Enzyme response type supramolecular PDRN-mini ECM liposome as well as preparation method and application thereof

Abstract

The invention provides an enzyme response type supramolecular PDRN-mini ECM liposome and a preparation method and application thereof.The enzyme response type supramolecular PDRN-mini ECM liposome comprises phospholipid, a membrane stabilizer, a supramolecular compound and an emulsifier, a water phase formed by the supramolecular compound and the emulsifier serves as an inner core, and the supramolecular compound is formed by PDRN and mini ECM through intermolecular acting force; the mini ECM is formed by self-assembly of collagen peptide, elastin and hyaluronic acid. The supramolecular PDRN-mini ECM liposome with uniform particle size and good stability is obtained through a microfluidic technology, the process is simple, the controllability is high, and amplification is easy.

Description

Technical Field

[0001] This invention belongs to the field of liposome preparation technology, specifically relating to an enzyme-responsive supramolecular PDRN-mini ECM liposome, its preparation method, and its applications. Background Technology

[0002] Liposomes, as a classic nanoparticle drug delivery system, are widely used to improve transdermal drug delivery efficiency due to their excellent biocompatibility and ability to encapsulate hydrophilic drugs. However, traditional liposomes (such as those prepared by thin-film dispersion and reverse-phase evaporation) often suffer from problems such as uneven particle size distribution, unstable encapsulation efficiency, and large batch-to-batch variability. More importantly, conventional liposomes lack intelligent responsiveness and cannot release drugs on demand after reaching the target site, which may lead to drug release in the superficial layers of the skin or premature clearance, making it difficult for them to penetrate deep into the dermis.

[0003] Polydeoxyribonucleotides (PDRNs) are polymers composed of deoxynucleotides that have been shown to possess a variety of biological activities, including promoting tissue repair, anti-inflammation, anti-apoptosis, and promoting collagen production, demonstrating great potential in the fields of dermatology and cosmetic medicine. However, PDRNs have a large molecular weight and strong hydrophilicity, making it difficult to penetrate the skin's stratum corneum barrier, resulting in low transdermal absorption efficiency and unsatisfactory bioavailability, which severely limits their application effects.

[0004] The extracellular matrix (ECM) is the microenvironment for cell survival, playing a crucial regulatory role in cell proliferation, differentiation, and migration. Mini ECMs can mimic the function of the natural ECM, providing essential support and signaling for skin cells. However, effectively combining PDRN with mini ECM components and precisely delivering it to the deep layers of the skin, while simultaneously responding to the overexpression of specific enzymes in the diseased skin microenvironment to achieve intelligent release, remains a pressing technical challenge in this field. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides an enzyme-responsive supramolecular PDRN-mini ECM liposome, its preparation method, and its applications. A supramolecular PDRN-mini ECM liposome with uniform particle size and good stability is obtained through microfluidic technology. This invention features a simple process, strong controllability, and easy scale-up. It can respond to hyaluronidase and matrix metalloproteinases in the skin microenvironment, achieving targeted drug release and significantly improving the transdermal permeability of PDRN.

[0006] The technical problem solved by this invention is achieved by the following technical solution: The first objective of this invention is to provide an enzyme-responsive supramolecular PDRN-mini ECM liposome, comprising the following components: Phospholipids, membrane stabilizers, supramolecular complexes, and emulsifiers, with an aqueous phase formed by the supramolecular complex and the emulsifier as the core, wherein the supramolecular complex is a supramolecular complex formed by PDRN and mini ECM through intermolecular forces; The mini ECM is formed by the self-assembly of collagen peptides, elastin, and hyaluronic acid.

[0007] Furthermore, the supramolecular PDRN-mini ECM liposome comprises the following components: 1-5% phospholipids, 20-40% membrane stabilizer, 0.2% supramolecular complex, 1-2% emulsifier, and the balance being deionized water.

[0008] Furthermore, the film stabilizer is composed of one or more polyols, wherein the polyols are selected from butanediol, 1,3-propanediol, 1,2-hexanediol, 1,2-pentanediol, and glycerol.

[0009] Furthermore, the phospholipid is selected from one of soybean lecithin, egg yolk lecithin, and hydrogenated lecithin.

[0010] Furthermore, the mass ratio of the PDRN to the mini ECM is 1:1 to 5:1.

[0011] Preferably, the mass ratio of the PDRN to the mini ECM is 1:1.

[0012] Furthermore, the mass ratio of the collagen peptides, elastin and hyaluronic acid is (14~17):(2~5):(1~2).

[0013] Furthermore, the emulsifier is selected from one or more of PEG-40 hydrogenated castor oil, polyglycerol-10 laurate, polyglycerol-10 myristate, polyglycerol-10 stearate, and polyglycerol-10 oleate.

[0014] Furthermore, the supramolecular PDRN-mini ECM liposomes have an average particle size of 40-60 nm, a polydispersity index (PDI) of less than 0.2, an absolute value of zeta potential greater than 30 mV, and an encapsulation efficiency of PDRN greater than 90%.

[0015] The second objective of this invention is to provide a method for preparing enzyme-responsive supramolecular PDRN-mini ECM liposomes, comprising the following steps: Phospholipids were dissolved in polyols to obtain the oil phase; PDRN and mini ECM were dissolved in deionized water in a certain ratio and incubated at 35-40°C to obtain a supramolecular complex; then an emulsifier was added and stirred until homogeneous to obtain an aqueous phase. The obtained oil phase and aqueous phase were injected into the microfluidic chip through injection pumps, respectively, so that the oil phase and aqueous phase were mixed in the chip and self-assembled to form liposome proemulsion; The liposome proemulsion flows out from the microfluidic chip outlet, and then the unencapsulated free PDRN, mini ECM and residual emulsifier are removed to obtain the supramolecular PDRN-mini ECM liposomes.

[0016] Furthermore, the flow rate ratio of the oil phase to the water phase when injected into the microfluidic chip is 1:1 to 1:5.

[0017] Furthermore, the total flow rate of the oil phase and water phase injected into the microfluidic chip is 4~12mL / min, and the injection temperature is controlled at 4±0.5℃.

[0018] Furthermore, the incubation conditions were 37°C for 1 hour of static incubation.

[0019] The third objective of this invention is to provide the use of enzyme-responsive supramolecular PDRN-mini ECM liposomes in the preparation of anti-inflammatory, anti-aging, or anti-allergic and antipruritic drugs for the skin.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention innovatively introduces a dual-response mechanism for hyaluronidase and matrix metalloproteinases into liposomes. When the liposomes penetrate into inflamed or aging skin areas, these areas highly express hyaluronidase and matrix metalloproteinases. Hyaluronic acid in the liposomes is degraded by hyaluronidase, and the mini ECM is cleaved by MMP-9, leading to the disruption of the liposome structure. This enables rapid and precise release of PDRN and mini ECM at the target site, significantly improving drug utilization efficiency and therapeutic efficacy. Compared to supramolecular PDRN liposomes, the addition of mini ECM enhances active targeting of the skin, interacts with skin cells, and improves skin penetration.

[0021] This invention pre-forms a supramolecular complex of PDRN and mini ECM, which not only improves the encapsulation efficiency and stability of PDRN in liposomes, but more importantly, the two can produce synergistic biological effects after release, achieving rapid release, prolonging the in vivo residence time, and exhibiting good biocompatibility.

[0022] This invention employs microfluidic technology to prepare liposomes, achieving precise control over liposome particle size, particle size distribution, and structure. The resulting liposomes exhibit uniform particle size, good stability, and high encapsulation efficiency. Furthermore, the production process is highly reproducible, facilitating large-scale production from laboratory to industrial applications and overcoming the drawbacks of traditional methods, such as significant batch-to-batch variations.

[0023] The uniform nanoscale particle size (40-60 nm) of this invention facilitates transdermal penetration. Simultaneously, the enzyme-responsive disruption of the liposome structure itself helps to break down the dense structure of the stratum corneum, thereby further promoting drug penetration.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0025] Figure 1 This is an appearance diagram of the supramolecular PDRN-mini ECM liposome in Example 1 of the present invention.

[0026] Figure 2 This is a transmission electron microscope image of the supramolecular PDRN-mini ECM liposomes of Example 1 of the present invention.

[0027] Figure 3 This is a comparison diagram of the in vitro enzyme response release of PDRN-mini ECM liposomes in Example 1 of the present invention with that in Comparative Examples 3 and 4.

[0028] Figure 4 These are skin permeation fluorescence images of Example 1 and Comparative Example 1 of the present invention at different times.

[0029] Figure 5 This is a comparison of the fluorescence intensity of skin penetration at different times between Example 1 and Comparative Example 1 of the present invention. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0031] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods. Example

[0032] A method for preparing enzyme-responsive supramolecular PDRN-mini ECM liposomes includes the following steps: Oil phase: Dissolve 2g of soybean lecithin and 20g of glycerol in 10g of butanediol and stir until completely dissolved to obtain the oil phase; Aqueous phase: Collagenase, elastin, and hyaluronic acid were prepared in a mass ratio of 16:3:1 to obtain miniECM for later use. 0.1g PDRN and 0.1g mini ECM were dissolved in 66.8g deionized water and incubated at 37℃ for 1h to allow them to form a supramolecular complex through intermolecular forces. Then, 1g PEG-40 hydrogenated castor oil was added and stirred evenly to obtain the aqueous phase. Microfluidic mixing: Using a microfluidic chip with a staggered herringbone mixing structure, the obtained oil and aqueous phases were injected into the microfluidic chip separately via injection pumps, with the temperature controlled at 4±0.5℃. This allowed the oil and aqueous phases to mix instantaneously within the chip's microchannels, self-assembling to form a liposome proemulsion. Purification: The obtained liposome proemulsion was flowed out from the microfluidic chip outlet and circulated through a 300 kDa tangential flow filter for 15 min to remove unencapsulated free PDRN, mini ECM and residual emulsifier, to obtain supramolecular PDRN-mini ECM liposomes.

[0033] Different embodiments were formed by adjusting the flow rates of the oil phase and the water phase, as detailed in Table 1.

[0034] Table 1 Comparative Example 1 A method for preparing a PDRN-mini ECM solution includes the following steps: Oil phase: Dissolve 2g of soybean lecithin and 20g of glycerol in 10g of butanediol and stir until completely dissolved to obtain the oil phase; Aqueous phase: Collagenase, elastin, and hyaluronic acid were prepared in a mass ratio of 16:3:1 to obtain miniECM for later use. 0.1g PDRN and 0.1g mini ECM were dissolved in 66.8g deionized water, and then 1g PEG-40 hydrogenated castor oil was added and stirred evenly to obtain the aqueous phase. Mixing: The oil phase is slowly added to the aqueous phase under high-speed stirring and stirring is continued to mix it evenly to obtain the PDRN-mini ECM solution.

[0035] Comparative Example 2 A method for preparing enzyme-responsive supramolecular PDRN-mini ECM liposomes includes the following steps: Oil phase: Dissolve 2g of soybean lecithin and 20g of glycerol in 10g of butanediol and stir until completely dissolved to obtain the oil phase; Aqueous phase: Collagenase, elastin, and hyaluronic acid were prepared in a mass ratio of 16:3:1 to obtain miniECM for later use. 0.1g PDRN and 0.1g mini ECM were dissolved in 66.8g deionized water and incubated at 37℃ for 1h to allow them to form a supramolecular complex through intermolecular forces, which was then used as the aqueous phase. Microfluidic mixing: Using a microfluidic chip with a staggered herringbone mixing structure, the obtained oil and aqueous phases were injected into the microfluidic chip at flow rates of 2 mL / min and 2 mL / min, respectively, using syringe pumps. The total flow rate was controlled at 4 mL / min, and the temperature was controlled at 4 ± 0.5 °C. This allowed the oil and aqueous phases to mix instantaneously within the chip's microchannels, self-assembling to form a liposome proemulsion. Purification: The obtained liposomal colostrum was flowed out from the microfluidic chip outlet and circulated through a 300 kDa tangential flow filter for 15 min to remove unencapsulated free PDRN and mini ECM, resulting in PDRN-mini ECM liposomes.

[0036] Comparative Example 3 A method for preparing enzyme-responsive supramolecular PDRN-mini ECM liposomes includes the following steps: Oil phase: Dissolve 2g of soybean lecithin and 20g of glycerol in 10g of butanediol and stir until completely dissolved to obtain the oil phase; Aqueous phase: 0.1g PDRN and 0.1g trehalose were dissolved in 66.8g deionized water and incubated at 37℃ for 1h to allow the supramolecular complex to be obtained through intermolecular forces; then 1g PEG-40 hydrogenated castor oil was added and stirred evenly to obtain the aqueous phase; Microfluidic mixing: Using a microfluidic chip with a staggered herringbone mixing structure, the obtained oil and aqueous phases were injected into the microfluidic chip at flow rates of 2 mL / min and 2 mL / min, respectively, using syringe pumps. The total flow rate was controlled at 4 mL / min, and the temperature was controlled at 4 ± 0.5 °C. This allowed the oil and aqueous phases to mix instantaneously within the chip's microchannels, self-assembling to form a liposome proemulsion. Purification: The obtained liposome proemulsion was flowed out from the microfluidic chip outlet and circulated through a 300 kDa tangential flow filter for 15 min to remove unencapsulated free PDRN, mini ECM and residual emulsifier, to obtain PDRN-mini ECM liposomes.

[0037] Comparative Example 4 A method for preparing enzyme-responsive supramolecular PDRN-mini ECM liposomes includes the following steps: Oil phase: Dissolve 2g of soybean lecithin and 20g of glycerol in 10g of butanediol and stir until completely dissolved to obtain the oil phase; Aqueous phase: Weigh 0.1g of a mixture of collagenase, elastin, and hyaluronic acid in a mass ratio of 16:3:1, then dissolve 0.1g of PDRN in 66.8g of deionized water, and then add 1g of PEG-40 hydrogenated castor oil and stir well to obtain the aqueous phase; Microfluidic mixing: Using a microfluidic chip with a staggered herringbone mixing structure, the obtained oil and aqueous phases were injected into the microfluidic chip at flow rates of 2 mL / min and 2 mL / min, respectively, using syringe pumps. The total flow rate was controlled at 4 mL / min, and the temperature was controlled at 4 ± 0.5 °C. This allowed the oil and aqueous phases to mix instantaneously within the chip's microchannels, self-assembling to form a liposome proemulsion. Purification: The obtained liposome proemulsion was flowed out from the microfluidic chip outlet and circulated through a 300 kDa tangential flow filter for 15 min to remove unencapsulated free PDRN, mini ECM and residual emulsifier, to obtain PDRN-mini ECM liposomes.

[0038] Comparative Example 5 A method for preparing enzyme-responsive supramolecular PDRN-mini ECM liposomes includes the following steps: Oil phase: Dissolve 2g of soybean lecithin and 20g of glycerol in 10g of butanediol and stir until completely dissolved to obtain the oil phase; Aqueous phase: Collagenase, elastin, and hyaluronic acid were prepared in a mass ratio of 16:3:1 to obtain miniECM for later use. 0.1g PDRN and 0.1g mini ECM were dissolved in 66.8g deionized water and incubated at 37℃ for 1h to allow them to form a supramolecular complex through intermolecular forces. Then, 1g PEG-40 hydrogenated castor oil was added and stirred evenly to obtain the aqueous phase. Microfluidic mixing: Using a microfluidic chip with a staggered herringbone mixing structure, the obtained oil and aqueous phases were injected into the microfluidic chip at flow rates of 2 mL / min and 12 mL / min, respectively, using a syringe pump. The total flow rate was controlled at 14 mL / min, and the temperature was controlled at 4 ± 0.5 °C. This allowed the oil and aqueous phases to mix instantaneously within the chip's microchannels, self-assembling to form a liposome proemulsion. Purification: The obtained liposome proemulsion was flowed out from the microfluidic chip outlet and circulated through a 300 kDa tangential flow filter for 15 min to remove unencapsulated free PDRN, mini ECM and residual emulsifier, to obtain PDRN-mini ECM liposomes.

[0039] Experimental Example 1 The liposomes obtained in each example and comparative example were analyzed using an Anton Paar laser particle size analyzer to determine the particle size, PDI, and Zeta potential. The PDRN encapsulation efficiency was determined using ultrafiltration centrifugation-UV spectrophotometry. The test results are shown in Table 2.

[0040] Table 2 As shown in Table 2, the microfluidic preparation method significantly improves encapsulation efficiency, particle size, PDI, and zeta potential compared to traditional methods. (Appendix) Figure 1 The image shown is an appearance diagram of Embodiment 1 of the present invention. The liquid is transparent and clear, with a pale blue opalescent color (significant Tyndall effect), and is uniformly dispersed without obvious aggregation or fusion. (Attached) Figure 2 The image shown is a transmission electron microscope image of Example 1 of the present invention. It exhibits a regular spherical or near-spherical structure with clear edges and typical lipid bilayer characteristics.

[0041] Compared with the conventional mixing method (Comparative Example 1), the microfluidic-prepared liposomes (Examples 1-5) have smaller particle size (40-60 nm), narrower particle size distribution (PDI < 0.2), higher absolute value of Zeta potential (> 30 mV) and higher PDRN encapsulation efficiency (> 90%), indicating that microfluidic technology can significantly improve the uniformity, stability and drug loading efficiency of liposomes.

[0042] Comparative Example 2 showed that the lack of emulsifiers led to increased particle size, increased PDI, decreased Zeta potential, and decreased encapsulation efficiency, confirming that emulsifiers are indispensable in maintaining the structural stability of liposomes and improving encapsulation efficiency.

[0043] Comparative Example 3 (trehalose replacing mini ECM) had a significantly larger particle size than Example 1, a slightly higher PDI, and a significantly lower encapsulation efficiency than Example 1. This indicates that the introduction of mini ECM helps to form smaller, more uniform liposome structures and is a key component in improving PDRN loading efficiency.

[0044] The encapsulation efficiency of Comparative Example 4 (unincubated) was lower than that of Example 1, and both the particle size and PDI increased, indicating that pre-incubation to form a supramolecular complex helps to improve the encapsulation efficiency of PDRN and the structural integrity of liposomes.

[0045] The flow rate ratio of the oil phase to the aqueous phase in the microfluidic process has a significant impact on the properties of liposomes. The optimal total flow rate range is 4-12 mL / min, and the flow rate ratio is preferably 1:1-1:5, indicating that the flow rate ratio has a regulatory effect on the liposome assembly process. Comparative Example 5 (total flow rate 14 mL / min, flow rate ratio 1:6) showed a significant increase in particle size, an increase in PDI, and a decrease in encapsulation efficiency, indicating that exceeding the optimal process range will lead to a decrease in liposome quality.

[0046] Experiment Example 2: In vitro release experiment In vitro release studies were conducted using ultrafiltration centrifugation. Equal amounts of supramolecular PDRN-mini ECM liposomes prepared in Comparative Examples 3, 4, and 1 were placed in different release media.

[0047] Grouped as follows: A. Enzyme-free PBS; B. PBS containing 1 μg / mL MMP-9; C. PBS containing 1 U / mL hyaluronidase; D. PBS containing 1 μg / mL MMP-9 and 1 U / mL hyaluronidase.

[0048] Each sample group was gently vortexed and incubated in a 37°C shaker for 2 hours, then immediately transferred to an ultrafiltration centrifuge tube (MWCO 300 kDa). The tubes were centrifuged at 3000×g for 30 minutes at room temperature, and the filtrate (i.e., the released PDRN) was collected. The cumulative release rate of PDRN was then determined.

[0049] See appendix Figure 3 In different release media, the supramolecular PDRN-mini ECM liposomes prepared in Example 1 exhibited significant enzyme-responsive drug release characteristics: in enzyme-free PBS, the cumulative release rate of PDRN was approximately 14.51% within 2 hours; in PBS containing 1 μg / mL MMP-9, the release rate increased to approximately 33.57%; in PBS containing 1 U / mL hyaluronidase, the release rate further increased to approximately 50.09%; and in the dual-enzyme medium containing both MMP-9 and hyaluronidase, the cumulative release rate exceeded 70% within 2 hours, significantly higher than the single-enzyme treatment group. These results indicate that MMP-9 can specifically degrade collagen peptides and elastin, while hyaluronidase can effectively cleave hyaluronic acid. The combined action of these two mechanisms can completely disrupt the structural integrity of the supramolecular complex, thereby causing a decrease in liposome membrane stability and promoting rapid PDRN release. This dual-enzyme response mechanism demonstrates a significant synergistic drug release effect, providing a key basis for the targeted and rapid drug release of the liposomes of this invention at skin lesions with upregulated enzyme activity due to inflammation and aging.

[0050] Comparative Example 3 replaced the core with a physical mixture of trehalose and PDRN. Trehalose is chemically stable and is not a substrate for matrix metalloproteinases or hyaluronidases. Therefore, the liposome core remains intact in the enzymatic environment and lacks the driving force for degradation. PDRN release depends solely on passive diffusion through the lipid bilayer, and its cumulative release rate is not significantly different from that in the enzyme-free medium.

[0051] Comparative Example 4 used the same components as Example 1, but without pre-incubation. The collagen peptides, elastin, hyaluronic acid, and PDRN in the core were merely physically mixed. Under these conditions, hyaluronidase and MMP-9 could only act on their respective dispersed substrates, resulting in isolated and incomplete degradation with limited impact on the overall core structure. Therefore, its PDRN release efficiency was significantly lower than in Example 1, and it failed to achieve the rapid and complete release effect resulting from the synergistic degradation of the supramolecular complex.

[0052] Experiment Example 3: In vitro skin penetration experiment The experiments were conducted using a Franz diffusion cell. PDRN was fluorescently labeled with fluorescein isothiocyanate (FITC), and fluorescently labeled supramolecular PDRN-mini ECM liposomes were prepared. Porcine back skin was used for the experiments. 1 mL of FITC-labeled supramolecular PDRN-mini ECM liposomes from Example 1 and the PDRN-mini ECM solution from Comparative Example 1 were added to the skin, and incubated for 4 h, 8 h, and 12 h. The skin was then harvested, wiped with physiological saline, and fixed in paraformaldehyde fixative for 4 h. The skin was then cut into uniformly thick slices using a cryostat, and the fluorescence distribution and intensity were observed and recorded using a fluorescence confocal microscope.

[0053] Comparative Example 1 was prepared using a traditional mixing process, which did not form liposome structures. The PDRN in the resulting PDRN-mini ECM solution existed primarily in free form. (See Appendix) Figure 4 The PDRN-mini ECM solution obtained in Comparative Example 1 showed weak fluorescence signals at 4 h, 8 h, and 12 h, and was always limited to the stratum corneum and epidermis, with overall dimness and blurred boundaries. In contrast, the supramolecular PDRN-mini ECM liposomes of Example 1 showed strong fluorescence intensity, with the fluorescence signal mainly concentrated in the stratum corneum and epidermis at 4 h, and gradually penetrating into the dermis at 8 h and 12 h as time progressed, with clear fluorescence images.

[0054] See appendix Figure 5 Table 3 shows that the fluorescence intensity of Example 1 at each time point was significantly higher than that of Comparative Example 1, increasing by approximately 1.74 times, 1.89 times, and 2.07 times at 4 h, 8 h, and 12 h, respectively. This indicates that liposome encapsulation can significantly enhance the skin penetration efficiency of PDRN. This is mainly attributed to the fact that the free PDRN molecules in Comparative Example 1 have a large particle size, making it difficult to effectively penetrate the stratum corneum, and lack a sustained-release carrier; while the present invention, by constructing supramolecular liposomes, utilizes the synergistic effect of mini ECM and PDRN to effectively reduce skin barrier resistance and significantly promote the transdermal delivery of PDRN.

[0055] Table 3. Fluorescence intensity of skin penetration at different time points The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0056] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An enzyme-responsive supramolecular PDRN-mini ECM liposome, characterized in that, Includes the following ingredients: Phospholipids, membrane stabilizers, supramolecular complexes, and emulsifiers, with an aqueous phase formed by the supramolecular complex and the emulsifier as the core, wherein the supramolecular complex is a supramolecular complex formed by PDRN and mini ECM through intermolecular forces; The mini ECM is formed by the self-assembly of collagen peptides, elastin, and hyaluronic acid.

2. The enzyme-responsive supramolecular PDRN-mini ECM liposome as described in claim 1, characterized in that: The film stabilizer is composed of one or more polyols, wherein the polyols are selected from butanediol, 1,3-propanediol, 1,2-hexanediol, 1,2-pentanediol, and glycerol.

3. The enzyme-responsive supramolecular PDRN-mini ECM liposome as described in claim 1, characterized in that: The phospholipid is selected from one of soybean lecithin, egg yolk lecithin, and hydrogenated lecithin.

4. The enzyme-responsive supramolecular PDRN-mini ECM liposome as described in claim 1, characterized in that: The mass ratio of PDRN to mini ECM is 1:1 to 5:

1.

5. The enzyme-responsive supramolecular PDRN-mini ECM liposome as described in claim 1, characterized in that: The mass ratio of collagen peptides, elastin and hyaluronic acid is (14~17):(2~5):(1~2).

6. The enzyme-responsive supramolecular PDRN-mini ECM liposome as described in claim 1, characterized in that: The supramolecular PDRN-mini ECM liposomes have an average particle size of 40-60 nm, a polydispersity index (PDI) of less than 0.2, an absolute value of zeta potential greater than 30 mV, and an encapsulation efficiency of PDRN greater than 90%.

7. The method for preparing an enzyme-responsive supramolecular PDRN-mini ECM liposome according to any one of claims 1-6, characterized in that, Includes the following steps: Phospholipids were dissolved in polyols to obtain the oil phase; PDRN and mini ECM were dissolved in deionized water in a certain ratio and incubated at 35-40°C to obtain a supramolecular complex; then an emulsifier was added and stirred until homogeneous to obtain an aqueous phase. The obtained oil phase and aqueous phase were injected into the microfluidic chip through injection pumps, respectively, so that the oil phase and aqueous phase were mixed in the chip and self-assembled to form liposome proemulsion; The liposome proemulsion flows out from the microfluidic chip outlet, and then the unencapsulated free PDRN, mini ECM and residual emulsifier are removed to obtain the supramolecular PDRN-mini ECM liposomes.

8. The method for preparing an enzyme-responsive supramolecular PDRN-mini ECM liposome as described in claim 7, characterized in that: The flow rate ratio of the oil phase to the water phase when injected into the microfluidic chip is 1:1 to 1:

5.

9. The method for preparing an enzyme-responsive supramolecular PDRN-mini ECM liposome as described in claim 7, characterized in that: The total flow rate of the oil and water phases injected into the microfluidic chip is 4~12 mL / min, and the injection temperature is controlled at 4±0.5℃.

10. Use of an enzyme-responsive supramolecular PDRN-mini ECM liposome according to any one of claims 1-6 in the preparation of anti-inflammatory, anti-aging, or anti-allergic and antipruritic drugs for the skin.

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