Stem cell-derived engineered nano-vesicle for promoting hair growth and preparation method of stem cell-derived engineered nano-vesicle

Abstract

The invention relates to stem cell-derived engineered nano-vesicles (e-NVs) for promoting hair growth, the engineered nano-vesicles are prepared from stem cells through engineering induction, apoptosis induction and programmed serial extrusion processes in sequence, and an inducer used for engineering induction is 3, 3 '-diindolylmethane. The particle size of the engineered nano vesicle is mainly distributed in a range of 50-300 nm, the polydispersity coefficient is 0.05-0.20, and the expression proportion of phosphatidylserine is 40%-50%. The rice vesicles can effectively improve the hair follicle microenvironment, adjust the hair growth cycle, promote proliferation and differentiation of hair follicle stem cells and remarkably increase the hair regeneration rate of androgenetic alopecia model mice, and a safe, convenient and efficient clinical strategy is provided for hair regeneration treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a stem cell-derived engineered nanovesicle for promoting hair growth and its preparation method. Background Technology

[0002] Hair loss is a common global disease characterized by hair loss. Androgenetic alopecia (AGA), the most common type of non-scarring hair loss, not only seriously affects patients' appearance but also has a significant negative impact on their mental health and social functioning, and has become an increasingly prominent public health problem.

[0003] Currently, the main clinical treatments for androgenetic alopecia include medication, surgery, and physical therapy, but these methods still have significant limitations. Topical medications, such as minoxidil, require continuous daily use, easily causing adverse reactions such as scalp itching and hirsutism, and some patients do not respond adequately. Finasteride can reduce dihydrotestosterone (DHT) levels in hair follicles by inhibiting type II 5α-reductase, but may cause systemic adverse reactions such as sexual dysfunction and mood swings, and hair is prone to fall out again after discontinuation. Low-intensity laser therapy requires long-term, regular irradiation, and its mechanism of action is not fully understood; efficacy also varies significantly among individuals. While autologous hair transplantation can improve local density, it is an invasive procedure, limited by donor site resources, expensive, and cannot improve the continuous degeneration of native hair follicles. In summary, existing treatment options still fall short of clinical needs in terms of safety, effectiveness, and long-term maintenance, necessitating the development of a safe, efficient, and patient-compliant new treatment strategy.

[0004] In recent years, stem cells and their exosomes have shown significant potential in hair regeneration research and have become a research hotspot in the field of biotherapy. Stem cell exosomes, carrying various bioactive molecules from the mother cell, can regulate the hair follicle microenvironment through paracrine effects, providing a novel approach to hair regeneration. However, conventional stem cell exosomes still face key bottlenecks in hair regeneration applications. For example, the content of hair regeneration-related factors in exosomes is low, and their targeting is insufficient, resulting in limited efficiency in activating the proliferation and differentiation of hair follicle stem cells. Simultaneously, the exosome preparation process suffers from low yield and difficulty in precisely controlling quality, hindering its large-scale production and clinical translation, and failing to fully meet the actual needs of hair regeneration therapy. Therefore, targeted engineering of stem cell vesicles and optimization of their preparation processes have become core directions for improving their hair regeneration efficacy and promoting clinical applications. Based on this, developing engineered stem cell nanovesicles (e-NVs) that are enriched with key hair regeneration factors, have a highly efficient and controllable preparation process, and exhibit excellent bioactivity has become a key breakthrough in overcoming the shortcomings of existing technologies. Summary of the Invention

[0005] Given the limitations of existing hair regeneration treatments, low levels of key regulatory signaling molecules in stem cell-derived vesicles, and uneven particle size distribution, this invention aims to provide a stem cell-derived engineered nanovesicle for promoting hair growth and its preparation method. This allows the vesicles to enrich key regulatory signaling molecules for hair regeneration and exert a highly efficient regeneration-promoting effect in the hair follicle microenvironment, thereby overcoming the shortcomings of existing treatments.

[0006] To address the aforementioned technical problems, this invention proposes engineered nanovesicles (e-NVs) derived from stem cells that promote hair growth. These e-NVs are engineered from mesenchymal stem cells through DIM (3,3'-diindolylmethane) induction, resulting in an enrichment of key regulatory signaling molecules for hair regeneration and a significant hair regeneration-promoting effect within the hair follicle microenvironment. Furthermore, the induced mesenchymal stem cells are processed using a tandem extrusion process, resulting in e-NVs with a more uniform particle size distribution.

[0007] The first aspect of the present invention provides a stem cell-derived engineered nanovesicles (e-NVs) for promoting hair growth. The engineered nanovesicles are prepared from stem cells through a sequential process of engineered induction, apoptosis induction, and programmed tandem extrusion. The inducing agent used for engineered induction is 3,3'-diindolemethane.

[0008] Preferably, the stem cells are selected from mesenchymal stem cells, induced pluripotent stem cells, epidermal stem cells, menstrual blood-derived stem cells, or umbilical cord blood-derived stem cells.

[0009] Preferably, the mesenchymal stem cells are selected from umbilical cord-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, or endometrial-derived mesenchymal stem cells. More preferably, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells. Even more preferably, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.

[0010] Preferably, the concentration of the inducer is 10-100 μM. More preferably, the concentration of the inducer is 25-75 μM. Even more preferably, the concentration of the inducer is 50 μM.

[0011] Preferably, the inducer is added to the stem cells in solution form, wherein the solvent is DMSO.

[0012] Preferably, each 1*106 -10*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 2*10 6 -9*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 3*10 6 -8*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4*10 6 -7*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4*10 6 -6*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4*10 6 -5*10 6 The amount of inducing agent added to each stem cell was 1 μmol.

[0013] Preferably, the apoptosis induction is induced by chemical substances, physical methods, or gene regulation.

[0014] Preferably, the chemical induction is hydrogen peroxide induction or asteroidin induction, and the physical induction method is ultraviolet irradiation induction.

[0015] Preferably, the intensity induced by the ultraviolet irradiation is 201000 mJ / cm. 2 More preferably, the intensity induced by the ultraviolet irradiation is 50500 mJ / cm. 2 More preferably, the intensity induced by the ultraviolet irradiation is 100-200 mJ / cm². 2 More preferably, the intensity induced by the ultraviolet irradiation is 100 mJ / cm². 2 .

[0016] Preferably, the ultraviolet irradiation induction time is 560 min. More preferably, the ultraviolet irradiation induction time is 1040 min. Even more preferably, the ultraviolet irradiation induction time is 2030 min. Even more preferably, the ultraviolet irradiation induction time is 20 min.

[0017] Preferably, the extrusion pressure of the tandem extrusion process is 0.21-0.0 MPa. More preferably, the extrusion pressure of the tandem extrusion process is 0.55-0 MPa. Even more preferably, the extrusion pressure of the tandem extrusion process is 0.52-0 MPa. Even more preferably, the extrusion pressure of the tandem extrusion process is 0.51-0 MPa.

[0018] Preferably, the tandem extrusion process involves sequentially passing through a large-pore porous membrane and a small-pore porous membrane. More preferably, it involves sequentially passing through a large-pore porous membrane, a medium-pore porous membrane, and a small-pore porous membrane.

[0019] Preferably, the pore size of the macroporous membrane is between 1 μm and 10 μm. More preferably, the pore size of the macroporous membrane is between 5 μm and 10 μm.

[0020] Preferably, the pore size of the mesopore membrane is between 0.5 μm and 5 μm. More preferably, the pore size of the mesopore membrane is between 1 μm and 2 μm.

[0021] Preferably, the pore size of the small-pore porous membrane is between 0.1 μm and 1 μm. More preferably, the pore size of the small-pore porous membrane is between 0.2 μm and 0.5 μm.

[0022] Preferably, the tandem extrusion process sequentially passes through a 5μm large-pore porous membrane, a 1μm medium-pore porous membrane, and a 0.4μm small-pore porous membrane.

[0023] Preferably, the engineered nanovesicles have an average particle size of 100-500 nm. More preferably, the engineered nanovesicles have an average particle size of 150-300 nm. Even more preferably, the engineered nanovesicles have an average particle size of 200-300 nm.

[0024] Preferably, the polydispersity index (PDI) of the engineered nanovesicles is 0.05-0.30. More preferably, the polydispersity index of the engineered nanovesicles is 0.05-0.25. Even more preferably, the polydispersity index of the engineered nanovesicles is 0.05-0.20.

[0025] Preferably, the expression rate of phosphatidylserine (PS) on the surface of the engineered nanovesicles is greater than 35%. More preferably, the expression rate of phosphatidylserine on the surface of the engineered nanovesicles is greater than 40%. Even more preferably, the expression rate of phosphatidylserine on the surface of the engineered nanovesicles is greater than 45%.

[0026] Preferably, the engineered nanovesicles have a morphology that is nearly circular or cup-shaped and possess a cell membrane-like structure.

[0027] A second aspect of the present invention provides a method for preparing the above-mentioned stem cell-derived engineered nanovesicles, the method comprising the following steps: (1) Use culture medium to culture stem cells. When the cell confluence reaches 70-90%, use 3,3'-diindolemethane solution to engineer the stem cells and obtain engineered MSCs. (2) Discard the culture supernatant of the engineered MSCs obtained in step (1), add serum-free culture medium, and induce apoptosis. Continue culturing after the induction is completed. (3) Collect the apoptosis-induced engineered MSCs obtained in step (2), and use a tandem extrusion device to perform programmed tandem extrusion of the suspension containing apoptosis-induced engineered MSCs to obtain the MSCs.

[0028] Preferably, the culture medium in step (1) is a DMEM / F12 medium containing 5% human platelet lysate (HPL).

[0029] Preferably, the stem cells in step (1) are selected from mesenchymal stem cells, induced pluripotent stem cells, epidermal stem cells, menstrual blood-derived stem cells, or umbilical cord blood-derived stem cells. More preferably, the mesenchymal stem cells are selected from umbilical cord-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, or endometrial-derived mesenchymal stem cells. Even more preferably, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells. Even more preferably, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.

[0030] Preferably, in step (1), when the cell confluence reaches 80-90%, the stem cells are engineered using a 3,3'-diindolemethane solution.

[0031] Preferably, the concentration of 3,3'-diindolemethane as an inducer in step (1) is 10-100 μM. More preferably, the concentration of the inducer is 25-75 μM. Even more preferably, the concentration of the inducer is 50 μM.

[0032] Preferably, in step (1), every 1*10 6 -10*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 2*10 6 -9*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 3*10 6 -8*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4*10 6 -7*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4*10 6 -6*10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4*106 -5*10 6 The amount of inducing agent added to each stem cell was 1 μmol.

[0033] Preferably, the apoptosis induction in step (2) is induced by chemical substances, physical methods, or gene regulation.

[0034] Preferably, the chemical induction is hydrogen peroxide induction or asteroidin induction, and the physical induction method is ultraviolet irradiation induction.

[0035] Preferably, the intensity induced by the ultraviolet irradiation is 201000 mJ / cm. 2 More preferably, the intensity induced by the ultraviolet irradiation is 50500 mJ / cm. 2 More preferably, the intensity induced by the ultraviolet irradiation is 100-200 mJ / cm². 2 More preferably, the intensity induced by the ultraviolet irradiation is 100 mJ / cm². 2 .

[0036] Preferably, the ultraviolet irradiation induction time is 560 min. More preferably, the ultraviolet irradiation induction time is 1040 min. Even more preferably, the ultraviolet irradiation induction time is 2030 min. Even more preferably, the ultraviolet irradiation induction time is 20 min.

[0037] Preferably, in step (2), the culture continues for 12-36 hours after induction. More preferably, in step (2), the culture continues for 16-32 hours after induction. Even more preferably, in step (2), the culture continues for 24 hours after induction.

[0038] Preferably, in step (2), after discarding the culture medium supernatant, the sample is washed with sterile PBS. More preferably, in step (2), after discarding the culture medium supernatant, the sample is washed 1-5 times with sterile PBS. Even more preferably, in step (2), after discarding the culture medium supernatant, the sample is washed 3 times with sterile PBS.

[0039] Preferably, the extrusion pressure of the tandem extrusion process in step (3) is 0.21-0.0 MPa. More preferably, the extrusion pressure of the tandem extrusion process is 0.55-0 MPa. Even more preferably, the extrusion pressure of the tandem extrusion process is 0.52-0 MPa. Even more preferably, the extrusion pressure of the tandem extrusion process is 0.51-0 MPa.

[0040] Preferably, the tandem extrusion process in step (3) involves passing the membrane through a large-pore porous membrane and a small-pore porous membrane in sequence. More preferably, it involves passing the membrane through a large-pore porous membrane, a medium-pore porous membrane, and a small-pore porous membrane in sequence.

[0041] Preferably, the pore size of the macroporous membrane is between 1 μm and 10 μm. More preferably, the pore size of the macroporous membrane is between 5 μm and 10 μm.

[0042] Preferably, the pore size of the mesopore membrane is between 0.5 μm and 5 μm. More preferably, the pore size of the mesopore membrane is between 1 μm and 2 μm.

[0043] Preferably, the pore size of the small-pore porous membrane is between 0.1 μm and 1 μm. More preferably, the pore size of the small-pore porous membrane is between 0.2 μm and 0.5 μm.

[0044] Preferably, the tandem extrusion process sequentially passes through a 5μm large-pore porous membrane, a 1μm medium-pore porous membrane, and a 0.4μm small-pore porous membrane.

[0045] A third aspect of the present invention provides stem cell-derived engineered nanovesicles obtained by the above preparation method.

[0046] The fourth aspect of the present invention provides the use of stem cell-derived engineered nanovesicles as described in the first or third aspect above in the preparation of medicaments for the prevention or treatment of hair loss or the promotion of hair regeneration.

[0047] Preferably, the hair loss is androgenetic alopecia, telogen effluvium, cicatricial alopecia, traction alopecia, lichen planus alopecia, or alopecia areata. Preferably, the dosage form of the drug is selected from injections, soluble microneedles, topical gels, or topical solutions.

[0048] The fifth aspect of the present invention provides the use of stem cell-derived engineered nanovesicles as described in the first or third aspect above in the preparation of a medicament that enhances the uptake capacity of dermal papilla cells.

[0049] The sixth aspect of the present invention provides the use of stem cell-derived engineered nanovesicles as described in the first or third aspect above in the preparation of a medicament for improving inflammatory damage to dermal papilla cells.

[0050] The beneficial effects of this invention are: This invention unexpectedly revealed that, compared to nanovesicles obtained through apoptosis induction alone and conventional exosomes, the nanovesicles obtained by sequentially treating stem cells with engineered induction (e.g., treatment with 3,3'-diindolemethane) and apoptosis induction (e.g., UV irradiation) can be more effectively taken up by dermal papilla cells (DPCs), exhibiting stronger protection against inflammatory damage to DPCs, and producing higher new hair coverage and faster hair regeneration speed in AGA mice. These experimental results confirm that engineered induction and apoptosis induction have a synergistic effect on the hair regrowth effect of stem cell-derived nanovesicles, significantly improving the efficiency, density, and stability of hair regeneration, providing a more efficient solution for the treatment of refractory hair loss such as androgenetic alopecia. Attached Figure Description

[0051] Figure 1 is a schematic diagram of the preparation process of the engineered stem cell nanovesicles (e-NVs) of the present invention; Figure 2 is a schematic diagram of cell viability detection described in step (3) of Example 2; Figure 3 shows the particle size distribution of e-NVs and NVs in this invention; Figure 4 is a transmission electron microscope image of the NVs of the present invention; Figure 5 is a transmission electron microscope image of the e-NVs of the present invention; Figure 6 shows the expression ratio of phosphatidylserine (PS) in e-NVs of this invention; Figure 7 shows the results of the in vitro anti-inflammatory ability test of the e-NVs of the present invention; Figure 8 shows the results of the quantitative uptake test of dermal papilla cells by the e-NVs of the present invention; Figure 9 shows the experimental results of the protective effect of the e-NVs of the present invention on inflammatory-damaged dermal papilla cells (DPCs); Figure 10 shows representative photographs of hair regeneration status in mice of each experimental group during treatment in Experiment 5; Figure 11 shows the time variation of the proportion of newly grown hair coverage area in each experimental group of mice in Experiment 5. Detailed Implementation

[0052] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope.

[0053] The experimental materials, reagents, and instruments used in the embodiments of this invention are all commercially available.

[0054] Example 1: Preparation and characterization of engineered stem cell nanovesicles (e-NVs) This embodiment provides a method for preparing e-NVs (see...). Figure 1 ), as detailed below: (1) Select human umbilical cord-derived mesenchymal stem cells (hU-MSCs), passage P3 cells continuously to P5, and use P5 MSCs for subsequent experiments. (2) The P5 generation MSCs obtained in step (1) were seeded into cell culture dishes containing DMEM / F12 medium supplemented with 5% human platelet lysate (HPL) and cultured in a carbon dioxide incubator until the cell confluence reached 80%-90%; (3) To step (2) containing 7*10 6 -8*10 6 Add 30 mL of 50 μM 3,3'-Diindolylmethane (DIM) to the culture medium of MSCs to induce MSCs for engineering and obtain engineered MSCs; (4) Discard the culture supernatant of the engineered MSCs obtained in step (3), wash three times with sterile PBS, add serum-free culture medium, and expose to ultraviolet light for 20 min to induce apoptosis (100 mJ / cm). 2 After induction, the cells were cultured for another 24 hours, and apoptotic engineered MSCs were collected using a cell scraper. (5) The apoptosis-engineered MSCs obtained in step (4) are extruded in sequence using a series extrusion device at a pressure of 0.5 MPa, and the cell suspension is extruded in sequence using porous membranes with pore sizes of 5 μm, 1 μm and 0.4 μm to obtain engineered stem cell nanovesicles (e-NVs).

[0055] The particle size distribution of the e-NVs prepared in this example was determined using a Malvern zetasizer at 25°C. The vesicle structure of the e-NVs was determined using a transmission electron microscope (TEM). The e-NVs were stained with the ANNEXIV / PI apoptosis staining kit in the dark for 15 min according to the instructions. The expression of phosphatidylserine (PS) on the surface of the e-NVs was analyzed using flow cytometry.

[0056] In this embodiment, the e-NVs particle size distribution results are as follows: Figure 3 As shown, the e-NVs have a particle size distribution of 50-300 nm and a PDI of 0.05-0.20, exhibiting high uniformity. Transmission electron microscopy (TEM) observations (see...) Figure 5The results showed that apoptotic vesicles exhibited a typical "cup-shaped" or "quasi-circular" morphology, with clearly visible membrane structures, indicating good vesicle integrity. The PS expression rate of e-NVs was 47.7% (see [link to original text]). Figure 6 This indicates that it can effectively retain obvious apoptotic vesicle characteristics, which is beneficial for macrophages to recognize and take up, thereby exerting their biological functions.

[0057] Comparative Example 1: Preparation and Characterization of Mesenchymal Stem Cell Nanovesicles (NVs) This comparative example provides the preparation and extraction method for NVs, as detailed below: (1) Select human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), passage P3 cells continuously to P5, and use P5 MSCs for subsequent experiments. (2) The P5 generation MSCs obtained in step (1) were seeded into cell culture dishes containing 5% human platelet lysate (HPL) DMEM / F12 medium and cultured in a carbon dioxide incubator until the cell confluence reached 80%-90%; (3) Discard the culture supernatant of the MSCs obtained in step (2), wash three times with sterile PBS, add serum-free culture medium, expose to ultraviolet light for 20 min to induce apoptosis, continue to culture for 24 h after induction, and collect apoptotic MSCs using a cell scraper. (4) The apoptotic MSCs obtained in step (3) are extruded in sequence using a tandem extrusion device at a pressure of 0.5 MPa, with membrane pore sizes of 5 μm, 1 μm and 0.4 μm, to obtain stem cell nanovesicles (NVs).

[0058] The particle size distribution of NVs was determined at 25°C using a Malvern zetasizer. The vesicle structure of the samples was determined using transmission electron microscopy (TEM). The samples were stained with the ANNEXIV / PI apoptosis staining kit in the dark for 15 min according to the instructions. The expression of phosphatidylserine (PS) on the surface of the test samples was analyzed using flow cytometry.

[0059] The particle size distribution results of the NVs prepared in this comparative example are as follows: Figure 3 As shown, the NVs have a particle size distribution of 50-300 nm and a PDI of 0.05-0.20, exhibiting high uniformity. Transmission electron microscopy (TEM) observations (see...) Figure 4 The results showed that apoptotic vesicles exhibited a typical "cup-shaped" or "quasi-circular" morphology, with clearly visible membrane structures, indicating good vesicle integrity. The PS expression rate of NVs was 44.2% (see...). Figure 6This indicates that it can effectively retain obvious apoptotic vesicle characteristics, which is beneficial for macrophages to recognize and take up, thereby exerting their biological functions.

[0060] Experimental Example 1: Screening Experiment for Engineering Drug Concentration in Mesenchymal Stem Cells 1. Test Methods Human umbilical cord mesenchymal stem cells (hUC-MSCs) in the logarithmic growth phase and in good growth condition were seeded in 6-well plates. 3,3'-diindolemethane (DIM) was dissolved in DMSO to prepare a DIM stock solution, which was then diluted with culture medium to the target dosage concentrations (0, 25, 50, 75, and 100 μM). When the stem cells reached 80%-90% aggregation, 30 mL of each of the target dosage concentrations of DIM solution was added to a medium containing 7*10... 6 -8*10 6 The mesenchymal stem cells were cultured in a culture medium for 24 hours. The treated mesenchymal stem cells were then digested and collected, and cell viability was assessed using AO / PI staining.

[0061] 2. Test Results Cell viability assay results after treatment with different DIM concentrations are shown in the figure. Figure 2 The experimental results showed that as the concentration of DIM increased, the viability of mesenchymal stem cells initially increased and then decreased, reaching its highest value at a DIM induction concentration of 50 μM. Therefore, a DIM concentration of 50 μM was selected as the induction concentration for the engineered apoptotic vesicles of this invention in subsequent experiments.

[0062] Experiment 2: Evaluation of the in vitro anti-inflammatory capacity of engineered stem cell nanovesicles (e-NVs) 1. Test Methods Using stem cell exosomes (Exo) and NVs as controls, the ability of e-NVs to regulate the inflammatory environment in vitro was evaluated by flow cytometry. The specific operation method is as follows: (1) e-NVs and NVs were prepared according to the preparation methods described in Example 1 and Comparative Example 1, respectively; the preparation method of Exo is as follows: take the culture supernatant of human umbilical cord mesenchymal stem cells, centrifuge at 300g and 2000g for 10 min to remove dead cells, take the supernatant and continue to centrifuge at 10000g for 30 min to remove large vesicles, collect the processed cell supernatant, and filter it through a 0.22μm filter membrane. The processed cell supernatant was transferred into an ultracentrifuge tube, centrifuged at 4℃ and 100000g for 70 min, the supernatant was removed, and the exosome precipitate was resuspended with an appropriate amount of PBS buffer.

[0063] (2) Take RAW 264.7 cells in the logarithmic growth phase and in good condition. Directly pipette the semi-adherent RAW 264.7 cells with pre-cooled PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the bottom cells in 1 mL of culture medium and count them, using 1×10⁻⁶ cells per cell. 5 Cells were seeded at a density of cells per well in 12-well cell culture plates; (3) After the cells adhered, the culture medium was discarded, and serum-free DMEM high-glucose medium with a lipopolysaccharide (LPS) concentration of 1 μg / mL was added to each well. The cells were then incubated at 37°C for 24 h to polarize the macrophages to the M1 phenotype. (4) After incubation, aspirate the culture medium containing LPS and replace it with fresh complete culture medium. Add drugs or controls according to the following groups: 1) PBS group; 2) Exo group; 3) NV group; 4) e-NVs group. The concentration of vesicle protein is 40 μg / mL. Incubate at 37℃ for 24 h.

[0064] (5) After incubation, the culture medium was aspirated, and the cells were washed three times with pre-cooled PBS. Then, the cells were directly pipetted off with pre-cooled PBS, centrifuged and resuspended, and 2 μL each of APC CD206 anti-mouse antibody and PE CD80 anti-mouse antibody were added. The cells were stained in the dark for 20 min, washed, and then CD80 was analyzed by flow cytometry. + CD206 + Macrophage level.

[0065] 2. Test Results The results of the in vitro anti-inflammatory ability of each experimental group are as follows: Figure 7 As shown, CD206 + / CD80 + Ratio analysis showed that the e-NVs group could effectively promote the repolarization of macrophages from the M1 phenotype to the M2 phenotype, and its ability to regulate the inflammatory environment in vitro far exceeded that of the NVs group and the Exo group, which were used as controls.

[0066] Experimental Example 3: Uptake of engineered stem cell nanovesicles (e-NVs) by dermal papilla cells (DPCs) 1. Test Methods Using Exo and NVs as controls, the uptake effect of e-NVs on dermal papilla cells was evaluated by flow cytometry. The specific procedure is as follows: (1) e-NVs and NVs were prepared according to the methods of Example 1 and Comparative Example 1, respectively; Exo was prepared according to the method of step (1) in Experimental Example 2. Take an appropriate amount of Exo, NVs and e-NVs, and incubate them with the above three types of cell exosomes / vesicles at 37°C in the dark for 30 min using cell membrane green fluorescent probe DiO (concentration of 10 μg / mL). After incubation, centrifuge at 40000g for 40 min at 4°C, discard the supernatant, add pre-cooled PBS and wash to remove excess dye, continue to centrifuge at 40000g for 40 min at 4°C, collect the precipitate, resuspend the vesicle precipitate with a certain amount of PBS to obtain the three types of DiO-labeled vesicles, and determine their concentration by protein content determination using a BCA kit.

[0067] (2) Take dermal papilla cells (DPCs) in the logarithmic growth phase and in good growth condition, digest adherent cells with 0.25% trypsin for 5 min, add complete culture medium to stop digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the bottom cells with 1 mL of culture medium and count them, and then count them at 1×10⁻⁶. 5 Cells were gently seeded into 12-well plates at a cell density of 1 cell per well. (3) After the cells adhere in step (2), discard the culture medium and add blank DMEM culture medium without FBS. Add drugs or controls according to the following groups: 1) PBS group; 2) Exo group; 3) NV group; 4) e-NVs group. The concentration of vesicle protein is 40 μg / mL. Incubate at 37℃ for 4 h.

[0068] (4) After incubation, adherent cells were digested with 0.25% trypsin for 5 min, digestion was stopped by adding complete culture medium, and cells were collected by centrifugation at 1000 rpm for 5 min. Then, the uptake efficiency of Exo, NVs and e-NVs was quantitatively detected by flow cytometry.

[0069] 2. Test Results The effective uptake of e-NVs by dermal papilla cells is an important prerequisite for them to exert their biological functions. To investigate the uptake capacity of dermal papilla cells for e-NVs, this experiment used the cell membrane green fluorescent probe DiO to stain and label Exo, NVs and e-NVs for tracing experiments, and the fluorescence intensity of DiO in dermal papilla cells after incubation for 4 hours was detected by flow cytometry.

[0070] The test results are as follows Figure 8 As shown, the uptake efficiency of e-NVs by dermal papilla cells was significantly higher than that of NVs and Exo. Since the effective uptake of vesicles by dermal papilla cells is an important prerequisite for their functional regulation, the above experimental results suggest that e-NVs can more efficiently regulate the function of dermal papilla cells, thus laying a key foundation for improving hair regeneration.

[0071] Example 4: Evaluation of the protective effect of e-NVs on inflammatory-damaged dermal papilla cells (DPCs) 1. Test Methods Using Exo and NVs as controls, the protective effect of e-NVs against inflammatory DPCs was evaluated by flow cytometry. The specific procedure is as follows: (1) e-NVs and NVs were prepared according to the methods of Example 1 and Comparative Example 1, respectively; Exo was prepared according to the method of step (1) in Experimental Example 2.

[0072] (2) Take DPCs in the logarithmic growth phase and in good growth condition, digest adherent cells with 0.25% trypsin for 5 min, add complete culture medium to stop digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the bottom cells with 1 mL of culture medium and count them, and then count them at 1×10⁻⁶. 5 Cells were gently seeded into 12-well plates at a cell density of 1 cell per well. (3) Take RAW 264.7 cells in the logarithmic growth phase and in good condition. Directly pipette the semi-adherent RAW 264.7 cells with pre-cooled PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the bottom cells in 1 mL of culture medium and count them, using 1×10⁻⁶ cells per cell. 5 Cells were seeded at a density of 1 cell per well in 12-well cell culture plates. After the cells adhered, the culture medium was discarded, and serum-free DMEM high-glucose medium with a lipopolysaccharide (LPS) concentration of 1 μg / mL was added to each well. The cells were then cultured at 37°C for 24 h to polarize the macrophages to the M1 phenotype. (4) Take the supernatant of the M1 phenotype macrophage culture medium from step (3), centrifuge at 2000g for 5min to remove cell debris, and obtain M1 macrophage conditioned medium (RAW Conditioned Medium, RAW-CM). (5) After the DPCs in step (2) adhere to the wall, discard the culture medium and divide into four groups for the experiment. Add DMEM high glucose medium containing 10% FBS to the blank control group. Add RAW-CM obtained in step (4) and DMEM high glucose medium containing 10% FBS to each well in a 1:1 ratio. Add drugs or controls according to the following groups: 1) Blank control (Control) group; 2) RAW-CM group; 3) RAW-CM+Exo group; 4) RAW-CM+NVs group; 5) RAW-CM+e-NVs group. The concentration of vesicle protein is 40 μg / mL. Incubate at 37℃ for 24 h.

[0073] (6) After incubation, wash DPCs three times with pre-cooled PBS, then digest DPCs with 0.25% trypsin for 5 min, add complete culture medium to stop digestion, centrifuge at 1000 rpm for 5 min to collect cells, stain with ANNEXIV / PI apoptosis staining kit in the dark for 15 min according to the instructions, wash cells and then analyze cell apoptosis level using flow cytometry.

[0074] 2. Test Results The results of the protective effect of e-NVs on inflammatory DPCs are as follows: Figure 9 As shown in the figure, the proportion of late apoptosis was observed in the e-NVs group, which was closest to the blank control group that was not placed in an inflammatory environment. Although the Exo group and NVs group, which served as controls, also showed some improvement, overall, e-NVs had the strongest protective effect against DPCs with inflammatory damage.

[0075] Experimental Example 5: Therapeutic Effect of Engineered Stem Cell Nanovesicles (e-NVs) on AGA Model Mice An AGA mouse model was established by applying testosterone (TES) topically daily. Mice were then divided into four groups: Model, Minoxidil, subcutaneous exosome injection (sc Exo), and subcutaneous engineered stem cell nanovesicles (sc e-NVs). Six mice were treated in each group. The Model group served as a blank control without treatment, while the Minoxidil group served as a positive control. 5% minoxidil solution (0.1 ml / cm²) was evenly applied to the hair-loss area on the back of the mice. Both the sc Exo and sc e-NVs groups received a single dose of 100 μg per mouse. Treatment began on day 1 after successful model establishment, with administration every two days for a total of five doses. The vesicles were subcutaneously injected into the hair-loss area using a 34G×1.5mm syringe. The 34G×1.5mm needle allowed for minimally invasive penetration of the stratum corneum, enabling rapid and precise follicle-targeting delivery of e-NVs. On day 14 post-treatment, mice in the Model group showed no hair regrowth, their skin remained pink, and hair growth was extremely limited. In contrast, both the Minoxidil and sc e-NVs groups exhibited significant hair regrowth, with the sc e-NVs group showing superior regrowth compared to the Minoxidil group. On day 21 post-treatment, except for the Model group, hair regrowth occurred in the Minoxidil, sc Exo, and sc e-NVs groups, but the sc e-NVs group showed the most significant hair regrowth. Figure 10 ).

[0076] Similar results were observed in the hair coverage of the mice in each experimental group. Figure 11 The sc e-NVs group showed significant hair coverage (11.7 ± 2.9%) as early as day 7. At day 14, the sc e-NVs group (31.8 ± 7.3%) had significantly higher new hair coverage than the Model group (1.3 ± 0.5%) and the sc Exo group (16.8 ± 1.8%). By day 21, hair coverage had increased in all experimental groups, with the sc e-NVs group showing the highest new hair coverage (63.5 ± 13.5%), followed by the Minoxidil group (61.6 ± 7.8%) and the sc Exo group (36.4 ± 4.7%), while the Model group (1.5 ± 0.5%) had the lowest.

[0077] Therefore, compared with the Model group, sc e-NVs treatment can significantly improve the new hair coverage and hair regeneration speed of AGA mice, and its effect is better than the positive control Minoxidil group and the vesicle control sc Exo group.

[0078] In summary, this invention systematically evaluated the effects of e-NVs on the biological function of damaged dermal papilla cells through the above-mentioned in vitro and in vivo experimental system. The results show that this strategy can not only significantly improve hair regeneration efficiency, but also restore the phenotype and biological function of damaged DPCs, overcoming the limitations of existing AGA treatment methods.

Claims

1. A stem cell-derived engineered nanovesicle for promoting hair growth, characterized in that, The engineered nanovesicles were prepared from stem cells through a sequential process of engineered induction, apoptosis induction, and programmed tandem extrusion. The inducing agent used in the engineered induction was 3,3'-diindolemethane.

2. The stem cell-derived engineered nanovesicles according to claim 1, characterized in that, Apoptosis induction can be induced by chemical substances, physical methods, or gene regulation.

3. The stem cell-derived engineered nanovesicles according to claim 1, characterized in that, The extrusion pressure of the tandem extrusion process is 0.2-10.0 MPa.

4. The stem cell-derived engineered nanovesicles according to claim 1, characterized in that, The tandem extrusion process involves sequentially passing the material through a large-pore porous membrane and a small-pore porous membrane.

5. The stem cell-derived engineered nanovesicles according to claim 1, characterized in that, The engineered nanovesicles have an average particle size of 100-500 nm.

6. The stem cell-derived engineered nanovesicles according to claim 1, characterized in that, The polydispersity index of the engineered nanovesicles is 0.05-0.

30.

7. The stem cell-derived engineered nanovesicles according to claim 1, characterized in that, The expression rate of phosphatidylserine on the surface of the engineered nanovesicles is greater than 35%.

8. The method for preparing stem cell-derived engineered nanovesicles according to any one of claims 1-7, characterized in that, The preparation method includes the following steps: (1) Stem cells were cultured using a culture medium. When the cell confluence reached 70-90%, the stem cells were engineered using 3,3'-diindolemethane solution to obtain engineered MSCs. (2) Discard the culture supernatant of the engineered MSCs obtained in step (1), add serum-free culture medium, and induce apoptosis. Continue culturing after the induction is completed. (3) Collect the apoptosis-induced engineered MSCs obtained in step (2), and use a tandem extrusion device to perform programmed tandem extrusion of the suspension containing apoptosis-induced engineered MSCs to obtain the MSCs.

9. The use of stem cell-derived engineered nanovesicles according to any one of claims 1-7 in the preparation of medicaments for the prevention or treatment of hair loss or the promotion of hair regeneration.

10. The application according to claim 9, characterized in that, The hair loss mentioned includes androgenetic alopecia, telogen effluvium, cicatricial alopecia, traction alopecia, lichen planus alopecia, or alopecia areata.

Images