A preparation process of pachyman extracellular vesicle hydrogel for androgenetic alopecia treatment

Abstract

This invention discloses a preparation process for Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia. By introducing SA, CMC-Na, and L-HA to construct an interpenetrating network hydrogel, it not only possesses excellent mechanical strength and anti-degradation ability but also effectively simulates the extracellular matrix environment. This invention significantly improves the structural stability of PCEVs through the loading of the interpenetrating network hydrogel, preventing premature inactivation during application. Simultaneously, leveraging the natural "medicinal and edible homology" properties of the raw materials, it ensures extremely high biosafety of the formulation, with no irritation or immunogenicity risks. This invention, through the synergistic effect of the "carrier-drug" combination, provides an innovative treatment solution for androgenetic alopecia with significant clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and provides a process for preparing Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia. Background Technology

[0002] Androgenetic alopecia (AGA) is the most common progressive hair loss disease, characterized by progressive miniaturization of hair follicles, where terminal hairs are gradually replaced by short, thin vellus hairs, leading to decreased hair density and a sparse appearance. Epidemiological data show that the prevalence of AGA increases significantly with age, reaching approximately 80% in men and 50% in women by age 70. Although AGA is not directly life-threatening, the resulting anxiety, depression, and social avoidance severely reduce patients' quality of life, making it a significant public health issue.

[0003] The development of AGA (Advanced Gynecogenic Aging) is primarily attributed to genetic susceptibility and abnormal activation of androgen-dependent signaling. Circulating testosterone is converted to the more potent dihydrotestosterone (DHT) by 5α-reductase. DHT binds with high affinity to androgen receptors (AR) on dermal papillary cells (DPCs), activating downstream transcriptional processes. This leads to a shortened anagen phase and an increased ratio of catagen to telogen phases, ultimately resulting in follicle miniaturization. Furthermore, DPC dysfunction, inhibition of regeneration signaling pathways (such as Wnt / β-catenin), and local microenvironmental inflammation also play crucial roles in the AGA process. Therefore, treatment strategies targeting only the androgen pathway often fail to achieve sustained and stable hair follicle regeneration.

[0004] Currently, the main clinical interventions for AGA include oral 5α-reductase inhibitors (such as finasteride) and topical minoxidil, as well as hair transplantation surgery for moderate to severe cases. However, these therapies have significant limitations: Finasteride: While it can delay hair loss by inhibiting DHT production, systemic administration is often accompanied by adverse reactions such as decreased libido and erectile dysfunction. Recent studies and regulatory warnings have also pointed out that it may increase the risk of depression and suicidal tendencies, and it is contraindicated in women of childbearing age, limiting its widespread use.

[0005] Minoxidil: Although it can promote hair follicles to enter the growth phase, it requires long-term continuous use, and relapse is very common after discontinuation. In addition, its solvent (such as propylene glycol) often causes side effects such as contact dermatitis, scalp itching, and localized hirsutism, resulting in poor patient compliance.

[0006] Hair transplantation: Although it can improve appearance, it is limited by the scarcity of donor hair follicles, large surgical trauma, and high economic costs, making it difficult to meet the needs of most patients with mild to moderate hair loss.

[0007] Therefore, developing a novel intervention strategy that combines high safety, local targeting, ability to reshape the hair follicle microenvironment, and sustained efficacy is an urgent need in the current AGA treatment field.

[0008] In recent years, extracellular vesicles (EVs) have become a research hotspot in tissue regenerative medicine due to their excellent biocompatibility, low immunogenicity, and multi-target regulatory capabilities. EVs can deliver bioactive payloads such as proteins, lipids, and nucleic acids, mediating intercellular communication and regulating stem cell fate and the inflammatory microenvironment. Compared to mammalian-derived EVs, plant-derived extracellular vesicles (PDEVs) have significant advantages in terms of large-scale preparation, cost control, and biosafety. Studies have confirmed that EVs derived from plants such as ginger and Polygonum multiflorum can promote DPC proliferation and hair growth by activating pathways such as Wnt / β-catenin.

[0009] Poria cocos, a classic "food and medicine homology" fungus, has a clinical application history of over two thousand years and boasts extremely high safety. Previous studies have shown that Poria cocos extract and its active components (polysaccharides, triterpenoids) possess anti-inflammatory, antioxidant, and immunomodulatory effects, and can promote the proliferation of DPCs. More notably, recent research suggests that the active components of Poria cocos have the potential to regulate stem cell differentiation and cytoskeleton remodeling, and EV-like nanoparticles derived from Poria cocos exhibit excellent bioactivity in tissue protection. However, current research on extracellular vesicles (PCEVs) derived from Poria cocos in the field of hair follicle regeneration is still in its early stages, particularly regarding their mechanisms for regulating the state of hair follicle stem cells, promoting hair follicle structural reconstruction, and long-term therapeutic effects, which remain unclear.

[0010] However, PDEVs face serious challenges in practical conversion applications: Poor stability: It is prone to structural degradation and loss of activity during in vitro storage and application.

[0011] Short local retention time: The scalp surface barrier and sebum secretion make it difficult for liquid preparations to penetrate, and once they enter the skin, they are easily cleared by the mononuclear phagocytic system.

[0012] Low bioavailability: There is a lack of effective delivery carriers, making it difficult to maintain effective therapeutic concentrations in the hair follicle microenvironment. Summary of the Invention

[0013] In view of this, the purpose of this invention is to provide a process for preparing Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia, so as to solve the technical problems existing in the treatment of androgenetic alopecia (AGA), such as large systemic side effects of oral drugs, short local retention time and low bioavailability of topical preparations, as well as the poor stability, easy degradation and difficulty in targeting the hair follicle microenvironment faced by plant-derived extracellular vesicles (PDEVs) in application.

[0014] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a process for preparing Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia, the specific steps of which are as follows: S1. Preparation of extracellular vesicles (PCEVs) from Poria cocos: Fresh Poria cocos fruiting bodies were used as raw materials. They were washed, chopped, homogenized with water, filtered, and the filtrate was initially clarified. The filtrate was then subjected to chromatography and filtration, enriched by tangential flow ultrafiltration (TFF), and dialysis to obtain a PCEV suspension. S2. Preparation of drug-loaded hydrogel PCEV@Gel: Sodium carboxymethyl cellulose (CMC-Na) aqueous solution and sodium alginate (SA) aqueous solution were mixed and stirred. Low molecular weight hyaluronic acid (L-HA) aqueous solution was added and gently mixed to obtain a polysaccharide mixture. Then, PCEV suspension was added to the polysaccharide mixture and gently mixed to avoid shearing and destroying the vesicles to obtain a hydrogel system. Calcium chloride and gluconate-δ-lactone (GDL) were then added, and the reaction was carried out at room temperature to induce endogenous gelation. After post-treatment, PCEV@Gel was obtained.

[0015] Preferably, in step S1, the method of adding water for homogenization is as follows: add ultrapure water and homogenize using a wall-breaking machine to obtain a homogenate; filter the homogenate through a filter screen to remove coarse tissue residues to obtain a filtrate; the ratio of Poria cocos fruiting body to ultrapure water is 200g:1L.

[0016] Preferably, in step S1, the preliminary clarification method is as follows: centrifuge the filtrate at 4°C and 10,000×g for 30 min, and collect the supernatant to remove cell debris and large particulate impurities.

[0017] Preferably, in step S1, the chromatography and filtration method is as follows: preliminary purification is performed by silica gel chromatography column, followed by filtration through a 0.22μm microporous membrane to remove residual particles and macromolecular polymers.

[0018] Preferably, in step S1, multi-step tangential flow ultrafiltration technology is used for concentration, using a polyethersulfone (PES) membrane with a molecular weight cutoff of 300 kDa, and the operating conditions are controlled as follows: room temperature, transmembrane pressure ≤ 1 bar, feed flow rate 30-60 mL / min, and permeate flow rate 4 mL / min.

[0019] Preferably, in step S1, dialysis replacement is performed using PBS solution (phosphate buffer). The PBS composition is: 135mM NaCl, 4.7mM KCl, 10mM Na2HPO4, 2mM NaH2PO4, with a pH of 7.3 ± 0.1. The PBS solution is sterilized and can be directly used for washing cells during cell culture or for other routine purposes.

[0020] Preferably, the PCEVs suspension obtained in step S1 is aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles.

[0021] Preferably, in step S2, the mass concentrations of sodium carboxymethyl cellulose aqueous solution, sodium alginate aqueous solution, and low molecular weight hyaluronic acid aqueous solution are 4%, 4%, and 0.9%, respectively, and the mass concentrations of sodium carboxymethyl cellulose, sodium alginate, and low molecular weight hyaluronic acid contained in the polysaccharide mixture are 4%, 4%, and 0.9%, respectively.

[0022] Preferably, in step S2, the volume ratio of the PCEVs suspension to the polysaccharide mixture is 1:10.

[0023] Preferably, in step S2, calcium chloride and gluconate-δ-lactone are respectively prepared into aqueous solutions with a mass concentration of 0.6%, mixed in equal volumes to obtain a premix, and then added to the hydrogel system; the volume ratio of the premix to the hydrogel system is 1:99.

[0024] Preferably, in step S2, the pH is controlled at 6.8 to 7.4 when inducing endogenous gelation.

[0025] Preferably, in step S2, the post-processing includes: rinsing the gel 2-3 times with PBS to remove free Ca. 2+ Store at 4℃ away from light for later use.

[0026] The present invention also provides a Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia, which is obtained by the aforementioned preparation process.

[0027] The present invention also provides the application of the aforementioned hydrogel in the preparation of a drug for treating androgenetic alopecia.

[0028] The beneficial effects of this invention are: This invention discloses a preparation process for Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia. By introducing SA, CMC-Na, and L-HA to construct an interpenetrating network hydrogel, it not only possesses excellent mechanical strength and anti-degradation ability but also effectively simulates the extracellular matrix environment. This invention significantly improves the structural stability of PCEVs through the loading of the interpenetrating network hydrogel, preventing premature inactivation during application. Simultaneously, leveraging the natural "medicinal and edible homology" properties of the raw materials, it ensures extremely high biosafety of the formulation, with no irritation or immunogenicity risks. This invention, through the synergistic effect of the "carrier-drug" combination, provides an innovative treatment solution for androgenetic alopecia with significant clinical application prospects.

[0029] Loaded hydrogel systems are considered ideal carriers for the local delivery of EVs due to their three-dimensional network structure, high water content, and controllable release characteristics. Among them, sodium carboxymethyl cellulose (CMC-Na) has good biocompatibility and degradability, and its abundant carboxyl and hydroxyl groups on its molecular chain can regulate network performance through various cross-linking mechanisms. However, single CMC-Na hydrogels suffer from insufficient mechanical strength, excessively rapid swelling, and easy disintegration. This invention utilizes a dual mechanism of ionic cross-linking and physical entanglement to significantly enhance the structural stability and anti-degradation ability of the hydrogel, prolong its local retention time, and further improve the moisturizing properties of the hydrogel by supplementing it with low molecular weight hyaluronic acid. Furthermore, by utilizing its specific interaction with cell surface receptors (such as CD44), it promotes the targeted enrichment of the carrier at the hair follicle site, synergistically optimizing the microenvironment for hair follicle regeneration.

[0030] Specifically, the present invention has the following advantages: 1. A highly stable and biocompatible delivery vector was constructed: Highly efficient loading and stable maintenance of active ingredients: This addresses the problem of structural disintegration and loss of activity of free PCEVs during in vitro storage and in the complex environment of the scalp. Physical encapsulation and protection of PCEVs are achieved through a three-dimensional hydrogel network.

[0031] Synergistic enhancement of local retention time and permeability: Overcoming the shortcomings of traditional liquid formulations that are easy to lose and difficult to penetrate on the scalp surface, the adhesion and sustained-release properties of interpenetrating polymer networks (IPN) are utilized to prolong the residence time of PCEVs in the hair follicle and promote their deep penetration.

[0032] 2. Achieved long-lasting sustained release and targeted enrichment of active ingredients: This invention utilizes tangential flow technology to achieve large-scale extraction of extracellular vesicles from Poria cocos cells. Through a dual mechanism of ionic crosslinking and physical entanglement, this hydrogel system can intelligently respond to the scalp microenvironment, achieving continuous and controllable release of PCEVs. This "reservoir effect" effectively prolongs the effective duration of drug action in the hair follicle, solving the problem of high-frequency drug administration required by traditional formulations; simultaneously, the introduction of hyaluronic acid (HA) enhances the specific interaction between the carrier and hair follicle cells, promoting the targeted delivery of PCEVs to the hair follicle stem cell niche.

[0033] 3. Demonstrates superior hair regrowth promotion efficacy and synergistic effects through multiple mechanisms: Experimental results show that this composite system can significantly reverse dihydrotestosterone (DHT)-induced hair follicle miniaturization and promote the transformation of resting follicles into anagen phases. This invention overcomes the limitations of single androgen suppression therapy. Its mechanism of action is not limited to simple nutrient supply, but also lies in deeply activating the Wnt / β-catenin key signaling pathway by delivering active payloads in PCEVs, reshaping the differentiation potential of hair follicle stem cells, and simultaneously regulating the local immune microenvironment (anti-inflammatory, antioxidant). This multi-regulatory mechanism of "carrier-drug" synergy makes it significantly more effective than single-drug therapy in promoting increased hair density, thicker hair shaft, and prolonged growth cycle.

[0034] 4. This invention provides a novel hair loss intervention solution with great translational potential: It combines tangential flow ultrafiltration separation technology with advanced biomaterial preparation processes, resulting in a clear, highly controllable process that is easy to scale up for production. This strategy provides a new technological path and application paradigm for developing next-generation hair loss treatment products that combine biomimetic characteristics, efficient delivery capabilities, and clinical safety.

[0035] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1Isolation and characterization of extracellular vesicles (PCEVs) from Poria cocos; (A) Schematic diagram of the isolation of PCEVs from fresh Poria cocos by tangential flow filtration (TFF). (B) Transmission electron microscopy (TEM) image of PCEVs. (C) Particle size distribution and concentration of PCEVs determined by nanoparticle tracking analysis (NTA). (D) Confocal microscopy image: results after 12 h of co-incubation. PKH26-labeled PCEVs show red fluorescence, WGA shows green fluorescence, and DAPI shows blue fluorescence. Scale bar: 50 μm.

[0037] Figure 2 Evaluation of in vitro biocompatibility and bioactivity of PCEVs; (A) Detection of dermal papillary cells (DPCs) viability using the CCK-8 assay. (B) Live / dead staining of DPCs. Live cells showed green fluorescence (Calcein-AM), and dead cells showed red fluorescence (PI). Scale bar: 200 μm. (C) Detection of changes in DPC cell viability using the CCK-8 assay. (D) Cell viability detection results after co-culturing different concentrations of PCEVs with DPCs for 24 h and 48 h under 50 μM DHT treatment. (E) Isolated mouse whisker hair follicles were cultured in vitro under different treatment conditions (Control, DHT, DHT + 10% PCEVs, DHT + 20% PCEVs), and representative images of hair follicle growth were recorded on days 0, 3, 6, 9, and 12. Scale bar: 1 mm. (F) Quantitative analysis results of hair-shaft elongation on day 12 (n = 5).

[0038] Figure 3 Construction and physicochemical characterization of the PCEV@CMC / SA hydrogel system; (A) Schematic diagram of the PCEV@CMC / SA hydrogel preparation process. (B) Comparison of compressive stress of different hydrogel formulations. (C) Representative compressive stress-strain curves of different hydrogel formulations. (D) Scanning electron microscope (SEM) images of the porous microstructure inside CMC / SA and PCEV@CMC / SA hydrogels. Scale bar: 100 μm and 20 μm. (E, F) Quantitative analysis of pore size distribution of CMC / SA and PCEV@CMC / SA hydrogels, respectively. (G) Representative photographs of the adhesion behavior of the hydrogel on different substrates (glass, rubber, skin, steel, and plastic). (H) In vitro cumulative release curve of PCEVs in PCEV@CMC / SA hydrogel within 24 h at room temperature.

[0039] Figure 4In vitro biocompatibility evaluation of CMC / SA and PCEV@CMC / SA hydrogels; (A) Cell viability of DPCs after co-culturing with different concentrations of hydrogel extracts for 24 h and 48 h, where the extract concentrations are low (25%), medium (50%), and high (100%), respectively. (B) Cell viability of HUVECs after treatment with different concentrations of hydrogel extracts for 24 h and 48 h. (C) Live / dead cell fluorescence staining images of DPCs and HUVECs after treatment with CMC / SA and PCEV@CMC / SA hydrogel extracts for 24 h. Scale bar: 100 μm.

[0040] Figure 5 A schematic diagram of the AGA mouse model establishment and subsequent treatment process.

[0041] Figure 6 Evaluation of the in vivo therapeutic effect of PCEV@CMC / SA in a mouse model of androgenetic alopecia (AGA); (A) Representative back photographs of mice in each group on days 0, 7, 15, and 21. Each group contained n = 6 biologically independent mice, and 3 representative mice from each group are shown for ease of presentation. (B) Quantitative analysis of the area of ​​hair regeneration on the back skin of mice in each treatment group on days 7, 15, and 21 (n = 6 / group). (C) Changes in body weight of mice in each group. (D) Representative H&E staining images of mouse back skin on day 22, scale bar: 200 μm. (EG) Quantitative analysis of the number of hair follicles (HFs), follicle diameter, and dermal thickness per unit field of view (n = 6 biologically independent mice / group).

[0042] Figure 7 Changes in serum hormone levels in mice in each treatment group; quantitative analysis of serum dihydrotestosterone (DHT) levels in mice in each group. (B) Quantitative analysis of serum 17β-estradiol (17β-E2) levels in mice in each group.

[0043] Figure 8 Flowchart. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments.

[0045] For the specific process of this invention, please refer to Figure 8 .

[0046] Example 1: Isolation and characterization of extracellular vesicles (PCEVs) derived from Poria cocos ( Figure 1 ) 1. Preparation of PCEVs (1) Raw material pretreatment: Take 200g of fresh Poria cocos fruiting body, wash thoroughly with ultrapure water and then chop. Add 1L of pre-cooled ultrapure water at 4℃ (the ratio of Poria cocos fruiting body to ultrapure water is 200g:1L), and homogenize using a wall-breaking machine. Filter the resulting homogenate through a filter screen to remove coarse tissue residue. (2) Preliminary clarification: Centrifuge the filtrate at 4°C and 10,000×g for 30 min, and collect the supernatant to remove cell debris and large particulate impurities. (3) Chromatography and filtration: The supernatant is preliminarily purified by passing it through a silica gel chromatography column (silica gel 60, AR, 200-300 mesh), and then filtered through a 0.45 μm microporous membrane to remove residual particles and macromolecular polymers. (4) Tangential flow ultrafiltration (TFF) enrichment: Take 500 mL of the clarified filtrate and concentrate it using multi-step TFF technology. Using a polyethersulfone (PES) membrane with a molecular weight cutoff of 300 kDa, the operating conditions were controlled as follows: room temperature, transmembrane pressure ≤1 bar, feed flow rate 30-60 mL / min, and permeate flow rate approximately 4 mL / min. (5) Dialysis replacement: The obtained retentate was dialyzed with 500 mL of phosphate-buffered saline (PBS). The above TFF concentration cycle was repeated twice to finally obtain a high-purity PCEVs concentrate with a volume of approximately 50 mL. After sterilization by filtration through a 0.22 μm microporous membrane in a clean bench, the concentrate was aliquoted. (6) Storage: After aliquoting, the samples were stored at -80°C to avoid repeated freeze-thaw cycles.

[0047] 2. Characterization of PCEVs (1) Particle size and concentration analysis: The particle size distribution and particle concentration of PCEVs were determined by nanoparticle tracking analysis (NTA). (2) Morphological observation: The morphological structure and size characteristics of PCEVs were observed by transmission electron microscopy (TEM) to confirm that they have a typical vesicle-like bilayer membrane structure.

[0048] Example 2: Isolation, culture and identification of primary dermal papilla cells (DPCs) 1. Cell isolation (1) Sample collection: Six-week-old male C57BL / 6J mice were euthanized by an overdose of sodium pentobarbital. Skin from the beard pad area was cut off, disinfected with 75% ethanol aqueous solution, and rinsed in sterile PBS. (2) Dissection: Under a dissecting microscope, the connective tissue around the hair follicle was dissected to expose the intact hair follicle. The hair bulb tissue containing the dermal papilla was excised from the lower part of the hair follicle and temporarily stored in DMEM medium at 4℃. (3) Enzyme digestion: The hair bulb tissue was transferred to a digestion solution containing 0.2% type I collagenase and digested at 37℃ for 30-60 min. During this period, the tissue was gently pipetted and mixed every 10 min until the tissue around the hair bulb was fully digested and the dermal papilla structure was clearly exposed. (4) Termination and inoculation: An equal volume of complete culture medium containing serum was added to terminate the reaction. After centrifugation and washing, the dermal papilla tissue was resuspended in DMEM medium containing 20% ​​fetal bovine serum (FBS) and 1% penicillin-streptomycin and inoculated into culture dishes. (5) Culture: Incubate at 37℃ in a 5% CO2 incubator. After tissue adhesion, replace with DMEM complete medium containing 10% FBS, changing the medium every 3 days. Around day 7, cell migration and aggregation can be observed. Passage is performed when cell confluence reaches 80-90%. Subsequent experiments use cells from passages 3 to 6 (P3-P6).

[0049] 2. Cell identification P3 generation DPCs were seeded in 24-well plates, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 5% goat serum for 30 min. Then, 200 μl of antibody working solution of α-SMA (Catalog No. 19245, Cell Signaling Technology), ALP (Catalog No. sc-398461, Santa Cruz Biotechnology), and β-Catenin (Catalog No. 8480, Cell Signaling Technology) primary antibodies (1:200 dilution) were added, and the plates were incubated overnight at 4°C. The next day, the plates were incubated with fluorescent secondary antibody (1:200) for 30 min, and the nuclei were counterstained with DAPI for 5 min. Images were acquired using an inverted fluorescence microscope to confirm the expression of cell-specific markers.

[0050] Example 3: Evaluation of the biocompatibility and proliferative activity of PCEVs ( Figure 2 ) 1. CCK-8 assay for cell proliferation (1) Grouping treatment: After sterile filtration of the PCEV suspension, it was serially diluted with complete culture medium to prepare treatment solutions with volume fractions of 1%, 5%, 10%, and 20%. (2) Cell seeding: DPCs were seeded at a concentration of 2 × 10⁻⁶ cells / mL.3 (3) Drug administration and detection: The medium was replaced with different concentrations of PCEVs and treated for 24 h. CCK-8 working solution was added and incubated in the dark for 2 h, and the absorbance (OD value) at 450 nm was measured. (4) Calculation: The cell proliferation rate was calculated according to the formula: cell proliferation rate = (ODa-ODc) / (ODb-ODc)×100%, where ODa is the treatment group, ODb is the control group, and ODc is the blank group.

[0051] 2. Live / dead cell staining DPCs were seeded into 24-well plates, and after reaching a density of 60%, they were treated with PCEVs at concentrations of 5%, 10%, and 20% for 24 h, respectively. The cells were stained using a Calcein-AM / PI double staining kit, and the distribution of live cells (green) and dead cells (red) was observed and recorded under a fluorescence microscope.

[0052] Example 4: Construction and optimization of CMC-Na / SA / HA composite hydrogel ( Figure 3 ) 1. Preparation of basic stock solution Prepare aqueous solutions of 0.9% (w / v) low molecular weight hyaluronic acid (L-HA, 10-30 kDa), 4% (w / v) CMC-Na, and 4% (w / v) SA, respectively. The L-HA solution was hydrated overnight at 4°C. The remaining solutions were stirred at room temperature until completely dissolved, degassed, and sterilized by filtration through a 0.22 μm filter membrane.

[0053] 2. Formulation screening and mechanical property testing: Four hydrogel systems with different SA to CMC-Na volume ratios (v:v) were constructed: Group A: 0:1 Group B: 0.5:1 Group C: 1:1 Group D: 2:1 Maintain the final HA concentration at 0.9% (w / v). Form each group of hydrogels into regular cylinders and perform uniaxial compression tests (rate 2 mm / min) using a universal testing machine. Record the stress-strain curves and maximum compressive strength, and select the ratio with the best mechanical properties (in this embodiment, group C, i.e., 1:1, is preferred).

[0054] 3. Preparation of drug-loaded hydrogel (PCEV@Gel) (1) Mixing: Mix CMC-Na and SA solution at a ratio of 1:1 (v / v) and stir for 30 min; add L-HA stock solution and gently mix on the lowest setting of the stirrer to achieve final concentrations of CMC-Na 2%, SA 2%, and HA 0.9%. (2) Drug loading: Under ice bath conditions, add 9 ml of PCEV suspension to 90 ml of the above polysaccharide mixture and gently mix on the lowest setting of the stirrer to avoid shearing and damaging the vesicles to obtain a hydrogel system. (3) In-situ gelation: Mix 0.6% (w / v) calcium chloride (CaCl2) and glucono-δ-lactone (GDL) in equal volumes, and add 1 mL of the mixture to the hydrogel system in (2) (1:99 volume ratio). React at room temperature for 1-4 hours to induce endogenous gelation (pH maintained at 6.8-7.4). (4) Post-treatment: Rinse the gel with PBS 2-3 times to remove free Ca. 2+ Store at 4°C away from light for later use.

[0055] 4. Microstructure and Adhesion Evaluation (1) SEM observation: The hydrogel was freeze-dried for 36 h and its microstructure was observed using a scanning electron microscope (SEM). The average pore size was calculated by randomly measuring 20 pores using ImageJ software. (2) Adhesion test: A qualitative adhesion experiment was conducted. The hydrogel was placed on the surfaces of glass, rubber, skin, stainless steel and plastic. Its adhesion stability under gravity was observed and photographed.

[0056] 5. Encapsulation efficiency and load efficiency determination The total number of PCEVs (Ntotal) before gelation and the number of free particles (Nfree) in the supernatant after gelation were determined using NTA.

[0057] Encapsulation efficiency (EE): EE (%) = [(Ntotal - Nfree) / Ntotal] × 100% EE (%) Loading efficiency (LE): LE = (Ntotal - Nfree) / Mhydrogel, where Mhydrogel is the wet weight of the hydrogel.

[0058] 6. In vitro release kinetics Immerse 0.1 mL of PCEV@Gel in 1 mL of PBS and incubate at room temperature. Collect all supernatant at time points of 1, 2, 4, 6, 8, 12, and 24 h and replenish with an equal volume of fresh PBS. Measure the particle concentration released at each time point using NTA (subtracting the background of the blank gel) and plot the cumulative release curve.

[0059] Example 5: Biosafety evaluation of the composite hydrogel system ( Figure 4 ) 1. Preparation of extract Blank CMC / SA hydrogels and PCEV@CMC / SA composite hydrogels were immersed in complete culture medium at a ratio of 1:10 (v / v) and extracted at 4°C in a shaker for 24 h. The extracts were sterilized by 0.22 μm filtration and prepared into a series of mass concentrations of 0%, 25%, 50%, and 100%.

[0060] 2. Cytotoxicity detection (1) CCK-8 assay: Human umbilical vein endothelial cells (HUVECs) (Haixing Biotechnology, Suzhou) and DPCs were seeded in 96-well plates. After the confluence reached 50%-60%, the extract was replaced with different concentrations of extract. After culturing for 24 h and 48 h, CCK-8 reagent was added to detect cell proliferation activity. (2) Live / dead staining: Cells were seeded in 24-well plates and treated with different concentrations of extract for 24 h. Then, Calcein-AM / PI double staining was performed, and cell morphology and viability were observed under a fluorescence microscope.

[0061] Figure 5 This diagram illustrates the establishment and subsequent treatment process for the AGA mouse model. Specifically, an appropriate amount of DHT powder was weighed and added to corn oil to prepare a working solution of 10 mg / mL. The solution was vortexed and gently heated in a water bath to promote dissolution. The prepared working solution was stored away from light and brought to room temperature before use, then thoroughly mixed. Mice in the DHT group and each treatment group were injected intraperitoneally daily at a dose of 1 mg / mouse, i.e., 100 μL of DHT working solution per mouse; the control group received an equal volume of corn oil intraperitoneally. During injection, the mice were immobilized, and the needle was inserted into the lower left or lower right quadrant of the abdomen, slowly injecting the solution into the peritoneal cavity. Injections were performed at relatively fixed times each day throughout the modeling period.

[0062] On day 2 of the experiment, depilatory cream was used to remove hair from the backs of mice to synchronize the hair follicle cycle and expose the back skin. Starting the day after hair removal, each treatment group received its corresponding intervention: the DHT+CMC / SA group received CMC / SA hydrogel applied to the hair removal area; the DHT+PCEV@CMC / SA group received CMC / SA composite hydrogel loaded with PCEVs; and the DHT+MXD group received minoxidil gel. All treatments were administered once daily, evenly applied to the hair removal area. The control group and DHT group received no topical medication. Hair growth was observed and photographed on the back skin of mice at days 0, 7, 15, and 21 after hair removal. Consistent lighting conditions, fixed shooting distances, and the same mouse position were maintained during photography to ensure consistent image acquisition. At the end of the experiment on day 22, mice were anesthetized, and blood was collected via the eyeball into centrifuge tubes containing anticoagulant. Mice were then euthanized by cervical dislocation, and their back skin tissue was immediately harvested. Some samples were fixed with 4% paraformaldehyde for subsequent histological observation, while the remaining samples were flash-frozen in liquid nitrogen and stored at -80 °C for further use. In addition, mouse whole blood was centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatant was collected as serum samples and stored at -80 °C for later use.

[0063] Hair coverage calculation To quantitatively assess hair growth, ImageJ software was used to analyze images of the back skin. The total area (A) of the hair removal zone was measured using the color thresholding function. total ) and hair coverage area (A hair ), and calculate hair growth coverage using the following formula: Hair coverage = A hair / A total *100% Hematoxylin-Eosin (H&E) staining Skin samples from the backs of mice were fixed by immersion in 4% paraformaldehyde solution for 24 h. Subsequently, the tissue blocks were dehydrated stepwise with ethanol, cleared with xylene, and finally embedded in paraffin. After paraffin embedding, the tissue was cut into continuous sections approximately 4 μm thick using a paraffin microtome. After spreading, the sections were lifted onto glass slides and baked overnight at 60 ℃ to enhance adhesion between the tissue and the slides.

[0064] Before staining, the sections were first dewaxed and rehydrated. The sections were immersed in xylene twice for dewaxing, each time for 20 minutes. Then, a gradient ethanol hydration process was performed: first, the sections were rinsed twice in anhydrous ethanol (5 minutes each), then transferred to a 75% ethanol solution for 5 minutes to gradually rehydrate the tissue. After rehydration, the sections were rinsed under running tap water to remove residual staining reagents. The sections were then immersed in hematoxylin staining solution for nuclear staining for 1-3 minutes. After staining, the sections were immediately treated with tap water for differentiation and blueing to enhance the clarity of the nuclear structure. After treatment, the sections were rinsed again with running water. Then, the sections were counterstained with eosin for about 1 minute to make the cytoplasm and interstitial tissue pink. After staining, the sections were rinsed with tap water to remove excess dye. After staining, the sections were dehydrated and cleared. The sections were sequentially placed in anhydrous ethanol for dehydration, then transferred to xylene for clearing. Finally, neutral resin was added for mounting, and a coverslip was placed on top. The sections were then allowed to dry at room temperature for later use.

[0065] The stained tissue sections were observed and images were acquired under a Leica optical microscope. Quantitative analysis of the histological images was performed independently by two researchers without knowledge of the grouping information. ImageJ software was used to measure indicators such as the number of hair follicles, hair follicle diameter, and dermal thickness to evaluate the effects of different treatment groups on hair follicle structure and skin tissue morphology.

[0066] Representative photographs of mice in each group on days 0, 7, 15, and 21 are shown below. Figure 6 A. Observation showed that immediately after hair removal, the backs of all mice were pink, with the hair follicle cycle starting point being basically the same, and all were in the resting phase. On day 7, the back skin was still predominantly pink, with no significant differences between groups. By day 15, the back skin of the control group, MXD group, and PCEV@CMC / SA group gradually darkened and showed obvious new hair growth; while the hair follicles in the DHT group and CMC / SA group remained in a prolonged resting phase. By day 21, most of the drug groups had relatively extensive hair coverage, while hair growth recovery in the DHT group and CMC / SA group was significantly delayed. Among them, the hair regeneration degree of the PCEV@CMC / SA group was further improved, and the overall appearance was significantly better than that of the DHT group and CMC / SA group, approaching the level of the MXD group. During the experiment, there were no obvious redness, swelling, or allergic reactions in the drug-treated areas of the mice.

[0067] The quantitative results of hair coverage were consistent with visual observation. Figure 6(B) On days 0 and 7, the backs of mice in all groups were in the early stage of post-hair loss, with hair coverage close to 0. By day 15, hair recovery was significant in the healthy control group, with coverage exceeding 50%; the hair coverage in the PCEV@CMC / SA group was significantly higher than that in the DHT and CMC / SA groups. By day 21, this difference widened further, with hair in the healthy control group almost completely recovered, reaching nearly 100% coverage. The hair coverage in the PCEV@CMC / SA group also increased significantly, reaching over 70%, similar to the MXD group and significantly higher than the DHT and blank CMC / SA groups. These results indicate that PCEV@CMC / SA can effectively improve DHT-induced hair regeneration inhibition.

[0068] Notably, the CMC / SA blank hydrogel group also showed some degree of hair recovery, suggesting that the hydrogel matrix itself may provide some support to the hair follicle microenvironment by maintaining a local moist environment. During the experiment, the body weight of mice in all groups steadily increased. Figure 6 (C) No obvious abnormal fluctuations were observed, indicating that the overall tolerance of each treatment regimen was good.

[0069] H&E staining results showed that the dorsal skin of mice in the DHT group exhibited typical AGA-like histological changes, including follicle miniaturization, reduced follicle number, and structural atrophy. Figure 6 (D) In ​​contrast, the control group showed intact hair follicle structure and a denser arrangement. Both the PCEV@CMC / SA and MXD groups exhibited relatively intact hair follicle morphology, with significantly improved follicle quantity and volume compared to the DHT group. Furthermore, although a small number of new hair follicles were observed in the CMC / SA group, their number and density were significantly lower than in the PCEV@CMC / SA group.

[0070] Further quantitative analysis showed that ( Figure 6 Compared to the DHT group, PCEV@CMC / SA treatment significantly increased the number of hair follicles, hair follicle diameter, and dermal thickness, with improvements approaching those of the MXD group. While the hair follicle diameter increased in the CMC / SA group, the number of hair follicles did not change significantly and remained lower overall than in the PCEV@CMC / SA group. These results further demonstrate that PCEV@CMC / SA can effectively improve DHT-induced hair follicle atrophy and promote hair follicle structure recovery and hair growth.

[0071] Measurement of serum DHT and 17β-E2 hormones in mice The levels of dihydrotestosterone (DHT) and estradiol (17β-E2) in serum were detected using enzyme-linked immunosorbent assay (ELISA), with all procedures strictly following the kit instructions. The main procedure was as follows: Standards and test serum were sequentially added to microplates pre-coated with antibodies, incubated, and then washed. Enzyme-labeled antibodies were then added and incubated together. After thorough washing, the chromogenic substrate was added, and the reaction was carried out for an appropriate time under dark conditions. Stop solution was immediately added to stop the color development. Finally, the absorbance (OD) values ​​of each well were read at 450 nm, and the levels of the two hormones were calculated using a standard curve. See details below. Figure 7 .

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A process for preparing Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia, characterized in that, The specific steps are as follows: S1. Preparation of extracellular vesicles (PCEVs) from Poria cocos: Fresh Poria cocos fruiting bodies were used as raw materials. They were washed, chopped, homogenized with water, filtered, and the filtrate was initially clarified. The filtrate was then subjected to chromatography and filtration, enriched by tangential flow ultrafiltration, and dialysis to obtain a PCEV suspension. S2. Preparation of drug-loaded hydrogel PCEV@Gel: Sodium carboxymethyl cellulose aqueous solution and sodium alginate aqueous solution were mixed and stirred, and low molecular weight hyaluronic acid aqueous solution was added and gently mixed to obtain a polysaccharide mixture; then PCEV suspension was added to the polysaccharide mixture and gently mixed to avoid shearing and destroying the vesicles to obtain a hydrogel system; then calcium chloride and gluconate-δ-lactone were added, and the reaction was carried out at room temperature to induce endogenous gelation. After post-treatment, PCEV@Gel was obtained.

2. The preparation process according to claim 1, characterized in that, In step S1, the method of adding water and homogenizing is as follows: add ultrapure water and homogenize using a high-speed blender to obtain a homogenate; filter the homogenate through a filter screen to remove coarse tissue residues to obtain a filtrate; the ratio of Poria cocos fruiting body to ultrapure water is 200g:1L.

3. The preparation process according to claim 1, characterized in that, In step S1, the preliminary clarification method is as follows: the filtrate is centrifuged at 4°C and 10,000×g for 30 min, and the supernatant is collected to remove cell debris and large particulate impurities.

4. The preparation process according to claim 1, characterized in that, In step S1, the chromatography and filtration methods are as follows: preliminary purification is performed through a silica gel chromatography column, followed by filtration through a 0.22μm microporous membrane to remove residual particles and macromolecular polymers.

5. The preparation process according to claim 1, characterized in that, In step S1, multi-step tangential flow ultrafiltration technology is used for concentration. A polyethersulfone membrane with a molecular weight cutoff of 300 kDa is used. The operating conditions are controlled as follows: room temperature, transmembrane pressure ≤1 bar, feed flow rate 30-60 mL / min, and permeate flow rate 4 mL / min.

6. The preparation process according to claim 1, characterized in that, In step S2, the mass concentrations of sodium carboxymethyl cellulose aqueous solution, sodium alginate aqueous solution, and low molecular weight hyaluronic acid aqueous solution are 4%, 4%, and 0.9%, respectively. The mass concentrations of sodium carboxymethyl cellulose, sodium alginate, and low molecular weight hyaluronic acid contained in the polysaccharide mixture are 4%, 4%, and 0.9%, respectively.

7. The preparation process according to claim 1, characterized in that, In step S2, the volume ratio of the PCEV suspension to the polysaccharide mixture is 1:

10. Calcium chloride and gluconate-δ-lactone were each prepared into 0.6% aqueous solutions, and then mixed in equal volumes to obtain a premix. This premix was then added to the hydrogel system. The volume ratio of the premix to the hydrogel system was 1:

99.

8. The preparation process according to claim 1, characterized in that, In step S2, the pH is controlled at 6.8–7.4 during the induction of endogenous gelation; Post-processing includes rinsing the gel 2-3 times with PBS to remove free Ca. 2+ Store at 4℃ away from light for later use.

9. A Poria cocos extracellular vesicle hydrogel for the treatment of androgenetic alopecia, characterized in that, It is obtained by the preparation process described in any one of claims 1 to 8.

10. The use of the hydrogel of claim 9 in the preparation of a medicament for treating androgenetic alopecia.

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