An engineered stem cell nanocaspaosome, and a preparation method and application thereof

Abstract

The present application relates to a kind of engineered stem cell nanometer apoptotic bodies, its preparation method includes the following steps: by 3,3'-diindolylmethane directional induction obtains the engineered stem cell rich in repair and anti-inflammatory signal molecules, and then the engineered stem cell is induced to apoptosis, and the supernatant is separated and obtained by collection, then the programmed tandem extrusion process is handled to the engineered apoptotic body, and the particle size uniformity of the engineered nanometer apoptotic body is prepared.The engineered nanometer apoptotic body of the present application can effectively improve the microenvironment around hair follicle and regulate hair growth cycle, significantly improve the hair density and regeneration rate of androgenetic alopecia model mice, provide a new strategy of safe, convenient and efficient for clinical hair regeneration treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered stem cell nanoparticle, its preparation method, and its application. Background Technology

[0002] Hair loss is a prevalent skin appendage disease worldwide, characterized by progressive hair loss, which seriously threatens patients' physical and mental health and quality of life. Androgenetic alopecia (AGA), the most common non-scarring subtype of hair loss, is characterized by progressive miniaturization of hair follicles, thinning of hair shaft diameter, and gradual decrease in hair density. It not only significantly impairs patients' appearance and self-identity but can also trigger psychological problems such as anxiety and depression, becoming an increasingly prominent public health and medical challenge globally.

[0003] Currently, common clinical treatments for AGA mainly include drug therapy, surgical treatment, and physical intervention. However, each approach has clear limitations and is difficult to achieve ideal clinical efficacy: ① Topical minoxidil, as a first-line treatment, requires regular daily application (treatment cycle is usually ≥ 6 months), demanding high compliance and experiencing adverse reactions such as scalp itching, contact dermatitis, and hirsutism. Furthermore, some patients exhibit low treatment response rates due to individual differences; ② Oral finasteride reduces the concentration of dihydrotestosterone (DHT) in the hair follicle microenvironment by specifically inhibiting type II 5α-reductase activity, thereby alleviating the toxic damage of DHT to hair follicles. However, this drug may cause systemic adverse reactions such as sexual dysfunction, mood swings, and abnormal breast development. Furthermore, the miniaturization process of hair follicles is prone to relapse after discontinuation of the drug, and the safety of long-term use still needs close monitoring. ③ Low-intensity laser therapy (such as low-energy laser therapy) requires long-term regular irradiation. Its efficacy has significant individual heterogeneity, and its mechanism of action has not been fully elucidated. It is speculated that it may be related to improving local blood circulation in hair follicles, regulating oxidative stress and inflammatory response, but the specific regulatory pathway still needs further verification. ④ Although autologous hair transplantation (such as follicular unit extraction) can quickly improve local hair density, its essence is "hair redistribution," which is an invasive surgery. It is limited by the amount of hair reserve in the donor area and has risks such as donor area scar formation and postoperative infection. At the same time, the treatment cost is high, and it cannot stop or reverse the progressive miniaturization process of native hair follicles.

[0004] In summary, existing treatment options have shortcomings in terms of safety, efficacy stability, long-term maintenance effects, and patient compliance, making it difficult to meet the clinical needs for precise treatment of AGA. Therefore, developing novel treatment strategies that combine high safety, a clear mechanism of action, long-term stable effects, and good patient compliance has become a research hotspot and an urgent clinical need in the field of hair regeneration.

[0005] In recent years, stem cells and their derived vesicles (especially exosomes) have shown significant potential in hair regeneration research. However, the clinical translation of exosomes is still limited by problems such as low yield, complex isolation / purification processes, easy degradation of membrane structures in vitro / in vivo, and lack of unified quality control standards, which restrict their large-scale production and stable drug delivery.

[0006] As a special type of cell-derived vesicle, apoptotic bodies (ABs) have natural advantages in terms of yield and bioactivity loading. ABs are vesicles generated during programmed cell death and generally have higher extraction efficiency compared to exosomes. Simultaneously, the AB membrane surface is rich in phosphatidylserine (PS) and related immunomodulatory signals, theoretically beneficial for inducing repair-related immune-repair responses, making them a potentially "practical" vesicle source for industrialization. However, natural ABs also have key limitations hindering their direct use in hair follicle-targeted therapy: natural ABs have a wide particle size distribution (50nm-5μm), which is unfavorable for quality control and interaction with target cells; although natural ABs are rich in components, they contain relatively few factors related to hair regeneration, limiting their application in the field of hair regeneration. Based on these shortcomings, engineered processing of ABs has become a necessary technical approach.

[0007] Therefore, to address the inherent defects of natural apoptotic extracellular vesicles in existing technologies, such as poor particle size uniformity, insufficient specificity, and lack of efficiency, this invention provides an engineered nanoapoptotic vesicle (e-nABs) enriched with key regulatory signaling molecules for hair regeneration. These e-nABs can effectively enhance their regulatory role in hair follicle regeneration, thereby specifically compensating for the aforementioned technical defects of natural apoptotic extracellular vesicles and providing a novel biological agent with greater clinical translational potential and application prospects for the field of hair regeneration treatment. Summary of the Invention

[0008] Given the limited efficacy of existing hair regeneration drugs and the low content of key regulatory signaling molecules for hair regeneration in stem cell-derived vesicles, this invention aims to provide an engineered nanoapoptotic body, its preparation method, and its application, so as to achieve the enrichment of key regulatory signaling molecules for hair regeneration in vesicles and their efficient regeneration-promoting effect in the hair follicle microenvironment, thereby overcoming the shortcomings of existing treatment methods.

[0009] To address the aforementioned technical problems, this invention proposes an engineered stem cell nanoapoptotic body (e-nABs). Experimental results show that this engineered nanoapoptotic body can effectively improve the local skin microenvironment and promote hair regeneration.

[0010] The first aspect of the present invention provides an engineered stem cell apoptotic nanobody, which is prepared from stem cells by a process of engineering induction, apoptosis induction and differential centrifugation, wherein the inducing agent used in the engineering induction is 3,3'-diindolemethane.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] Preferably, the inducer is added to the stem cells in solution form, wherein the solvent is DMSO.

[0015] Preferably, each 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 stem cells... 6 -8×10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4 × 10⁴ stem cells... 6 -7×10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4 × 10⁴ stem cells... 6 -6×10 6 The amount of inducing agent added to each stem cell is 1 μmol. More preferably, per 4 × 10⁴ stem cells... 6 -5×10 6 The amount of inducing agent added to each stem cell was 1 μmol.

[0016] Preferably, the apoptosis induction is induced by chemical substances, physical methods, or gene regulation.

[0017] Preferably, the chemical induction is hydrogen peroxide induction or asteroidin induction, and the physical induction method is ultraviolet irradiation induction.

[0018] 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 .

[0019] 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.

[0020] Preferably, the differential centrifugation step involves sequentially removing intact cells and cell debris through low-speed centrifugation and medium-speed centrifugation, followed by harvesting engineered apoptotic bodies through ultracentrifugation.

[0021] Preferably, the low-speed centrifugation is 250-350g for 3-8 minutes, the medium-speed centrifugation is 1800-2200g for 8-12 minutes, and the ultra-speed centrifugation is 30000-60000g for 40-80 minutes.

[0022] Preferably, the low-speed centrifugation is 300g for 5 minutes, the medium-speed centrifugation is 2000g for 10 minutes, and the ultra-speed centrifugation is 40000g for 60 minutes.

[0023] Preferably, the differential centrifugal separation further includes a programmed tandem extrusion step.

[0024] Preferably, the extrusion pressure of the programmed tandem extrusion process is 0.21-0.0 MPa. More preferably, the extrusion pressure of the programmed tandem extrusion process is 0.55-0 MPa. Even more preferably, the extrusion pressure of the programmed tandem extrusion process is 0.52-0 MPa. Even more preferably, the extrusion pressure of the programmed tandem extrusion process is 0.51-0 MPa.

[0025] Preferably, the programmed 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.

[0026] 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 1 μm and 5 μm.

[0027] 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 0.5 μm and 2 μm.

[0028] 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.

[0029] Preferably, the programmed tandem extrusion process sequentially passes through a 3μm large-pore porous membrane, a 0.8μm medium-pore porous membrane, and a 0.4μm small-pore porous membrane.

[0030] Preferably, the engineered apoptotic nanoparticles have an average particle size of 100-500 nm. More preferably, the engineered apoptotic nanoparticles have an average particle size of 150-300 nm. Even more preferably, the engineered apoptotic nanoparticles have an average particle size of 200-300 nm.

[0031] Preferably, the polydispersity index (PDI) of the engineered apoptotic nanobody is 0.05-0.30. More preferably, the polydispersity index of the engineered apoptotic nanobody is 0.05-0.25. Even more preferably, the polydispersity index of the engineered apoptotic nanobody is 0.05-0.20.

[0032] Preferably, the positivity rate of phosphatidylserine (PS) on the surface of the engineered apoptotic nanobody is greater than 50%. More preferably, the positivity rate of phosphatidylserine on the surface of the engineered apoptotic nanobody is greater than 60%. Even more preferably, the positivity rate of phosphatidylserine on the surface of the engineered apoptotic nanobody is greater than 70%. Even more preferably, the positivity rate of phosphatidylserine on the surface of the engineered apoptotic nanobody is greater than 80%.

[0033] A second aspect of the present invention provides a method for preparing the above-mentioned engineered stem cell nanoparticles, the method comprising the following steps: (1) Use culture medium to culture stem cells. When the cell confluence reaches 80-90%, 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 culture supernatant obtained in step (2) and obtain engineered apoptotic bodies (e-ABs) by differential centrifugation. (4) Prepare a suspension from the engineered apoptotic bodies collected in step (3), and perform a programmed series extrusion of the suspension to obtain the final product.

[0034] Preferably, the culture medium in step (1) is a DMEM / F12 medium containing 5% human platelet lysate (HPL).

[0035] 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.

[0036] 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.

[0037] 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*10 6 -5*10 6 The amount of inducing agent added to each stem cell was 1 μmol.

[0038] Preferably, the apoptosis induction in step (2) is induced by chemical substances, physical methods, or gene regulation.

[0039] Preferably, the chemical induction is hydrogen peroxide induction or asteroidin induction, and the physical induction method is ultraviolet irradiation induction.

[0040] 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 .

[0041] 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.

[0042] 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.

[0043] Preferably, the differential centrifugation step (3) involves sequentially removing intact cells and cell debris by low and medium speeds, followed by harvesting e-ABs by ultracentrifugation.

[0044] Preferably, the low-speed centrifugation is 250-350g for 3-8 minutes, the medium-speed centrifugation is 1800-2200g for 8-12 minutes, and the ultra-speed centrifugation is 30000-60000g for 40-80 minutes.

[0045] Preferably, the low-speed centrifugation is 300g for 5 minutes, the medium-speed centrifugation is 2000g for 10 minutes, and the ultra-speed centrifugation is 40000g for 60 minutes.

[0046] Preferably, the extrusion pressure of the programmed tandem extrusion process in step (4) is 0.21-0.0 MPa. More preferably, the extrusion pressure of the programmed tandem extrusion process is 0.55-0 MPa. Even more preferably, the extrusion pressure of the programmed tandem extrusion process is 0.52-0 MPa. Even more preferably, the extrusion pressure of the programmed tandem extrusion process is 0.51-0 MPa.

[0047] Preferably, the programmed tandem extrusion process in step (4) 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.

[0048] 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 1 μm and 5 μm.

[0049] 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 0.5 μm and 2 μm.

[0050] 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.

[0051] Preferably, the tandem extrusion process sequentially passes through a 3μm large-pore porous membrane, a 0.8μm medium-pore porous membrane, and a 0.4μm small-pore porous membrane.

[0052] A third aspect of the present invention provides a pharmaceutical composition loaded with the above-described engineered stem cell nanoapoptotic bodies.

[0053] Preferably, the dosage form of the pharmaceutical composition is selected from injections, soluble microneedles, topical gels, or topical solutions.

[0054] The fourth aspect of the present invention provides the role of the above-described engineered stem cell nanoapoptotic bodies or the above-described pharmaceutical compositions in the preparation of medicaments for promoting hair regeneration or treating hair loss.

[0055] Preferably, the hair loss is androgenetic alopecia, telogen effluvium, cicatricial alopecia, traction alopecia, lichen planus alopecia, or alopecia areata. The beneficial effects of this invention are: 1. Active remodeling and anti-inflammatory regulation of the immune microenvironment: Utilizing the "eat me" signal of natural phosphatidylserine (PS) retained on the surface of e-nABs, local macrophages are actively driven to recognize and phagocytose them, inducing macrophage polarization from pro-inflammatory M1 type to anti-inflammatory M2 type (e.g., Figure 5 (As shown). This process significantly inhibits the chronic inflammatory state of local tissues by increasing the secretion of anti-inflammatory factors, creating a favorable immune microenvironment for hair follicle repair and regeneration, and solving the problem of hair follicles entering the growth phase due to persistent inflammation.

[0056] 2. Synergistic Multidimensional Biological Effects: This technology overcomes the limitations of existing techniques, such as insufficient activation efficiency and poor regeneration regulation of hair follicle stem cells. e-nABs can exert the core biological function of targeted regulation of hair follicle regeneration, precisely driving the transformation of hair follicles from the resting phase to the anagen phase at the molecular level. This synergistic mechanism of "hair follicle stem cell activation - growth cycle initiation" significantly accelerates the hair regeneration process. Attached Figure Description

[0057] Figure 1 A schematic diagram of the preparation method of the engineered nanoapoptotic bodies (e-nABs) of this invention.

[0058] Figure 2 This is a schematic diagram of cell viability detection in Experiment Example 1.

[0059] Figure 3 Transmission electron microscopy images of ABs and e-nABs prepared in Comparative Example 1 and Example 1.

[0060] Figure 4 PS flow cytometry results for ABs, Exo, and e-nABs prepared in Comparative Examples 1-2 and Example 1.

[0061] Figure 5 The image shows the uptake efficiency fluorescence quantification graphs for each experimental group in Experiment Example 2.

[0062] Figure 6 CD206 in each experimental group of Experiment Example 3 + / CD80 + Ratio bar chart.

[0063] Figure 7 The bar chart shows the percentage of late-stage apoptotic dermal papilla cells (DPCs) in each experimental group in Experiment Example 4.

[0064] Figure 8 Macroscopic photograph of e-nABs@MN prepared in Experimental Example 5.

[0065] Figure 9 This is a schematic diagram of the experimental procedure for the AGA mouse model in Experiment Example 6.

[0066] Figure 10 These are representative photographs of the hair regeneration status of AGA mice in each experimental group during treatment in Experiment 6.

[0067] Figure 11 This is a graph showing the time-varying proportion of newly grown hair coverage area in each experimental group of AGA mice in Experiment Example 6. Detailed Implementation

[0068] 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.

[0069] The experimental materials, reagents, and instruments used in the embodiments of this invention are all commercially available.

[0070] Example 1: Preparation and characterization of engineered nano-apoptotic bodies (e-nABs) This embodiment provides a method for preparing e-nABs (see...). Figure 1 The specific preparation method is as follows: (1) Human umbilical cord mesenchymal stem cells (hU-MSCs) in the logarithmic growth phase and in good growth condition were seeded in 150 mm cell culture dishes containing DMEM / F12 medium supplemented with 5% human platelet lysate (HPL); (2) When the cell confluence reaches 80%-90%, add 7*10 6 -8*10 6 Add 30 mL of 50 μM 3,3'-diindolemethane (DIM) to the culture medium of each MSC and induce culture for 24 h to obtain engineered mesenchymal stem cells (eMSCs). (3) Take the eMSCs obtained in step (2), aspirate the supernatant and replace it with serum-free DMEM / F12 medium, and expose it to ultraviolet light for 20 min to induce apoptosis (100 mJ / cm). 2 ); (4) After induction, continue culturing for 24 hours, collect the culture supernatant, and remove intact cells and cell debris step by step by centrifuging at 300g for 5 min and 2000g for 10 min at 4℃. Take the last supernatant and centrifuge at 40000×g for 60 min, discard the supernatant and collect the precipitate, which is the engineered apoptotic bodies (e-ABs). Resuspend in pre-cooled PBS for later use. (5) The e-ABs collected by differential centrifugation were resuspended in sterile PBS to prepare a 1-5 mg / mL suspension. The suspension was then extruded in sequence using a series extrusion device at a pressure of 0.5 MPa through porous membranes with pore sizes of 5 μm, 1 μm, and 0.4 μm to obtain engineered stem cell nanoapoptotic bodies.

[0071] The particle size and zeta potential of e-nABs were determined at 25°C using a Malvern zetasizer; vesicle structure was determined using transmission electron microscopy (TEM); and the ANNEXIV / PI apoptosis staining kit was used to stain the samples in the dark for 15 min according to the instructions. The expression of phosphatidylserine (PS) on the surface of the test samples was analyzed by flow cytometry.

[0072] The particle size potential results of e-nABs are detailed in Table 1. Compared with ABs, e-nABs have smaller and more uniform particle sizes; their potentials are basically the same, with no significant difference. Transmission electron microscopy (TEM) observation results (see...) Figure 3 The results showed that e-nABs exhibited a typical "cup-shaped" or "quasi-circular" morphology, with clearly visible membrane structures, indicating good vesicle integrity. PS expression ratio results showed that the expression ratio of phosphatidylserine (PS) was close to that of the ABs described in Comparative Example 1, and both were greater than 85% (see...). Figure 4 (As shown in Table 1), this result effectively verifies that the sample consists of apoptotic bodies generated by programmed cell death. These results indicate that drug-induced engineering and tandem extrusion operations have no significant effect on the physicochemical properties and surface protein expression of e-nABs.

[0073] Table 1. Particle size, potential, PDI and PS positivity rate of ABs, e-nABs and Exo (n=3) Comparative Example 1: Preparation and Characterization of Stem Cell Apoptotic Bodies This comparative example provides a method for preparing and extracting stem cell apoptotic bodies, the specific scheme of which is as follows: (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 placed in a carbon dioxide incubator for culture until the cell confluence reached 80%-90%; (3) Use ultraviolet irradiation (20 min, 100 mJ / cm) 2 Apoptosis was induced in the MSCs described in step 2), and the induction was continued for 24 h after the induction was completed. The culture supernatant was then collected. (4) The cells and cell debris were removed step by step by centrifuging at 300g for 5 min and 2000g for 10 min at 4℃. The last supernatant was centrifuged at 40000×g for 60 min to collect the apoptotic bodies (ABs) secreted in the culture supernatant obtained in step (3).

[0074] The particle size distribution and potential of ABs were determined at 25°C using a Malvern Zetasizer; the vesicle structure of the samples was determined using a transmission electron microscope (TEM); the samples were stained with the ANNEXIV / PI apoptosis staining kit in the dark for 15 min according to the instructions; and the expression of phosphatidylserine (PS) on the surface of the ABs was analyzed by flow cytometry.

[0075] The particle size, potential, and PDI of ABs in this comparative example are shown in Table 1. Transmission electron microscopy (TEM) observation results (see...) Figure 3 The results showed that the apoptotic bodies exhibited a typical "cup-shaped" or "round" morphology, with clearly visible membrane structures, indicating good integrity of the apoptotic bodies; the PS expression rate was 92.7 ± 1.7%. Figure 4 (Table 1) It exhibits obvious apoptotic body characteristics.

[0076] Comparative Example 2: Preparation and Characterization of Stem Cell Exosomes Methods for preparing exosomes mainly include ultracentrifugation, density gradient centrifugation, ultrafiltration, precipitation reagent method, immunoaffinity capture method, and microfluidic technology. Ultracentrifugation is the most commonly used basic method; therefore, this invention uses ultracentrifugation to extract and prepare stem cell exosomes as a control. The specific method is as follows: Culture supernatant of human umbilical cord-derived mesenchymal stem cells was collected and centrifuged at 300 g and 2000 g for 10 min to remove dead cells and cell debris. The supernatant was then centrifuged at 10000 g for 30 min to remove large vesicles. The processed cell supernatant was collected and filtered through a 0.22 μm filter membrane. The processed cell supernatant was then transferred to an ultracentrifuge tube and centrifuged at 100000 g for 70 min at 4 °C. The supernatant was removed, and the precipitate was resuspended with an appropriate amount of PBS buffer to obtain stem cell exosomes (Exo).

[0077] The particle size and zeta potential of Exo were determined at 25°C using a Malvern zetasizer. The ANNEXIV / PI apoptosis staining kit was used to stain the samples for 15 min in the dark according to the instructions. The expression of phosphatidylserine (PS) on the surface of the test samples was analyzed by flow cytometry.

[0078] The Exo particle size, potential, and PDI in this comparative example are shown in Table 1. The PS expression ratio was 0.72 ± 0.3%, indicating that PS expression was essentially nonexistent (see Table 1). Figure 4 (and Table 1).

[0079] 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.

[0080] 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.

[0081] Example 2: Evaluation of the uptake effect of engineered nanoparticles of apoptosis by dermal papilla cells (DPCs) 1. Test Methods Using Exo and ABs as controls, the DPC uptake effect of e-nABs was evaluated by flow cytometry. The specific procedure is as follows: (1) e-nABs, ABs and Exo were prepared according to the preparation methods described in Example 1 and Comparative Examples 1-2. The three types of cell vesicles were incubated with the cell membrane green fluorescent probe DiO (concentration of 10 μg / mL) at 37°C in the dark for 30 min. After incubation, the cells were centrifuged at 40,000 g for 40 min at 4°C. The supernatant was discarded, and the cells were washed with pre-cooled PBS to remove excess dye. The cells were then centrifuged at 40,000 g for 40 min at 4°C. The precipitate was collected and resuspended with a certain amount of PBS to obtain the three types of DiO-labeled vesicles. The protein content was determined by BCA kit to determine their concentration.

[0082] (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 in 1 mL of culture medium and count them, and then count them at 1 × 10⁻⁶. 5Cells were gently seeded into 12-well plates at a cell density of 1 cell per well. (3) After the DPCs in step (2) adhere to the wall, discard the culture medium and add blank DMEM culture medium without FBS and administer the drug according to the following groups: 1) PBS group; 2) Exo group; 3) ABs group; 4) e-nABs, the vesicle protein concentration is 40 μg / mL, and incubate at 37℃ for 4 h.

[0083] (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, ABs and e-nABs was quantitatively detected by flow cytometry.

[0084] 2. Test Results The effective uptake of e-nABs 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-nABs, ABs, Exo, and e-nABs were stained and labeled with the cell membrane green fluorescent probe DiO for tracing experiments. The fluorescence intensity of DiO in dermal papilla cells after incubation for 4 h was detected by flow cytometry.

[0085] The experimental results showed that the uptake efficiency of e-nABs by dermal papilla cells was significantly higher than that of Exo and ABs (see...). Figure 5 Since efficient uptake of vesicles by dermal papilla cells is an important prerequisite for vesicles to exert their functional regulatory role, this suggests that e-nABs can more efficiently regulate dermal papilla cell function, thus laying a key foundation for improving hair regeneration.

[0086] Experimental Example 3: Evaluation of the in vitro inflammatory regulation ability of engineered apoptosis nanobody 1. Test Methods Using Exo and ABs as controls, the ability of e-nABs to regulate the inflammatory environment in vitro was evaluated by flow cytometry. The specific procedure is as follows: (1) e-nABs, ABs and Exo were prepared according to the preparation methods described in Example 1 and Comparative Examples 1-2, respectively; (2) Take RAW 264.7 cells in the logarithmic growth phase and in good condition. Directly pipette the partially adherent RAW 264.7 cells off the culture vessel 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. Then, use 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, the culture medium containing LPS was aspirated and replaced with fresh complete culture medium. The following groups were used for drug administration: 1) PBS group; 2) Exo group; 3) ABs group; 4) e-nABs group. The concentration of vesicle protein was 40 μg / mL. The groups were incubated at 37℃ for 24 h.

[0087] (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. After washing the cells, CD80 was analyzed by flow cytometry. + CD206 + Macrophage level.

[0088] 2. Test Results Results of e-nABs' in vitro anti-inflammatory ability: Figure 6 As shown, CD206 + / CD80 + Ratio analysis showed that the e-nABs group could effectively promote the repolarization of macrophages from the M1 phenotype to the M2 phenotype; its ability to regulate the inflammatory environment in vitro far exceeded that of the vesicle control ABs group and the Exo group.

[0089] Experimental Example 4: Protective effect of engineered nano-apoptotic bodies on inflammatory-damaged dermal papilla cells (DPCs) 1. Test Methods Using Exo and ABs as controls, the protective effect of e-nABs against inflammatory DPCs was evaluated by flow cytometry. The specific procedure is as follows: (1) e-nABs, ABs and Exo were prepared according to the preparation methods described in Example 1 and Comparative Examples 1-2, respectively; (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 5 × 10⁻⁶. 4 Cells were gently seeded into 24-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, and then count them at 5 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell per well in 24-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 2000 rpm for 5 min to remove cell debris, and obtain M1 macrophage conditioned medium (RAW 264.7 Conditioned Medium, RAW-CM). (5) After the DPCs in step (2) adhere to the wall, discard the culture medium and divide into five groups for experiments. The blank control group is added with DMEM high glucose medium containing 10% FBS. The other groups are added to each well with RAW-CM obtained in step (4) and DMEM high glucose medium containing 10% FBS at a ratio of 1:1. The specific drug administration groups are as follows: 1) Blank control (Control) group; 2) RAW-CM group; 3) RAW-CM+Exo group; 4) RAW-CM+ABs group; 5) RAW-CM+e-nABs group. The concentration of vesicle protein is 40 μg / mL. They are incubated at 37℃ for 24 h.

[0090] (6) After incubation, adherent cells were digested with 0.25% trypsin for 5 min, and complete culture medium was added to stop the digestion. Cells were collected by centrifugation at 1000 rpm for 5 min. The cells were stained with the ANNEXIV / PI apoptosis staining kit in the dark for 15 min according to the instructions. After washing the cells, the apoptosis level was analyzed by flow cytometry.

[0091] 2. Test Results The results of the protective effect of e-nABs on inflammatory DPCs are as follows: Figure 7 As shown, the proportion of late apoptosis was observed in the RAW-CM+e-nABs group, which was closest to the blank control group that was not placed in an inflammatory environment; the other vesicle administration groups all showed some improvement. Overall, e-nABs had the strongest protective effect against DPCs with inflammatory damage.

[0092] Example 5: A method for preparing a soluble microneedle patch (e-nABs@MN) loaded with engineered nanoapoptotic bodies. This experimental example provides a method for preparing e-nABs@MN, which includes the following steps: (1) Preparation of PDMS negative mold: PDMS negative mold was made using a metal positive mold. After the mold was made, the PDMS mold was soaked in 75% ethanol for 30 min, and then cleaned three times with anhydrous ethanol in an ultrasonic cleaner for 30 min each time. After cleaning, it was dried at 37°C for later use. (2) Preparation of needle solution: After uniformly mixing HA powder with PVP K30 powder of the corresponding mass ratio, dissolve it in PBS to prepare a needle solution with a concentration of 2.5% w / v. Swell at room temperature for 2 h, continuously add e-nABs, stir and mix well, remove bubbles under vacuum for 10 min, and store at 4℃ for later use. (3) Microneedle forming: The prepared needle body solution is injected into the PDMS negative mold, and the needle tip is completely filled by vacuuming. The needle tip is pre-dried at 37°C for 1 h to form the needle tip. (4) Formation of backing layer: Add 10% PVA solution that has been degassed beforehand as a backing layer, and centrifuge at 4000 rpm for 10 min to form a uniform backing layer; (5) Drying and storage: After drying at 37℃ for 12 h, demold, vacuum package, and store at 4℃ for later use.

[0093] like Figure 8 As shown, the macroscopic morphology of e-nABs@MN prepared by this method is a square patch of 12.7 mm × 12.7 mm, which includes 225 conical microneedles with a tip diameter of 340 μm, a needle height of 650 μm, and an arrangement spacing of 700 μm, which is conducive to uniformly piercing the skin barrier.

[0094] Experimental Case 6: e-nABs@MN applied to the treatment of androgenetic alopecia 1. Test Methods Exo@MN and ABs@MN were prepared as controls according to the preparation method described in Experiment Example 5. The therapeutic effect of e-nABs on androgenetic alopecia was evaluated by applying e-nABs@MN to animals with androgenetic alopecia. The specific experimental procedure is as follows: like Figure 9As shown, an AGA mouse model was established by topical application of testosterone (TES). Mice were then divided into four groups: Model, Minoxidil, Blank, Exo@MN, ABs@MN, and e-nABs@MN, with six mice 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. The Exo@MN, ABs@MN, and e-nABs@MN groups received a single dose of 100 μg / patch / mouse. Treatment began on day 1 after successful model establishment, with administration every two days for a total of five treatments. Microneedle patches were applied to the hair-loss area, allowing for minimally invasive penetration of the stratum corneum through the needle tip of the soluble microneedle patch, achieving targeted, rapid, and precise delivery to the hair follicles.

[0095] 2. Test Results Figure 10 The study detailed the hair regeneration status of mice in each group at different time points (D-7, D0, D7, D14). After different treatment regimens, mice in the Model group showed no new hair growth on D14, and their skin remained pink. In contrast, both the Minoxidil group and the e-nABs@MN group showed significant hair regeneration, with the e-nABs@MN group exhibiting better hair regeneration than the Minoxidil group. The e-nABs@MN group showed significant hair regeneration as early as D7, validating the successful model construction and the hair growth-promoting effects of each treatment.

[0096] Hair coverage ( Figure 11 Similar results were also observed. At D7, the new hair coverage of the e-nABs@MN group (32.7 ± 10.3%) was significantly higher than that of the Model group (1.3 ± 0.5%), the Blank MN group (8.8 ± 1.5%), and the positive control Minoxidil group (15.9 ± 5.9%), and also higher than that of the ABs@MN group (15.1 ± 4.8%) and the Exo@MN group (9.7 ± 2.6%). Hair coverage increased in all groups on day 14, but showed the same trend. Except for the Healthy group, the e-nABs@MN group had the highest new hair coverage (75.4 ± 8.5%), followed by the Minoxidil group (53.6 ± 8.3%), ABs@MN group (51.5 ± 7.6%), and Exo@MN group (36.4 ± 8.6%). The Blank MN group (21.3 ± 3.9%) and Model group (1.6 ± 0.7%) had the lowest coverage.

[0097] In summary, compared to the Model group, e-nABs@MN treatment significantly improved the new hair coverage and hair regeneration rate in AGA mice, and its effect was superior to the positive control Minoxidil group and the ABs@MN control group. Furthermore, Blank MN also promoted hair regeneration, but its new hair coverage was relatively low and the hair regeneration process was slower.

[0098] This invention proposes an engineered mesenchymal stem cell nanoparticle apoptosis body (e-nABs) and constructs it into soluble microneedles (e-nABs@MN) loaded with engineered mesenchymal stem cell nanoparticles. Through a series of in vitro and in vivo experiments, this invention systematically evaluated the effects of e-nABs@MN on the biological function of damaged dermal papilla cells and explored its mechanism of action in promoting AGA hair regeneration. The results show that this strategy can significantly improve hair regeneration efficiency, restore the phenotype and biological function of damaged DPCs, and overcome the limitations of existing AGA treatment methods.

Claims

1. An engineered stem cell nanoparticle apoptosis body, characterized in that, The engineered apoptotic nanobody was prepared from stem cells through a process of engineering induction, apoptosis induction, and differential centrifugation. The inducing agent used in the engineering induction was 3,3'-diindolemethane.

2. The engineered stem cell nanoparticle apoptosis body according to claim 1, characterized in that, 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.

3. The engineered stem cell nanoparticle apoptosis body according to claim 1, characterized in that, The concentration of the inducer is 10-100 μM.

4. The engineered stem cell nanoparticle apoptosis body according to claim 1, characterized in that, Apoptosis induction can be induced by chemical substances, physical methods, or gene regulation.

5. The engineered stem cell nanoapoptotic body according to claim 1, characterized in that, The differential centrifugation process involves sequentially removing intact cells and cell debris through low-speed centrifugation and medium-speed centrifugation, followed by harvesting engineered apoptotic bodies through ultracentrifugation.

6. The engineered stem cell nanoparticle apoptosis body according to claim 1, characterized in that, The differential centrifugal separation is further followed by a programmed tandem extrusion step.

7. The method for preparing engineered stem cell nanoparticles according to any one of claims 1-6, characterized in that, The method includes the following steps: (1) Use culture medium to culture stem cells. When the cell confluence reaches 80-90%, 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 culture supernatant obtained in step (2) and obtain engineered apoptotic bodies by differential centrifugation; (4) Prepare a suspension from the engineered apoptotic bodies collected in step (3), and perform a programmed series extrusion of the suspension to obtain the final product.

8. A pharmaceutical composition loading the engineered stem cell nanoparticles according to any one of claims 1-6.

9. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition is selected from injections, soluble microneedles, topical gels, or topical solutions.

10. The role of the engineered stem cell nanoapoptotic bodies according to any one of claims 1-6 or the pharmaceutical composition according to any one of claims 8-9 in the preparation of a medicament for promoting hair regeneration or treating hair loss.

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