A method for preparing MMP-13 responsive nanoparticles loaded with PDRN

Abstract

The application relates to a preparation method of MMP-13 response nanoparticles loaded with PDRN, and belongs to the technical field of biological medicines. In order to solve the problems of low transdermal efficiency of polydeoxyribonucleotide, poor stability under conventional storage conditions, and the fact that a traditional delivery system cannot distinguish between aging and normal tissues, leading to insufficient concentration at a target site and low bioavailability, the application provides MMP-13 response nanoparticles loaded with PDRN. By introducing a natural and safe response unit and a cross-linking strategy, an innovative full-chain solution and solid data support are provided for the development of the next generation of intelligent anti-aging cosmetics which are precise in targeting, efficient in anti-aging, and excellent in stability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for preparing PDRN-loaded MMP-13 responsive nanoparticles. Background Technology

[0002] Skin aging is a complex biological process, one of its core pathological features being the imbalance between the degradation and synthesis of the extracellular matrix (ECM) in the dermis. Matrix metalloproteinase-13 (MMP-13), a key member of the collagenase family, is specifically highly expressed in photoaged and naturally aging skin, exhibiting highly efficient cleavage activity against type I collagen—the most important supporting protein in the dermis. Therefore, MMP-13 is not only a key biomarker of the skin aging process but also constitutes a unique pathological microenvironment signal that can be used for precise intervention. It is worth noting that in in vitro studies, UVB irradiation has been widely confirmed as a classic model for inducing skin photoaging and its typical molecular characteristics. A recognized core event is the significant upregulation of the expression of various matrix metalloproteinases (including MMP-1, MMP-3, MMP-9, and MMP-13) in dermal fibroblasts, thereby driving the degradation of the extracellular matrix.

[0003] Polydeoxyribonucleic acid (PDRN), as a bioactive ingredient, has been shown to effectively promote fibroblast proliferation and migration and upregulate the synthesis of type I and type III collagen by activating the adenosine A2A receptor pathway, demonstrating clear anti-aging potential. However, PDRN has low transdermal efficiency, and traditional delivery systems (such as ordinary liposomes) cannot distinguish between aging areas and normal tissues, resulting in insufficient drug concentration at the target site, low bioavailability, and potential irritation or waste in non-target areas.

[0004] Therefore, those skilled in the art are eager to develop an intelligent targeted delivery system that responds to the pathological microenvironment with high MMP-13 expression, so as to achieve precise and efficient delivery of PDRN. Summary of the Invention

[0005] To address the problems of low transdermal efficiency and poor stability of polydeoxyribonucleic acid under conventional storage conditions in existing technologies, and the inability of traditional delivery systems to distinguish between aging and normal tissues, resulting in insufficient target concentration and low bioavailability, this invention provides a method for preparing PDRN-loaded MMP-13 responsive nanoparticles.

[0006] One objective of this invention is to provide a method for preparing PDRN-loaded MMP-13 responsive nanoparticles, the method comprising the following steps: S1: Dipalmitoylphosphatidylethanolamine, cholesterol, and 1,2-dioleoyl-3-trimethylammonium-propane were weighed and dissolved in anhydrous ethanol to form the organic phase; polydeoxyribonucleotides were weighed and dissolved in phosphate buffer preheated to 60°C to form the aqueous phase; the organic phase was injected into the aqueous phase at a constant rate using a syringe pump under constant temperature magnetic stirring at 500 rpm, and stirring was continued for 30 min after injection to obtain a crude suspension; the obtained crude suspension was transferred to a high-pressure microfluidic homogenizer and homogenized 5 times under ice-water bath cooling at 150 MPa pressure; then, the liposomes were purified by centrifugation at 4°C and 4000×g for 20 min using ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa, repeated 3 times to obtain drug-loaded liposomes; S2: Weigh out the hydrolyzed collagen peptides and dissolve them in phosphate buffer to prepare a 5 mg / mL hydrolyzed collagen peptide stock solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4°C for later use. S3: The drug-loaded liposomes prepared in S1 were placed under magnetic stirring at 300 rpm and room temperature, and phosphate buffer containing 1 mg / mL chondroitin A sodium salt was added dropwise. After the addition was complete, stirring was continued for 30 min to form a primary complex based on electrostatic interaction. S4: Add the hydrolyzed collagen peptide stock solution prepared in S2 to the primary complex prepared in S3 to obtain a mixture; then, add freshly prepared tannic acid aqueous solution dropwise to a final concentration of 0.5 mg / mL, adjust the pH of the system to 7.4 using 0.1 M NaOH, and stir the reaction at 200 rpm, room temperature, and in the dark for 2 hours to obtain a mixed system; S5: The mixture obtained in S4 was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and centrifuged at 4℃ and 4000×g for 20 min. The filtrate was discarded, and the precipitate was resuspended with phosphate buffer. This washing process was repeated 3 times. The mixture was then filtered through a 0.45 μm sterile filter membrane to obtain PDRN-loaded MMP-13 responsive nanoparticles.

[0007] In a preferred embodiment of the present invention, the mixing ratio of dipalmitoylphosphatidylethanolamine, cholesterol and 1,2-dioleoyl-3-trimethylammonium-propane in S1 is as follows: the total lipid mass is kept at 100 μmol and the addition ratio of 1,2-dioleoyl-3-trimethylammonium-propane is 5 mol.

[0008] In a preferred embodiment of the present invention, the mixing ratio of the polydeoxyribonucleotide and phosphate buffer in S1 is 10 mg: 9 mL.

[0009] In a preferred embodiment of the present invention, the rate at which the organic phase is injected into the aqueous phase in S1 is 0.5 mL / min.

[0010] In a preferred embodiment of the present invention, the mixing ratio of the hydrolyzed collagen peptide and phosphate buffer in S2 is 5 mg: 1 mL; the molecular weight of the hydrolyzed collagen peptide is 1-3 kDa, and the hydrolyzed collagen peptide is derived from fish scales.

[0011] In a preferred embodiment of the present invention, the mixing volume ratio of the drug-loaded liposomes to the phosphate buffer in S3 is 1:4.

[0012] In a preferred embodiment of the present invention, the final concentration of the hydrolyzed collagen peptide stock solution in the mixture in S4 is 2 mg / mL; and the concentration of the tannic acid aqueous solution is 10 mg / mL.

[0013] A second objective of this invention is to provide a PDRN-loaded MMP-13 responsive nanoparticle, which is obtained by the above-described preparation method.

[0014] A third objective of this invention is to provide the application of the above-mentioned PDRN-loaded MMP-13 responsive nanoparticles in the preparation of anti-aging cosmetics.

[0015] The fourth objective of this invention is to provide an anti-aging serum, which is composed of the following ingredients by weight percentage: 10% of the above-mentioned PDRN-loaded MMP-13 responsive nanoparticles, 5% of hydrolyzed Chlorella extract, 5% of glycerin, 10% of sodium hyaluronate solution, 1% of panthenol, 0.8% of phenoxyethanol, and the balance being deionized water; the concentration of the sodium hyaluronate solution is 0.2%.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention proposes and constructs a new paradigm of intelligent drug delivery based on "pathological signal response." Its core concept lies in cleverly utilizing MMP-13, which is overexpressed in aging skin areas, as a "molecular trigger." A nanoparticle with a "core-shell" structure is designed, whose shell integrates a natural response unit that can be specifically recognized and cleaved by MMP-13, thereby achieving on-demand release of PDRN at the target site. This invention innovatively uses hydrolyzed collagen peptides (ColP), widely used in the cosmetics field with a molecular weight range of 1-3 kDa, as the response substrate and intelligent release switch for MMP-13. The hydrolyzed collagen peptides contain a characteristic amino acid sequence recognizable by MMP-13, and their enzymatic activity has been experimentally verified in this study. Simultaneously, natural plant polyphenol tannic acid (TA) is used as a green cross-linking agent, whose antioxidant activity synergistically enhances the repair-promoting activity of PDRN. A stable smart shell is constructed through multiple non-covalent interactions. Its rich catechol structure forms a multi-layered, dynamic non-covalent cross-linked network with chondroitin sulfate (SCS) polysaccharide chains and collagen peptide chains. This physical cross-linking strategy based on hydrogen bonds and hydrophobic interactions, compared with traditional chemical covalent cross-linking (such as using EDC / NHS), ensures shell stability while avoiding the formation of an overly dense and rigid network. This preserves the necessary spatial accessibility for MMP-13 enzyme molecules to approach and cleave the collagen peptides in the shell, which is one of the key designs for achieving efficient enzyme response release.

[0017] In normal skin (low MMP-13 environment), the shell network constructed based on multiple non-covalent interactions remains intact, forming an effective physical barrier for the drug. The nanoparticle structure is stable, and the drug is tightly encapsulated. When the nanoparticles reach the dermis of aging skin (high MMP-13 environment), MMP-13 acts as a "key" to specifically cleave collagen peptides in the shell, causing the peptides, which are key connection points in the network, to break down. This triggers the dynamic dissociation of the entire cross-linked network, resulting in shell dissociation and rapid drug release at the target site. The PDRN-loaded MMP-13 responsive nanoparticles provided by this invention precisely couple drug release kinetics with lesion-specific biological signals (MMP-13 concentration), achieving a shift from "passive diffusion" to "active triggering" delivery mode. This fundamentally solves the industry problem of the inability to simultaneously achieve transdermal efficiency and tissue targeting in traditional transdermal delivery systems. Furthermore, all raw materials comply with cosmetic safety regulations, demonstrating excellent industrialization prospects.

[0018] Systematic experiments have demonstrated that the PDRN-loaded MMP-13 responsive nanoparticles provided in this invention utilize cosmetic-grade hydrolyzed collagen peptides as natural responsive units and employ natural plant polyphenols and tannins for green cross-linking, forming a distinct "core-shell" structure and highly efficient MMP-13 enzyme-responsive release characteristics. Furthermore, tannins themselves possess antioxidant properties, synergistically complementing the repair-promoting effects of PDRN to jointly exert anti-aging effects; moreover, this core-shell structure effectively protects PDRN, significantly improving its storage stability. The serum products developed based on this technology exhibit stable physicochemical properties and can respond to MMP-13 signals in the skin microenvironment under topical application conditions, achieving enzyme-triggered release and highly efficient transdermal delivery of active ingredients, with transdermal efficiency approximately 2.5 times higher than traditional liposomes. At the cellular level, the PDRN-loaded MMP-13 responsive nanoparticles provided in this invention demonstrate good biocompatibility and can exert remarkable anti-aging effects by significantly promoting collagen synthesis and effectively resisting oxidative stress, synergistically with tannins.

[0019] In summary, this invention not only systematically elucidates the preparation, characterization, and core function verification of the intelligent nanoparticles, but also successfully formulates them into an anti-aging serum with good stability and user experience. Furthermore, it comprehensively evaluates the physicochemical stability, transdermal behavior, and in vitro cell efficacy of the final product. Thus, it constructs a complete technology chain from intelligent material design to stable formulation development and efficacy verification, providing a solid data foundation and innovative solutions for developing next-generation, highly efficient, safe, and precise intelligent anti-aging cosmetics. Attached Figure Description

[0020] Figure 1 Figure 1 shows the optimization of the cationic liposome core and the construction of core-shell nanoparticles in the physicochemical property characterization experiment; Figure 2 shows the change of zeta potential with the proportion of DOTAP; Figure 3 shows the change trend of PDRN encapsulation efficiency; Figure 4 shows the change of particle size and polydispersity index (PDI); Figure 5 shows the particle size detection after the construction of core-shell nanoparticles; Figure 6 shows the zeta potential detection after the construction of core-shell nanoparticles. Figure 2 Transmission electron microscopy (TEM) images of Lipo-PDRN@SCS-TA-ColP used in physicochemical characterization experiments; Figure 3 Figure 1 shows the results of the optimization of the cationic liposome core and the evaluation of the stability of the core-shell nanoparticles in the physicochemical characterization experiment; (a) is 4℃, (b) is 25℃, and (c) is 40℃. Figure 4 The fluorescence kinetics analysis of MMP-13 enzyme digestion of hydrolyzed collagen peptides in the physicochemical characterization experiment is shown. Figure 5The graph shows the accelerated stability evaluation of the anti-aging serum; A is the change in nanoparticle size during storage; B is the change in nanoparticle polydispersity index (PDI) during storage; C is the retention rate of PDRN content during storage; D is the change in product physicochemical properties (pH value) during storage; E is the change in product physicochemical properties (viscosity) during storage; and F is the state of the serum after centrifugation. Figure 6 This study illustrates the in vitro enzyme-responsive release behavior of different nano-formulations in an in vitro enzyme-responsive release experiment. Figure 7 This is a diagram showing the results of an in vitro transdermal experiment; Figure 8 This is a graph showing the cytotoxicity evaluation of Lipo-PDRN@SCS-TA-ColP nanoparticles in cell experiments. Figure 9 The image shows a Western blotting analysis of type I collagen expression in a cellular anti-aging efficacy evaluation experiment. A represents representative Western blotting bands of type I collagen (~139 kDa) and internal control GAPDH (~36 kDa) in cells treated with Control, Model, free PDRN, free TA, PDRN+TA Mix, Lipo-PDRN, and Lipo-PDRN@SCS-TA-ColP. B is a semi-quantitative analysis of the band grayscale values. Figure 10 The diagram shows the effect of MMP-13 responsive smart nanoparticles on improving the oxidative stress state of UVB-induced photoaging human dermal fibroblasts in the cellular level anti-aging efficacy evaluation experiment; A is a quantitative analysis diagram of intracellular reactive oxygen species (ROS) levels in the Control, Model, free PDRN, free TA, PDRN+TA Mix, Lipo-PDRN, and Lipo-PDRN@SCS-TA-ColP treatment groups; B is a quantitative analysis diagram of intracellular superoxide dismutase (SOD) activity in each treatment group. Detailed Implementation

[0021] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0023] All experiments in the following examples were independently repeated at least 3 times. Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA was used for comparisons among multiple groups. If the variances were homogeneous, Tukey's post-hoc test was used for pairwise comparisons. * p<0.05, ** p<0.01, *** p < 0.001 is considered statistically significant.

[0024] Example 1: Preparation of PDRN-loaded MMP-13 responsive nanoparticles S1: Weigh dipalmitoylphosphatidylethanolamine (DPPE), cholesterol, and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dissolve them in 2 mL of anhydrous ethanol (maintaining a total lipid mass of 100 μmol and DOTAP proportions of 0, 2.5, 5, 7.5, and 10 mol%) to form an organic phase; Weigh 20 mg of polydeoxyribonucleic acid (PDRN) and dissolve it in 18 mL of phosphate-buffered saline (PBS, 10 mM, pH 7.4) preheated to 60 °C to form the aqueous phase; Under constant temperature magnetic stirrer at 500 rpm, the organic phase was injected into the aqueous phase at a constant rate of 0.5 mL / min using a syringe pump. After injection, stirring was continued for 30 min to obtain a crude suspension. The obtained crude suspension was transferred to a high-pressure microfluidic homogenizer and homogenized 5 times at 150 MPa pressure under ice-water bath cooling. Subsequently, purification was performed by centrifugation at 4℃ and 4000×g for 20 min using ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa. This was repeated 3 times to completely remove unencapsulated PDRN and residual ethanol. Finally, the volume was adjusted to 10 mL with PBS to obtain drug-loaded liposomes, denoted as Lipo-PDRN-X (X represents the molar percentage of DOTAP). S2: Select cosmetic-grade hydrolyzed collagen peptides with a molecular weight of 1-3 kDa as the MMP-13 response unit. Weigh 50 mg of hydrolyzed collagen peptides (derived from fish scales) and dissolve them in 10 mL of phosphate buffer (PBS, 10 mM, pH 7.4) to prepare a 5 mg / mL hydrolyzed collagen peptide stock solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4℃ for later use. S3: Place 2 mL of the drug-loaded liposomes prepared in S1 under magnetic stirring at 300 rpm and room temperature, and add 8 mL of phosphate buffer (PBS, 10 mM, pH 6.5) containing 1 mg / mL chondroitin A sodium salt (SCS). After the addition is complete, continue stirring for 30 min to form a primary complex based on electrostatic interaction, denoted as Lipo-PDRN@SCS. This step utilizes the electrostatic attraction between the positively charged liposome core and the negatively charged SCS to provide a stable primary composite substrate for subsequent more complex multi-component cross-linking. S4: Add the hydrolyzed collagen peptide stock solution prepared in S2 to the primary complex prepared in S3 to obtain a mixture with a hydrolyzed collagen peptide stock solution concentration of 2 mg / mL; then, add freshly prepared tannic acid aqueous solution (10 mg / mL) dropwise to a final concentration of 0.5 mg / mL, adjust the pH of the system to 7.4 using 0.1 M NaOH, and stir the reaction at 200 rpm, room temperature, and in the dark for 2 hours to obtain a mixed system; In this process, multiple catechol / galloyl groups of tannic acid molecules form a dense hydrogen bond network with the hydroxyl and carboxyl groups on the SCS polysaccharide chain, and interact with the amino and carboxyl groups of collagen peptides. Simultaneously, hydrophobic and π-π stacking also occurs between tannic acid molecules. This multi-layered, synergistic non-covalent interaction constructs a dynamic three-dimensional network structure that physically entangles and fixes SCS and collagen peptides, immobilizing the responsive peptides on the nanoparticle shell. Importantly, this physically cross-linked network exhibits a degree of dynamic reversibility; its structural rigidity is lower than that of covalent cross-links. This is considered beneficial for the diffusion of external enzyme molecules (MMP-13) into the network interior and closer to its substrate (collagen peptides). S5: Transfer the mixture obtained in S4 to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. Centrifuge at 4℃ and 4000×g for 20 min, discard the filtrate, and resuspend the precipitate with phosphate buffer. Repeat this washing process three times to thoroughly remove unreacted free peptides and tannins. Finally, bring the volume to 5 mL with PBS and filter through a 0.45 μm sterile filter membrane to obtain PDRN-loaded MMP-13 responsive nanoparticles, abbreviated as: Lipo-PDRN@SCS-TA-ColP.

[0025] Comparative Example 1: The difference between this comparative example and Example 1 is that hydrolyzed collagen peptide stock solution and tannic acid are not added (step S4 is not performed), while the other steps are the same as in Example 1. MMP-13 responsive nanoparticles loaded with PDRN are prepared, abbreviated as: Lipo-PDRN@SCS.

[0026] Comparative Example 2: The difference between this comparative example and Example 1 is that hydrolyzed collagen peptide stock solution (used to verify the necessity of hydrolyzed collagen peptide) is not added. The other steps are the same as in Example 1, and PDRN-loaded MMP-13 responsive nanoparticles, abbreviated as: Lipo-PDRN@SCS-TA, are prepared.

[0027] Comparative Example 3: Weigh out dipalmitoylphosphatidylethanolamine (DPPE), cholesterol, and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dissolve them in 2 mL of anhydrous ethanol (maintaining a total lipid mass of 100 μmol and DOTAP proportions of 0, 2.5, 5, 7.5, and 10 mol%) to form an organic phase; Weigh 20 mg of polydeoxyribonucleic acid (PDRN) and dissolve it in 18 mL of phosphate-buffered saline (PBS, 10 mM, pH 7.4) preheated to 60 °C to form the aqueous phase; Under constant temperature magnetic stirrer at 500 rpm, the organic phase was injected into the aqueous phase at a constant rate of 0.5 mL / min using a syringe pump. After injection, stirring was continued for 30 min to obtain a crude suspension. The obtained crude suspension was transferred to a high-pressure microfluidic homogenizer and homogenized 5 times at 150 MPa pressure under ice-water bath cooling. Subsequently, purification was performed by centrifugation at 4℃ and 4000×g for 20 min using ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa. This process was repeated 3 times to completely remove unencapsulated PDRN and residual ethanol. Finally, the volume was adjusted to 10 mL with PBS to obtain drug-loaded liposomes, abbreviated as Lipo-PDRN.

[0028] Comparative Example 4: Based on the Lipo-PDRN@SCS prepared in Comparative Example 1, the following operations were performed: Hydrolyzed collagen peptide stock solution (final concentration 2 mg / mL) was added to the Lipo-PDRN@SCS suspension. Then, a mixed aqueous solution of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) (final concentrations of 0.5 mg / mL and 0.25 mg / mL, respectively) was added. The reaction system was adjusted with 0.1 M MES buffer (pH 6.0). The reaction was carried out with gentle stirring at room temperature in the dark for 4 hours. After the reaction, the mixture was purified three times by ultrafiltration centrifugation at 4000×g using a 100 kDa molecular weight cutoff tube at 4°C to completely remove unreacted cross-linking agents and free peptides. Finally, the volume was adjusted with PBS to obtain Lipo-PDRN@SCS-ColP (EDC). A chemical cross-linking control group was used to compare the cross-linking strategies.

[0029] Effect Experiment: 1. Physicochemical characterization of nanoparticle raw materials (1) Determination of particle size, polydispersity index and zeta potential The Lipo-PDRN@SCS-TA-ColP samples prepared in Example 1, the Lipo-PDRN@SCS samples prepared in Comparative Example 1, and the Lipo-PDRN nanoparticle samples prepared in Comparative Example 3 were diluted with PBS to a suitable concentration (the suitable concentration is such that the count rate of the dynamic light scattering instrument is between 200-300 kcps). Measurements were performed at 25°C using a Malvern Zetasizer Nano ZS dynamic light scattering instrument. The Z-mean particle size and polydispersity index (PDI) were obtained using the "Size Measurement" mode; the Zeta potential was measured using the "ZetaPotential Measurement" mode. Each sample was automatically measured three times, and the results are expressed as mean ± standard deviation.

[0030] (2) Determination of encapsulation efficiency and drug loading Using dextran gel column chromatography, 200 μL of the Lipo-PDRN@SCS-TA-ColP sample prepared in Example 1 was precisely pipetted and loaded onto the top of a Sephadex G-50 column (1.0 × 30 cm) fully equilibrated with PBS. Elution was performed using PBS as the mobile phase at a constant flow rate of 0.5 mL / min, with fractions automatically collected. The elution curve was monitored at 259 nm using an online UV detector. The eluent from the first elution peak (nanoparticle peak) was combined, and the PDRN concentration in the eluent was determined by high performance liquid chromatography (HPLC), which was the amount of encapsulated drug. An equal volume of the original sample was taken, and 1% (v / v) Triton X-100 was added for demulsification, followed by determination of the total drug amount using the same method.

[0031] HPLC conditions: Agilent 1260 system, ZORBAX SB-C18 column (4.6 × 150 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (15:85, v / v); flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 259 nm; injection volume: 20 μL.

[0032] Calculation formula: Encapsulation efficiency (%) = (Encapsulated drug amount / Total drug amount) × 100%; Drug loading (%) = (Drug loading / Total weight of nanoparticles) × 100%.

[0033] (3) Morphological observation by transmission electron microscopy Take 10 μL of the Lipo-PDRN@SCS-TA-ColP sample prepared in Example 1 and drop it onto the copper grid of the carbon support film. After standing for 1 min, use filter paper to absorb the excess liquid. Add 2% (w / v) phosphotungstic acid aqueous solution for negative staining for 1 min, absorb the staining solution, and air dry at room temperature. Observe and take images using a transmission electron microscope at an accelerating voltage of 100 kV.

[0034] The results are as follows Figure 1 As shown, when the DOTAP addition ratio is 5 mol%, the prepared Lipo-PDRN core exhibits suitable positive charge (Zeta potential: +24.7 ± 1.5 mV). Figure 1 Part A) and the highest drug encapsulation rate (68.5±2.3%) Figure 1 Part B of the middle section has an average particle size of 105.3 ± 3.2 nm and a polydispersity index (PDI) of 0.158. Figure 1 (Part C), therefore, the DOTAP addition ratio was selected as 5 mol% for subsequent assembly.

[0035] When the DOTAP addition ratio was 5 mol%, after SCS encapsulation and covalent crosslinking, the particle size of the resulting Lipo-PDRN@SCS-TA-ColP increased to 135.6±4.1 nm, the PDI was 0.175, and the Zeta potential reversed to a negative value (-29.1±1.8 mV). This charge reversal phenomenon confirmed that the negatively charged SCS shell had been successfully coated on the surface of the positively charged core. Figure 1 (Part DE in the middle).

[0036] Transmission electron microscopy observation results as follows Figure 2As shown, Lipo-PDRN@SCS-TA-ColP exhibits a typical core-shell structure, with a dark core of high electron density surrounded by a light-colored halo (shell). This clear core-shell structure, especially the presence of the shell, forms the physical basis for subsequent controlled drug release and enzyme-responsive behavior. The successful reversal of the zeta potential not only demonstrates the SCS shell coating but also foreshadows potential behavioral changes in the nanoparticles during subsequent transdermal processes (such as interactions with the stratum corneum).

[0037] (4) Stability evaluation Lipo-PDRN@SCS-TA-ColP suspension samples (PDRN-loaded nanoparticle group) prepared in Example 1 with the same PDRN concentration and PBS solution containing 0.1 mg / mL PDRN (free PDRN group) were taken respectively. The two groups of samples were stored at 4℃, 25℃ and 40℃ in the dark for 3 months. The PDRN content retention rate was measured at the end of 0, 1, 2 and 3 months. The free PDRN group was directly filtered and measured according to the above HPLC conditions. The nanoparticle group was demulsified and measured according to the above HPLC conditions.

[0038] The results are as follows Figure 3 As shown, the retention rate of free PDRN at all temperatures gradually decreased with prolonged storage time, with the most significant decrease at 40℃; while the nanoparticle group maintained a high retention rate at all temperatures, with a much smaller decrease than that of the free PDRN group. These results indicate that free PDRN has poor stability under conventional storage conditions, while the core-shell structure of this invention can effectively protect PDRN and significantly improve its storage stability.

[0039] (5) Verification of enzyme digestion activity of response unit The fluorescence substrate kinetics method was used to verify the activity of hydrolyzed collagen peptides cleaved by MMP-13. 1 mg / mL hydrolyzed collagen peptide solution (experimental group) and 20 ng / mL active MMP-13 were mixed in reaction buffer (50 mM Tris-HCl, 10 mM CaCl2, 0.05% Brij-35, pH 7.4) in black 96-well plates. An equimolar concentration of commercially available MMP-13-specific fluorescent substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) was used as a positive control, and an enzyme-free peptide solution was used as a negative control. The reaction plate was immediately placed in a 37℃ multi-mode microplate reader, and kinetics were monitored at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Fluorescence intensity was read every 5 min for 3 h. By comparing the fluorescence intensity trends of the experimental group and the positive control group over time, the activity of hydrolyzed collagen peptides cleaved by MMP-13 was indirectly quantitatively verified.

[0040] like Figure 4 As shown, under the condition of containing 20 ng / mL active MMP-13, the fluorescence intensity of the experimental group (hydrolyzed collagen peptide + MMP-13) showed a significant and continuous increase over time (the cumulative fluorescence intensity reached 15240±580 RFU after 3 hours), and its growth trend was consistent with that of the positive control group (commercial MMP-13 fluorescent substrate + MMP-13), and there was no statistically significant difference in the slope of the curves between the two groups (p>0.05).

[0041] In contrast, the fluorescence signal of the negative control group (hydrolyzed collagen peptides, without enzyme) remained at a low baseline throughout the monitoring period (fluorescence intensity of only 1250 ± 210 RFU after 3 hours). This result directly confirms that the cosmetic-grade hydrolyzed collagen peptides selected in this invention can be effectively recognized and cleaved by MMP-13, with enzymatic cleavage kinetics comparable to the standard substrate, fully meeting the functional requirements as a smart response unit. This provides a reliable molecular basis for the subsequent construction of an enzyme-triggered release system based on this peptide.

[0042] 2. Preparation of anti-aging serum (final product) The anti-aging serum, by weight percentage, consists of the following ingredients: 10% Lipo-PDRN@SCS-TA-ColP prepared in Example 1, 5% hydrolyzed Chlorella extract, 5% glycerin, 10% sodium hyaluronate solution, 1% panthenol, 0.8% phenoxyethanol, and the balance being deionized water; the concentration of the sodium hyaluronate solution is 0.2%.

[0043] Preparation process: Glycerin, sodium hyaluronate solution, panthenol, and hydrolyzed Chlorella extract were sequentially dissolved in an appropriate amount of deionized water at a low temperature of 4℃, and stirred until completely dissolved and homogeneous. Under continuous low-temperature stirring, Lipo-PDRN@SCS-TA-ColP suspension raw material was slowly added to ensure uniform mixing. Finally, preservatives were added, and deionized water was added to the total volume. The mixture was stirred at low speed at 4℃ for 30 min. The pH of the system was adjusted to 6.0-6.5 with dilute citric acid solution. After filtration through a 0.45 μm filter membrane, the mixture was filled into bottles to obtain the final product, denoted as Gel-Lipo-PDRN@SCS-TA-ColP.

[0044] Meanwhile, a blank matrix essence without Lipo-PDRN@SCS-TA-ColP nanoparticles was prepared, abbreviated as Gel-Blank; and a control essence containing ordinary liposome Lipo-PDRN was prepared, abbreviated as Gel-Lipo-PDRN.

[0045] 3. Stability evaluation of the final product, the serum. (1) Accelerated stability test The serum samples from each of the Gel-Lipo-PDRN@SCS-TA-ColP, Gel-Blank, and Gel-Lipo-PDRN groups were aliquoted and placed in constant temperature and humidity chambers at 4℃ (refrigeration), 25℃ (room temperature), and 40℃ (accelerated conditions), respectively. Samples were taken at the end of months 0, 1, 2, and 3 for the following tests: Appearance: Observe the color, transparency, and uniformity, and record whether there is any layering, sedimentation, or discoloration; pH value: Measured using a precision pH meter; Viscosity: Measured using a rotational viscometer at 25°C; Nanoparticle stability: Take an appropriate amount of sample, dilute it with deionized water, and use a Malvern Zetasizer NanoZS dynamic light scattering instrument to determine the particle size and PDI of the nanoparticles at 25℃. Stability of active ingredient content: PDRN was extracted from the essence using a solvent (such as methanol) extraction method. After centrifugation and filtration, the PDRN content was determined by the HPLC method described above, and the content retention rate (relative to the content on day 0) was calculated.

[0046] (2) Centrifugal stability test Take about 2 mL of the prepared Gel-Lipo-PDRN@SCS-TA-ColP essence sample and place it in a centrifuge tube. Centrifuge at 4000 rpm for 30 min and observe whether precipitation, layering or oil rings appear at the bottom of the tube to evaluate its physical stability.

[0047] The results are as follows Figure 5 As shown, a transparent and uniformly textured Gel-Lipo-PDRN@SCS-TA-ColP serum was successfully prepared; accelerated stability study results are as follows. Figure 5 As shown, after 3 months of storage at 4℃ and 25℃, the product's appearance, pH value (maintained at 6.0-6.5), viscosity, nanoparticle size, and PDI did not change significantly, and the PDRN content retention rate exceeded 95%, indicating that the product has good stability under normal storage conditions. When stored at 40℃ under accelerated conditions for 3 months, the product viscosity decreased slightly, but no stratification or precipitation occurred; the nanoparticle size increased slightly (about 10%), but the PDI remained less than 0.25, maintaining a monodisperse state.

[0048] Therefore, it is evident that the serum matrix formulation incorporating PDRN-loaded MMP-13 responsive nanoparticles provided by this invention (such as using sodium hyaluronate) can effectively protect the intelligent nanoparticles with complex core-shell structures, preventing their aggregation or structural damage, thus ensuring the product's shelf life. In centrifugation tests, no precipitation or stratification was observed in any of the samples, further confirming their excellent physical stability. These results demonstrate that this invention not only constructs functional nanoparticles but also successfully solves the key technical problem of their stable integration into actual cosmetic dosage forms, removing obstacles to industrial application.

[0049] 4. In vitro enzyme-responsive release experiment To verify the core intelligent function of the nanoparticle raw material—MMP-13-triggered drug release—and to systematically evaluate the necessity of each structural component, an in vitro release study was conducted using the dialysis bag method.

[0050] (1) Sample preparation Accurately measure the following nanoparticle sample suspensions containing an equal amount of PDRN (1 mg): Lipo-PDRN@SCS-TA-ColP (the core smart nanoparticle prepared in Example 1, with a DOTAP addition ratio of 5 mol%). Lipo-PDRN@SCS-TA (a non-responsive unit control prepared in Comparative Example 2, used to verify the necessity of collagen peptides). Lipo-PDRN@SCS-ColP (EDC) (a chemical crosslinking control group prepared in Comparative Example 4, used to compare crosslinking strategies). Lipo-PDRN (a shell-free control prepared in Comparative Example 3, used to evaluate the role of the shell in controlling burst release).

[0051] (2) Release test method Each of the above samples was placed in a pretreated dialysis bag (molecular weight cutoff 8-14 kDa) (boiled in deionized water for 5 min and equilibrated in release medium for 30 min). The dialysis bag was completely immersed in 100 mL of release medium, which was prepared as follows: The experimental media for Lipo-PDRN@SCS-TA-ColP and Lipo-PDRN@SCS-TA were Tris-HCl buffer (50 mM, pH 7.4, containing 5 mM CaCl2) containing active MMP-13 enzyme (20 ng / mL).

[0052] Lipo-PDRN@SCS-ColP (EDC) and Lipo-PDRN control group media: the same buffer without MMP-13 enzyme.

[0053] The entire release system was placed in a 37℃ constant-temperature water bath shaker and oscillated at a constant rate of 100 rpm. At preset time points (0.5, 1, 2, 4, 8, 12, 24, 48 h), all receiving media were collected, and fresh corresponding media of equal temperature and volume were immediately replenished. The collected samples were filtered through a 0.22 μm filter membrane, and the concentration of PDRN in the filtrate was determined according to the above HPLC conditions. The cumulative release rate was calculated, and release curves were plotted. All experiments were independently repeated three times (n=3), and results are expressed as mean ± standard deviation.

[0054] The results are as follows Figure 6 As shown, the release kinetics of different nano-formulations exhibited significant differences. Lipo-PDRN@SCS-TA-ColP demonstrated strictly MMP-13-dependent release. In MMP-13-containing media, its release curve exhibited a typical lag-rapid release characteristic: the cumulative release rate was 22.3±1.5% in the first 4 hours, then rapidly increased to 82.3±3.5% at 24 hours and 85.5±3.0% at 48 hours. In contrast, in the control medium without MMP-13, release remained slow throughout, with a cumulative release rate of only 28.3±2.5% at 48 hours. Its mechanism of action lies in the presence of MMP-13-specific cleavage sites (such as -Gly-Leu-) integrated into the shell layer of hydrolyzed collagen peptides. In a low-enzyme environment, the peptide, tannic acid, and SCS form a complete cross-linked network structure through multiple non-covalent interactions, which can effectively block the diffusion of the drug (PDRN). When in a high-MMP-13 environment, the enzymatic cleavage reaction destroys the peptide, leading to the disintegration of the cross-linked network and a sharp increase in shell permeability, thereby achieving rapid, triggered release of the drug.

[0055] The release curves of the control sample Lipo-PDRN@SCS-TA (without collagen peptides) highly overlapped under MMP-13 conditions (24-hour release rates were 83.5±3.2% and 83.3±3.1%, respectively), and its release rate was significantly faster than that of Lipo-PDRN@SCS-TA-ColP under enzyme-free conditions (4-hour release rate: 78.5±3.0% vs 16.8±1.3%). This comparison clearly demonstrates that the SCS shell cross-linked solely by tannins lacks MMP-13 responsiveness, and its release is simply passive diffusion; while the introduction of hydrolyzed collagen peptides not only acts as a responsive "smart switch," but also participates in cross-linking and synergistically forms a denser network structure with tannins, thus exhibiting stronger drug sealing ability under enzyme-free conditions. This reflects the "dual functional and structural role" played by the responsive unit in the system.

[0056] Lipo-PDRN@SCS-ColP (EDC) (chemical crosslinking control) also showed accelerated release in enzyme-containing media (78.5 ± 3.0% at 24 hours), but its release kinetics differed from that of Lipo-PDRN@SCS-TA-ColP; for example, its initial hysteresis phase might be shorter or the shape of the release rate curve might be different. This suggests that chemical crosslinking based on EDC / NHS may form a more rigid and homogeneous covalent network. Although it can also be cleaved by enzymes, its accessibility to enzymes and the disintegration mode of the network after cleavage may differ from that of the dynamic physical network based on tannic acid. This indirectly supports the potential advantages of the green crosslinking strategy using tannic acid in constructing an "enzyme-friendly" smart shell.

[0057] The shell-less Lipo-PDRN exhibited a severe burst release effect, with a release rate of 78.9±3.2% within 4 hours and as high as 92.1±2.5% within 24 hours. This highlights the fundamental role of shell construction in controlling drug burst release and maintaining an effective drug reservoir.

[0058] In summary, the Lipo-PDRN@SCS-TA-ColP nanoparticles constructed in this study successfully achieved the following effects: ① Precise response to the pathological microenvironment: The release behavior is precisely coupled with the concentration of MMP-13, a key biomarker of aging skin, enabling on-demand drug release at the target site and improving the precision and safety of intervention. ② Efficient "sealing-trigger" bidirectional regulation: The drug can be stably sealed in normal tissue (low MMP-13) for a long time, reducing delivery loss; at the target site (high MMP-13), it can respond rapidly and release a large amount of drug, thus simultaneously solving the problem of long circulation and high target site accumulation that traditional delivery systems cannot simultaneously address. ③ Significantly improved drug utilization: By inhibiting burst release and achieving target-triggered release, the effective local concentration and retention time of the active ingredient (PDRN) in the aging dermis can be significantly increased, providing a pharmaceutical basis for better anti-aging efficacy. ④ Excellent raw material safety and process feasibility: The core functional units (hydrolyzed collagen peptides and tannic acid) are all natural and approved cosmetic raw materials, and the preparation process is mild and green, with clear prospects for industrialization.

[0059] Therefore, this MMP-13 responsive smart nanoparticle changes the drug release mode from passive diffusion to active regulation by pathological signals, providing an innovative solution for developing next-generation efficient and precise transdermal anti-aging delivery systems.

[0060] 5. In vitro transdermal test This experiment used a Franz vertical diffusion cell (effective diffusion area 1.77 cm²). 2Using isolated full-thickness mouse dorsal skin (0.3-0.5 mm thick) as a transdermal model, the transdermal properties of the final product serum and its MMP-13 responsiveness were evaluated.

[0061] 200 mg of each of the following samples were weighed: Gel-Lipo-PDRN@SCS-TA-ColP, Gel-Blank+DiR (a blank matrix essence loaded with the fluorescent probe DiR), Gel-Lipo-PDRN, and PBS solution containing free DiR (all samples had the same DiR loading). The samples were evenly spread onto the stratum corneum of the skin. The receiving cell was filled with 15 mL of PBS pre-warmed to 37°C and divided into two groups: the experimental group contained active MMP-13 (20 ng / mL), and the control group contained no enzyme. After incubation at 37°C in the dark for 24 hours, all receiving solutions were collected. The fluorescence intensity of DiR was measured using a fluorescence spectrophotometer (excitation / emission wavelength: 748 / 780 nm). The concentration was calculated based on the standard curve and further converted to the cumulative transdermal dose per unit skin area (μg / cm²). 2 All experiments were independently repeated 3 times (n=3).

[0062] The results are as follows Figure 7 As shown, the serum product developed based on MMP-13-responsive smart nanoparticles can effectively respond to MMP-13 signals in the skin microenvironment under topical application conditions, significantly promoting the transdermal penetration of active ingredients. Gel-Lipo-PDRN@SCS-TA-ColP (smart serum) exhibits significant MMP-13-dependent transdermal behavior; under MMP-13-containing absorption conditions, its 24-hour cumulative transdermal absorption reaches as high as 25.8 ± 2.1 μg / cm³. 2 The transdermal absorption rate was significantly higher than that of the same product under enzyme-free conditions (8.5 ± 1.2 μg / cm³). 2 (p<0.001). The transdermal enhancement factor was approximately 3.0-fold. In contrast, the transdermal behavior of the ordinary liposome serum Gel-Lipo-PDRN was not affected by MMP-13, and its transdermal absorption rate showed no significant difference between enzyme-containing and enzyme-free conditions (enzyme content: 10.3 ± 1.5 μg / cm³). 2 Enzyme-free: 9.8 ± 1.4 μg / cm², p>0.05. Similarly, the transdermal transdermal transfusion rates of Gel-Blank + DiR (blank matrix) and the free DiR control group were not affected by the presence or absence of MMP-13. In a cross-sectional comparison, under conditions containing MMP-13, the transdermal transfusion rate of the intelligent serum was significantly higher than that of the ordinary liposome serum (25.8 vs 10.3 μg / cm²). 2(p<0.01), indicating a 2.5-fold increase in transdermal efficiency. Simultaneously, its transdermal absorption rate was significantly higher than the control serum (25.8 vs 5.2 μg / cm³). 2 (p<0.001) and the free DiR control group (25.8 vs 4.1 μg / cm). 2 (p<0.001). Under enzyme-free conditions, the transdermal penetration of all four groups of samples was at a low level with no statistically significant difference.

[0063] Therefore, it is evident that the intelligent anti-aging essence of this invention successfully transforms the enzymatic response characteristics of nanoparticles into functional advantages of the final product. Its enhanced transdermal efficiency does not stem from a simple permeation-enhancing effect, but rather from triggering nanoparticle shell dissociation and drug release in response to MMP-13 overexpression signals at aging sites of the skin. This creates a high concentration gradient locally, driving the drug to efficiently cross the skin barrier. This provides a reliable dosage form basis for achieving efficient enrichment of active ingredients at aging target sites.

[0064] 6. Cell experiments The potential toxicity of Lipo-PDRN prepared in Comparative Example 3 and Lipo-PDRN@SCS-TA-ColP nanoparticles prepared in Example 1 to human dermal fibroblasts and HaCaT keratinocytes was evaluated using the CCK-8 assay; cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. After 24 hours of culture and adherence, complete culture medium containing different concentrations (0, 20, 50, 100 μg / mL, based on lipid content) of Lipo-PDRN or Lipo-PDRN@SCS-TA-ColP was added to each well. A control well containing only complete culture medium was also set up (100% cell viability). Cells were cultured for another 24 and 48 hours. At the end of the specified time, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 1–4 hours. The absorbance was measured at 450 nm. The relative cell viability was calculated using the control wells as a reference. The experiment was independently repeated three times.

[0065] The results of the CCK-8 experiment are as follows: Figure 8 As shown, within the effective concentration range (20-100 μg / mL, based on lipids), Lipo-PDRN@SCS-TA-ColP and Lipo-PDRN treatment for 24 and 48 hours had no significant effect on the relative survival rate of human dermal fibroblasts and HaCaT keratinocytes (both >90%), demonstrating that both have good biocompatibility. Furthermore, the introduction of the smart shell composed of hydrolyzed collagen peptides and tannins did not introduce additional cytotoxicity, which provides a basic guarantee for the long-term external use safety of the product.

[0066] 7. Evaluation of anti-aging efficacy at the cellular level (1) Establishment of photoaging cell model When human dermal fibroblasts reach 80% confluence, they are gently washed with PBS, the PBS is removed, a small amount of fresh PBS is added to cover the cells, and they are placed under a UVB light source and irradiated once at a dose of 30 mJ / cm² to induce an in vitro photoaging model. Immediately after irradiation, the cells are replaced with complete culture medium and cultured for another 24 hours.

[0067] (2) Experimental grouping and treatment Control group: Normal control group, not irradiated; UVB group: Model control group, irradiated with UVB, without drug addition; Free PDRN group: UVB irradiation, with an equal amount of free PDRN added to the culture medium; Free TA (tannic acid) group: UVB irradiation, with an equal amount of free tannic acid added to the culture medium; PDRN+TA Mix group: PDRN+TA (tannic acid) physical mixture group, UVB irradiation, the culture medium is added with a mixture of free PDRN and free TA (tannic acid); Lipo-PDRN group: UVB irradiation, with the addition of Lipo-PDRN prepared in Comparative Example 3; Lipo-PDRN@SCS-TA-ColP group: UVB irradiation, with the addition of Lipo-PDRN@SCS-TA-ColP prepared in Example 1; In all the above-mentioned drug administration groups, the final concentration of PDRN was 20 μg / mL, and the cells in each group were treated for 48 hours.

[0068] (3) Detection of type I collagen expression (Western Blot) After the above treatment, cells were washed with pre-cooled PBS and lysed on ice with RIPA lysis buffer (containing protease inhibitors). The supernatant was collected by centrifugation, and protein concentration was determined using the BCA method. 30 μg of total protein was subjected to 10% SDS-PAGE electrophoresis, followed by wet transfer to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated overnight at 4°C with anti-type I collagen primary antibody (1:1000) and anti-GAPDH primary antibody (1:5000). After washing with TBST, the membrane was incubated at room temperature for 1 h with HRP-labeled secondary antibody and developed with ECL chemiluminescence buffer. ImageJ software was used to analyze the band gray values, and semi-quantitative analysis was performed using the ratio of type I collagen to GAPDH gray values.

[0069] Western Blot results are as follows: Figure 9As shown, UVB irradiation significantly downregulated type I collagen expression in the model control group, and all treatment groups were able to reverse collagen degradation to varying degrees. The collagen expression level in the free TA group was significantly higher than that in the Model group (p<0.05), confirming that tannic acid itself has collagen-promoting activity; the free PDRN group was also significantly higher than that in the Model group (p<0.05), and the collagen expression level in the PDRN+TA Mix group was further increased, significantly higher than that of either alone (p<0.05), indicating a physical synergistic effect between PDRN and TA; the collagen expression level in the Lipo-PDRN group was not significantly different from that in the PDRN+TA Mix group (p>0.05).

[0070] Importantly, in a pathological microenvironment created by UVB irradiation and accompanied by elevated levels of endogenous MMP-13, the Lipo-PDRN@SCS-TA-ColP group exhibited the most significant collagen synthesis-promoting effect. Its collagen expression level was not only significantly higher than the model control group (p<0.001), but also significantly superior to the free PDRN group and the ordinary liposome Lipo-PDRN group (p<0.01). This result strongly suggests that the smart nanoparticles of this invention can respond to and utilize the overexpressed MMP-13 signal of photoaged cells to achieve more efficient and targeted drug delivery and release, thereby demonstrating superior efficacy over traditional delivery systems in the key anti-aging pathway of collagen synthesis.

[0071] (4) Detection of intracellular reactive oxygen species (ROS) levels The ROS levels of cells in the Control group, UVB group, free PDRN group, free TA group, PDRN+TA Mix group, Lipo-PDRN group, and Lipo-PDRN@SCS-TA-ColP group were detected using the DCFH-DA fluorescent probe method. After treatment, the culture medium was discarded, and the cells were washed with PBS. The DCFH-DA probe diluted to 10 μM with serum-free culture medium was added, and the cells were incubated at 37°C in the dark for 30 min. After thorough washing with PBS, the fluorescence intensity was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The relative fluorescence intensity was used to characterize the intracellular ROS level.

[0072] (5) Detection of superoxide dismutase (SOD) activity The SOD activity of cells in the Control group, UVB group, free PDRN group, free TA group, PDRN+TA Mix group, Lipo-PDRN group, and Lipo-PDRN@SCS-TA-ColP group was detected using the Solarbio BC5610-50T / 24S Superoxide Dismutase (SOD) Activity Assay Kit (WST-1 method). After cell collection and lysis, the supernatant was collected and the absorbance was measured at 450 nm according to the instructions. The SOD activity inhibition rate was calculated according to the formula and converted into SOD enzyme activity units per milligram of protein (U / mg prot).

[0073] The results are as follows Figure 10 As shown in Part A, UVB irradiation significantly increased the level of reactive oxygen species (ROS) in the cells of the model control group. Both the free TA group and the free PDRN group significantly reduced the ROS level (p<0.05), and the PDRN+TA Mix group was even more effective than either of them alone (p<0.05), indicating that PDRN and TA have a physical synergistic antioxidant effect. The Lipo-PDRN group was comparable to the PDRN+TA mixture group, while the Lipo-PDRN@SCS-TA-ColP group was significantly more effective than the PDRN+TA mixture group and the Lipo-PDRN group (p<0.01), indicating that the Lipo-PDRN@SCS-TA-ColP treatment could most effectively remove ROS and reduce it to a level close to that of the normal group.

[0074] Correspondingly, the activity of the key intracellular antioxidant enzyme, superoxide dismutase (SOD), was inhibited in the model group, while Lipo-PDRN@SCS-TA-ColP treatment most significantly restored and enhanced SOD activity. Figure 10 (As shown in Part B).

[0075] In summary, the PDRN-loaded MMP-13 responsive nanoparticles provided by this invention can not only target and promote the synthesis of structural proteins (collagen), but also efficiently deliver PDRN. At the same time, tannic acid and PDRN can exert a synergistic antioxidant effect, effectively improving the overall oxidative stress state of cells, that is, reducing oxidative damage and enhancing endogenous antioxidant defense.

[0076] The specific embodiments of the present invention disclosed above are only for illustrating the present invention. These specific embodiments do not describe all details exhaustively, nor do they limit the invention to only the described embodiments. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention.

Claims

1. A method for preparing PDRN-loaded MMP-13 responsive nanoparticles, characterized in that, The preparation method includes the following steps: S1: Dipalmitoylphosphatidylethanolamine, cholesterol, and 1,2-dioleoyl-3-trimethylammonium-propane were weighed and dissolved in anhydrous ethanol to form the organic phase; polydeoxyribonucleotides were weighed and dissolved in phosphate buffer preheated to 60°C to form the aqueous phase; the organic phase was injected into the aqueous phase at a uniform rate using a syringe pump under constant temperature magnetic stirring at 500 rpm, and stirring was continued for 30 min after injection to obtain a crude suspension; the obtained crude suspension was transferred to a high-pressure microfluidic homogenizer and homogenized 5 times under ice-water bath cooling at 150 MPa pressure; then, the liposomes were purified by centrifugation at 4°C and 4000×g for 20 min using ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa, repeated 3 times to obtain drug-loaded liposomes; S2: Weigh out the hydrolyzed collagen peptides and dissolve them in phosphate buffer to prepare a 5 mg / mL hydrolyzed collagen peptide stock solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4°C for later use. S3: The drug-loaded liposomes prepared in S1 were placed under magnetic stirring at 300 rpm and room temperature, and phosphate buffer containing 1 mg / mL chondroitin A sodium salt was added dropwise. After the addition was complete, stirring was continued for 30 min to form a primary complex based on electrostatic interaction. S4: Add the hydrolyzed collagen peptide stock solution prepared in S2 to the primary complex prepared in S3 to obtain a mixture; then, add freshly prepared tannic acid aqueous solution dropwise to a final concentration of 0.5 mg / mL, adjust the pH of the system to 7.4 using 0.1 M NaOH, and stir the reaction at 200 rpm, room temperature, and in the dark for 2 hours to obtain a mixed system; S5: The mixture obtained in S4 was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and centrifuged at 4℃ and 4000×g for 20 min. The filtrate was discarded and the precipitate was resuspended with phosphate buffer. This washing process was repeated 3 times. The mixture was then filtered through a 0.45 μm sterile filter membrane to obtain PDRN-loaded MMP-13 responsive nanoparticles. The hydrolyzed collagen peptides in S2 have a molecular weight of 1-3 kDa and are derived from fish scales.

2. The preparation method according to claim 1, characterized in that, The mixing ratio of dipalmitoylphosphatidylethanolamine, cholesterol and 1,2-dioleoyl-3-trimethylammonium-propane in S1 is as follows: the total lipid mass is kept at 100 μmol and the addition ratio of 1,2-dioleoyl-3-trimethylammonium-propane is 5 mol.

3. The preparation method according to claim 1, characterized in that, The mixing ratio of polydeoxyribonucleotides and phosphate buffer described in S1 is 10 mg: 9 mL.

4. The preparation method according to claim 1, characterized in that, The rate at which the organic phase is injected into the aqueous phase as described in S1 is 0.5 mL / min.

5. The preparation method according to claim 1, characterized in that, The mixing ratio of hydrolyzed collagen peptides to phosphate buffer in S2 is 5 mg: 1 mL.

6. The preparation method according to claim 1, characterized in that, The volume ratio of drug-loaded liposomes to phosphate buffer as described in S3 is 1:

4.

7. The preparation method according to claim 1, characterized in that, The final concentration of the hydrolyzed collagen peptide stock solution in the mixture described in S4 is 2 mg / mL; the concentration of the tannic acid aqueous solution is 10 mg / mL.

8. A PDRN-loaded MMP-13 responsive nanoparticle, characterized in that, The PDRN-loaded MMP-13 responsive nanoparticles were obtained using the preparation method described in any one of claims 1 to 7.

9. The application of the PDRN-loaded MMP-13 responsive nanoparticles of claim 8 in the preparation of anti-aging cosmetics.

10. An anti-aging serum, characterized in that, The anti-aging essence comprises, by weight percentage, the following components: 10% of the PDRN-loaded MMP-13 responsive nanoparticles as described in claim 8, 5% of hydrolyzed Chlorella extract, 5% of glycerin, 10% of sodium hyaluronate solution, 1% of panthenol, 0.8% of phenoxyethanol, and the balance being deionized water; the concentration of the sodium hyaluronate solution is 0.2%.

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