androgen-releasing therapy

Abstract

This invention discloses a sustained-release therapeutic agent for androgenetic alopecia, wherein the androgenetic alopecia sustained-release therapeutic agent is an extracellular vesicle of *Drynaria fortunei*; or an extracellular vesicle of *Drynaria fortunei* combined with minoxidil. This androgenetic alopecia sustained-release therapeutic agent can effectively inhibit dihydrotestosterone-induced apoptosis and oxidative stress in human hair follicle cells, and has excellent hair follicle targeting and safety; this invention belongs to the field of pharmaceutical technology.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a sustained-release therapeutic agent for androgenetic alopecia. Background Technology

[0002] Androgenetic alopecia typically manifests as a receding hairline in men and a widening of the parting on the crown in women. It seriously affects people's pursuit of beauty and mental health, and its incidence increases with age.

[0003] Androgens, especially dihydrotestosterone (DHT), are the core contributing factor. In susceptible individuals, the activity of 5α-reductase in hair follicles of the scalp is elevated, converting testosterone into DHT. DHT binds to androgen receptors on hair follicle cells, inducing follicle miniaturization, characterized by a shortened anagen phase and a prolonged telogen phase, resulting in thinner and softer hair, eventually leading to follicle atrophy and disappearance, resulting in hair loss. Even with normal androgen levels, high sensitivity of hair follicles to DHT can still cause hair loss. The changes in the hair follicle microenvironment caused by DHT are also accompanied by increased inflammation and oxidative stress, leading to impaired function of human dermal papillary cells (HDPCs).

[0004] Therefore, the treatment of androgenetic alopecia has always been a hot topic of research. Existing therapies, such as finasteride, can inhibit 5α-reductase activity and thus reduce DHT production, but they carry risks of side effects such as sexual dysfunction. Topical minoxidil has limited efficacy and requires long-term maintenance, with a high relapse rate after discontinuation. Furthermore, some patients experience scalp irritation. Therefore, there is an urgent need to develop novel, highly effective, low-toxicity, and precisely targeted hair follicle therapies.

[0005] *Davallia trichomanoides* Blume, commonly known as hairy ginger, is a fern belonging to the genus *Davallia* in the family Drynariaceae. It is a traditional Chinese medicine widely recorded in ancient herbal texts as an important remedy for bone injuries. The rhizome is used medicinally; it is bitter and warm in nature, and has the effects of tonifying the kidneys and strengthening bones, promoting healing, and relieving pain. In traditional clinical applications for treating hair loss, some remedies involve soaking *Davallia trichomanoides* in alcohol and applying the resulting liquid. This can directly stimulate the scalp and accelerate hair growth. However, alcohol is extremely irritating to the skin and hair follicles and should not be used frequently. Furthermore, there is a risk of allergic reactions.

[0006] Therefore, scholars further studied and found that Drynaria fortunei alcohol extract (DTEE) has a therapeutic effect on cyclophosphamide-induced alopecia areata model in mice because it contains abundant flavonoids, which can effectively inhibit inflammation. However, DTEE contains too many other components, making it impossible to effectively isolate individual flavonoids. Furthermore, its application in the treatment of androgenetic alopecia has not been studied, and the flavonoid components have poor stability and low bioavailability in vivo, making it difficult to achieve continuous and effective targeted delivery to hair follicles. Summary of the Invention

[0007] To address the above-mentioned problems, the purpose of this invention is to provide a sustained-release therapeutic agent for androgenetic alopecia, which has a significant proliferative effect on DHT-induced HDPCs and effectively inhibits HDPC apoptosis and oxidative stress.

[0008] Therefore, the first technical solution provided by this invention is as follows:

[0009] A sustained-release treatment for androgenetic alopecia includes Drynaria fortunei extracellular vesicles (DTNVs).

[0010] Furthermore, the aforementioned sustained-release treatments for androgenetic alopecia also include minoxidil.

[0011] Furthermore, in the aforementioned sustained-release treatment for androgenetic alopecia, the mass ratio of *Drynaria fortunei* extracellular vesicles to minoxidil is 1.25000-5.00000: 0.03275-0.13100.

[0012] Furthermore, in the aforementioned sustained-release treatment for androgenetic alopecia, the main components of the extracellular vesicles of *Drynaria fortunei* are naringenin chalcone and sphingomyelin.

[0013] Furthermore, in the aforementioned sustained-release treatment for androgenetic alopecia, the *Drynaria fortunei* extracellular vesicles are prepared by the following method:

[0014] 1) Mix fresh Drynaria fortunei and PBS at a material-to-liquid ratio of 1:2-4, and then pulverize with stirring to obtain the filtrate;

[0015] 2) Filter the filtrate from step 1) with gauze, collect the filtrate and let it stand in a refrigerator at 4°C for 8-12 hours, then take the supernatant.

[0016] 3) Centrifuge the supernatant obtained in step 2) at 4℃ and 5000 g for 30 min, and take the supernatant after centrifugation;

[0017] 4) Centrifuge the supernatant obtained in step 3) at 4℃ and 10000 g for 60 min. After centrifugation, take the supernatant and filter it using a 0.22 µm filter membrane, and collect the filtrate.

[0018] 5) Centrifuge the filtrate from step 4) at 4℃ and 150,000 g for 90 min. After centrifugation, take the precipitate to obtain crude extract of Drynaria fortunei extracellular vesicles.

[0019] 6) The crude extract of *Drynaria fortunei* extracellular vesicles prepared in step 5) is suspended in PBS buffer and then centrifuged at 4°C and 150,000g for 90 min to obtain *Drynaria fortunei* extracellular vesicles. These vesicles are then stored in PBS buffer at -80°C.

[0020] Furthermore, the above-mentioned androgenic alopecia treatment agent undergoes filtration twice in step 4).

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. The extracellular vesicles (DTNVs) of Drynaria fortunei provided by this invention have good biocompatibility, have a specific proliferative effect on HDPCs, and can significantly inhibit DHT-induced apoptosis and oxidative stress of HDPCs and promote the migration of HDPCs.

[0023] 2. The extracellular vesicles (DTNVs) of Drynaria fortunei provided by this invention mainly contain naringenin chalcone, of which the content of naringenin chalcone is 31.57%, which has anti-inflammatory effects and can promote the proliferation of HDPCs and inhibit their apoptosis; the content of sphingomyelin is 18.2%, indicating that the extracted substances have a membrane structure, thereby reducing the cellular metabolic stress caused by the direct exposure of plant active ingredients to cells, continuously releasing active ingredients, and prolonging the duration of action.

[0024] 3. The technical solution provided by this invention scientifically combines extracellular vesicles of Drynaria fortunei with minoxidil. The combined use of DTNVs and minoxidil can significantly reduce the dosage of each drug, while enhancing the proliferative effect on HDPCs, achieving synergistic effects and realizing multi-target intervention. Attached Figure Description

[0025] Figure 1 LC-MS compositional analysis spectrum (positive and negative ion scanning spectrum) of extracellular vesicles of Drynaria fortunei cells;

[0026] Figure 2 LC-MS component analysis spectrum (positive and negative ion scanning spectrum) of the alcohol extract of Drynaria fortunei;

[0027] Figure 3 Transmission electron microscopy (TEM) images of the morphology and physical properties of extracellular vesicles from *Drynaria fortunei* cells;

[0028] Figure 4 Graph showing the safety evaluation of extracellular vesicles and alcohol extracts of *Drynaria fortunei* on three cell types;

[0029] Figure 5 Comparative figure showing the effect of extracellular vesicles of Drynaria fortunei on the proliferation activity of DHT-induced HDPCs;

[0030] Figure 6 Figure showing the inhibitory effect of extracellular vesicles of *Drynaria fortunei* cells on DHT-induced apoptosis in HDPCs;

[0031] Figure 7 Figure showing the inhibitory effect of extracellular vesicles of *Drynaria fortunei* on DHT-induced oxidative stress in HDPCs;

[0032] Figure 8 This describes the uptake of extracellular vesicles of *Drynaria fortunei* by HDPCs.

[0033] Figure 9 The effect of extracellular vesicles of *Drynaria fortunei* on the migration ability of DHT-induced HDPCs is shown in the figure. Detailed Implementation

[0034] The present invention will be further explained below with reference to embodiments and accompanying drawings, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Example 1

[0036] This embodiment provides a sustained-release treatment for androgenetic alopecia, which is a Drynaria fortunei extracellular vesicle (DTNV).

[0037] Its preparation method includes the following steps:

[0038] S1. Wash the fresh Drynaria fortunei with pure water and air dry at room temperature. Weigh 50g of fresh Drynaria fortunei, add 150ml of PBS (solid-to-liquid ratio 1:3) and mix. Then, grind the mixture using a juicer. Filter the mixture through gauze, place the filtrate in a refrigerator at 4℃ and let it stand for 12 hours. Take the supernatant.

[0039] S2. Centrifuge the supernatant obtained in S1 at low speed (5000 g, 30 min, 4℃) once. After centrifugation, take the supernatant.

[0040] S3. Centrifuge the supernatant obtained in S2 twice at low speed (10000 g, 60 min, 4℃), and take the supernatant after centrifugation.

[0041] S4. Filter the supernatant obtained in S3 twice using a 0.22 µm filter membrane.

[0042] S5. The liquid obtained in S4 was centrifuged at high speed (150,000 g, 90 min, 4℃). After centrifugation, the precipitate was collected to obtain crude DTNVs. After collection with 10 mL PBS, a second round of high-speed centrifugation was performed (150,000 g, 90 min, 4℃) to obtain DTNVs. These were collected with 1 mL PBS, and the concentration of DTNVs was determined using a BCA kit. The solution was then stored at -80℃.

[0043] The chemical composition of the DTNVs prepared above was determined by LC-MS, and the LC-MS spectra are shown below. Figure 1As shown in Table 1, the specific components include naringin chalcone (31.57%), which has been reported to have anti-inflammatory effects, and sphingomyelin (18.2%), indicating that the extracted substance may have a membrane structure. Figure 3 As shown in Figure A, the extracted DTNVs are pale yellow, and the morphology of the DTNVs obtained by transmission electron microscopy is typical of exosomes. Figure 3 B), The physical properties of DTNVs were characterized using nanoparticles and a Zeta potential analyzer. Figure 3 C), the measured DTNVs showed a negative Zeta potential, indicating good stability. Their particle size range was 129.23 nm ± 1.75 nm, which is consistent with the nanoparticle size range of exosomes. The polydispersity index < 0.3 indicates that the DTNVs extracted by this method have a relatively uniform particle size distribution, which is beneficial to the consistency and reproducibility of drug delivery.

[0044] Table 1: Components of DTNVs detected by LC-MS

[0045]

[0046] Comparative Example 1

[0047] This comparative example provides a Drynaria fortunei alcohol extract (DTEE), the preparation method of which includes the following steps:

[0048] S1. Wash the fresh Drynaria fortunei with pure water, let it air dry at room temperature, and then crush it.

[0049] S2. Take 50 g of freshly crushed Drynaria fortunei and mix it with 400 mL of anhydrous ethanol (solid-to-liquid ratio 1:8). Transfer the mixture to a round-bottom flask, connect a constant pressure funnel to a condenser, and reflux at 70-80°C for 4 h in a heating mantle. The reaction system should be kept at a gentle boil. After the reaction is complete, collect the filtrate.

[0050] S3. The filtrate obtained in step S2 is concentrated by evaporation using a rotary evaporator to obtain pure DTEE in paste form.

[0051] The chemical composition of the DTEE prepared above was determined by LC-MS, and the LC-MS spectrum is shown below. Figure 2 As shown in Table 2, the specific components are as follows. It can be seen that, according to LC-MS analysis, DTEE contains 39 chemical components, compared to the 12 chemical components of DTNVs. Therefore, DTEE has a more complex chemical composition and lower efficiency in separating the effective components.

[0052] Table 2: LC-MS Detection of DTEE Components

[0053]

[0054]

[0055]

[0056] To verify the effectiveness of the technical solution provided in this application, experimental test results are provided below:

[0057] I. The safety of DTNVs provided in Example 1 and DTEE provided in Comparative Example 1 for three common skin cell types (HACATs, HUVECs, and HDPCs) was tested.

[0058] 1. Experimental subjects: HACATs, HUVECs, HDPCs, Example 1 DTNVs, Comparative Example 1 DTEE.

[0059] 2. Experimental Methods: First, a single-cell suspension was prepared (three types of adherent cells were washed twice with PBS, then digested with trypsin and centrifuged). The cell concentration was adjusted to 5 × 10⁶ cells / mL using whole culture (DMEM / F12, 10% fetal bovine serum, 1% penicillin-dextrose antibody). 4 Cells / mL were seeded at 100 μL per well in a 96-well plate (three replicates for each concentration). After pre-culturing the three cell types in whole culture for 24 h, the cells were washed twice with PBS, diluted with DMEM / F12 to form Example 1 and Comparative Example 1, and co-incubated with the cells. After 48 h, the cells were washed twice with PBS, and culture medium containing 10% CCK-8 solution was added to each well. The cells were incubated at 37°C for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader (P* < 0.05, P** < 0.01).

[0060] 3. Results and Discussion: The proliferative effects and safety of DTNVs on three cell types were evaluated using the CCK-8 assay. Figure 4 The results showed that DTNVs exhibited good biocompatibility with all three cell types and no significant cytotoxicity was observed within the experimental concentration range. Furthermore, DTNVs showed a significant specific proliferative effect on HDPCs, and this effect was concentration-dependent. As the concentration of DTNVs increased, the proliferation rate of HDPCs increased significantly, suggesting that it may have the potential to target and regulate the physiological activity of HDPCs. In contrast, although traditional DTEE had a certain proliferative effect on HDPCs, the proliferation activity of HDPCs decreased in the same high concentration range. Moreover, compared with DTNVs, DTEE was more cytotoxic to HACATs and HUVECs.

[0061] In summary, DTNVs demonstrated superior safety compared to DTEE in all three cell systems. This enhanced safety may stem from the unique biomembrane structure of extracellular vesicles, which protects plant active ingredients and provides a sustained-release effect, thereby reducing the cellular metabolic stress caused by direct exposure of plant active ingredients to cells. Furthermore, the specific activation of HDPCs by DTNVs offers insights for developing regenerative therapies targeting hair follicles.

[0062] II. The effect of DTNVs provided in Example 1 on the proliferation of DHT-induced HDPCs was tested.

[0063] 1. Experimental subjects: HDPCs and DTNVs from Example 1.

[0064] 2. Experimental Methods: First, a single-cell suspension was prepared (HDPCs were washed twice with PBS, then digested with trypsin and centrifuged). The cell concentration was adjusted to 5 × 10⁶ cells / year using whole culture (DMEM / F12, 10% fetal bovine serum, 1% penicillin-dextrose antibody). 4 The culture medium was prepared at a concentration of 100 μL / mL in a 96-well plate. After 24 h of pre-culture, the plates were washed twice with PBS and diluted with DMEM / F12. The plates were divided into a blank group (containing only DMEM / F12), a model group (10 nM DHT), and an experimental group (three concentrations of Example 1 + 10 nM DHT). Each group was divided into three replicates. After 48 h, the plates were washed twice with PBS and culture medium containing 10% CCK-8 solution was added to each well. The plates were incubated at 37 °C for 2 h. The absorbance at 450 nm was measured using a microplate reader (P* < 0.05, P** < 0.01).

[0065] 3. Results and Discussion: (e.g.) Figure 5 As shown, compared with the control group, the number of cells in the model group was significantly reduced (P<0.05), indicating that DHT has a significant inhibitory effect on HDPC proliferation. After intervention with DTNVs, the cell number increased in a dose-dependent manner; 40 μg / mL DTNVs reversed the inhibitory effect of DHT, restoring it to a level comparable to the control group; while 80 μg / mL DTNVs significantly promoted HDPC proliferation. In conclusion, DTNVs can reverse the inhibitory effect of DHT on HDPCs, laying the foundation for its development as a treatment for androgenetic alopecia.

[0066] III. The inhibitory effect of DTNVs provided in Example 1 on DHT-induced apoptosis of HDPCs was tested.

[0067] 1. Experimental subjects: HDPCs and DTNVs from Example 1.

[0068] 2. Experimental Methods: Single-cell suspensions were prepared (HDPCs were washed twice with PBS, then digested with trypsin and centrifuged). The cell concentration was adjusted to 1×10⁶ cells / cells using whole culture (DMEM / F12, 10% fetal bovine serum, 1% penicillin-dextrose antibody). 5 HDPCs were seeded at a rate of 1 mL / well in a 12-well plate and pre-cultured for 24 h. The plates were then washed twice with PBS. Example 1 was diluted with DMEM / F12 to create a blank group (containing only DMEM / F12), a model group (10 nM DHT), and an experimental group (80 μg / mL Example 1 + 10 nM DHT). After a total of 48 h of incubation, apoptosis of HDPCs was detected using Annexin V / PI double staining. Collect the supernatant from each well, digest with EDTA-free trypsin, and terminate digestion with the original supernatant. Centrifuge at 500 g for 5 min and discard the supernatant. Wash once with PBS and resuspend in pre-cooled 1× Binding Buffer. Add 5 μL Annexin V-FITC to each tube and incubate in the dark for 20 min. Then add 10 μL PI dye and incubate in the dark for 5 min. Add 300 μL PBS to resuspend and detect the cells using flow cytometry within 1 hour. Blank tubes, single-stained tubes, and negative control tubes were set up simultaneously for voltage adjustment and compensation. Each group was repeated three times to ensure accurate results (P* < 0.05, P** < 0.01).

[0069] 3. Results and Discussion: Compared with the blank control group, the apoptosis rate of HDPCs in the DHT-treated model group was significantly increased (P* < 0.05). Flow cytometry analysis using Annexin V / PI also confirmed a significant increase in the total number of early and late apoptotic cells, indicating that DHT can effectively induce programmed cell death in HDPCs and disrupt the physiological homeostasis of dermal papilla cells. This result is consistent with... Figure 5 The observation that DHT inhibits cell proliferation corroborates each other, revealing that DHT damages HDPC function through multiple pathways, thereby driving hair follicle degeneration and miniaturization. After DTNV intervention, the level of DHT-induced HDPC apoptosis significantly decreased (P** < 0.01), and flow cytometry results showed that the apoptosis rate was close to or even lower than that in the control group. In the process of androgenetic alopecia, excessive apoptosis of HDPCs mediated by DHT leads to a reduction in dermal papilla volume and attenuation of induction signals, ultimately causing progressive miniaturization of hair follicles and a shortened growth phase. DTNV, by strongly inhibiting apoptosis, maintains the stability of the HDPC population, providing the necessary cellular basis for hair follicle cycle restart and morphological reconstruction.

[0070] IV. The inhibitory effect of DTNVs provided in Example 1 on DHT-induced HDPCs on oxidative stress was tested.

[0071] 1. Experimental subjects: HDPCs and DTNVs from Example 1.

[0072] 2. Experimental Methods: Single-cell suspensions were prepared (HDPCs were washed twice with PBS, then digested with trypsin and centrifuged), and the cell concentration was adjusted to 1×10⁶ cells / cells using whole culture (DMEM / F12, 10% fetal bovine serum, 1% penicillin-dextrose antibody). 5 Cells were seeded at a rate of 1 mL / well in 12-well plates and pre-cultured for 24 h. Afterward, the cells were washed twice with PBS. Example 1 was diluted with DMEM / F12 to create a blank group (containing only DMEM / F12), a model group (10 nM DHT), and an experimental group (80 μg / mL Example 1 + 10 nM DHT). After a total of 48 h of incubation, the cells were washed twice with PBS to completely remove the culture medium. DCFH-DA was diluted 1:1000 with serum-free medium, and the diluted DCFH-DA probe was added to each well and incubated for 20 min. The wells were then washed three times with serum-free medium to remove any probes that had not entered the cells. The cell nuclei were stained with Hoechst 33342 staining solution for 5 min, followed by washing three times with serum-free medium. Finally, the fluorescence signal intensity was observed and recorded under a fluorescence microscope; the signal strength reflects the reactive oxygen species (ROS) level. The relative ROS levels of each group were analyzed using ImageJ software (P** < 0.01).

[0073] 3. Results and Discussion: Figure 7 This study revealed the significant inhibitory effect of DTNVs on DHT-induced oxidative stress in HDPCs. Compared with the blank control group, the level of reactive oxygen species (ROS) in HDPCs in the DHT-treated group was significantly increased (P** < 0.01), and DCFH-DA fluorescence detection showed a significant increase in fluorescence intensity, suggesting that DHT can strongly induce intracellular oxidative stress response and disrupt the dynamic balance of the oxidation-antioxidant system. After co-treatment with DTNVs, the excessive accumulation of DHT-induced ROS was effectively curbed, and the fluorescence signal intensity was significantly reduced (P** < 0.01), demonstrating that DTNVs have a strong free radical scavenging ability and antioxidant system remodeling function. Existing research has found that oxidative stress, as a key link between DHT accumulation and HDPC dysfunction in the process of androgenetic alopecia, can directly damage DNA, protein, and lipid structures, and indirectly activate inflammation and apoptosis-related pathways such as MAPK and NF-κB, accelerating senescence and signal exhaustion of dermal papilla cells. The intervention of DTNVs protects HDPCs from oxidative attack and provides a stable internal environment. Therefore, the outstanding performance of DTNVs in inhibiting oxidative stress is not only an important precursor mechanism of their anti-apoptotic effect, but also reflects their deep regulatory value in delaying hair follicle miniaturization and promoting hair follicle homeostasis reconstruction, providing strong experimental evidence for the development of targeted antioxidant therapy strategies based on nanocarriers.

[0074] V. Detect the uptake of DTNVs provided in Example 1 by HDPCs.

[0075] 1. Experimental subjects: HDPCs and DTNVs from Example 1.

[0076] 2. Experimental Methods: Single-cell suspensions were prepared (HDPCs were washed twice with PBS, then digested with trypsin and centrifuged). The cell concentration was adjusted to 1×10⁶ cells / cells using whole culture (DMEM / F12, 10% fetal bovine serum, 1% penicillin-dextrose antibody). 4 HDPCs were seeded at a rate of 1 mL / mL into each confocal microscopy dish and pre-cultured in whole culture for 24 h, followed by washing twice with PBS. DTNVs were labeled using the BeyoExo™ Exosome Labeling and Tracing Kit (PKH67). PKH67-DTNVs were obtained by high-speed centrifugation and diluted to a concentration of 40 μg / mL using DMEM / F12. The PKH67-DTNVs were then added to the confocal microscopy dishes. HDPCs were imaged using a laser confocal microscope at 0 h, 12 h, and 24 h. Before imaging, the cells were washed three times with PBS to remove free PKH67-DTNVs. The cell nuclei were then stained with Hoechst 33342 for 10 min, followed by washing three times with PBS to remove any remaining staining solution. Finally, 1 mL of PBS was added to the confocal microscopy dishes to prevent the HDPCs from drying out.

[0077] 3. Results and Discussion: Figure 8 The results showed that HDPCs could effectively endocytose DTNVs, rather than simply adhering to the cell surface. Furthermore, the accumulation of DTNVs within the cell increased over time, supporting the view that they possess sustained-release properties. These results indicate that DTNVs have potential applications as single therapeutic agents or drug delivery carriers.

[0078] VI. The effect of DTNVs provided in Example 1 on the cell migration of DHT-induced HDPCs was tested.

[0079] 1. Experimental subjects: HDPCs and DTNVs from Example 1.

[0080] 2. Experimental Methods: HDPCs were cultured in six-well plates until fully confluent. A 200 μL yellow pipette tip was used to gently streak a line vertically across the central region of the cell growth area. Cell debris from the streak was then removed by washing with PBS. Three groups were set up: a control group (containing only DMEM / F12), a model group (10 nM DHT), and an experimental group (80 μg / mL of Example 1 + 10 nM DHT). Images were taken at the same field of view at 0 h and 24 h. The cell migration rate in the streak area was compared among the different groups to determine the effect of DTNVs on the migration ability of HDPCs.

[0081]

[0082] 3. Results and Discussion: Figure 9 The results showed that, compared with the control group, the cell migration rate of the DHT-treated model group was significantly reduced (P** < 0.01), indicating that DHT can effectively inhibit the migration ability of HDPCs, consistent with the proliferation inhibition results. Figure 5 The migration rate in the DTNVs group was restored and even exceeded that in the control group, indicating that DTNVs could completely reverse the migration-inhibiting effect of DHT. The migration ability of HDPCs directly affects hair follicle morphological reconstruction. In androgenetic alopecia, DHT leads to hair follicle miniaturization, while the directional migration of HDPCs can promote dermal papilla-epithelial cell interaction and restore the normal structure of hair follicles.

[0083] Example 2

[0084] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of bone fragmentation cells and minoxidil in a mass ratio of 5:0.03275.

[0085] Example 3

[0086] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 2.5:0.262.

[0087] Example 4

[0088] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 2.5:0.131.

[0089] Example 5

[0090] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 2.5:0.0655.

[0091] Example 6

[0092] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 2.5:0.03275.

[0093] Example 7

[0094] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 1.25:0.262.

[0095] Example 8

[0096] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 1.25:0.131.

[0097] Comparative Example 2

[0098] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 5:0.262.

[0099] Comparative Example 3

[0100] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 5:0.131.

[0101] Comparative Example 4

[0102] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 5:0.0655.

[0103] Comparative Example 5

[0104] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 1.25:0.0655.

[0105] Comparative Example 6

[0106] This embodiment provides a sustained-release therapeutic agent for androgenetic alopecia, which is composed of extracellular vesicles of Drynaria fortunei and minoxidil in a mass ratio of 1.25:0.03275.

[0107] The extracellular vesicles of *Drynaria fortunei* used in Examples 2-8 and Comparative Examples 2-5 above were the extracellular vesicles of *Drynaria fortunei* prepared in Example 1.

[0108] To verify the effectiveness of the technical solutions provided in Examples 2-8 of this application, the following experimental verification of the synergistic effect of HDPCs, Examples 2-8, and Comparative Examples 2-5 are presented.

[0109] 2. Experimental Methods: Clinical co-operation (CI) is an important indicator used to assess whether the combined use of two or more drugs produces a synergistic effect. CI values ​​can help researchers understand the effects of different drug combinations, thus providing a scientific basis for clinical drug use.

[0110] First, a single-cell suspension was prepared (HDPCs were washed twice with PBS, then digested with trypsin and centrifuged). The cell concentration was adjusted to 5 × 10⁶ cells / year using whole culture (DMEM / F12, 10% fetal bovine serum, 1% penicillin-dextrose antibody). 4DTNVs were seeded at 100 μL per well in a 96-well plate. After pre-culturing for 24 h, the plates were washed twice with PBS. Examples 2, 3, and 4 were diluted with culture medium to create a blank group (containing only DMEM / F12), a model group (10 nM DHT), and an experimental group. DTNVs were added in a gradient along the X-axis of the 96-well plate using a two-fold dilution method to achieve final concentrations of 1.25–5 µg / mL. Similarly, MXD was added along the Y-axis to achieve final concentrations of 0.0655–0.262 µg / mL. Each well in the experimental group contained 10 nM DHT. After 48 h, the plates were washed twice with PBS, and culture medium containing 10% CCK-8 solution was added to each well. The plates were incubated at 37°C for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader. CI was calculated using the following formula:

[0111]

[0112] Wherein, (DTNVs) combined and (MXD) combined refer to the actual doses of DTNVs and MXD in the combination groups that reached the blank level. (DTNVs) alone and (MXD) alone refer to the doses required to reach the blank level by single drugs (previous experiments verified that DTNVs alone is 40 µg / mL and MXD alone is 0.262 µg / mL).

[0113] 3. Judgment criteria: CI < 0.9, synergistic effect; 0.9 ≤ CI ≤ 1.1, additive effect; CI > 1.1, antagonistic effect.

[0114] 4. The results are shown in Table 3.

[0115] Table 3: CI Calculation Results of DTNVs and MXD

[0116]

[0117] As shown in Table 3, the CI values ​​for all combinations in Examples 2 to 8 were significantly lower than 0.9, indicating that DTNVs and minoxidil (MXD) produced a significant synergistic effect at specific ratios. In particular, the results of Example 8 confirmed that when the mass ratio of DTNVs to MXD was 152.67:1 (5:0.03275), the cell proliferation capacity inhibited by DHT was restored to a level comparable to the Control group. Based on this, the calculated CI value was as low as 0.25, demonstrating a synergistic effect (less than 0.9). This indicates that the addition of DTNVs not only effectively overcame the inhibitory effect of DHT on HDPC proliferation but also significantly reduced the MXD dose required to achieve equivalent or even better therapeutic effects, thus reducing the potential side effects of MXD. Conversely, the CI values ​​of Comparative Examples 2 to 4 were all greater than 1.1, indicating an antagonistic effect. Among them, Comparative Example 2 (DTNVs 5.00000 µg / mL + MXD 0.26200 µg / mL) had the highest CI value (1.12500), suggesting that the combination of high concentrations of MXD and DTNVs may weaken the therapeutic effect.

[0118] Furthermore, Comparative Examples 5 and 6 could not be calculated for CI values ​​because they did not recover to the Control group level, further confirming that excessively low MXD doses (e.g., 0.03275 µg / mL) could not effectively reverse DHT damage at specific DTNV ratios. This demonstrates the synergistic advantage of DTNVs:MXD mass ratios of 5:0.03275 to 1.25:0.0655 in restoring HDPC function, while combinations deviating from this range exhibited antagonistic or ineffective effects.

[0119] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, these obvious variations or modifications derived from the essential spirit of the present invention still fall within the scope of protection of the present invention.

Claims

1. A sustained-release therapeutic agent for androgenetic alopecia, characterized in that, Composed of extracellular vesicles of Drynaria fortunei cells and minoxidil; The mass ratio of *Drynaria fortunei* extracellular vesicles to minoxidil is 1.25000-5.00000:0.03275-0.13100; and the concentration of *Drynaria fortunei* extracellular vesicles is 1.25000-5.00000 µg / mL; the concentration of minoxidil is 0.03275-0.13100 µg / mL. The aforementioned extracellular vesicles of *Drynaria fortunei* were prepared by the following method: 1) Mix fresh Drynaria fortunei and PBS at a material-to-liquid ratio of 1:2-4, and then pulverize with stirring to obtain the filtrate; 2) Filter the filtrate from step 1) with gauze, collect the filtrate and let it stand in a refrigerator at 4°C for 8-12 hours, then take the supernatant. 3) Centrifuge the supernatant obtained in step 2) at 4℃ and 5000 g for 30 min, and take the supernatant after centrifugation; 4) Centrifuge the supernatant obtained in step 3) at 4℃ and 10000 g for 60 min. After centrifugation, take the supernatant and filter it using a 0.22 µm filter membrane, and collect the filtrate. 5) Centrifuge the filtrate from step 4) at 4°C and 150,000 g for 90 min. After centrifugation, take the precipitate to obtain crude extract of Drynaria fortunei extracellular vesicles. 6) The crude extract of *Drynaria fortunei* extracellular vesicles prepared in step 5) is suspended in PBS buffer and then centrifuged at 150,000 g for 90 min at 4°C to obtain *Drynaria fortunei* extracellular vesicles. These vesicles are then stored in PBS buffer at -80°C.

2. The androgen-induced hair loss sustained-release therapeutic agent according to claim 1, characterized in that, The main components of the extracellular vesicles of *Drynaria fortunei* are naringenin chalcone and sphingomyelin.

3. The androgen-induced hair loss sustained-release therapeutic agent according to claim 1, characterized in that, Step 4) The filtering is performed twice.

Images