PDRN-loaded umbilical cord mesenchymal stem cell source nano-vesicle and preparation method thereof

Abstract

The invention discloses a PDRN-loaded umbilical cord mesenchymal stem cell source nano-vesicle and a preparation method thereof, and belongs to the technical field of biological medicines. The preparation method provided by the invention comprises the following steps: mixing an umbilical cord mesenchymal stem cell suspension with a PDRN solution, and carrying out freeze thawing treatment; incubating the mixed solution subjected to freeze thawing treatment; extruding the incubated mixed solution for multiple times by using a filter membrane; and collecting the extruded product, and separating and purifying to obtain the PDRN-loaded umbilical cord mesenchymal stem cell source nano-vesicle. According to the preparation method disclosed by the invention, the encapsulation efficiency of PDRN is remarkably improved, meanwhile, secondary damage of a traditional post-loading technology to a preformed vesicle structure is avoided, the membrane integrity and biological activity of a final product are better maintained, the yield is high, and the preparation process is rapid; the introduction of exogenous impurities is avoided from the source, and the high purity and biological safety of the obtained nano-vesicle component are ensured.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicle and its preparation method. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the fields of biomedical engineering and nanomedicine, small extracellular vesicles (sEVs) are considered promising novel drug delivery platforms due to their natural nanoscale size, low immunogenicity, good biocompatibility, and potential for delivery across biological barriers. However, the process of isolating and purifying them from cell culture supernatants faces significant technical bottlenecks. Currently mainstream methods such as ultracentrifugation, size exclusion chromatography, immunoaffinity capture, and polymer precipitation generally have unavoidable drawbacks, including physical damage to the vesicle structure due to high shear forces, difficulty in balancing separation purity and efficiency, introduction of exogenous impurities or antibody components, and complex, time-consuming, and difficult-to-standardize large-scale production. These limitations severely restrict the practical application and clinical translation of sEVs as a therapeutic carrier.

[0004] Meanwhile, bioactive macromolecules such as polydeoxyribonucleotides (PDRNs) with tissue repair functions face inherent delivery challenges when applied directly. Their large molecular weight limits their ability to penetrate biological barriers, and they are easily and rapidly degraded by nucleases in vivo, resulting in short in vivo half-lives and low bioavailability, which greatly limits their therapeutic efficacy.

[0005] To overcome the challenge of isolating natural sEVs, alternative strategies for preparing biomimetic nanovesicles through mechanical extrusion and other methods have been developed. However, existing strategies still have limitations in efficiently loading therapeutic molecules, especially nucleic acid substances like PDRN, into vesicles. Whether through endogenous loading via genetically engineered parental cells or exogenous loading via electroporation, sonication, or incubation of isolated vesicles, the processes are often cumbersome, have limited loading efficiency, are costly, and may adversely affect the integrity and bioactivity of the vesicle membrane. Therefore, developing a method for simultaneously and efficiently producing and loading PDRN into biomimetic vesicles is of great significance for advancing the development of related drug delivery systems. Summary of the Invention

[0006] In view of this, the present invention provides a PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicle and its preparation method. The present invention achieves efficient in-situ loading of PDRN during the nanovesicle preparation process by mixing umbilical cord mesenchymal stem cells with PDRN and then subjecting them to freeze-thaw treatment, incubation and multi-stage polycarbonate membrane extrusion in sequence, which simplifies the process steps and better maintains the integrity of the vesicle structure.

[0007] In a first aspect, the present invention provides a method for preparing PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles, comprising the following steps: The suspension of umbilical cord mesenchymal stem cells was mixed with PDRN solution and subjected to freeze-thaw treatment; The mixture after freeze-thaw treatment is incubated; The incubated mixture was squeezed multiple times using a filter membrane. The product after extrusion was collected, separated, and purified to obtain PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles.

[0008] Preferably, the umbilical cord mesenchymal stem cells are 4th to 9th generation cells; the solvent for the umbilical cord mesenchymal stem cell suspension is phosphate-buffered saline (PBS).

[0009] Preferably, the cell concentration in the umbilical cord mesenchymal stem cell suspension is 1×10⁻⁶. 6 ~4×10 6 The concentration of the PDRN solution is 5-15 mg / mL; the volume ratio of the umbilical cord mesenchymal stem cell suspension to the PDRN solution is 1:(0.5-1.5).

[0010] Preferably, the freeze-thaw treatment is performed 1 to 3 times, and each freeze-thaw treatment is performed by treating in liquid nitrogen for 2 to 5 minutes, followed by thawing in a water bath at 35 to 38°C for 2 to 5 minutes.

[0011] Preferably, the incubation is carried out at 22~32℃ for 15~30 minutes.

[0012] Preferably, the step of repeatedly pressing the incubated mixture with a filter membrane specifically involves: passing the incubated mixture through a filter membrane with decreasing pore size in multiple stages for repeated pressing.

[0013] Furthermore, the filter membranes with decreasing pore sizes sequentially include: a 5~10μm pore size filter membrane, a 0.4~1μm pore size filter membrane, and a 0.08~0.2μm pore size filter membrane; the number of compression cycles for each filter membrane stage is 3~10 times.

[0014] Preferably, the separation and purification is performed by ultracentrifugation.

[0015] Furthermore, the conditions for ultracentrifugation are: 100,000~120,000×g, 2~5℃, 60~90 minutes.

[0016] Furthermore, before ultracentrifugation, the product is allowed to stand for 5 to 20 minutes.

[0017] Secondly, the present invention provides PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles prepared by the above preparation method.

[0018] Thirdly, the present invention provides the application of the above-mentioned PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles in the preparation of drugs or cosmetics for promoting skin repair.

[0019] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) This invention creatively combines the drug loading process with the vesicle generation process by mixing umbilical cord mesenchymal stem cells with PDRN and then sequentially subjecting the mixture to freeze-thaw treatment, incubation, and multi-stage membrane extrusion. This integrated process utilizes freeze-thaw to promote the efficient internalization of PDRN into the cell, and then mechanically extrudes the entire cell into nanovesicles, thereby achieving in-situ encapsulation of PDRN at the source. This method significantly improves the encapsulation rate of PDRN, while avoiding secondary damage to the pre-formed vesicle structure caused by traditional post-loading techniques, better maintaining the membrane integrity and bioactivity of the final product; and it also offers high yield and rapid preparation.

[0020] (2) The freeze-thaw process used in this invention is a mild physical permeation method that does not require the addition of chemical permeation agents or transfection reagents, thus avoiding the introduction of exogenous impurities from the source and ensuring the high purity and biosafety of the obtained nanovesicle components. Combined with subsequent multi-stage polycarbonate membrane extrusion, the particle size distribution of the generated nanovesicles can be precisely controlled to obtain a uniformly sized vesicle population that meets the requirements for drug delivery, thereby improving the standardization of the product and batch-to-batch consistency.

[0021] (3) The PDRN-loaded nanovesicles prepared in this invention retain the bioactivity of vesicles derived from umbilical cord mesenchymal stem cells and possess the tissue repair and regeneration function of PDRN. PDRN is encapsulated within the vesicles and is effectively protected by the vesicle lipid bilayer, thereby significantly enhancing its resistance to nuclease degradation and prolonging its half-life and local retention time in vivo. Simultaneously, as a natural delivery carrier, the nanovesicles can promote the crossing of biological barriers and efficient uptake by target cells of PDRN, ultimately achieving synergistic therapeutic effects in applications such as skin repair. Attached Figure Description

[0022] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0023] Figure 1 This is the standard curve for protein content detection in the experimental examples of this invention; Figure 2 This is the transmission electron microscopy imaging result of PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles prepared in Example 1 of this invention; Figure 3 This is a graph showing the particle size distribution of PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles prepared in Example 1 of this invention. Figure 4 This refers to the cell migration rate results of the human skin fibroblast scratch repair experiment in the experimental examples of this invention; Figure 5 These are cell migration images from the human skin fibroblast scratch repair experiment in the experimental examples of this invention; Figure 6 This is a graph showing the absorbance values ​​at 450 nm for different culture days in the human keratinocyte CCK-8 proliferation experiment of the present invention. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] This invention provides a method for preparing PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles, comprising the following steps: The suspension of umbilical cord mesenchymal stem cells was mixed with PDRN solution and subjected to freeze-thaw treatment; The mixture after freeze-thaw treatment is incubated; The incubated mixture was squeezed multiple times using a filter membrane. The product after extrusion was collected, separated, and purified to obtain PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles.

[0026] The present invention first employs a freeze-thaw process to prepare a suspension of cells and PDRN. This process utilizes the physical stress generated during the rapid freezing and thawing of cells, resulting in a transient and reversible change in the permeability of the cell membrane lipid bilayer. This increased membrane permeability provides a direct physical channel for the passive diffusion of PDRN macromolecules into the cytoplasm, which is a prerequisite for achieving high-efficiency loading.

[0027] The subsequent incubation step provides a sufficient time window for the diffusion and distribution of PDRN molecules. Under suitable isothermal conditions, PDRN that has already passed through the cell membrane can further diffuse into the cytoplasm and may become stable through interactions with endogenous cellular substances, thus laying the foundation for subsequent steps.

[0028] Subsequently, a controlled mechanical shear force is applied to the incubated mixture through a filter membrane. As the cell suspension is forced through the micropores of the membrane, the cells experience enormous membrane tension, ultimately leading to the fragmentation and reorganization of the entire cell structure. During this dynamic process, the cell's membrane structure (including the plasma membrane and part of the endometrial system) is torn and reassembles, encapsulating a portion of the cytoplasmic contents (including internalized PDRN) to form uniformly sized, membrane-closed nanoscale vesicles. Essentially, this step reconstructs a complete, drug-loaded cell into a large number of drug-loaded nanoscale vesicles.

[0029] Finally, through separation and purification of the extrusion product, intact PDRN-loaded nanovesicles were selectively separated and enriched to obtain the final target product.

[0030] In an optional embodiment of the present invention, the umbilical cord mesenchymal stem cells are passage 4 to 9 cells. During in vitro culture, the biological characteristics of umbilical cord mesenchymal stem cells change with the number of passages. While lower passage cells exhibit stronger stemness, their proliferative activity, total cell count, and batch-to-batch stability may be insufficient. Higher passage cells are more prone to senescence, exhibiting slowed proliferation, functional decline, and morphological changes. Passage 4 to 9 cells are in the logarithmic growth phase, maintaining good proliferative activity and stem cell characteristics, and exhibiting a stable phenotype—an ideal range that ensures the cells used as raw materials have vigorous metabolic activity, thereby forming high-quality vesicles after extrusion.

[0031] In an optional embodiment of the present invention, the solvent of the umbilical cord mesenchymal stem cell suspension is phosphate buffered saline (PBS); the pH value and ionic strength of PBS are consistent with the human body's internal environment, which can maintain the integrity of the vesicle membrane structure to the greatest extent and avoid vesicle rupture or aggregation caused by improper osmotic pressure or pH, thereby maintaining the stability of nanovesicles.

[0032] In an optional embodiment of the present invention, the cell concentration in the umbilical cord mesenchymal stem cell suspension is 1×10⁻⁶.6 ~4×10 6 Cells / mL. Excessively high cell concentrations may lead to uneven ice crystal formation during freeze-thaw cycles, preventing cells within cell clumps from fully contacting PDRN, and causing filter blockage during subsequent compression, resulting in uneven shear force distribution and affecting vesicle uniformity. The concentration of the PDRN solution is 5-15 mg / mL; the volume ratio of umbilical cord mesenchymal stem cell suspension to PDRN solution is 1:(0.5-1.5), more preferably 1:(0.8-1.2); ensuring sufficient PDRN molecules are available for cell uptake to meet the loading required for the final nanovesicles to achieve an effective therapeutic dose. Insufficient PDRN content will result in inadequate loading, affecting subsequent biological efficacy. Excessively high PDRN content may significantly alter the system's osmotic pressure or viscosity, adversely affecting cell stability during freeze-thaw cycles; furthermore, due to limited loading capacity, excessively high content will also lead to raw material waste.

[0033] In an optional embodiment of the present invention, the freeze-thaw treatment is performed 1 to 3 times, with each freeze-thaw treatment consisting of: treatment in liquid nitrogen for 2 to 5 minutes, followed by thawing in a water bath at 35 to 38°C for 2 to 5 minutes. The rapid freezing process in liquid nitrogen causes the cell suspension to quickly cross the ice crystal formation point within a very short time, forming tiny ice crystals inside and outside the cell. These ice crystals physically pierce or disturb the phospholipid bilayer of the cell membrane, forming temporary, nanoscale pores or defects, thereby significantly increasing membrane permeability. Rapid rewarming melts the ice crystals, and some membrane pores may reclose due to membrane fluidity, but a large number of PDRN molecules have already diffused into the cytoplasm through the pores driven by the concentration gradient. The rapid temperature change cycle (freeze-thaw treatment) exacerbates the disturbance of the membrane structure.

[0034] In an optional embodiment of the present invention, the incubation is carried out at 22-32°C, preferably 25-30°C, and most preferably 30°C. At this temperature, cell membrane lipids are in a normal liquid crystal state with good fluidity, which is conducive to the further transmembrane transport of PDRN molecules that have entered the intermembrane space or partially penetrated, and their more uniform distribution in the cytoplasm. The incubation time is 15-30 minutes to allow PDRN to complete diffusion and distribution.

[0035] In an optional embodiment of the present invention, the step of repeatedly squeezing the incubated mixture through a filter membrane specifically involves: repeatedly squeezing the incubated mixture through a series of filter membranes with decreasing pore sizes. Further, the filter membranes with decreasing pore sizes sequentially include: a 5-10 μm pore size filter membrane, a 0.4-1 μm pore size filter membrane, and a 0.08-0.2 μm pore size filter membrane. The primary function of the first stage (5-10 μm) is to break up large cell clumps and cell aggregates, and to initially squeeze and deform and break down the cells, forming larger membrane structures. The second stage (0.4-1 μm) further refines the primary fragmentation products, forming submicron-sized membrane vesicles or liposome precursors. The third stage (0.08-0.2 μm) ultimately squeezes the membrane structure through pores close to the target vesicle size. Under enormous shear force, the membrane structure ruptures and self-assembles, forming uniformly sized, membrane-sealed nanovesicles. During this process, the cytoplasmic contents (including internalized PDRN) are encapsulated within. The number of compressions per filter membrane stage is 3 to 10, more preferably 4 to 8; multiple compressions ensure that all materials pass through the filter membrane fully and evenly, completing the complete size remodeling process and improving vesicle yield and uniformity.

[0036] This invention does not impose any special restrictions on the material of the filter membrane; any filter membrane commonly used in the field can be used. This invention preferably uses a polycarbonate filter membrane, whose pores are straight cylindrical with precise pore size, which is conducive to obtaining vesicles with uniform particle size.

[0037] In an optional embodiment of the present invention, the separation and purification is performed by ultracentrifugation. The ultracentrifugation conditions are: 100,000~120,000 × g, 2~5 °C, 60~90 minutes. Under the strong centrifugal force, the target vesicles will precipitate. Free PDRN, small molecule impurities, and some soluble proteins are mainly retained in the supernatant. Low temperature operation (2~5 °C, more preferably 4 °C) is used to inhibit vesicle degradation and microbial growth, and to maintain product stability.

[0038] In an optional embodiment of the present invention, the product is allowed to stand for 5-20 minutes before ultracentrifugation. The product after extrusion is complex in composition, containing target nanovesicles, incompletely broken organelles, large membrane fragments, and free PDRN. Short-term room temperature standing facilitates the natural sedimentation of some large, unstable aggregates or fragments, simplifying the subsequent ultracentrifugation step and preventing contamination of the vesicle precipitation. Simultaneously, after extrusion, the vesicle membrane undergoes significant deformation and shear force upon passing through the micropores; therefore, the newly formed vesicle membrane may be in a metastable or defective state. Standing allows phospholipid molecules to rearrange into a more stable and complete bilayer structure.

[0039] The present invention preferably includes a resuspension step after the ultracentrifugation step, preferably using an appropriate amount of PBS to resuspend the precipitate, thereby obtaining a PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicle suspension.

[0040] This invention also provides PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles prepared by the above-described method. The nanovesicles have an average particle size of 80-230 nm and a PDRN encapsulation efficiency higher than 15%.

[0041] The present invention also provides the application of the above-mentioned PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles in the preparation of drugs or cosmetics for promoting skin repair.

[0042] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0043] Example 1 This embodiment provides a method for preparing PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles.

[0044] (1) Resuscitate 5th generation umbilical cord mesenchymal stem cells at a concentration of 7000 cells / cm 2 The cells were seeded at a density in cell culture flasks and cultured in an incubator at 37°C and 5% CO2.

[0045] (2) When the cell confluence reaches 80%, digest and harvest the cells with trypsin, wash the cells three times with sterile phosphate-buffered saline (PBS), and finally adjust the cell concentration to 1×10⁻⁶ cells with PBS. 6 The concentration of cells / mL was increased to obtain a suspension of umbilical cord mesenchymal stem cells.

[0046] (3) The cell suspension was mixed with a PDRN solution with a concentration of 15 mg / mL at a volume ratio of 1:1, so that the final concentration of PDRN in the mixture was 7.5 mg / mL. The mixture was placed in liquid nitrogen for 3 minutes, and then immediately transferred to a 37°C water bath for 3 minutes for rapid thawing, thus completing one freeze-thaw treatment.

[0047] (4) Place the freeze-thawed mixture at room temperature (25°C) and let it stand for 20 minutes.

[0048] (5) Using a squeezing device equipped with a polycarbonate membrane, the incubated mixture is continuously squeezed at room temperature. The mixture is drawn with a 1 mL syringe and squeezed 5 times through a polycarbonate filter membrane with a 5 μm pore size, then squeezed 5 times through a polycarbonate filter membrane with a 0.6 μm pore size, and finally squeezed 5 times through a polycarbonate filter membrane with a 0.1 μm pore size.

[0049] (6) Collect all extruded products and let them stand at room temperature for 10 minutes.

[0050] (7) Transfer the product after standing to an ultracentrifuge tube and centrifuge at 4°C and 100,000 × g for 70 minutes. Carefully discard the supernatant to remove free PDRN and soluble impurities.

[0051] (8) Gently resuspend the precipitate at the bottom of the tube with an equal volume of phosphate-buffered saline (PBS) to obtain a suspension of PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles (NV@PDRN).

[0052] Example 2 This embodiment provides a method for preparing PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles.

[0053] (1) Resuscitate 5th generation umbilical cord mesenchymal stem cells at a concentration of 7000 cells / cm 2 The cells were seeded at a density in cell culture flasks and cultured in an incubator at 37°C and 5% CO2.

[0054] (2) When the cell confluence reaches 80%, digest and harvest the cells with trypsin, wash the cells three times with sterile PBS, and finally adjust the cell concentration to 3×10⁻⁶ cells with PBS. 6 The concentration of cells / mL was increased to obtain a suspension of umbilical cord mesenchymal stem cells.

[0055] (3) The cell suspension was mixed with a PDRN solution with a concentration of 10 mg / mL at a volume ratio of 1:1, so that the final concentration of PDRN in the mixture was 5 mg / mL. The mixture was placed in liquid nitrogen for 3 minutes, and then immediately transferred to a 37°C water bath and allowed to stand for 3 minutes for rapid thawing. One freeze-thaw treatment was completed, and a total of two freeze-thaw treatments were completed.

[0056] (4) Place the freeze-thawed mixture at 30°C and incubate for 20 minutes.

[0057] (5) Using a squeezing device equipped with a polycarbonate membrane, the incubated mixture is continuously squeezed at room temperature. The mixture is drawn with a 1 mL syringe and squeezed 5 times through a polycarbonate filter membrane with a 5 μm pore size, then squeezed 5 times through a polycarbonate filter membrane with a 0.6 μm pore size, and finally squeezed 5 times through a polycarbonate filter membrane with a 0.1 μm pore size.

[0058] (6) Collect all extruded products and let them stand at room temperature for 15 minutes.

[0059] (7) Transfer the product after standing to an ultracentrifuge tube and centrifuge at 4°C and 120,000 × g for 60 minutes. Carefully discard the supernatant to remove free PDRN and soluble impurities.

[0060] (8) Gently resuspend the precipitate at the bottom of the tube with an equal volume of phosphate-buffered saline (PBS) to obtain a suspension of PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles.

[0061] Comparative Example 1 This comparative example provides a method for preparing PDRN-loaded nanovesicles, which employs a co-incubation followed by loading method, including the following steps: (1) Resuscitate 5th generation umbilical cord mesenchymal stem cells at a concentration of 7000 cells / cm 2 The cells were seeded at a density in cell culture flasks and cultured in an incubator at 37°C and 5% CO2.

[0062] (2) When the cell confluence reaches 80%, digest and harvest the cells with trypsin, wash the cells three times with sterile PBS, and finally adjust the cell concentration to 1×10⁻⁶ cells with PBS. 6 The concentration of cells / mL was increased to obtain a suspension of umbilical cord mesenchymal stem cells.

[0063] (3) Using an extrusion device equipped with a polycarbonate membrane, the mixture after freeze-thaw treatment is continuously extruded at room temperature. The mixture is drawn with a 1 mL syringe and extruded 5 times through a polycarbonate filter membrane with a 5 μm pore size, then 5 times through a polycarbonate filter membrane with a 0.6 μm pore size, and finally 5 times through a polycarbonate filter membrane with a 0.1 μm pore size.

[0064] (4) Collect all the extruded product and let it stand at room temperature for 10 minutes. Transfer the product after standing to an ultracentrifuge tube and centrifuge at 4°C and 100,000 × g for 70 minutes. Carefully discard the supernatant and gently resuspend the precipitate at the bottom of the tube with an equal volume of phosphate-buffered saline (PBS) to obtain an empty nanovesicle (NV) suspension.

[0065] (5) The empty nanovesicle suspension was mixed with a PDRN solution with a concentration of 15 mg / mL at a volume ratio of 1:1, so that the final concentration of PDRN in the mixture was 7.5 mg / mL. The mixture was then incubated at 4°C for 12 hours.

[0066] (6) Transfer the co-incubated mixture to an ultracentrifuge tube and centrifuge at 4°C and 100,000 × g for 70 minutes to remove free PDRN. Carefully discard the supernatant and resuspend the precipitate with PBS to obtain PDRN-loaded nanovesicles.

[0067] Comparative Example 2 This comparative example provides a method for preparing PDRN-loaded nanovesicles using a membrane extraction-extrusion method, comprising the following steps: (1) Take the same amount of fifth-generation umbilical cord mesenchymal stem cells as in Example 1, digest and clean them, and then resuspend the cells in a hypotonic lysis buffer containing protease inhibitors.

[0068] (2) The cell suspension was subjected to two freeze-thaw cycles between liquid nitrogen and a 37°C water bath to fully lyse the cells, followed by sonication (300W power, 2s sonication, 2s interval, total duration 2 minutes).

[0069] (3) Centrifuge the lysate at 4°C and 800×g for 10 minutes and collect the supernatant; then centrifuge the supernatant at 4°C and 10000×g for 30 minutes and collect the supernatant; finally, centrifuge the supernatant at 4°C and 100000×g for 2 hours and discard the supernatant. The resulting precipitate is the preliminarily purified cell membrane component.

[0070] (4) Resuspend the cell membrane pellet in PBS and mix it with PDRN solution to achieve a final PDRN concentration of 7.5 mg / mL.

[0071] (5) Extrude this mixture using the same extrusion device and parameters as in Example 1.

[0072] (6) Transfer the extruded product to an ultracentrifuge tube and centrifuge at 4°C and 100,000 × g for 70 minutes to remove free PDRN. Carefully discard the supernatant and resuspend the precipitate with PBS to obtain PDRN-loaded nanovesicles.

[0073] Test case 1. Quality characterization of nanovesicles (1) Protein content detection: The protein concentration of the PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicle suspension from Example 1 was detected using the BCA protein detection kit, and the protein concentration was found to be 0.06 mg / mL. The protein content detection standard curve is shown in [reference needed]. Figure 1 .

[0074] (2) Electron microscopic morphological observation: 10 μL of PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicle suspension from Example 1 was dropped onto a copper grid and precipitated for 1 min. The floating liquid was then absorbed with filter paper. 10 μL of uranium acetate was dropped onto the copper grid and precipitated for 1 min. The floating liquid was then absorbed with filter paper. The mixture was dried at room temperature for several minutes. Electron microscopy was performed at 80 kV to obtain transmission electron microscopy (TEM) imaging results. The TEM results are as follows: Figure 2 As shown, it exhibits a typical cup-shaped or spherical membrane structure with clear boundaries, consistent with the morphological characteristics of exosome-like vesicles.

[0075] (3) Particle size and concentration detection: According to NTA (ZetaView) analysis, the particle concentration in the PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicle suspension sample of Example 1 was 8.3 × 10⁻⁶. 9Particles / mL, average particle size 125.3 nm. Particle size measurement results are shown below. Figure 3 .

[0076] 2. Calculation of PDRN encapsulation efficiency in nanovesicles The nanovesicle products were completely lysed using a lysis buffer to release the internal PDRN. The PDRN in the lysis buffer was quantified using a micro spectrophotometer to calculate the amount of PDRN loaded into the vesicles. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency = (Amount of PDRN loaded into the vesicles / Total initial PDRN) × 100%. The PDRN encapsulation efficiency of the nanovesicles was calculated. The calculation results are summarized in Table 1.

[0077] Table 1. Encapsulation efficiency of PDRN in nanovesicles of Examples 1-2 and Comparative Examples 1-2 serial number PDRN encapsulation rate serial number PDRN encapsulation rate Example 1 16.1% Comparative Example 1 3.2% Example 2 18.4% Comparative Example 2 2.3% As can be seen from Table 1, the embodiment adopts an in-situ extrusion process at the cellular level. After the cell suspension is directly co-incubated with the target nucleic acid, the cells are forced to efficiently encapsulate nucleic acid molecules during the generation of nanovesicles by physical extrusion, resulting in a high PDRN encapsulation rate.

[0078] 3. Nanovesicle efficacy testing (a) Human skin fibroblast scratch repair experiment (1) Cell pretreatment: P3 human skin fibroblasts in the logarithmic growth phase were digested into a single-cell suspension and subjected to 2×10 5 Cells were seeded per well in 12-well cell culture plates, with 2 mL of complete culture medium added to each well. When the cells reached 70-80% confluence, a scratch assay was performed.

[0079] (2) Scratch treatment: Scratch treatment with a 1000μL pipette tip.

[0080] (3) Washing: Aspirate the culture supernatant, wash 3 times with PBS to remove the cells that have been cut off.

[0081] (4) Group treatment: 2 mL of DMEM was added to the control group, and 1.5 mL of DMEM culture medium was added to each well of the experimental group, and 0.5 mL of the following sample was added respectively: (i) empty cell vesicles (NV, 8.3 × 10⁻⁶) prepared by the method of Comparative Example 1. 9 (ii) Free PDRN (with the same concentration as the effective PDRN concentration in NV@PDRN of Example 1), and (iii) a physical mixture of empty cell vesicles and free PDRN prepared by the method of Comparative Example 1 (NV+PDRN, with the NV concentration controlled at 8.3 × 10⁻⁶). 9(iv) Product prepared in Example 1 (NV@PDRN, 8.3 × 10⁻⁶ Particles / mL, PDRN concentration is the same as the effective PDRN concentration in NV@PDRN of Example 1) 9 (Particles / mL), and incubate in an incubator.

[0082] (5) Take photos: Take photos at 0h, 24h, 48h and 72h to observe cell migration.

[0083] (6) Results Analysis: The mean change in cell scratch area was calculated. Results are as follows: Figure 4 and Figure 5 As shown.

[0084] The scratch assay results showed that, compared with the blank control group, treatment with PDRN alone, NV alone, NV+PDRN and the product of Example 1 could significantly promote the migration of HDF cells, and the product of the present invention had the most significant cell migration promoting effect.

[0085] (II) Human keratinocyte CCK-8 proliferation assay (1) Cell pretreatment: P3 human keratinocytes in the logarithmic growth phase were digested into a solution with a concentration of 2.5 × 10⁻⁶. 4 Single-cell suspension of cells / mL. 120 μL of cell suspension was seeded into each well of a 96-well cell culture plate, resulting in 3000 cells per well. The control group received no cells, only 120 μL of culture medium.

[0086] (2) Group treatment: 30 μL of DMEM medium was added to the control group, and less than 30 μL of sample was added to each well of the experimental group: (i) empty cell vesicles (NV, 8.3 × 10⁻⁶) prepared by the method of Comparative Example 1. 9 (ii) Free PDRN (with the same concentration as the effective PDRN concentration in NV@PDRN of Example 1), and (iii) a physical mixture of empty cell vesicles and free PDRN prepared by the method of Comparative Example 1 (NV+PDRN, with the NV concentration controlled at 8.3 × 10⁻⁶). 9 (iv) Product prepared in Example 1 (NV@PDRN, 8.3 × 10⁻⁶ Particles / mL, PDRN concentration is the same as the effective PDRN concentration in NV@PDRN of Example 1) 9 (Particles / mL), and incubate in an incubator.

[0087] (3) Add CCK-8 working solution: After incubation for 0, 24, 48, 72, 96, 120, 144 and 168 h respectively, add 15 μL of CCK-8 solution to each well and incubate the culture plate in an incubator for 2 h.

[0088] (4) Absorbance measurement: The absorbance (OD) at 450 nm was measured using an ELISA reader. 450 ).

[0089] (5) Results analysis: OD of all holes 450 The blank group was subtracted from all results to correct for background absorbance from the culture medium and reagents. Experimental results are as follows: Figure 6 As shown.

[0090] Cell proliferation experiments confirmed that free PDRN, empty nanovesicles (NV), a physical mixture of NV and PDRN, and the product of Example 1 of this invention all promoted HDF cell proliferation. Among them, the product of Example 1 of this invention showed significantly better proliferation-promoting effects than the other groups.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles, characterized by, The method comprises the following steps: mixing the umbilical cord mesenchymal stem cell suspension with the PDRN solution and performing freeze-thaw treatment; incubating the mixture after the freeze-thaw treatment; performing multiple extrusions on the incubated mixture using filter membranes; separating and purifying the extruded product to obtain the PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles.

2. The production method according to claim 1, wherein The umbilical cord mesenchymal stem cells are cells of the 4th to 9th generation, and the solvent of the umbilical cord mesenchymal stem cell suspension is phosphate buffer.

3. The production method according to claim 1, wherein The concentration of the umbilical cord mesenchymal stem cell suspension is 1x10 6 4x10 6 The concentration of the PDRN solution is 5-15 mg / mL; and the volume ratio of the umbilical cord mesenchymal stem cell suspension to the PDRN solution is 1:(0.5-1.5).

4. The production method according to claim 1, wherein The freeze-thaw treatment is performed 1 to 3 times, each time for 2 to 5 minutes in liquid nitrogen and then for 2 to 5 minutes in a 35 to 38℃ water bath for thawing.

5. The production method according to claim 1, wherein The incubation is performed at 22 to 32℃ for 15 to 30 minutes.

6. The production method according to claim 1, wherein The multiple extrusions on the incubated mixture using filter membranes are performed by sequentially passing the incubated mixture through multiple filter membranes with decreasing pore sizes, wherein the filter membranes with decreasing pore sizes include, in sequence, filter membranes with pore sizes of 5 to 10μm, 0.4 to 1μm and 0.08 to 0.2μm, and the extrusion is performed 3 to 10 times for each filter membrane.

7. The production method according to claim 1, wherein The separation and purification are performed by ultracentrifugation under the following conditions: 100000 to 120000×g, 2 to 5℃, 60 to 90 minutes.

8. The production method according to claim 7, wherein Before the ultracentrifugation, the extruded product is allowed to stand for 5 to 20 minutes.

9. The PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles prepared by the method of any one of claims 1 to 8.

10. Use of the PDRN-loaded umbilical cord mesenchymal stem cell-derived nanovesicles of claim 9 in the preparation of a medicine or cosmetic for promoting skin repair.

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