Cyclization transdermal peptide-pdrn supramolecular complex and preparation method and application thereof

Abstract

The application discloses a kind of cyclization transdermal peptide-PDRN supramolecular complex and its preparation method and application.Cyclization transdermal peptide-PDRN supramolecular complex includes cyclization transdermal peptide and PDRN;Wherein, PDRN is wrapped in the inside of cyclization transdermal peptide by electrostatic adsorption;Cyclization transdermal peptide includes cation amino acid residue and at least two cysteine residues, and disulfide bond is formed between cysteine residue and constitutes cyclization structure.The cyclization structure of complex can be wrapped in the inside of cyclic peptide PDRN, and disulfide bond can be broken under intracellular reducing environment, realize the intelligent release of PDRN.Therefore, the cyclization transdermal peptide-PDRN supramolecular complex of the application has triple functions of efficient transdermal delivery, structural stable protection and intracellular intelligent release, so as to have excellent skin repair effect.

Description

Technical Field

[0001] This invention belongs to the field of bioactive molecule delivery technology, specifically relating to a complex of a cyclized transdermal peptide and polydeoxyribonucleotide (PDRN), its preparation method, and the application of pharmaceutical or cosmetic compositions containing the complex in skin repair. Background Technology

[0002] Transdermal peptides are a class of short peptides that help large molecular drugs such as proteins and nucleic acids cross the skin barrier. They can efficiently carry large molecular drugs, achieving non-damaging and reversible transdermal delivery, and significantly improving drug efficacy. They have shown great promise in the field of drug delivery, especially in the transdermal delivery of large molecular drugs.

[0003] Polydeoxyribonucleotides (PDRNs) possess biological functions such as activating adenosine A2A receptors, promoting fibroblast proliferation, and accelerating collagen synthesis. Studies have shown that PDRNs can effectively alleviate wrinkles caused by skin aging by stimulating collagen and elastin fiber proliferation, help improve pigmentation, accelerate wound healing, and alleviate skin problems such as acne scars and marks, making them of significant application value in the field of skin repair. However, PDRNs face key technical challenges in practical applications, including difficulties in transdermal absorption, poor stability, and uncontrollable release, which remain to be solved. Summary of the Invention

[0004] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention provides a cyclized transdermal peptide-PDRN supramolecular complex, its preparation method, and its application in skin repair. The cyclized transdermal peptide-PDRN supramolecular complex of this invention utilizes a 13-amino acid transdermal peptide sequence, which binds to PDRN via electrostatic adsorption and then forms a cyclized structure through disulfide bonds, encapsulating the PDRN within. The disulfide bonds in the cyclized structure break under intracellular reducing conditions, achieving intelligent release of PDRN. Therefore, the complex of this invention can significantly improve transdermal efficiency, enhance serum stability, and achieve intelligent intracellular release, thus possessing excellent skin repair effects. It can be used to prepare cosmetics, pharmaceuticals, or skin repair products, showing promising application prospects.

[0005] It should be noted that this invention was completed by the applicant based on the following work: Based on the 11-amino acid transdermal peptide TD-1 (SEQ ID NO:1), the applicant designed six 13- or 14-peptide sequences by adding 2-3 cationic amino acids near the C-terminus. Surprisingly, through experimental screening, the supramolecular complex prepared using the SEQ ID NO:3 sequence exhibited the best overall performance, demonstrating unexpected technical effects. Extensive experiments by the applicant revealed that the supramolecular complex prepared using SEQ ID NO:3 showed significantly improved transdermal efficiency, serum stability, and intracellular release rate compared to free PDRN, demonstrating excellent skin repair effects.

[0006] Therefore, in a first aspect, the present invention provides a cyclized transdermal peptide-PDRN supramolecular complex, comprising: a cyclized transdermal peptide and PDRN; wherein the PDRN is encapsulated within the cyclized transdermal peptide by electrostatic adsorption; the cyclized transdermal peptide comprises cationic amino acid residues and at least two cysteine ​​residues, wherein disulfide bonds are formed between the cysteine ​​residues to constitute a cyclized structure; the amino acid sequence of the cyclized transdermal peptide is shown in SEQ ID NO: 3. Thus, the supramolecular complex of the present invention possesses a triple function of highly efficient transdermal penetration, in vitro stable protection, and intelligent intracellular release, thereby exhibiting excellent skin repair effects.

[0007] In some embodiments, the molecular weight of the PDRN is 50~3000 kDa.

[0008] In some embodiments, the cyclized transdermal peptide-PDRN supramolecular complex satisfies at least one of the following: (1) the particle size of the cyclized transdermal peptide-PDRN supramolecular complex is 50~200 nm; (2) the Zeta potential of the cyclized transdermal peptide-PDRN supramolecular complex is +15~+35 mV; (3) the encapsulation efficiency of the cyclized transdermal peptide-PDRN supramolecular complex for PDRN is 60~90%; (4) the disulfide bond cyclization efficiency of the cyclized transdermal peptide-PDRN supramolecular complex is 85~98%.

[0009] In a second aspect, the present invention provides a method for preparing the cyclized transdermal peptide-PDRN supramolecular complex described in the first aspect. The method includes the following steps: S1: mixing a linear transdermal peptide with PDRN to obtain a peptide-DNA complex; S2: subjecting the peptide-DNA complex to a cyclization reaction to form disulfide bonds between cysteine ​​residues, thereby obtaining the cyclized transdermal peptide-PDRN supramolecular complex. Thus, the preparation method of the present invention not only achieves efficient encapsulation and protection of PDRN but also endows it with high transdermal efficiency and intelligent intracellular release capability, thereby enabling its application in skin repair.

[0010] In some embodiments, the mass ratio of the linear transdermal peptide to the PDRN is 1:1 to 1:1000; the pH of the mixing treatment is 6.5 to 8.0, the temperature is 4 to 37°C, and the time is 0.5 to 4 h; the cyclization reaction method includes one or more of air oxidation, dimethyl sulfoxide oxidation, and glutathione redox reaction; in some embodiments, the pH of the cyclization reaction is 7.0 to 8.5, the temperature is 4 to 25°C, and the reaction time is 2 to 24 h; in some embodiments, the amino acid sequence of the linear transdermal peptide is as shown in SEQ ID NO: 3.

[0011] In a third aspect, the present invention provides a pharmaceutical composition comprising the cyclized transdermal peptide-PDRN supramolecular complex described in the first aspect. Thus, the pharmaceutical composition of the present invention can effectively promote skin repair, collagen synthesis, and wound healing.

[0012] In some embodiments, the concentration of the cyclized transdermal peptide-PDRN supramolecular complex in the pharmaceutical composition is 0.01-5% w / v; the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0013] In a fourth aspect, the present invention proposes the application of the cyclized transdermal peptide-PDRN supramolecular complex described in the first aspect and the pharmaceutical composition described in the third aspect in the preparation of skin repair drugs; the skin repair includes one or more of the following: post-cosmetic surgery skin repair, sensitive skin repair, photodamaged skin repair, wound healing promotion, skin barrier reconstruction, and anti-skin aging. Therefore, the cyclized transdermal peptide-PDRN supramolecular complex of the present invention, when applied to skin repair drugs, will have excellent skin repair effects and can be widely used in skin repair.

[0014] In a fifth aspect, the present invention provides a cosmetic composition comprising: the cyclized transdermal peptide-PDRN supramolecular complex described in the first aspect. Therefore, the cosmetic composition of the present invention will possess excellent skin repair and protection properties.

[0015] In a sixth aspect, the present invention proposes the application of the cyclized transdermal peptide-PDRN supramolecular complex described in the first aspect in the preparation of cosmetics, pharmaceuticals, or products for skin repair. Therefore, the complex of the present invention can be widely used in skin repair products and exhibits excellent skin repair efficacy.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a comparison of the transdermal efficiency of different polypeptide sequences in Example 1 of the present invention; Figure 2 This is the transdermal efficiency change curve of Example 3 of the present invention; Figure 3 This is the reduction-responsive release curve of Example 4 of the present invention; Figure 4 This is a comparison of serum stability in Example 5 of the present invention; Figure 5 This is a comparison of the cell proliferation effect in Example 6 of the present invention; Figure 6 This is a human efficacy evaluation diagram from Embodiment 7 of the present invention; Figure 7 This is a graph showing the accelerated stability test results of Embodiment 8 of the present invention; Figure 8 This is a graph showing the cytotoxicity evaluation results of Example 9 of the present invention. Detailed Implementation

[0018] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0019] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0022] In this article, the term "transdermal peptide" refers to a class of short peptides that can temporarily and reversibly open the skin barrier, thereby facilitating the transdermal absorption of macromolecular drugs.

[0023] In this article, the term "polydeoxyribonucleic acid (PDRN)" refers to a low molecular weight DNA fragment extracted from fish reproductive cells that possesses various pharmacological activities such as promoting tissue repair and anti-inflammation, and has attracted much attention in the medical and cosmetic fields.

[0024] In a first aspect of the present invention, the present invention provides a cyclized transdermal peptide-PDRN supramolecular complex comprising: a cyclized transdermal peptide and PDRN; wherein, PDRN is encapsulated within the cyclized transdermal peptide by electrostatic adsorption; the cyclized transdermal peptide comprises cationic amino acid residues and at least two cysteine ​​residues, wherein disulfide bonds are formed between the cysteine ​​residues to constitute a cyclized structure; the amino acid sequence of the cyclized transdermal peptide is shown in SEQ ID NO: 3.

[0025] The cyclic transdermal peptide of this invention contains cationic amino acid residues that carry a strong positive charge at physiological pH, while PDRN, as a polynucleotide, has a strong negative charge on its phosphate backbone. The two attract each other through positive and negative charges, encapsulating the negatively charged PDRN within the peptide to form a stable supramolecular complex. The cysteine ​​residues in the transdermal peptide sequence form disulfide bonds that cross-link to create a cyclic structure that encapsulates PDRN. This cyclic structure acts like a "cage," more firmly and completely encapsulating PDRN within the cyclic peptide, preventing premature dissociation or degradation before it crosses the skin barrier, further endowing the complex with mechanical stability and resistance to enzymatic degradation. The disulfide bonds can break under intracellular reducing conditions, releasing PDRN to exert its function, ensuring that PDRN reaches the target cells in an intact and highly active form.

[0026] The cyclized transdermal peptide amino acid sequence according to embodiments of the present invention is: Alanyl-Cys-Arg-Ser-Ser-Ser-Pro-Ser-Ser-Lys-His-Cys-Lysyl-Histyl-Cys-Gly-Arg (ACRSSSPSKHCGR). Its moderate length of 13 amino acids is sufficient to encapsulate PDRN without hindering transdermal absorption due to excessive molecular weight. Furthermore, the positions of the two cysteine ​​residues (Cys) are rationally designed, ensuring a sufficiently tight cyclization cage to encapsulate PDRN and form a stable "peptide cage," which is key to improving cyclization efficiency and further enhancing the structural stability of the supramolecular complex. Therefore, the supramolecular complex of the present invention possesses a triple function of highly efficient transdermal absorption, in vitro stable protection, and intelligent intracellular release, resulting in excellent skin repair effects.

[0027] In some embodiments of the present invention, the molecular weight of PDRN is 50-3000 kDa. According to embodiments of the present invention, a molecular weight of PDRN of 50-3000 kDa is beneficial for improving its encapsulation stability, enhancing the structural stability of the supramolecular complex, and simultaneously achieving a synergistic effect of rapid anti-inflammatory and long-lasting regeneration, providing the complex with multi-layered skin repair effects. Therefore, the supramolecular complex of the present invention exhibits excellent skin repair effects.

[0028] In some embodiments of the present invention, the cyclized transdermal peptide-PDRN supramolecular complex satisfies at least one of the following: (1) The particle size of the cyclized transdermal peptide-PDRN supramolecular complex is 50~200 nm; According to embodiments of the present invention, when the particle size of the complex is 50~200 nm, it is beneficial for the complex to efficiently carry PDRN across the skin barrier, thereby further improving the transdermal efficiency. (2) The Zeta potential of the cyclized transdermal peptide-PDRN supramolecular complex is +15~+35 mV; According to embodiments of the present invention, a Zeta potential of +15~+35 mV is a basic guarantee for ensuring the colloidal stability of the complex, enabling it to be stored stably for a long time in the form of solutions, gels, etc., thereby further improving the stability of the supramolecular complex. (3) The encapsulation rate of PDRN in the cyclized transdermal peptide-PDRN supramolecular complex is 60-90%; the encapsulation rate is the proportion of PDRN successfully encapsulated inside the cyclized transdermal peptide to the total amount of feed. According to the embodiments of the present invention, an encapsulation rate of 60%-90% ensures that each unit of peptide can carry enough PDRN, which can ensure that PDRN is completely encapsulated inside the cyclic peptide to form a dense and stable "peptide cage" structure, while avoiding the surge in process costs caused by excessively high encapsulation rates. Thus, the transdermal efficiency and stability of the supramolecular complex can be further improved. (4) The disulfide bond cyclization efficiency of the cyclized transdermal peptide-PDRN supramolecular complex is 85-98%; according to the embodiments of the present invention, a disulfide bond cyclization efficiency of 85%-98% is beneficial for the supramolecular complex to achieve a highly uniform structure, high encapsulation protection, and highly intelligent release.

[0029] In a second aspect of the present invention, the present invention provides a method for preparing a cyclized transdermal peptide-PDRN supramolecular complex, the method comprising the following steps: S1: mixing a linear transdermal peptide with PDRN to obtain a peptide-DNA complex; S2: subjecting the peptide-DNA complex to a cyclization reaction to form disulfide bonds between cysteine ​​residues to obtain a cyclized transdermal peptide-PDRN supramolecular complex.

[0030] According to the preparation method of this invention, a flexible linear peptide is first mixed with PDRN to achieve sufficient electrostatic binding, molecular recognition, and self-assembly, forming a tightly structured peptide-DNA complex. Subsequently, cyclization is performed to fix the binding state. This yields a supramolecular complex with uniform structure, stable properties, and complete function. Therefore, the preparation method of this invention not only achieves efficient encapsulation and protection of PDRN but also endows it with high transdermal efficiency and intelligent intracellular release capability, thereby enabling its application in skin repair.

[0031] In some embodiments of the present invention, the mass ratio of linear transdermal peptide to PDRN is 1:1 to 1:1000; the pH of the mixing treatment is 6.5 to 8.0, the temperature is 4 to 37°C, and the time is 0.5 to 4 h; the cyclization reaction method includes one or more of air oxidation, dimethyl sulfoxide oxidation, and glutathione redox methods. This allows the linear transdermal peptide to form a stable complex structure with PDRN; the pH of the cyclization reaction is 7.0 to 8.5, the temperature is 4 to 25°C, and the reaction time is 2 to 24 h; the amino acid sequence of the linear transdermal peptide is shown in SEQ ID NO: 3.

[0032] According to embodiments of the present invention, by adjusting the mass ratio of the linear transdermal peptide to the PDRN supramolecular complex, the transdermal efficiency, stability, and intracellular targeted release of the supramolecular complex are further improved, thereby enhancing its skin repair effect. Simultaneously, the applicant has obtained the aforementioned optimal experimental conditions through extensive experimental verification. This allows the linear transdermal peptide to form a stable cyclic structure, encapsulating PDRN to form a stable supramolecular complex, further improving its transdermal efficiency and structural stability.

[0033] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising: the cyclized transdermal peptide-PDRN supramolecular complex of the first aspect. The pharmaceutical composition according to the embodiments of the present invention can effectively activate fibroblasts and promote skin repair, collagen synthesis, and wound healing.

[0034] In some embodiments of the present invention, the concentration of the cyclized transdermal peptide-PDRN supramolecular complex in the pharmaceutical composition is 0.01~5% w / v; the pharmaceutical composition further includes a pharmaceutically acceptable carrier. The pharmaceutical compositions according to embodiments of the present invention further enhance their skin repair effects by adjusting the mass concentration of the cyclized transdermal peptide-PDRN supramolecular complex.

[0035] In some embodiments of the present invention, the dosage form of the pharmaceutical composition includes one or more of the following: solution, gel, cream, mask, serum, spray, and patch.

[0036] In a fourth aspect of the present invention, the present invention proposes the application of the cyclized transdermal peptide-PDRN supramolecular complex described in the first aspect and the pharmaceutical composition described in the third aspect in the preparation of skin repair drugs; skin repair includes one or more of the following: post-cosmetic surgery skin repair, sensitive skin repair, photodamaged skin repair, wound healing promotion, skin barrier reconstruction, and anti-skin aging.

[0037] Those skilled in the art will understand that the features and advantages described above for the cyclized transdermal peptide-PDRN supramolecular complex and pharmaceutical composition also apply to this use, and will not be repeated here.

[0038] Those skilled in the art will understand that the features and advantages described above for the cyclized transdermal peptide-PDRN supramolecular complex and pharmaceutical composition also apply to this use, and will not be repeated here.

[0039] In a fifth aspect of the present invention, a cosmetic composition is provided, comprising: a cyclized transdermal peptide-PDRN supramolecular complex as described in the first aspect. Therefore, the cosmetic composition of the present invention exhibits excellent skin repair effects and is safe for human use.

[0040] In a sixth aspect of the present invention, the present invention proposes the application of the cyclized transdermal peptide-PDRN supramolecular complex of the first aspect in the preparation of cosmetics, pharmaceuticals or products for skin repair.

[0041] Those skilled in the art will understand that the features and advantages described above for the cyclized transdermal peptide-PDRN supramolecular complex also apply to this application, and will not be repeated here.

[0042] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0043] Example 1: Design and Screening of Transdermal Peptides 1.1 Experimental Methods (1) Sequence design: Based on TD-1 (SEQ ID NO:1, 11 amino acids), two cationic amino acids were added near the C-terminus to design the following 6 transdermal peptide sequences: • SEQ ID NO:1 (TD-1): Ala-Cys-Ser-Ser-Ser-Pro-Ser-Lys-His-Cys-Gly (control) • SEQ ID NO:2 (TDP-1): Ala-Cys-Lys-Ser-Ser-Ser-Pro-Ser-Lys-His-Cys-Gly-Lys • SEQ ID NO:3 (TDP-2):Ala-Cys-Arg-Ser-Ser-Ser-Pro-Ser-Lys-His-Cys-Gly-Arg • SEQ ID NO:4 (TDP-3):Ala-Cys-Arg-Lys-Ser-Ser-Ser-Pro-Ser-Lys-His-Cys-Gly • SEQ ID NO:5 (TDP-4): Ala-Cys-Arg-Arg-Ser-Ser-Ser-Pro-Ser-Lys-His-Cys-Gly-Arg • SEQ ID NO:6 (TDP-5): Ala-Cys-Lys-Lys-Arg-Ser-Ser-Ser-Pro-Ser-Lys-His-Cys-Gly (2) Peptide synthesis: The Fmoc solid-phase synthesis method was used and the following steps were performed on the CS Bio peptide synthesizer (CS BioCS336Xt).

[0044] ① Resin activation: Take 2 g of Wang resin, swell it with NMP for 2 h, filter and dry.

[0045] ② Coupling reaction: amino acids are coupled sequentially from the C-terminus to the N-terminus according to the sequence. Coupling conditions: 4 times excess Fmoc-amino acids, HBTU / DIEA (1:2 molar ratio), dissolved in NMP, and reacted at room temperature for 30 min.

[0046] ③ Fmoc deprotection: Treat with 20% piperidine / NMP solution for 20 min to remove the N-terminal Fmoc protecting group.

[0047] ④ Repeat steps ② and ③ until all amino acid coupling is completed.

[0048] ⑤ Lysis: Add lysis buffer (TFA:EDT:TIS:H2O=90:2.5:2.5:5), stir at room temperature for 3 hours, filter and collect the filtrate, precipitate with ice-cold ether, centrifuge to collect the precipitate, and vacuum dry to obtain crude peptide.

[0049] ⑥ Purification: The target peak was collected by preparative HPLC (Agilent 1260, C18 column, mobile phase A: 0.1% TFA aqueous solution, B: acetonitrile, gradient 0-60% B for 40 min), and the pure product was obtained by freeze drying.

[0050] ⑦ Mass spectrometry confirmation: The molecular weight was confirmed using MALDI-TOF mass spectrometry (Bruker Microflex LT).

[0051] (3) Preparation of TDP-PDRN: Take 2 mg of each sequence peptide and dissolve it in 1 mL of PBS (10 mM, pH 7.4) to prepare a 2 mg / mL peptide solution. Take 200 mg of PDRN and dissolve it in PBS to prepare a 1 mg / mL solution. Under magnetic stirring, add the peptide solution dropwise to the PDRN solution at a rate of 10 μL / min (peptide:PDRN mass ratio of 1:100), and stir at room temperature for 2 hours to obtain a linear peptide-DNA complex solution.

[0052] (4) Transdermal efficiency determination: The transdermal efficiency of each complex was determined using the Franz diffusion cell method (see Example 3).

[0053] 1.2 Experimental Results Characterization data and transdermal efficiency results of linear polypeptide-DNA complexes for each sequence are shown in Table 1 and 2. Figure 1 .

[0054] Table 1. Characterization data and transdermal efficiency of linear peptide-DNA complexes ; Test results showed that TDP-2 (SEQ ID NO:3) had the best overall performance, with a transdermal efficiency of 62.8 ± 5.4 μg / cm³. 2 The particle size was 82±12 nm, and the zeta potential was +25±3 mV. TDP-2 was selected as the optimal sequence for subsequent studies.

[0055] Example 2: Preparation and characterization of cyclized transdermal peptide-PDRN supramolecular complex 2.1 Preparation steps of cyclized transdermal peptide-PDRN supramolecular complex: ① PDRN solution preparation: Take 100 mg of PDRN raw material (Bioact from Korea, purity ≥98%), dissolve it in 50 mL of PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) to prepare a 2 mg / mL PDRN solution, and filter it through a 0.22 μm filter membrane.

[0056] ② Preparation of TDP-2 peptide solution: Take 2 mg of TDP-2 peptide (self-made) and dissolve it in 1 mL of PBS (10 mM, pH 7.4) to prepare a 2 mg / mL peptide solution.

[0057] ③ Self-assembly: The TDP-2 solution was loaded into a syringe and slowly added to the PDRN solution at a rate of 10 μL / min under magnetic stirring (500 rpm). After the addition was completed, the solution was stirred and incubated at room temperature for 2 hours to allow the linear transdermal peptide and PDRN to self-assemble into a peptide-DNA complex through electrostatic adsorption.

[0058] ④ Cycling reaction: Add GSH / GSSG (Sigma-Aldrich) redox pair (GSH 1 mM + GSSG 1 mM, molar ratio 1:1) to the above peptide-DNA complex solution, adjust the pH to 8.0 with 0.1 M NaOH, and let it stand at 4°C for 12 hours to oxidize the thiol group of cysteine ​​residues to form a disulfide bond and complete the cyclization.

[0059] ⑤ Purification: The reaction solution was placed into a dialysis bag (MWCO 3.5 kDa) and dialyzed with PBS for 24 hours (changing the solution 3 times in the first 4 hours). The dialysate was collected.

[0060] ⑥ Sterilization and drying: Sterilize by filtration through a 0.22 μm filter membrane, dispense, and freeze-dry (-50°C, 0.01 mbar, 48 hours) to obtain a white powdery cyclized transdermal peptide-PDRN supramolecular complex.

[0061] 2.2 Characterization methods: ① Particle size and Zeta potential: Take 1 mL of cyclized transdermal peptide-PDRN supramolecular complex solution and measure the particle size distribution and Zeta potential (Zetasizer Nano ZS) using DLS / ELS at a temperature of 25°C and a scattering angle of 90°.

[0062] ②PDRN encapsulation efficiency: The transdermal peptide-PDRN supramolecular complex was prepared by mixing FITC-labeled PDRN and unlabeled PDRN at a ratio of 1:100 using a fluorescent labeling method. Free PDRN was separated by centrifugation, and the fluorescence intensity of the supernatant and precipitate was measured to calculate the encapsulation efficiency.

[0063] ③ Cyclization efficiency: The free thiol content was determined using the Ellman reagent method (Sigma-Aldrich). 0.5 mL of sample solution was taken, and 0.5 mL of Ellman reagent (4 mg / mL) was added. The mixture was reacted at room temperature for 15 minutes, and the absorbance at 412 nm was measured. The free thiol content was calculated based on the standard curve. Cyclization efficiency = (1 - measured thiol groups / theoretical thiol groups) × 100%.

[0064] 2.3 Experimental Results The physicochemical properties of the transdermal peptide-PDRN supramolecular complex are shown in Table 2.

[0065] Table 2. Characterization results of the physicochemical properties of the transdermal peptide-PDRN supramolecular composite. ; As shown in Table 2, the transdermal peptide-PDRN supramolecular complex of the present invention possesses excellent physicochemical properties. Through the dual stabilization strategy of "electrostatic compounding + chemical crosslinking", it maintains a nanoscale dispersion state while possessing sufficient structural integrity to resist interference from the physiological environment. Therefore, the cyclized transdermal peptide-PDRN supramolecular complex of the present invention has excellent structural stability.

[0066] Example 3: Transdermal Efficiency Experiment 3.1 Experimental Methods (1) Skin treatment: Take frozen pig skin, thaw at room temperature, cut a circular skin piece with a diameter of 25 mm, ensuring a thickness of about 500±50 μm, remove the subcutaneous fat layer, and rinse 3 times with physiological saline.

[0067] (2) Assembly of diffusion cell: Fix the skin patch between the supply cell and the receiving cell of the Franz diffusion cell, with the stratum corneum facing the supply cell. Add 12 mL of PBS receiving solution to the receiving cell and remove all air bubbles.

[0068] (3) Drug administration: Add 1 mL of each of the following samples to the supply tank: • Control group: Free PDRN solution (1 mg / mL) • Experimental group: TDP-2 complex solution (containing PDRN 1 mg / mL) • Parallel groups: TDP-1 complex, TDP-3 complex (containing PDRN 1 mg / mL) Each group has 6 parallels.

[0069] (4) Transdermal test: The diffusion cell was placed in a constant temperature water bath at 32±0.5°C and the magnetic stirring speed was 600 rpm.

[0070] (5) Sampling: 0.5 mL samples were taken from the receiving pool at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours, and an equal amount of fresh PBS preheated at 37°C was added at the same time.

[0071] (6) Content determination: The sample was filtered through a 0.22 μm filter membrane, and the absorbance at 260 nm was measured by ultraviolet spectrophotometry. The content was determined according to the PDRN standard curve (concentration 0-100 μg / mL, R 2 Calculate the PDRN concentration using >0.999.

[0072] (7) Data processing: The cumulative transmittance is calculated according to the formula Q=(Cn×V+ΣCi×Vs) / A, where Q is the cumulative transmittance, Cn is the concentration of the nth sampling, V is the volume of the receiving cell, ΣCi×Vs is the total amount of the first n-1 samplings, and A is the effective diffusion area.

[0073] 3.2 Experimental Results The results of the transdermal efficiency changes over time for each group are shown in Table 3 and... Figure 2 .

[0074] Table 3. Results of transdermal efficiency changes over time ; Experimental results showed that the 24-h transdermal efficiency of TDP-2-PDRN was 62.8 ± 5.4 μg / cm³. 2 Compared with free PDRN (13.6±1.8 μg / cm), 2 The efficiency was increased by 4.62 times, a statistically significant difference (p<0.001). Therefore, the cyclized transdermal peptide-PDRN supramolecular complex of the present invention exhibits highly efficient transdermal delivery performance.

[0075] Example 4: Reduction-responsive release experiment 4.1 Experimental Methods (1) Solution preparation: Take TDP2-PDRN and prepare a solution containing PDRN 1 mg / mL with PBS. Prepare 10 mL of solutions containing 0, 10 μM, 100 μM, 1 mM and 10 mM GSH respectively. The GSH concentration design is based on: 0 μM (simulating extracellular fluid), 10-100 μM (simulating normal intracellular fluid), and 1-10 mM (simulating intracellular fluid of tumor cells or cells under high oxidative stress).

[0076] (2) Release experiment: Each GSH concentration sample was placed in a constant temperature shaker at 37°C (100 rpm), and 0.2 mL of sample was taken at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours respectively.

[0077] (3) Separation: The released free PDRN and complex were separated by centrifugal ultrafiltration (14000 rpm × 15 min), and the filtrate (containing free PDRN) was collected.

[0078] (4) Determination: The PDRN content in the filtrate was determined by PicoGreen fluorescence method. Take 100 μL of sample, add 100 μL of PicoGreen working solution, react at room temperature in the dark for 5 minutes, and measure the fluorescence intensity at the emission wavelength of 525 nm (excitation wavelength of 480 nm). Calculate the PDRN concentration according to the PDRN standard curve.

[0079] (5) Calculation: Release rate (%) = (Amount of PDRN released / Total amount of PDRN) × 100%.

[0080] 4.2 Experimental Results The results of PDRN release rates at different GSH concentrations are shown in Table 4 and Figure 3 .

[0081] Table 4. PDRN release rate results at different GSH concentrations ; Experimental results show that TDP-2-PDRN exhibits a significant GSH concentration-dependent release characteristic, with the release rate increasing with increasing GSH concentration. The release rate reached 89.2±10.5% after 24 h at 10 mM GSH, compared to only 5.2±0.9% at GSH 0. This is because PDRN is encapsulated within the transdermal peptide via electrostatic adsorption, and the cysteine ​​residues in the transdermal peptide form disulfide bonds, creating a cyclic structure that encapsulates PDRN within the cyclic peptide. These disulfide bonds can break under intracellular reducing conditions, thereby releasing PDRN. Therefore, this invention demonstrates that it can achieve intelligent responsive release of PDRN.

[0082] Example 5: Serum stability test 5.1 Experimental Methods (1) Sample preparation: Take TDP-2-PDRN and free PDRN, and add them to 50% FBS PBS solution to make the final concentration of PDRN 100 μg / mL.

[0083] (2) Incubation: Place the sample in a constant temperature shaker at 37°C (100 rpm) for incubation.

[0084] (3) Sampling: Take 0.1 mL samples at 0, 2, 4, 8, 12, 24 and 48 hours respectively, and store them immediately at -20°C.

[0085] (4) Measurement: The content of intact PDRN was determined by PicoGreen fluorescence method (degraded fragments cannot be effectively stained by PicoGreen).

[0086] (5) Calculation: PDRN residual rate (%) = (measured concentration / initial concentration) × 100%.

[0087] 5.2 Experimental Results Serum stability results are shown in Table 5 and Figure 4 .

[0088] Table 5. Results of serum stability test ; Experimental results showed that TDP-2-PDRN retained 75.2±3.5% after 48 h, which was approximately 9.2 times higher than that of free PDRN (8.2±2.5%) (p<0.001), significantly improving serum stability. Therefore, the cyclized transdermal peptide-PDRN supramolecular complex of this invention exhibits excellent serum stability.

[0089] Example 6 Cell proliferation experiment 6.1 Experimental Methods (1) Cell culture: HDF cells were cultured in a 37°C, 5% CO2 incubator (Thermo Fisher) using complete culture medium, and cells in the logarithmic growth phase were used for experiments.

[0090] (2) Inoculation: 1×10 4 Cells were seeded at a density of 1 / well in 96-well plates with 100 μL of complete culture medium per well and cultured for 24 hours to allow the cells to adhere.

[0091] (3) Drug administration: Discard the culture medium and add 100 μL of serum-free culture medium containing different concentrations of PDRN (0, 5, 10, 25, 50, 100, 200 μg / mL) to each group, with 6 parallel wells in each group. At the same time, a blank control group (cell-free) and a negative control group (containing cells but not drugged) were set up.

[0092] (4) Cultivation: Continue cultivation for 48 hours.

[0093] (5) MTT assay: Add 20 μL of MTT solution to each well and continue culturing for 4 hours. Discard the culture medium, add 150 μL of DMSO to each well, and shake for 10 minutes to fully dissolve the formazan crystals.

[0094] (6) Measurement: The absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader (reference wavelength 630 nm).

[0095] (7) Calculation: Cell proliferation rate (%) = (OD of experimental group / OD of negative control group) × 100%.

[0096] 6.2 Experimental Results The cell proliferation results are shown in Table 6 and Figure 5 .

[0097] Table 6 Cell proliferation results ; The experimental results showed that at 100 μg / mL, the proliferation rate of the TDP-2-PDRN group was 178.3±11.2%, which was 24.9% higher than that of the free PDRN group (142.8±9.1%) (p<0.01), significantly promoting cell proliferation.

[0098] Example 7: Human Efficacy Evaluation Experiment 7.1 Preparing the serum The serum formula containing 0.5% cyclized transdermal peptide-PDRN supramolecular molecules is as follows (w / w%): TDP-2-PDRN complex: 0.5% Sodium hyaluronate (molecular weight 1000 kDa): 0.1% Nicotinamide: 2.0% Glycerin: 5.0% Butylene glycol: 3.0% Carbomer 940: 0.2% Triethanolamine: Appropriate amount (adjust pH to 6.8) Phenoxyethanol: 0.5% Disodium ethylenediaminetetraacetate: 0.05% Purified water: Add to 100% 7.2 Skin Function Instruments Skin moisture content meter: Corneometer CM825 (C+K Company) Transdermal water loss meter: Tewameter TM300 (C+K Company) Skin erythema measuring instrument: Mexameter MX18 (C+K Company) Skin elastometer: Cutometer MPA580 (C+K Company) 7.3 Experimental Methods (1) Subject screening: Inclusion criteria: Women aged 25-45 with sensitive skin.

[0099] Exclusion criteria: pregnant or breastfeeding women, those allergic to the test ingredients, those who have recently used hormonal drugs, and patients with severe skin diseases.

[0100] Ten participants were eventually included, with a mean age of 34.6 ± 6.8 years.

[0101] (2) Method of use: After the subject cleansed his face, he took 0.5 mL of the essence and applied it evenly to his face. He used it once in the morning and once in the evening for 28 consecutive days.

[0102] (3) Testing time points: before use (D0), 3 days after use (D3), 7 days (D7), 14 days (D14), 21 days (D21), 28 days (D28).

[0103] (4) Detection method: (a) Skin moisture content: The skin moisture content of the forehead was measured using a CM825 at room temperature of 22±2°C and relative humidity of 45±5%. Each site was measured 3 times and the average value was taken.

[0104] (b) Transdermal water loss (TEWL): The TEWL value of the forehead was measured using the TM300. The probe was placed vertically on the skin surface and the value was read after stabilization. Each site was measured 3 times.

[0105] (c) Erythema Index: The facial erythema index was measured using an MX18, with three measurements taken for each area and the average value taken.

[0106] (d) Skin elasticity: The skin elasticity parameter (R2 value) of the cheek was measured using a Cutometer MPA580, with three measurements taken for each area.

[0107] (e) Subject satisfaction: assessed through a questionnaire survey using a 5-point scale (1 = very dissatisfied, 5 = very satisfied), and the satisfaction rate (4-5 points percentage) was calculated.

[0108] (5) Data processing: Each indicator is expressed as the rate of change relative to D0, and the data is presented in... The results indicate that SPSS 26.0 was used for statistical analysis, and p < 0.05 was considered statistically significant.

[0109] 7.4 Experimental Results The results of the human efficacy evaluation are shown in Table 7 and Figure 6 .

[0110] Table 7 Results of Human Efficacy Evaluation ; Experimental results showed that after 28 days, skin moisture increased by 48.2±8.6%, TEWL decreased by 38.5±7.2%, erythema decreased by 42.1±9.5%, and elasticity improved by 42.5±8.5%. The subject satisfaction rate reached 93.3% (28 / 30), indicating that the cyclized transdermal peptide-PDRN supramolecular complex of the present invention has a significant skin repair effect.

[0111] Example 8 Accelerated Stability Experiment 8.1 Experimental Methods The TDP-2-PDRN complex was aliquoted into transparent glass bottles and placed in constant temperature and humidity chambers at 4°C (refrigeration), 25°C (room temperature), and 40°C±2°C / 75%±5% RH (accelerated conditions). Samples were taken at 0, 7, 14, 21, and 28 days to determine the particle size and encapsulation efficiency.

[0112] 8.2 Experimental Results The stability test results are shown in Table 8 and Figure 7 .

[0113] Table 8 Stability Test Results ; Experimental results show that the transdermal peptide-PDRN supramolecular complex has good stability within 28 days at 4°C and 25°C; the particle size increases and the encapsulation efficiency decreases at 40°C. It is recommended to store it at 4-25°C in the dark, and the shelf life is not less than 24 months.

[0114] Example 9 Cytotoxicity Evaluation 9.1 Experimental Methods (1) Cell culture: HDF cells were cultured in a 37°C, 5% CO2 incubator using complete culture medium.

[0115] (2) Inoculation: 1×10 4 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours to allow the cells to adhere.

[0116] (3) Drug administration: Discard the culture medium and add serum-free culture medium containing TDP-2 peptide, complex and free PDRN (concentration 0, 10, 50, 100, 200 and 500 μg / mL respectively), with 6 parallel wells in each group.

[0117] (4) Cultivation: Continue cultivation for 24 hours.

[0118] (5) CCK-8 detection: Add 10 μL of CCK-8 solution to each well, continue to incubate at 37°C for 2 hours, and measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0119] (6) Calculation: Cell viability (%) = (OD of experimental group / OD of negative control group) × 100%.

[0120] 9.2 Experimental Results The results of the cytotoxicity evaluation are shown in Table 9 and Figure 8 .

[0121] Table 9 Cytotoxicity Evaluation Results ; Experimental results showed that cell viability was >75% at all concentrations, indicating that neither the TDP-2 peptide nor the TDP-2-PDRN complex had significant cytotoxicity and good safety. This demonstrates that the cyclized transdermal peptide-PDRN supramolecular complex of the present invention can be used for skin repair with good safety.

[0122] Example 10 Skin Irritation Evaluation 10.1 Experimental Methods (1) Subjects: Ten healthy subjects (5 males and 5 females) aged 18-65 years were selected.

[0123] (2) Skin treatment: Select a hairless and lesionless skin area on the back of the subject, with an area of ​​2.5 cm × 2.5 cm.

[0124] (3) Spot test: Apply 0.2 mL of solution containing 0.5% TDP-2-PDRN complex evenly to the skin surface, cover with gauze, fix with 3M tape, and apply for 4 hours.

[0125] (4) Observation: After removing the patch, the skin reaction was observed at 1, 4, 24, 48 and 72 hours.

[0126] (5) Scoring: Erythema and edema were scored according to the Draize scoring criteria: 0 points = no erythema / no edema, 1 point = very mild erythema / very mild edema, 2 points = mild erythema / mild edema, 3 points = moderate erythema / moderate edema, 4 points = severe erythema / severe edema.

[0127] (6) Calculation: Stimulation index = (erythema score + edema score) / 2.

[0128] 10.2 Experimental Results The results of the skin irritation test are shown in Table 10.

[0129] Table 10 Results of Skin Irritation Test ; Experimental results show that the stimulation index of the cyclized transdermal peptide-PDRN supramolecular complex of the present invention is <1, which means it is non-irritating or very mildly irritating and can be used in cosmetics and topical drug preparations.

[0130] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0131] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cyclized transdermal peptide-PDRN supramolecular complex, characterized in that, include: Cyclic transdermal peptides and PDRN; The PDRN is encapsulated within the cyclized transdermal peptide through electrostatic adsorption. The cyclized transdermal peptide comprises cationic amino acid residues and at least two cysteine ​​residues, wherein disulfide bonds are formed between the cysteine ​​residues to constitute a cyclized structure; the amino acid sequence of the cyclized transdermal peptide is shown in SEQ ID NO:

3.

2. The cyclized transdermal peptide-PDRN supramolecular complex according to claim 1, characterized in that, The molecular weight of the PDRN is 50 to 3000 kDa.

3. The cyclized transdermal peptide-PDRN supramolecular complex according to claim 1 or 2, characterized in that, The cyclized transdermal peptide-PDRN supramolecular complex satisfies at least one of the following: (1) The particle size of the cyclized transdermal peptide-PDRN supramolecular complex is 50~200 nm; (2) The zeta potential of the cyclized transdermal peptide-PDRN supramolecular complex is +15~+35 mV; (3) The cyclized transdermal peptide-PDRN supramolecular complex has an encapsulation rate of 60-90% for PDRN; (4) The disulfide bond cyclization efficiency of the cyclized transdermal peptide-PDRN supramolecular complex is 85~98%.

4. A method for preparing the cyclized transdermal peptide-PDRN supramolecular complex according to any one of claims 1 to 3, characterized in that, The method includes the following steps: S1: The linear transdermal peptide is mixed with PDRN to obtain a peptide-DNA complex; S2: The peptide-DNA complex is cyclized to form disulfide bonds between cysteine ​​residues, resulting in a cyclized transdermal peptide-PDRN supramolecular complex.

5. The method according to claim 4, characterized in that, The mass ratio of the linear transdermal peptide to the PDRN is 1:1 to 1:1000; the mixing treatment is carried out at a pH of 6.5 to 8.0, a temperature of 4 to 37°C, and a time of 0.5 to 4 h; the cyclization reaction method includes one or more of air oxidation, dimethyl sulfoxide oxidation, and glutathione redox; the cyclization reaction is carried out at a pH of 7.0 to 8.5, a temperature of 4 to 25°C, and a reaction time of 2 to 24 h; the amino acid sequence of the linear transdermal peptide is shown in SEQ ID NO:

3.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: the cyclized transdermal peptide-PDRN supramolecular complex according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, characterized in that, The concentration of the cyclized transdermal peptide-PDRN supramolecular complex in the pharmaceutical composition is 0.01~5% w / v; the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

8. The use of the cyclized transdermal peptide-PDRN supramolecular complex according to any one of claims 1 to 3, or the pharmaceutical composition according to any one of claims 6 or 7, in the preparation of a skin repair drug; wherein the skin repair includes one or more of the following: post-cosmetic surgery skin repair, sensitive skin repair, photodamaged skin repair, wound healing promotion, skin barrier reconstruction, and anti-skin aging.

9. A cosmetic composition, characterized in that, The cosmetic composition comprises: the cyclized transdermal peptide-PDRN supramolecular complex according to any one of claims 1 to 3.

10. The use of the cyclized transdermal peptide-PDRN supramolecular complex according to any one of claims 1 to 3 in the preparation of cosmetics, pharmaceuticals or products for skin repair.

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