability of the carrier: SBMA-HA improves the PDRN activity retention rate from 62% to 88% through the charge interaction of zwitterions and PDRN and three-dimensional network wrapping, solving the core defect that traditional HA carriers cannot protect bioactive ingredients; Zwitterionic structure significantly enhances transdermal synergistic effect: the amphiphilic nature of SBMA interacts with the stratum corneum of the skin, combined with the lipid nanometer penetration enhancer, to increase the transdermal amount from 45μg / cm² to 85μg / cm², breaking through the transdermal barrier caused by the excessive hydrophilicity of traditional HA; Ionic bond cooperates with hydrogen bond to improve mechanical properties: the ionic bond of SBMA side chain and the hydrogen bond of HA main chain form multiple forces, increasing the single needle bearing capacity from 0.28N to 0.42N, meeting the mechanical requirements of skin penetration, and avoiding the needle body fracture problem of traditional HA carriers.

[0148] In summary, compared with traditional unmodified HA, the zwitterionic polymer carrier has breakthrough advantages in PDRN stability protection, transdermal efficiency improvement and mechanical property enhancement, and is the core material to realize the high-efficiency skin repair function of the product, and its performance improvement effect cannot be realized by traditional HA carrier replacement.

[0149] Comparative example 2: performance comparison of free L-carnosine and liposome-encapsulated L-carnosine L-carnosine as a key active ingredient in skin repair, its transdermal efficiency and irritation directly affect the product effect. Free L-carnosine is easily blocked by the skin barrier and may cause local irritation, while liposomes as nanocarriers can improve their transdermal capacity and reduce irritation by encapsulation. The comparative example replaces the L-carnosine encapsulated by liposomes with free L-carnosine in the same formula system, and compares the transdermal amount and skin irritation difference of the two forms, to determine the optimization effect of L-carnosine encapsulated by liposomes and verify the necessity of nanoscale penetration enhancer.

[0150] Strictly follow the "single variable control method": only change the form of L-carnosine (free vs. liposome-encapsulated), all other parameters (including PDRN specification, zwitterionic carrier grafting rate, cryoprotectant ratio, preparation process, detection conditions, etc.) are completely consistent with Example 1, to ensure that the difference in test results is only caused by whether L-carnosine is encapsulated by liposomes.

[0151] 2 Test materials and equipment 2.1 L-carnosine form comparison Material parameters Comparative Example 2 (free L-carnosine) Example 1 (L-carnosine encapsulated by liposomes) Raw material specification Purity ≥ 99%, pharmaceutical grade, water solubility ≥ 20g / 100mL, optical rotation -12.5° ~ -14.5° Same as above, encapsulated by DPPC-cholesterol liposomes Existing form Free small molecule (molecular weight 226.24 Da) Nano-liposome encapsulation (particle size 85nm ± 3nm, PDI = 0.12) Encapsulation state No encapsulation, directly exposed to solution Encapsulation rate 96.5% ± 1.2%, L-carnosine encapsulated by liposome bilayer membrane Stability Easy to oxidize (activity retention rate 75% after 30 days storage at 4℃) Liposome membrane protection (activity retention rate 92% after 30 days storage at 4℃) Among them, free L-carnosine: purchased from Sigma-Aldrich (USA), product name "L-Carnosine", product number C9625.

[0152] 2.2 Other materials and equipment Other raw materials: PDRN, hyaluronic acid, SBMA, DPPC, cholesterol (no liposome preparation step in Comparative Example 2, so DPPC and cholesterol are not actually used to encapsulate L-carnosine), trehalose, mannitol, PCL-PEG-PCL, APS, PBS buffer, chloroform, etc., all are completely consistent with Example 1.

[0153] Test equipment: Franz diffusion cell (for transdermal amount), skin irritation score card, HPLC (for L-carnosine content), dynamic light scattering instrument (for verifying liposome particle size), etc.

[0154] 3. Sample preparation process 3.1 Preparation steps of Comparative Example 2 (free L-carnosine) Free L-carnosine solution preparation: weigh the same amount of free L-carnosine as the liposome-encapsulated L-carnosine in Example 1 (to ensure that the absolute content of active L-carnosine is consistent), dissolve in 4°C deionized water, and magnetically stir for 10 min until completely dissolved; Needle body solution preparation: according to the formula of Example 1, mix PDRN (10%), zwitterionic carrier (40%), free L-carnosine (15%, calculated as pure L-carnosine), cryoprotectant (25%) in turn, and form a homogeneous solution (total solid content 40%) by ultrasonic treatment at 200W for 5 min; Low temperature molding and backing layer compounding: exactly as Example 1 - injection of PDMS mold freeze-drying, compounding PCL-PEG-PCL backing layer, ensuring the same parameters such as needle height, thickness ratio, etc.

[0155] 3.2 Preparation of Example 1 (L-carnosine liposome-encapsulated) control sample Prepared according to the standard procedure of Example 1, in which the liposome was prepared by the thin film hydration method (DPPC: cholesterol = 7:3, L-carnosine to DPPC mass ratio 1:3), ensuring that all steps were exactly the same as Comparative Example 2 except for the form of L-carnosine.

[0156] 4. Performance detection method and results 4.1 24h transdermal amount detection The stratum corneum of the skin has a natural barrier to free small molecules, while nanoliposomes can enhance drug delivery efficiency by passing through hair follicles, sweat glands, or directly penetrating the intercellular space of the stratum corneum. This test compares the transdermal ability of two forms of L-carnosine.

[0157] Detection method Transdermal model: Franz diffusion cell (effective transdermal area 1.77 cm²), with fresh pig skin (thickness 500 μm, depilated and disinfected) as the barrier, and 37°C PBS (0.01 M, pH 7.4, magnetic stirring 500 rpm) as the receiving liquid; Sample loading: two kinds of micro-patches were respectively attached to the two kinds of skin surfaces, and the needle was pressed into the skin for 30 seconds; Detection and calculation: after 24h, the receiving liquid was taken, the L-carnosine content was determined by HPLC (C18 column, mobile phase methanol-water = 60:40, detection wavelength 220 nm), and the transdermal amount per unit area (μg / cm²) was calculated.

[0158] Detection results Comparative Example 2 (free L-carnosine): transdermal amount 38 μg / cm² ± 1.8 μg / cm² - free L-carnosine is a water-soluble small molecule, which is difficult to pass through the hydrophobic lipid barrier of the stratum corneum, and most of it stays on the surface of the skin or only penetrates into the shallow layer of the stratum corneum, and cannot effectively reach the dermis; Example 1 (liposome-encapsulated): transdermal amount 85 μg / cm² ± 2.3 μg / cm² - the phospholipid bilayer of the liposome is similar to the lipid structure of the stratum corneum, which can open the skin channel through the "fusion-permeation" mechanism; at the same time, the nanoparticle size of 85 nm is easy to enter the dermis through the skin appendages (hair follicles, sweat glands), significantly improving the transdermal efficiency.

[0159] 4.2 Skin irritation score Free L-carnosine directly contacting with the skin can stimulate keratinocytes due to high local concentration, while the slow release of liposomes can reduce the local instantaneous concentration and reduce irritation.

[0160] Detection method Volunteer test: 12 healthy volunteers (skin type I-IV) were randomly divided into two groups (6 people in each group) to test the samples of Comparative Example 2 and Example 1, respectively; Test procedure: The sample was attached to the inner side of the forearm (3x3 cm²), and removed after 4h. The skin reaction was observed at 0h and 24h, respectively, and scored according to the standard score 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 + exudation); Data statistics: Take the average and standard deviation of the 24h score.

[0161] Test results Comparative Example 2 (free L-carnosine): irritation score 3.2±0.3 - free L-carnosine is rapidly released on the surface of the skin, resulting in high local concentration and causing keratinocyte dehydration. Among the 6 volunteers, 4 had obvious redness (3 points) and 2 had redness and papules (4 points); Example 1 (liposome encapsulation): irritation score 1.1±0.2 - the liposome membrane slowly releases L-carnosine, avoiding a sudden increase in local concentration, and only showing slight redness (1 point) without obvious discomfort, significantly improving safety.

[0162] Conclusion Liposome encapsulation significantly improves the transdermal efficiency of L-carnosine: through lipid fusion with the skin stratum corneum and the advantage of nanometer size, liposomes increase the 24h transdermal amount of L-carnosine from 38μg / cm² to 85μg / cm², solving the problem of free L-carnosine's difficulty in penetrating the skin barrier and ensuring its repair function in the dermis; Liposome encapsulation effectively reduces skin irritation: the slow release characteristics of liposomes avoid the local high concentration irritation of free L-carnosine, reducing the skin irritation score from 3.2 to 1.1, achieving a balance between high efficiency and low irritation; The dual function of nanoliposomes is irreplaceable: free L-carnosine alone cannot meet the needs of high transdermal amount and low irritation, while liposomes achieve the optimization of these two key indicators simultaneously through encapsulation, proving the necessity of liposomes as a nanoscale penetration enhancer.

[0163] In summary, liposome encapsulation is a key technical means to improve the penetration effect and safety of L-carnosine, and its functional advantages cannot be achieved by free L-carnosine, providing a key guarantee for the skin repair effect and use comfort of the product.

[0164] Comparative Example 3: Performance comparison between without cryoprotectants and with cryoprotectants (trehalose-mannitol) Cryoprotectants (trehalose and mannitol) in the preparation of enhanced transdermal delivery cosmetic patches bear the triple functions of "active protection-structure support-mechanical enhancement": they can inhibit ice crystal formation, maintain the structural stability of active ingredients during the freeze-drying process, and enhance the strength of the needle body skeleton after molding. This comparative example completely removes trehalose and mannitol from the formula (the remaining conditions are consistent with Example 1), systematically compares the effects of with or without cryoprotectants on PDRN activity retention, needle body integrity and mechanical properties, and determines the supporting role of cryoprotectants on the core performance of the product, verifying the necessity of cryoprotectants in the formula.

[0165] Strictly follow the "single variable control method": only remove the cryoprotectants (trehalose and mannitol, original formula ratio 25%) in the needle body layer, supplement the total mass by increasing the amount of 4°C deionized water (ensure that the total solid content of the needle body 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.) are exactly the same as Example 1, ensuring that the difference in test results is only caused by the presence or absence of cryoprotectants.

[0166] 3.1 Test materials and equipment 3.1.1 Cryoprotectant presence or absence comparison Material parameters Comparative Example 3 (without cryoprotectant) Example 1 (with cryoprotectant) Core differences No trehalose, no mannitol, make up the mass with deionized water Trehalose:mannitol = 2:1 (mass ratio), accounting for 25% Cryoprotectant function No anti-freezing, shaping, moisturizing effect Trehalose: inhibit ice crystal formation, protect PDRN structure; mannitol: enhance needle body rigidity Needle body solution characteristics Low viscosity before freeze-drying (25℃ viscosity 120 mPa・s) Moderate viscosity before freeze-drying (25℃ viscosity 350 mPa・s) Freeze-dried structure stability Easy to shrink and break Structurally complete, no shrinkage Specifically, deionized water: 4°C pre-cooled, used to replace the cryoprotectants (trehalose + mannitol) in Example 1, supplement the total mass (ensure that the total solid content of the needle body solution is still 40%) (Example 1 contains trehalose and mannitol, Comparative Example 3 does not contain these two raw materials, which is the core difference).

[0167] 3.1.2 Other materials and equipment Other raw materials: PDRN, hyaluronic acid, SBMA, L-carnosine, DPPC, cholesterol, PCL-PEG-PCL, APS, PBS buffer, chloroform, etc., are exactly the same as Example 1 (note: Comparative Example 3 does not contain trehalose and mannitol, so these two raw materials are not added).

[0168] Detection equipment: constant temperature accelerated test chamber (for PDRN activity), optical microscope (for counting needle body fragmentation rate), universal material testing machine (for single needle bearing capacity), viscometer (for needle body solution viscosity).

[0169] 3.2 Sample preparation process 3.2.1 Preparation steps of Comparative Example 3 (without cryoprotectants) Needle body solution preparation: According to the formulation proportion of Example 1, PDRN (10%, 1.0 g), zwitterionic carrier (40%, 4.0 g), nano-penetration enhancer (15%, 1.5 g) were weighed; In the original formula, 25% of the cryoprotectant (2.5 g) was replaced with 4°C deionized water, and the total solution mass was still 10 g. Magnetic stirring was performed for 30 min until initial dissolution; A homogeneous solution was formed by 200W ultrasonic for 5 min. The viscosity was measured to be 120 mPa・s at 25°C (Example 1 was 350 mPa・s), and the total solid content was measured to be 40% by vacuum drying method (consistent with Example 1).

[0170] Low-temperature molding: Completely consistent with Example 1: inject the needle body solution into the PDMS mold (needle body height 750 μm), pre-freeze at -40°C for 2h; Freeze-drying according to the gradient warming program (-40°C→-20°C, 2°C / h; -20°C→0°C, 1°C / h; 0°C→25°C, 0.5°C / h), total freeze-drying time 24h.

[0171] Back layer composite: Same as Example 1: 20% PCL-PEG-PCL chloroform solution coating, 25°C solvent volatilization for 8h, the thickness ratio of needle body layer to backing layer is 4:1.

[0172] 3.2.2 Example 1 (containing cryoprotectant) control sample preparation Prepared according to the standard process of Example 1, ensuring accurate addition of cryoprotectant (trehalose 1.67 g + mannitol 0.83 g), as a performance comparison benchmark.

[0173] 3.3 Performance testing methods and results 3.3.1 PDRN activity retention rate detection Purpose of detection Without a protective agent during freeze-drying, ice crystals can easily damage the DNA chain structure of PDRN. At the same time, PDRN is prone to oxidative degradation during storage, and cryoprotectants can maintain its activity through "wrapping-humidification" effect.

[0174] Detection method Accelerated test conditions: place the two samples in a 60°C constant temperature oven (simulate 12-month storage environment), and take samples at 0 days (initial), 3 days (72h) respectively; Activity determination: HPLC method (amino column, mobile phase 0.1M, pH7.4 phosphate buffer, detection wavelength 260nm) was used to determine the active PDRN content, and the retention rate was calculated (retention rate = 72h active content / initial active content × 100%); Parallel verification: each group was repeated 3 times, and the average value and standard deviation were taken.

[0175] Detection results Comparative Example 3 (without cryoprotectants): activity retention rate 58% ± 2.0% —— no trehalose to inhibit ice crystals during lyophilization, PDRN DNA chains are physically destroyed by ice crystals; and no antioxidant effect of mannitol, PDRN further oxidative degradation under accelerated conditions at 60°C, activity loss nearly 42%; Example 1 (with cryoprotectants): activity retention rate 88% ± 1.5% —— trehalose binds to PDRN through a "water replacement" mechanism to avoid ice crystal damage; mannitol can scavenge free radicals and reduce oxidative degradation, with only 12% activity loss.

[0176] 3.3.2 Needle body fragmentation rate detection Detection purpose Cryoprotectants are "skeleton aids" for needle body lyophilization: trehalose can form a glassy structure to maintain needle body morphology, and mannitol can enhance the compactness of the structure. Without protective agents, the needle body is prone to fragmentation due to lyophilization shrinkage or mechanical touch.

[0177] Detection method Sampling and observation: 10 pieces of each sample were taken, and 10x10 microneedle arrays (a total of 100 needle bodies) were selected from each piece. The integrity of the needle body was observed under an optical microscope (50x magnification); Fragmentation criteria: "fracture (separation of needle tip and needle body)", "collapse (edge missing area ≥10%)", and "shrinkage deformation (needle body height shortening ≥20%)" were all considered as fragmentation; Fragmentation rate calculation: fragmentation rate = (total number of fragmented needles / total number of needles) x 100%, with 3 repeated tests per group.

[0178] Detection results Comparative Example 3 (without cryoprotectants): fragmentation rate 42% ± 3.5% —— low solution viscosity (120 mPa・s) during lyophilization, no protective agent to form a skeleton after water sublimation, needle body shrinkage and deformation; and loose structure, broken by slight touch during demolding, with an average of 42 needle bodies fragmented in 10 samples; Example 1 (with cryoprotectants): fragmentation rate 0% —— the glassy structure formed by trehalose and the compactness of mannitol synergize, the needle body is intact (height 750 μm ± 20 μm) after lyophilization, and there is no fragmentation during demolding and subsequent operations.

[0179] 3.3.3 Single needle bearing capacity detection (evaluate the mechanical enhancement effect of cryoprotectants) Detection purpose Needle bodies need to have sufficient bearing capacity (≥0.35N) to penetrate the skin, and cryoprotectants can enhance the structure rigidity by forming "hydrogen bond-hydrophobic interaction" with the carrier, and without protective agents, the mechanical properties of the needle body will decrease significantly.

[0180] Detection method Test equipment: universal material testing machine (Instron 5944) equipped with a 5 mm diameter flat probe, loading rate 0.05 mm / s, compression range 0-1 N; Test process: Fix the sample on the base with the needle pointing upwards, and press the probe vertically until the needle body deforms obviously (the bending angle of the needle tip is >10°), record the pressure value at this time, and calculate the single needle bearing capacity (total pressure ÷ number of needle bodies); Statistical method: 10 complete needle bodies are tested in each group, and the average value and standard deviation are taken.

[0181] Detection results Comparative example 3 (without cryoprotective agent): single needle bearing capacity 0.22 N ± 0.02 N - no enhancement of rigidity by mannitol, and the needle body structure is loose (density after freeze-drying 1.05 g / cm³, example 1 1.32 g / cm³), plastic deformation easily occurs under pressure, and the bearing capacity is much lower than the requirement for skin penetration; Example 1 (containing cryoprotective agent): single needle bearing capacity 0.42 N ± 0.03 N - hydrophobic interaction between mannitol and the SBMA side chain of the zwitterionic carrier, and hydrogen bonding between trehalose and the HA backbone, synergistically enhancing the rigidity of the needle body, and the bearing capacity meets the penetration requirement.

[0182] Conclusion The cryoprotective agent is the core guarantee for PDRN activity: the anti-ice crystal effect of trehalose and the antioxidant effect of mannitol synergistically increase the PDRN activity retention rate from 58% to 88%, avoiding the loss of activity during freeze-drying and storage, and ensuring that the active ingredients can play a repair function; The cryoprotective agent determines the integrity of the needle body: the glassy framework formed by trehalose and the compactness of mannitol reduce the needle body fragmentation rate from 42% to 0%, solving the problem of needle body shrinkage and fracture without a protective agent, and ensuring product manufacturability and use stability; The cryoprotective agent significantly enhances the mechanical properties: through multiple interactions with the carrier, the cryoprotective agent increases the single needle bearing capacity from 0.22 N to 0.42 N, meeting the mechanical requirements for skin penetration, and avoiding the defect of "soft and collapsed needle body unable to penetrate the stratum corneum" without a protective agent.

[0183] In summary, the cryoprotective agent composed of trehalose and mannitol is not simply a "freeze-drying aid", but a key component that simultaneously guarantees PDRN activity, needle body integrity, and mechanical properties, and its function cannot be replaced by other components, which is a necessary condition to achieve the core performance of the product.

[0184] 3.4 Verification of detection method The accuracy, repeatability and applicability of the detection method are the core of ensuring the controllability of product quality. The following is for the determination of PDRN content (HPLC method) and the determination of L-carnosine cumulative penetration (Franz diffusion cell method), supplemented with key operation details, condition selection basis and complete method validation to ensure reliable and reproducible test results.

[0185] 3.4.1 PDRN content determination (HPLC method) Method principle PDRN is a DNA fragment from salmon sperm (a negatively charged polar molecule). An amino column is selected to 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 a detection wavelength of 260nm (characteristic absorption peak of nucleic acid substances). Finally, the content is calculated by external standard method.

[0186] Reagent and instrument preparation (1) Reagent preparation 0.1M PBS buffer (pH 7.4): accurately weigh potassium dihydrogen phosphate (KH2PO4) 0.68g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 1.42g, dissolve in 1000mL ultrapure water (resistivity ≥18.2MΩ·cm), adjust pH to 7.4±0.02 with 0.1M sodium hydroxide (NaOH) or 0.1M hydrochloric acid (HCl), filter through a 0.22μm water filter membrane, ultrasonic degassing for 15min (power 300W) and store for use; PDRN standard stock solution (1000μg / mL): take PDRN standard (purity ≥99%, molecular weight 200kDa) 10mg, accurately weigh, dissolve in 0.1M PBS buffer and dilute to 10mL, store at 4℃ in the dark, valid for 7 days; PDRN standard working solution: dilute the stock solution with 0.1M PBS buffer to 10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL, 200μg / mL series of working solutions, prepare and use immediately.

[0187] (2) Instrument and consumable specifications High performance liquid chromatograph: equipped with ultraviolet detector (model Agilent1260); Chromatographic column: amino-bonded silica gel column (250mm×4.6mm, 5μm, brand: Waters XBridge Amide); Ultrasonic instrument: power 200W, frequency 20kHz (model: SCIENTZ-IID); Filter membrane: 0.22μm water-based polyether sulfone filter membrane (diameter 25mm).

[0188] Sample pretreatment Sample sampling: Take one piece of enhanced transdermal delivery cosmetic patch, cut it into fine particles of 1 mm x 1 mm with a sterile scissors (ensure sufficient dissolution), and accurately weigh the mass (recorded as m, unit: g); Extraction and dissolution: Transfer the fine particles to a 50 mL centrifuge tube, add 10 mL of 0.1 M PBS buffer (pH 7.4), and place it in an ultrasonic instrument. Set the parameters: power 200 W, frequency 20 kHz, work 3 s / intermittent 5 s, total ultrasonic time 30 min (preliminary experiment verification: 20 min dissolution rate is only 85%, 30 min dissolution rate reaches 99.5%, so 30 min is determined); Purification and filtration: After ultrasonic, centrifuge (5000 rpm, 10 min), take the supernatant and filter it through a 0.22 μm water filter membrane, discard the first filtrate 1 mL, and take the subsequent filtrate as the test solution; Blank control: Take blank enhanced transdermal delivery cosmetic patch without PDRN (other components are consistent with the sample), prepare the blank control solution according to the above steps, and use it to exclude matrix interference.

[0189] Chromatographic conditions Chromatographic parameters Set value Screening basis Chromatographic column Amino column (250mm × 4.6mm, 5μm) Comparative C18 column (PDRN retention is weak, peak shape is tailing), amino column has strong retention for polar PDRN, peak shape is symmetrical (tailing factor 1.05-1.15) Mobile phase 0.1M PBS (pH 7.4) Comparative methanol-PBS mixed phase (separation degree ≤1.2), pure PBS buffer can completely separate PDRN and impurities (separation degree ≥1.5) Flow rate 1.0mL / min Flow rate 0.8mL / min, retention time is too long (15min), 1.2mL / min, peak shape is broadened, 1.0mL / min takes into account efficiency and peak shape Column temperature 30℃ Retention time fluctuation ± 0.5min at room temperature (25℃), constant temperature at 30℃ can make the retention time RSD ≤0.3% Detection wavelength 260nm Scan 200-400nm ultraviolet spectrum, PDRN has maximum absorption at 260nm, and the blank matrix has no interference Injection volume 10 μL 5 μL response value is low, 20 μL is easy to overload, 10 μL peak area and concentration linear relationship is best Methodology verification (1) System suitability test: Take 100 μg / mL PDRN standard working solution for injection, record the chromatogram: theoretical plate number (N): ≥3000 (measured 3850); separation degree (R): PDRN peak and adjacent impurity peak separation degree ≥1.5 (measured 2.2); tailing factor (T): 0.95-1.15 (measured 1.08); retention time (tR): stable at 8.5±0.2 min (measured 8.45 min), meeting the system suitability requirements.

[0190] (2) Linearity: Take 10-200 μg / mL series of standard working solution for injection, take PDRN concentration (C, μg / mL) as the abscissa and peak area (A) as the ordinate, and perform linear regression: regression equation: A=12563C+452 (n=5); correlation coefficient (R²): 0.9998 (R²>0.999, meeting the linear requirements); linear range: 10-200 μg / mL, covering the expected concentration of PDRN in the sample (8-12 μg / mL, diluted to fall within the linear range).

[0191] (3) Precision repeatability: take the same test solution (PDRN concentration 9.8 μg / mL), continuously sample 6 times, measure the peak area: peak area RSD = 0.8% (RSD < 2%, good repeatability); intermediate precision: by 2 experimenters in different days (Day 1, Day 2), using different instruments (Agilent 1260, Waters e2695) to measure the same sample: twice measurement results RSD = 1.0% (RSD < 2%, good intermediate precision).

[0192] (4) Accuracy (recovery test) using "spiked recovery method", take the sample with known content (PDRN content 9.8%), respectively, add low, medium and high three levels of PDRN standard (spiked amount is 80%, 100%, 120% of the amount of PDRN in the sample), prepare and measure according to the sample pretreatment steps: PDRN amount (μg) in the spiked level sample, PDRN amount (μg) added, total PDRN amount (μg) measured, 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).

[0193] (5) Stability of test solution: take the test solution, respectively, at 0h, 4h, 8h, 12h, 24h, sample, measure the peak area: 24h peak area RSD = 1.1% (RSD < 2%, the test solution is stable at 4°C in the dark for 24h); standard solution stability: take 100 μg / mL standard working solution, store at 4°C in the dark for 7 days, measure the content: content RSD = 1.3% within 7 days (RSD < 2%, the standard solution is stable within 7 days).

[0194] (6) Specificity Take the blank control solution, test solution, PDRN standard solution respectively: the blank control solution has no chromatographic peak at the PDRN retention time (no matrix interference); the PDRN peak in the test solution is completely separated from the impurity peak (separation degree 2.2), with good specificity. 4.2.1 Method principle 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 (keratin layer structure close to human body) is used as the barrier, 37°C constant temperature simulates the physiological temperature of the skin, magnetic stirring ensures uniform concentration of the receiving liquid; the content of L-carnosine in the receiving liquid at different times is measured by HPLC, the 24h cumulative permeation amount is calculated, and the penetration enhancing effect is evaluated.

[0195] Reagents and instrument preparation (1) Reagent preparation 0.01M PBS buffer (pH 7.4): weigh potassium dihydrogen phosphate 0.068 g, dipotassium hydrogen phosphate 0.142 g, dissolve in 1000 mL ultrapure water, pH calibrated to 7.4 ± 0.02, 0.22 μm filter membrane filtration, degassed for standby (receiving solution); L-carnosine standard stock solution (1000 μg / mL): take L-carnosine standard (purity ≥ 99%) 10 mg, dissolve in 0.01M PBS buffer to 10 mL, store at 4°C in the dark; L-carnosine standard working solution: gradient dilution to 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, prepared and used immediately.

[0196] (2) (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°C (model: HH-S4); magnetic stirrer: speed accuracy ± 10 rpm (model: IKARCTbasic); skin barrier: fresh pig skin (taken from the abdomen of pigs, thickness 500 ± 50 μm, stored at 4°C after depilation and disinfection, used within 24 hours); HPLC instrument: equipped with C18 column (model: Agilent ZORBAX SB-C18, 250 mm x 4.6 mm, 5 μm).

[0197] 4.2.3 Sample pretreatment and diffusion cell setup (1) Pig skin treatment (key step, avoid barrier difference) Depilation: use an electric shaver to remove the hair on the surface of the pig skin, avoiding damage to the stratum corneum; washing and disinfection: rinse with normal saline (0.9% NaCl) 3 times to remove surface dirt; wipe the surface with 0.1% new jie'er solution, disinfect for 10 min, then rinse with normal saline 3 times to remove the disinfectant residue; thickness control: use a skin thickness gauge (accuracy 0.01 mm) to randomly measure 5 points to ensure the thickness is 500 ± 50 μm, and gently thin the over-thick part with a scalpel (only thin the subcutaneous tissue, leaving the stratum corneum intact).

[0198] (2) Diffusion cell assembly and receiving chamber filling: 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 stratum corneum with the treated surface facing up between the donor and receiving chambers using a clamp to ensure that the skin is tightly sealed against the cell wall (to prevent leakage of the receiving solution); sample addition: Take one piece of the enhanced transdermal delivery cosmetic patch, remove the backing, and attach the needle layer to the surface of the porcine stratum corneum with the needle layer facing down, and gently press for 30 seconds (pressure of about 20 N / cm2, simulating the pressing force during human use), to ensure that the needles penetrate the skin; seal the donor chamber with parafilm to prevent water evaporation.

[0199] Sampling and detection Sampling time points: Set 1 h, 4 h, 8 h, 12 h, and 24 h as the sampling time points (preliminary experiments showed that L-carnosine began to penetrate within 1 h, the penetration rate tended to be stable after 8 h, and the penetration reached equilibrium at 24 h, so these time points were selected); Sampling operation: Each time, 1 mL of the receiving solution was precisely pipetted (denoted as Vsample), and 1 mL of fresh 0.01 M PBS buffer preheated at 37°C was immediately added (denoted as Vadd), to ensure that the volume of the receiving chamber was constant; HPLC detection: After the sample solution was filtered through a 0.22 μm water filter, the L-carnosine content was determined according to the following chromatographic conditions: chromatographic column: Agilent ZORBAX SB-C18 (250 mm x 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°C; detection wavelength: 220 nm; injection volume: 10 μL.

[0200] Cumulative penetration amount calculation Since fresh receiving solution was added after each sampling, the cumulative penetration amount needed to be corrected, and the formula was as follows:

[0201] Q t : cumulative penetration amount at t time point (μg / cm2); C t : L-carnosine concentration in the receiving solution at t time point (μg / mL); V0: initial volume of the receiving chamber (6.5 mL); C i : L-carnosine concentration in the receiving solution at t-1 time point (μg / mL); V s : volume of each sampling (1 mL); A: effective transdermal area (1.77 cm2).

[0202] Methodology validation (1) System suitability test: 50 pg / mL L-carnosine standard solution was injected: theoretical plate number ≥ 3000 (4200 actually measured); separation degree ≥ 1.5 (2.5 actually measured); tailing factor 0.95-1.15 (1.05 actually measured); retention time was stable at 6.8±0.2 min (6.78 min actually measured), and the system suitability was qualified.

[0203] (2) Linearity: 5-100 pg / mL series of standard solution was injected, and 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 pg / mL).

[0204] (3) Precision and repeatability: the same receiving solution sample (L-carnosine concentration 7.5 pg / mL) was continuously injected for 6 times, and the peak area RSD was 1.1% (RSD<2%); intermediate precision: different experimenters and different instruments were used for determination, and the result RSD was 1.3% (RSD<2%).

[0205] (4) Accuracy (recovery test): low, medium and high levels of L-carnosine standard (5 pg / mL, 10 pg / mL, 20 pg / mL) were added to the blank receiving solution, and the detection steps were determined: Spiked level Amount (μg) Measured amount (μg) Recovery rate (%) Average recovery rate (%) RSD (%) Low 5.0 4.89 97.8 99.9 1.5 Medium 10.0 9.98 99.8 / / High 20.0 20.46 102.3 / / The average recovery rate is 99.9%, and the RSD is 1.5% (which meets the requirements of 95%-105%, and the accuracy is good). / / / / / (5) Skin integrity verification After the experiment, the pig skin barrier was smeared with 0.1% methylene blue solution, and after 10 min, the observation was made: No blue penetration on the skin (proving that the skin barrier is complete and there is no leakage); Small blue spots were visible at the microneedle puncture site (proving that the needle body effectively penetrated and the transdermal path was normal).

[0206] Conclusion PDRN content determination (HPLC method): good linearity in the range of 10-200 pg / mL (R²=0.9998), precision (RSD≤1.0%), accuracy (recovery rate 99.8%), and stability (24 h RSD=1.1%) all meet the requirements, and the content of PDRN in the enhanced transdermal delivery cosmetic patch can be accurately determined; L-carnosine cumulative penetration determination (Franz diffusion cell method): linear range 5-100 pg / mL (R²=0.9997), precision and accuracy meet the requirements, and the pig skin barrier integrity is controllable, which can reliably evaluate the transdermal penetration effect of L-carnosine; The operation details of the two detection methods are clear and reproducible, which can provide scientific basis for product quality control and performance evaluation.

[0207] The present application focuses on the core goal of "high-efficiency active PDRN delivery + skin repair function enhancement". Through component screening, structure optimization and process innovation, the enhanced transdermal delivery cosmetic patch has achieved a comprehensive breakthrough in effectiveness, stability and practicality, as follows: 1. Component synergy, guaranteeing core function: The optimal component combination is selected - salmon testis-derived PDRN with a molecular weight of 150-250 kDa as the core active ingredient, SBMA-HA zwitterionic carrier with a grafting rate of 28% to improve its stability (active retention rate of 88% after 60°C acceleration), DPPC and cholesterol 7:3 ratio liposome to enhance L-carnosine penetration (24h transdermal amount of 85μg / cm²), and trehalose and mannitol 2:1 complex low-temperature protective agent to avoid needle body fragmentation (fragmentation rate of 0%), and then 0.2% tranexamic acid + 3% glutathione + 6% asiaticoside to form an "anti-inflammatory-whitening-barrier repair" closed loop, with an 8-week post-acne hyperpigmentation improvement rate of 92%.

[0208] 2. Structure optimization, improving user experience: The design of needle body height 750μm, needle body layer and backing layer thickness ratio 4:1, combined with 35μm height backing layer micron-level convex, prolongs the fitting time to 6.8h, reduces the shedding rate to 8%, and improves the drug utilization rate to 92%, while the single needle bearing capacity is 0.38N and the 37℃ physiological saline dissolution time is 2.5min, meeting the skin penetration and component rapid release requirements.

[0209] 3. Process controllable, ensuring quality stability: Through the standardized process of "SBMA-HA copolymerization (50°C reaction for 7h) - liposome film hydration (35°C rotary evaporation) - gradient freeze-drying (-40°C pre-freezing followed by stepwise warming) - backing layer coating and embossing", the parameters at each link are controllable, ensuring consistent performance between product batches, and the solvent residue is ≤0.1%, meeting safety requirements.

[0210] 4. Performance breakthrough, highlighting creativity: Compared with traditional solutions, the present application replaces unmodified HA with zwitterionic carriers, increasing PDRN active retention rate from 62% to 88%; encapsulates free L-carnosine with liposomes, increasing transdermal amount from 38μg / cm² to 85% and reducing irritation to 1.1 points; and adding a low-temperature protective agent, the single needle bearing capacity is increased from 0.22N to 0.38N, solving the pain points of traditional microneedles "low activity, poor transdermal penetration, and easy fragmentation".

[0211] All experimental data are based on 3 parallel tests with an error range of ≤2%, and the operation details of each step are clear, which can be completely reproduced by those skilled in the art, meeting the "full disclosure" requirement of patents, and providing a new efficient and safe solution for post-acne hyperpigmentation treatment and skin barrier repair.

[0212] The above detailed description of the embodiments of the present application is only a preferred embodiment of the present application, and should not be considered as limiting the scope of the present application. Any equivalent changes and improvements made according to the scope of the present application should still belong to the patent scope of the present application.

Claims

1. An enhanced transdermal delivery cosmetic patch containing highly active PDRN (DNA sodium) characterized in that: Composed of a needle body layer and a backing layer, wherein: The needle body layer contains the following components by mass percentage: PDRN with a molecular weight of 50-300 kDa, content of 5%-15%; Zwitterionic polymer carrier copolymerized from methacryloyl ethyl sulfobetaine and hyaluronic acid, content of 30%-50%; L-carnosine encapsulated by liposomes with a particle size of no more than 100 nm as a nanoscale penetration enhancer, content of 10%-20%; Cryoprotectant composed of trehalose and mannitol in a mass ratio of 1:1-3:1, content of 15%-25%; The backing layer is a block copolymer PCL-PEG-PCL of degradable polycaprolactone with a molecular weight of 8000-12000 Da and polyethylene glycol.

2. The enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA) according to claim 1, wherein: The molecular weight of the PDRN is 150-250 kDa, and the loading amount in the needle body layer is 8%-12%; The grafting rate of the sulfobetaine monomer of the zwitterionic polymer carrier is 20%-35%; The encapsulation rate of the L-carnosine encapsulated by liposomes is no less than 95%.

3. The enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA) according to claim 1, wherein: The needle body height is 600-900 μm, and the single needle bearing capacity is no less than 0.35 N; The complete dissolution time in physiological saline at 35-37 ℃ is no more than 3 min; After 48-72 h of accelerated test at 50 ℃-60 ℃, the PDRN activity retention rate is no less than 85%.

4. The enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA) according to claim 1, wherein: The needle body layer further contains 0.1%-0.3% of tranexamic acid and 2%-4% of glutathione by mass percentage; The backing layer is added with 5%-7% of centella asiatica extract by mass percentage.

5. The enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA) according to claim 1, wherein: The PDRN is a DNA fragment from salmon sperm, with a length of 50-500 bp; The liposome contains dipalmitoyl phosphatidylcholine DPPC and cholesterol, with a molar ratio of 6.5:3.5-7.5:2.

5.

6. The enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA) according to claim 1, wherein: The thickness ratio of the needle body layer to the backing layer is 3:1-5:1; The surface of the backing layer has micron-level protruding structures with a height of 20-50 μm.

7. The method of preparation of the enhanced transdermal delivery cosmetic patch containing PDRN (DNA sodium) with high activity according to any one of claims 1-6, characterized in that, The steps include: (1) Synthesis of zwitterionic polymer carrier: Dissolve methacryloyl ethyl sulfobetaine and hyaluronic acid with a molecular weight of 50-100 kDa in PBS buffer at a molar ratio of 1:3-1:5, add 0.08%-0.12% ammonium persulfate initiator by mass percentage, and react at 48-52 ℃ for 6-8 h, then purify by dialysis; (2) Preparation of nanoscale penetration enhancer: L-carnosine was mixed with dipalmitoyl phosphatidylcholine DPPC at a mass ratio of 1:2-1:4, and liposomes were prepared by the thin film hydration method, and sterilized by 0.20-0.24 μm filter membrane; (3) Preparation of needle body solution: PDRN, zwitterionic polymer carrier, nano penetration enhancer, and cryoprotectant were dissolved in deionized water at 4±2℃, and a homogeneous solution was formed by ultrasonic treatment at a power of 180-220 W for 4-6 min; (4) Cryogenic forming: The needle body solution was injected into the PDMS mold, pre-frozen at-45 to-35℃ for 1.5-2.5 h, and then freeze-dried at a vacuum degree of 0.08-0.12 mPa and a temperature of-30 to-20℃ for 22-26 h; (5) Backing layer compounding: A PCL-PEG-PCL chloroform solution with a mass-volume ratio of 18-22% was coated on the back of the needle body layer, and after the solvent was volatilized at 23-27℃, it was peeled off and formed.

8. The preparation method of the enhanced transdermal delivery cosmetic patch containing high-efficiency active PDRN (sodium DNA) according to claim 7, characterized in that: The total solid content of the solution in step (3) is 35%-45%; The freeze-drying program includes: From-45 to-35℃, the temperature is raised to-25 to-15℃ at a rate of 1.8-2.2℃ / h, and the temperature is kept for 3-4 h; From-25 to-15℃, the temperature is raised to-5 to 5℃ at a rate of 0.8-1.2℃ / h, and the temperature is kept for 4-5 h; From-5 to 5℃, the temperature is raised to 23-27℃ at a rate of 0.4-0.6℃ / h, and the temperature is kept for 2-3 h.

9. Use of the enhanced transdermal delivery cosmetic patch according to claim 1 for the preparation of a skin repair medical device, characterized in that: For the treatment of post-acne pigmentation and for skin barrier repair.

10. A method of detecting the presence of the enhanced transdermal delivery cosmetic patch of claims 1-6, wherein Comprises: The PDRN content is determined by high-performance liquid chromatography: the chromatographic column is a C18 column, the mobile phase is 0.08-0.12 M, pH 7.2-7.6 phosphate buffer, the flow rate is 0.8-1.2 mL / min, the detection wavelength is 260 nm, and the injection volume is 10-20 μL; The cumulative penetration amount of L-carnosine is determined by Franz diffusion cell: the effective penetration area of the diffusion cell is 1.5-2.5 cm², the receiving liquid is PBS buffer at pH 7.2-7.4, and the 24 h penetration amount is not less than 75-85 μg / cm².

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