Exosome mixture for treating alopecia and preparation method and application thereof

Abstract

The invention relates to the technical field of alopecia treatment, and discloses an exosome mixture for treating alopecia, which comprises: (a) exosome from gene edited hair follicle progenitor cells; (b) nano-selenium particles; and (c) a pH and glutathione dual responsive mineralization layer coated on the surface of the exosome, the gene-edited hair follicle progenitor cell overexpresses miR-25-5p and miR-205 through a CRISPR / Cas9 technology, and integrates a Wnt3a gene to a regulation region of exosome membrane protein CD63, and the mineralization layer is composed of calcium phosphate and selenite. According to the invention, a scheme of combining a gene editing exosome compound and nano-selenium is adopted, so that synchronous activation of three channels of regeneration promotion, anti-inflammation and anti-apoptosis of hair follicles is realized, the beta-catenin signal intensity is improved, the growth period of the hair follicles is prolonged, and the bottleneck of the curative effect of the traditional therapy is broken through.

Description

Technical Field

[0001] The present invention relates to the technical field of hair loss treatment, and specifically to an exosome mixture for treating hair loss, and a preparation method and application thereof. Background Art

[0002] In the field of hair loss treatment, existing technologies mainly include: 1) chemical drugs (such as minoxidil and finasteride) that delay hair loss by regulating local blood flow or hormone levels; 2) growth factor therapies (such as FGF and VEGF) that directly stimulate hair follicle cell proliferation; 3) exosome delivery systems that use stem cell-secreted vesicles to deliver active ingredients; and 4) transdermal delivery technologies (such as liposomes and nanoemulsions) to enhance drug permeability through the skin. These technologies focus on a single mechanism of action or optimizing delivery efficiency, but fail to systematically address the multi-pathway imbalance and dynamic adaptation of delivery and release during the hair loss process.

[0003] Existing technologies have significant limitations: chemical drugs cannot reverse hair follicle miniaturization, and exosome activity is easily destroyed by freeze-drying and oxidation; single-target gene editing is difficult to cover the coordinated regulation of Wnt / apoptosis / inflammation multiple pathways; traditional transdermal systems rely on passive diffusion, with a targeting efficiency of less than 20%, and cannot achieve deep delivery to hair follicles; sustained-release preparations lack the ability to dynamically respond to the pathological stages of hair loss (acute inflammatory stage-chronic atrophy stage), resulting in delayed efficacy or excessive release; the low-oxygen amplification and stability protection technologies in the exosome preparation process are separated, and industrial storage faces the problem of rapid activity decay. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides an exosome mixture for treating hair loss, a preparation method and application thereof, which solves the problems of the existing technology such as lack of multi-pathway coordinated regulation, low transdermal efficiency, insufficient dynamic response capability and weak cross-stage stability control.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: an exosome mixture for treating hair loss, comprising:

[0006] (a) Exosomes derived from gene-edited hair follicle progenitor cells;

[0007] (b) Selenium nanoparticles (SeNPs);

[0008] (c) A pH and glutathione (GSH) dual-responsive mineralization layer coated on the surface of the exosomes.

[0009] The fundamental innovation of this invention lies in the selection of exosome sources and their enhanced functionality. Compared to conventional mesenchymal stem cells (MSCs) or dermal papilla cells (DPCs), this invention uses hair follicle progenitor cells (HFPCs) differentiated from induced pluripotent stem cells (iPSCs) as a source of exosomes. HFPCs naturally express signaling molecules associated with hair follicle regeneration (Wnt10b and SHH), and the exosomes they secrete have the ability to regulate the hair follicle microenvironment.

[0010] Gene editing of HFPCs using CRISPR / Cas9 technology achieves dual enhancement of exosome function:

[0011] Overexpression of miR-25-5p and miR-205: miR-25-5p directly promotes the proliferation of hair follicle stem cells by inhibiting the Wnt pathway antagonist DKK1 and activating β-catenin signaling; miR-205 reduces the release of hair follicle degeneration-related inflammatory factors (IL-6) by downregulating the EGFR pathway.

[0012] Integration of the Wnt3a gene into the CD63 regulatory region of the exosome membrane protein: Wnt3a is a key protein for initiating the hair follicle cycle. Through gene editing, it is co-expressed with the CD63 membrane protein to ensure that exosomes continuously release Wnt3a during the delivery process, breaking through the limitation of traditional exosomes that only rely on the passive release of contents.

[0013] Gene editing not only enhances the natural activity of exosomes, but also gives them a dual-driving mechanism of "content (miRNA) + membrane protein (Wnt3a)", forming a synergistic stimulation effect on hair follicle stem cells.

[0014] The introduction of nano-selenium is not a simple physical mixture, but a functional coupling formed by in-situ reduction and integration with the mineralized layer. The core function of nano-selenium is to inhibit the pathway related to hair follicle apoptosis:

[0015] Targeted inhibition of the TGF-β / Smad3 pathway: SeNPs upregulate Smad7 expression, blocking TGF-β1-induced apoptosis signals in hair follicle stem cells, complementing the pro-proliferation effect of exosomes.

[0016] Synergistic anti-oxidative stress: The semiconductor properties of SeNPs enable them to generate electron-hole pairs under blue light catalysis, eliminating reactive oxygen species (ROS) around hair follicles, while the SOD2 enzyme carried by exosomes further enhances the antioxidant capacity, forming a double barrier.

[0017] Nano-selenium and exosomes cover the core pathology of hair loss through the dual pathways of "inhibiting apoptosis + promoting regeneration", and the photocatalytic process simultaneously activates the protection mechanism of exosomes, breaking through the destruction of biological activity by traditional chemical reduction methods.

[0018] The design of the mineralized layer is the core of the invention to achieve precise delivery and long-term stability. The mineralized layer is composed of calcium phosphate (CaP) and selenite (SeO3 2- ) gradient doping structure, its response logic and structural characteristics are as follows:

[0019] pH response mechanism: The weakly acidic environment (pH 5.0-5.5) around the hair follicle triggers the preferential dissolution of the high-concentration selenite in the outer layer, releasing SeNPs to inhibit the TGF-β pathway while exposing the inner bionic bone structure (Ca / P=1.67).

[0020] GSH response mechanism: Glutathione (GSH ≥ 5 mM) highly expressed in hair follicle stem cells further decomposes the inner mineralized structure, releases exosomes and Wnt3a protein, and activates the Wnt signaling pathway.

[0021] Through the spatiotemporal sequential control of "pH (triggering SeNPs release) + GSH (triggering exosome release)", the drug can be precisely enriched in the hair follicle area to avoid non-targeted loss on the skin surface.

[0022] Preferably, the gene-edited hair follicle progenitor cells overexpress miR-25-5p and miR-205 through CRISPR / Cas9 technology, and integrate the Wnt3a gene into the regulatory region of the exosomal membrane protein CD63.

[0023] Coordinated overexpression of miR-25-5p and miR-205:

[0024] Target selection basis: miR-25-5p and miR-205 have complementary roles in hair follicle regeneration. Their coordinated overexpression can cover the multi-pathway regulation of hair loss:

[0025] Function of miR-25-5p: By directly binding to the mRNA 3'UTR region of the Wnt pathway antagonist DKK1, it inhibits its translation, relieves the inhibitory effect of DKK1 on Wnt / β-catenin signaling, and thus activates the proliferation of hair follicle stem cells.

[0026] Function of miR-205: By downregulating the expression of epidermal growth factor receptor (EGFR), it reduces the miniaturization of hair follicles and the release of inflammatory factors (IL-6, TNF-α) caused by excessive activation of EGFR, thereby delaying the process of hair follicle degeneration.

[0027] Traditional technologies usually only regulate a single miRNA or growth factor, while the present invention achieves the dual effects of "proliferation promotion (Wnt pathway) + anti-inflammation (EGFR pathway)" through the collaborative design of dual miRNAs, significantly improving the comprehensive therapeutic effect of exosomes.

[0028] Wnt3a gene integration into the CD63 regulatory region:

[0029] Integration site design: CD63 is a transmembrane protein highly expressed on the exosome membrane. Its gene regulatory region (promoter region) was selected as the insertion site for Wnt3a. The specific advantages are:

[0030] Targeted delivery guarantee: CD63 protein is enriched on the surface of the exosome membrane. After integrating the Wnt3a gene, its expression product (Wnt3a protein) can be directly anchored on the exosome membrane, ensuring the local high-concentration release of Wnt3a when delivered to hair follicle stem cells.

[0031] Continuous activity maintenance: The CD63 promoter is constitutively active (not restricted by the cell cycle), allowing Wnt3a to be continuously expressed during exosome production, overcoming the problem of easy degradation of traditional exosome contents (free Wnt3a protein).

[0032] In the prior art, Wnt3a usually exists as exosome contents and is rapidly inactivated after release; however, the present invention uses targeted integration of membrane proteins to enable Wnt3a to exist in a stable form and be delivered in a targeted manner, increasing its active half-life to more than three times that of traditional methods.

[0033] Synergistic enhancement of gene editing and exosome function:

[0034] Synergistic mechanism: miRNA overexpression and Wnt3a integration are not designed independently, but form a synergistic network through pathway intersection:

[0035] miR-25-5p enhances the Wnt3a effect: After miR-25-5p inhibits DKK1, the sensitivity of hair follicle stem cells to Wnt3a is significantly increased, forming a positive feedback.

[0036] Wnt3a activates miR-205 expression: Wnt3a binds to the miR-205 promoter through the β-catenin / TCF4 complex, further upregulating its expression and enhancing the anti-inflammatory effect.

[0037] The gene editing design achieves bidirectional synergy of "miRNA-Wnt3a" through pathway interaction, forming a self-reinforcing therapeutic network and breaking through the efficacy bottleneck of traditional single-pathway regulation.

[0038] Preferably, the mineralized layer is composed of calcium phosphate and selenite, and the concentration of selenite in the outer mineralized liquid is 1.5-3 times that of the inner layer.

[0039] Gradient doping design of calcium phosphate and selenite:

[0040] The mineralized layer is composed of calcium phosphate (CaP) and selenite (SeO3 2-), and the concentration of selenite in the outer mineralization solution (4mM) is 1.5-3 times that of the inner layer (1.5-2mM). This gradient design is based on the precise correspondence between the characteristics of the hair follicle microenvironment and the drug release requirements:

[0041] High selenium doping in the outer layer: High concentration of selenite forms a selenium-rich area in the outer layer, which preferentially responds to the weakly acidic environment of the hair follicle (pH 5.0-5.5), quickly releases selenium nanoparticles (SeNPs), and inhibits hair follicle apoptosis caused by the TGF-β pathway.

[0042] Inner layer biomimetic calcium phosphate: The Ca / P ratio of the inner layer simulates the natural bone mineral phase and needs to be triggered by glutathione (GSH ≥ 5mM) highly expressed by hair follicle stem cells to decompose, release exosomes and Wnt3a protein, and activate hair follicle regeneration signals.

[0043] Traditional mineralized layers are usually homogeneous structures and cannot achieve spatiotemporal control of drug release. The gradient design of the present invention solves the problem of drug loss in non-target areas through "external layer speed control (SeNPs rapid release) + inner layer target control (exosome precise release)".

[0044] Synergy between component gradient and response logic:

[0045] The gradient distribution of the mineralized layer forms a double match with the microenvironmental triggering conditions:

[0046] pH response priority: The outer high-selenium area dissolves rapidly in an acidic environment, ensuring that SeNPs are enriched at the entrance of the hair follicle and blocking TGF-β signaling.

[0047] GSH response specificity: The decomposition of inner calcium phosphate depends on GSH-mediated reduction reaction, and the GSH concentration around hair follicle stem cells (5-10mM) is significantly higher than that in the epidermis (<1mM), thus ensuring that exosomes are released only in the target area.

[0048] By coupling component gradients with microenvironmental biomarkers (pH, GSH), three-dimensional precise control of drug release in terms of "position-time-dose" is achieved, breaking through the blind release defects of traditional delivery systems (liposomes, microneedles).

[0049] Mechanisms for improving the stability of mineralized layers:

[0050] The biomimetic properties of the calcium phosphate skeleton and the antioxidant function of selenite synergistically enhance the stability of the mineralized layer:

[0051] Calcium phosphate skeleton protection: The crystal structure of CaP (hydroxyapatite phase) provides a physical barrier for exosomes, isolating them from degradation by external oxidases (MPO).

[0052] Antioxidant properties of selenite: SeO3 2-It can scavenge free radicals in the mineralized layer and reduce the risk of lipid peroxidation in the exosome membrane (experiments show that 85% of its activity is retained after 28 days of storage at room temperature).

[0053] Traditional exosome protection technologies (lyophilization, lipid encapsulation) require sacrificing activity or increasing process complexity, while the present invention achieves a balance between activity and stability through the material properties of the mineralized layer itself.

[0054] Preferably, the thickness of the mineralized layer is 20-50 nm, and the pH-responsive triggering condition is pH ≤ 5.5 and GSH concentration is 1-10 mM.

[0055] The mineralization layer thickness range (20-50nm) is set based on the balance between exosome protection needs and release efficiency:

[0056] Lower limit (20nm): Ensure the formation of a continuous and dense mineralized layer on the surface of exosomes to block the degradation of external enzymes (nucleases, proteases) while avoiding local defects caused by excessive thinness.

[0057] Upper limit (50 nm): Limit the excessive growth of the mineralized layer to prevent the release of exosome contents (miRNA, Wnt3a) from being hindered by excessive thickness, while maintaining reasonable fluid resistance during transdermal delivery.

[0058] Traditional mineralization processes have difficulty controlling thickness uniformity (CV>20%), but the present invention uses dynamic regulation of the microfluidic electric field to reduce the thickness fluctuation (CV) to <5%, significantly improving batch stability.

[0059] Targeted aptamers with pH-responsive triggering conditions (pH ≤ 5.5):

[0060] The pH trigger threshold (≤5.5) is set based on the pH difference between the hair follicle microenvironment and the skin surface:

[0061] The hair follicle area is weakly acidic: the pH value of the hair follicle infundibulum and stem cell microenvironment is 5.0-5.5, while the pH value of the skin surface is 4.5-5.5 (affected by sweat and sebum).

[0062] Specific distinction: The high-selenium area in the outer layer of the mineralized layer selectively dissolves at pH ≤ 5.5, but although the pH on the skin surface is close, the lack of GSH (described in detail later) cannot trigger the release of the inner layer, thereby achieving a hair follicle-specific response.

[0063] Innovative mechanism: traditional pH-responsive materials It is impossible to distinguish between similar pH regions. The present invention achieves precise targeting through the logical "and" relationship between pH and GSH (which must be met at the same time).

[0064] Biological matching of GSH concentration (1-10mM):

[0065] The GSH triggering concentration range (1-10mM) is highly consistent with the metabolic characteristics of hair follicle stem cells:

[0066] High GSH expression in hair follicle stem cells: The GSH concentration in the hair follicle bulge area stem cells can reach 5-10mM, which is much higher than that in epidermal keratinocytes (<1mM), ensuring that the inner layer of calcium phosphate is decomposed only in the target area.

[0067] Reduction reaction drives decomposition: GSH, as a strong reducing agent, can break the Ca-OP bond in calcium phosphate crystals, releasing exosomes and Wnt3a protein, while the release of nanoselenium (SeNPs) is not affected by GSH, forming a sequential release logic.

[0068] Traditional controlled-release systems rely on a single stimulus (pH or enzyme) and are susceptible to interference from non-target tissues. The present invention uses a dual biomarker combination of "pH (outer layer) + GSH (inner layer)" to target hair follicles as the only effective release site.

[0069] The coordinated design of mineralization layer thickness and triggering conditions forms a "structure-function" coupling:

[0070] The outer thin region (20-30nm) is high in selenium: it responds quickly to pH triggers, releases SeNPs at the entrance of the hair follicle, and blocks TGF-β signaling;

[0071] High calcium in the inner thick area (30-50nm): A higher GSH concentration is required to drive decomposition, ensuring that the exosomes penetrate deep into the hair follicle stem cell niche and are released to increase local drug concentration.

[0072] By spatially matching the thickness gradient with the component gradient, the drug release can be achieved through step-by-step penetration from the outside to the inside.

[0073] A preparation method based on the above-mentioned exosome mixture is characterized by comprising the following steps:

[0074] (1) Differentiating induced pluripotent stem cells into hair follicle progenitor cells and performing gene editing to overexpress hair growth-promoting miRNA and Wnt3a protein;

[0075] Differentiation of iPSCs into hair follicle progenitor cells (HFPCs): Compared with directly using mature hair follicle cells, HFPCs differentiated from iPSCs have stronger proliferation ability and secretory activity, and their exosomes are naturally enriched in hair follicle regeneration-related molecules (FGF7, VEGF), providing basic activity for subsequent treatment.

[0076] Dual gene editing targets: CRISPR / Cas9 technology simultaneously overexpresses miR-25-5p / miR-205 and integrates the Wnt3a gene into the CD63 locus, creating a complementary interaction between the exosome contents (miRNA) and the membrane protein (Wnt3a). MiR-25-5p inhibits DKK1 to relieve Wnt pathway inhibition, while Wnt3a on the exosome membrane directly activates β-catenin signaling, synergistically enhancing the hair follicle stem cell response.

[0077] Traditional methods rely on single gene editing or natural components of exosomes, while the present invention uses dual regulation of "differentiation + editing" to enable exosomes to have the ability to actively enhance their functions.

[0078] (2) isolating exosomes secreted by the hair follicle progenitor cells;

[0079] 3D suspension bioreactor culture: Compared with traditional 2D culture, the 3D environment (pore size 3-10μm) simulates the hair follicle microenvironment, which increases the production of exosomes secreted by HFPCs and makes the exosome particle size distribution more uniform.

[0080] Differential centrifugation combined with tangential flow filtration (TFF): After removing cell debris and apoptotic bodies by gradient centrifugation, TFF (100kDa membrane package) is used to achieve efficient exosome concentration, with a removal rate of impurity proteins >95%, avoiding nonspecific adsorption in subsequent mineralization reactions.

[0081] Traditional ultracentrifugation (above 100,000×g) easily leads to exosome aggregation and inactivation. However, the present invention achieves large-scale preparation while ensuring activity through the combination of 3D culture and TFF.

[0082] (3) forming a selenium-containing mineralized layer on the surface of the exosomes through a biomimetic mineralization process;

[0083] Pulsed electric field regulation of ion adsorption: An alternating electric field (5-15 Hz, 3-10 V / cm) is applied to the microfluidic chip, and the negative charge on the surface of exosomes (zeta potential -20 to -30 mV) is used to guide the Ca 2- / PO4 3- Ion adsorption forms an asymmetric mineralized layer that is thick on the outside and thin on the inside.

[0084] Selenite gradient doping: SeO3 in the outer mineralization solution 2- The concentration (4 mM) is twice that of the inner layer (2 mM), forming a concentration gradient from outside to inside, matching the subsequent pH / GSH-triggered release logic.

[0085] The traditional mineralization method (co-precipitation) cannot control the layer thickness and component distribution, while the present invention achieves precise structural design and uniform thickness of the mineralized layer through dynamic regulation of the electric field.

[0086] (4) in situ reduction of selenite in the mineralized layer into nano-selenium particles;

[0087] Blue light catalytic reduction of SeO3 2- :450nm blue light excites SeO3 2- The electron transition of the exosomes is reduced to SeNPs (particle size 10-30nm) with the assistance of vitamin C, while avoiding the damage of the strong reducing agent (NaBH4) to the exosome membrane.

[0088] Heat shock protein (HSP70) activation: Blue light synchronously activates the expression of HSP70 in exosomes, enhancing their antioxidant and heat stress resistance.

[0089] Traditional chemical reduction methods destroy the integrity of the exosome membrane, while the present invention achieves mild and controllable SeNPs synthesis through photocatalysis and simultaneously improves the stability of exosomes.

[0090] (5) Purification and preparation of freeze-dried preparations.

[0091] Trehalose-mannitol composite protective agent: Trehalose (3-8%) forms a rigid matrix through glass transition to prevent the lipid phase change of the exosome membrane; mannitol (2-5%) acts as a filler to reduce ice crystal damage. The two work together to make the moisture content of the freeze-dried powder ≤3%.

[0092] Multi-stage microfluidic purification: Pore size screening (30-100nm) and charge screening (pH 5.8-6.2) are used to remove unmineralized impurities before lyophilization to ensure a formulation purity >95% and avoid aggregation effects caused by impurities during the lyophilization process.

[0093] The traditional freeze-drying process causes exosome aggregation and inactivation (activity loss > 50%), while the present invention achieves long-term storage at room temperature by optimizing the protective agent and combining it with pre-purification.

[0094] Preferably, the biomimetic mineralization process is carried out in a microfluidic chip, and a pulsed alternating electric field with a frequency of 5-15 Hz and an intensity of 3-10 V / cm is applied to form an asymmetric mineralized layer structure.

[0095] Microfluidic chips (Y-shaped channels, 100-300 μm in width) achieve efficient mixing and orderly assembly of exosomes and mineralized fluid through laminar flow and confined space effects:

[0096] Laminar interface regulation: Exosome suspension and mineralization solution are injected in parallel at a volume ratio of 1:2-1:4, forming a stable ion concentration gradient at the laminar interface, guiding Ca 2+ / PO4 3- / SeO3 2- Ions are adsorbed in a targeted manner on the surface of exosomes, avoiding random aggregation caused by traditional stirring methods.

[0097] Confined space effect: The narrow space of the microchannel (depth 50-100μm) enhances the collision probability between exosomes and mineralized ions, thereby increasing the mineralization reaction rate and reducing reagent consumption.

[0098] Microfluidic technology solves the problems of reaction heterogeneity (CV>30%) and low efficiency in traditional mineralization processes through spatial constraints and fluid dynamics optimization.

[0099] Apply a pulsed alternating electric field with a frequency of 5-15Hz and an intensity of 3-10V / cm to achieve the asymmetric structure of the mineralized layer through the synergy of charge force and electric field force:

[0100] Charge reversal induces asymmetric adsorption: the zeta potential of the exosome surface is -20 to -30 mV, and charge reversal occurs periodically in an alternating electric field (Ca adsorption in the positive half cycle) 2+ , negative half cycle adsorption PO4 3- ), forming an outer Ca-rich 2+ (Thickness 50nm), PO4-rich inside 3- (thickness 20nm) gradient structure.

[0101] The electric field frequency matches the ion migration: the frequency range of 5-15 Hz matches Ca 2+ Migration rate (10 -8 m 2 / (V·s)), ensuring that ions complete directional migration and adsorption within the electric field cycle, avoiding ion accumulation caused by high frequency or reaction lag caused by low frequency.

[0102] Traditional mineralization relies on passive diffusion, while the present invention actively regulates the ion migration path through an electric field to achieve programmable design of the mineralization layer thickness and component distribution.

[0103] Asymmetric structure (thick outside / thin inside) optimizes drug release and transdermal delivery efficiency through physical properties:

[0104] Outer thick layer (50nm): High-density calcium phosphate and selenite form a rigid barrier to resist skin surface friction and enzymatic hydrolysis, protecting the integrity of exosomes during transdermal penetration.

[0105] Inner thin layer (20nm): The low-density porous structure facilitates GSH penetration in the hair follicle area, triggering the rapid decomposition of the inner layer to release exosomes, shortening the drug's onset time.

[0106] The asymmetric structure realizes the functional zoning of "outer layer protection + inner layer controlled release", breaking through the contradiction between the protection and release efficiency of the traditional homogeneous mineralized layer.

[0107] Preferably, in (4), blue light with a wavelength of 440-460 nm is used to catalyze the reduction of selenite, thereby synchronously activating the expression of heat shock protein HSP70 in exosomes.

[0108] The selection of blue light with a wavelength of 440-460nm is based on the energy of its photons and the wavelength of selenite (SeO3 2- )’s electronic transition energy level matching:

[0109] Photocatalytic reduction pathway: Blue light photon energy (2.75-2.82eV) excites SeO3 2- The non-bonding electrons in the * →Se-O), resulting in the breaking of the Se-O bond, releasing the Se nucleus and forming zero-valent selenium (Se 0 ) nanoparticles (particle size 10-30nm).

[0110] Ascorbic acid (AA) synergistic effect: the added AA acts as an electron donor by transferring electrons to the photoexcited SeO3 2- , accelerating the reduction reaction process while avoiding the damage to exosome membrane lipids caused by the use of strong reducing agents (NaBH4).

[0111] Traditional chemical reduction methods result in a loss of exosome membrane integrity of >30%, while blue photocatalysis enables efficient synthesis of SeNPs under mild conditions (room temperature, neutral pH), and the activity of exosomes is fully preserved.

[0112] While catalyzing the synthesis of SeNPs, blue light activates the expression of HSP70 in exosomes through the mitochondrial signaling pathway:

[0113] Mitochondrial reactive oxygen species (ROS) signal: Blue light (450 nm) penetrates the exosome membrane, stimulating mitochondrial complex III to produce trace amounts of ROS (H2O2 concentration 0.1-1 μM), which acts as a second messenger to activate heat shock factor 1 (HSF1), thereby upregulating HSP70 gene transcription.

[0114] Functional enhancement of HSP70: HSP70 acts as a molecular chaperone to maintain the correct folding of membrane proteins (CD63-Wnt3a) during the freeze-drying process of exosomes and inhibit lipid peroxidation induced by oxidative stress.

[0115] Traditional exosome protection technology (adding exogenous HSP70) requires complex processes and is costly. However, the present invention regulates the expression of endogenous HSP70 through light, achieving the simultaneous completion of self-protection function and SeNPs synthesis.

[0116] In this step, blue light energy is split into two uses, forming a "material synthesis-biological activity" synergistic network:

[0117] Energy distribution optimization: about 70% of light energy is used for SeO3 2- Reduction, 30% light energy activates HSP70 through mitochondrial ROS signaling, avoid high-intensity light (> 100mW / cm 2 ) caused by thermal damage to exosomes.

[0118] Timing matching design: The photocatalytic reaction time (15-30 minutes) and the peak expression time of HSP70 (24 hours after irradiation) form a staggered synergy to ensure that HSP70 is fully expressed and exerts a protective effect before freeze-drying.

[0119] Traditional technology requires separate steps for SeNPs synthesis and activity protection, while the present invention achieves dual functional integration through single blue light stimulation, shortening the process time.

[0120] Preferably, the purification adopts multi-stage microfluidic screening, including a ceramic membrane with a pore size of 30-100 nm and charge screening at pH 5.8-6.2.

[0121] The sieving design of pore size gradient ceramic membranes is based on the size difference between exosome complexes and impurities:

[0122] Protection of target products: The average particle size of the exosome-mineralized layer complex is 80-120nm, and the pore size range of 30-100nm can accurately intercept the target product while allowing free protein (<30nm), unmineralized selenite (<10nm) and solvent ions to pass through.

[0123] Minimizing shear force: The laminar flow characteristics of microfluidic sieving (flow rate 0.1-0.5mL / min) keep the shear force below 10Pa, avoiding damage to the exosome membrane or peeling of the mineral layer caused by traditional ultrafiltration (shear force > 50Pa).

[0124] Traditional ultracentrifugation methods cause loss of exosome activity due to high shear forces, while the present invention achieves both high purity and high activity simultaneously under mild conditions through microfluidic pore screening.

[0125] The charge screening conditions of pH 5.8-6.2 are based on the biological properties of the exosome surface zeta potential:

[0126] Isoelectric point compatibility: The isoelectric point (pI) of the mineralized exosome complex is 5.3-5.5, and the surface has a weak negative charge (zeta potential -5 to -15 mV) at pH 5.8-6.2, while the residual impurities (unreacted Ca 2+ , free SeO3 2- ) is positively charged (+10 to +30 mV) at this pH and selectively adsorbs impurities through the anion exchange membrane.

[0127] Dynamic charge compensation: Anion / cation exchange membranes are alternately arranged in the microfluidic channel (with a spacing of 200-500μm), and the charge difference between impurities and target products is used to achieve dynamic separation, avoiding the risk of stationary phase clogging in traditional chromatography columns.

[0128] Traditional purification relies on a single ion exchange chromatography, which requires multiple elutions (taking 4-6 hours); the present invention achieves continuous flow purification through microfluidic charge screening without the need to change the buffer system.

[0129] The "pore size-charge" dual-stage screening is not simply connected in series, but forms functional complementarity through the integrated design of the microfluidic chip:

[0130] Pre-pore screening: removes large particle size impurities (cell fragments, mineralized layer fragments) and small molecule interfering substances, reducing the risk of membrane contamination in subsequent charge screening.

[0131] Post-charge screening: Precisely remove residual metal ions (Ca 2+ 、Na + ) and free SeO3 2- , so that the conductivity of the product can be reduced.

[0132] Traditional purification technology (ultrafiltration + dialysis) requires the use of multiple devices and has a low yield (<60%). However, the present invention improves the yield and ensures batch-to-batch consistency through microfluidic integrated technology.

[0133] Preferably, the lyophilized preparation contains 3-8% trehalose and 2-5% mannitol, and after reconstitution, is mixed with the poloxamer 407 thermosensitive gel to form a transdermal delivery vehicle.

[0134] The ratio of trehalose (3-8%) and mannitol (2-5%) is designed based on their complementary effects during the freeze-drying process:

[0135] Trehalose's glass protection: Trehalose forms a hydrogen bond network with water molecules through hydroxyl groups, forming an amorphous glassy matrix (glass transition temperature Tg ≥ 60°C) during the freeze-drying process, which encapsulates exosomes and inhibits ice crystal growth, making the moisture content of the freeze-dried powder ≤ 3% (traditional freeze-dried preparations > 8%).

[0136] Crystallization regulation of mannitol: Mannitol, as a crystalline filler, preferentially forms micron-sized needle-shaped crystals (length 1-5 μm), filling the gaps in the trehalose glassy state and reducing the mechanical damage to the exosome membrane caused by freeze-drying stress.

[0137] Traditional single protective agents (trehalose only) cannot take into account both vitrification and stress resistance requirements. However, the present invention uses two phases (glassy state + crystal) to synergize, allowing exosomes to maintain high activity after long-term storage at room temperature.

[0138] After the freeze-dried powder is reconstituted, it is mixed with Poloxamer 407 (20-25%) to achieve intelligent transdermal delivery by utilizing its temperature responsiveness:

[0139] Sol-gel phase transition properties: Poloxamer 407 forms a low-viscosity sol (viscosity <100 cP) at room temperature (25°C), making it easy to apply. Upon contact with the skin (32-35°C), it forms a semi-solid gel (viscosity >10,000 cP), extending its retention time to 4-6 hours (compared to <2 hours for traditional hydrogels).

[0140] Enhanced microenvironment penetration: The gel releases water during the phase change process, causing the stratum corneum to temporarily hydrate and swell (hydration level increased by 30%), promoting the penetration of the exosome-mineralized layer complex through the hair follicle opening.

[0141] Traditional liposome or microneedle transdermal systems have difficulty delivering macromolecular complexes (exosomes). However, the present invention achieves >50% exosome targeting to the hair follicle area through the synergistic effect of viscosity gradient change of thermosensitive gel and physical penetration enhancement.

[0142] The combination of lyophilized preparation and thermosensitive gel forms a "storage-release" functional chain:

[0143] Stability of lyophilized powder: The trehalose-mannitol matrix maintains exosome activity during storage and completely disperses within 30 seconds after reconstitution (0.9% NaCl solution), avoiding aggregation during reconstitution of traditional lyophilized powder.

[0144] Controlled-release delivery of gel: Poloxamer 407 gel forms a "reservoir effect" on the skin surface, slowly releasing exosome complexes, so that the drug concentration in the hair follicle area is maintained at the effective threshold for a long time.

[0145] Traditional transdermal preparations (creams) cannot simultaneously meet the requirements of long-term storage and efficient delivery. However, the present invention achieves the dual goals of "ready-to-use and active" and "long-term sustained release" through a freeze-drying-gel step-by-step design.

[0146] An application based on the above-mentioned exosome mixture is characterized in that it is used to prepare a pharmaceutical composition for treating hair loss, and the drug is delivered to the hair follicle area through a transdermal drug delivery system.

[0147] The present invention provides an exosome mixture for treating hair loss, and its preparation method and application. It has the following beneficial effects:

[0148] 1. The present invention uses a gene-edited exosome complex (miR-25-5p / miR-205+Wnt3a) in combination with nano-selenium to achieve the simultaneous activation of the three pathways of hair follicle "promoting regeneration, anti-inflammation, and anti-apoptosis". The existing technology relies on single-target drugs (minoxidil only acts on K +The present invention increases the β-catenin signal intensity by 2-3 times, extending the hair follicle growth period by 2 times, breaking through the efficacy bottleneck of traditional therapies.

[0149] 2. This invention, through the synergistic design of a poloxamer thermosensitive gel and a cell-penetrating peptide (TAT), achieves a drug concentration in the hair follicle region that is 10 times higher than that achieved with systemic administration. Existing transdermal technologies (liposomes) rely on passive diffusion, resulting in a targeting efficiency of less than 20%. This invention triples the transdermal efficiency, achieving a cumulative release of 89% over 6 hours, addressing the core drawbacks of conventional delivery systems, such as short retention and poor penetration.

[0150] 3. This invention utilizes a mineralization gradient response (pH / GSH dual triggering) to achieve spatiotemporal sequential release of SeNPs and exosomes. Existing sustained-release formulations (microspheres) are unable to meet the dynamic demands of the hair loss process. This invention blocks 70% of apoptotic signals in the acute phase (2 hours) and continuously activates regenerative pathways in the chronic phase (6-24 hours), achieving efficacy throughout the entire hair loss cycle.

[0151] 4. This invention utilizes hypoxic amplification coupled with a photocatalytic-HSP70 protective network, enabling freeze-dried powder to retain >85% activity after six months of room-temperature storage. Conventional exosome preparations lose >50% activity after one month of storage due to oxidative aggregation and inactivation. The particle size stability (D50 = 108 ± 4 nm) and oxidation rate (11.4%) of this invention meet clinical-grade quality control standards, addressing the challenges of industrial storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1 Schematic diagram of the method of the present invention. DETAILED DESCRIPTION

[0153] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0154] Example 1: Preparation of highly active exosome complexes

[0155] Hair follicle progenitor cell differentiation and gene editing:

[0156] Induced pluripotent stem cells (iPSCs) were cultured in 3D suspension (60 rpm) in DMEM / F12 medium containing BMP4 (50 ng / mL) and FGF7 (20 ng / mL). After 7 days, they differentiated into hair follicle progenitor cells (HFPCs) with a differentiation efficiency of >90%.

[0157] The CRISPR / Cas9 system was used to simultaneously overexpress miR-25-5p / miR-205 (sgRNA targeting sequences: miR-25-5p: 5′-GCCCTGAGACCTTAATTGCC-3′; miR-205: 5′-TCCTTCATTCCACCGGA GTCT-3′), and the Wnt3a gene was integrated into the CD63 promoter region (homologous arm length 800 bp), with a transfection efficiency of >85%.

[0158] Exosome isolation:

[0159] The HFPCs culture supernatant was subjected to differential centrifugation (300×g for 10 min→2000×g for 20 min→10,000×g for 30 min) to remove cell debris and then concentrated 10-fold by tangential flow filtration (TFF, 100 kDa membrane package). The exosome yield was ≥1×10^11 particles / mL, with a particle size distribution of 80-120 nm (DLS detection).

[0160] Bionic mineralization:

[0161] Exosome suspension and mineralization solution (Ca 2+ :10mM, PO4 3- :6mM, SeO3 2- :4mM), applied 10Hz, 5V / cm alternating electric field, mineralization time 30min, formed the outer 50nm (SeO3 2- concentration 4mM), inner 20nm (SeO3 2- The asymmetric mineralized layer with a concentration of 2 mM has a thickness CV of <5%.

[0162] In situ synthesis of nano-selenium:

[0163] Blue light (450nm, intensity 50mW / cm 2 ) The mineralized exosome suspension was irradiated and ascorbic acid (5 mM) was added. The reaction was continued for 20 min. The SeNPs had a particle size of 20 ± 5 nm and the exosome activity was retained > 95%.

[0164] Purification and lyophilization:

[0165] Multi-stage microfluidic screening (50nm pore size ceramic membrane → pH 6.0 charge screening) results in a final product conductivity of ≤80μS / cm. The freeze-dried product contains 5% trehalose and 3% mannitol as freeze-dried protective agents. The water content after freeze-drying is 2.5%, and the reconstituted activity is >90%.

[0166] Example 2: Highly stable mineralized exosome preparation

[0167] Gene Editing Optimization:

[0168] Lentiviral vectors (MOI = 20) were used to deliver miR-25-5p / miR-205 and Wnt3a genes. After transfection, HFPCs proliferated under hypoxic conditions (5% O2), and exosome secretion increased by 40%.

[0169] Mineralization process enhancement:

[0170] Mineralization fluid gradient design: outer layer (SeO3 2- 5mM, Ca 2+ 12mM)→Inner layer (SeO3 2- 1.5 mM, Ca 2+ 8mM), alternating electric field parameters 15Hz, 8V / cm, mineralized layer thickness 60nm (CV<3%).

[0171] Combination of photocatalysis and protective agent:

[0172] When irradiated with blue light (460nm), glutathione (2mM) was added simultaneously to activate HSP70 expression by 3 times. After freeze-drying and storage at room temperature for 6 months, the activity retention was >85%.

[0173] Transdermal carrier optimization:

[0174] The lyophilized powder was reconstituted in poloxamer 407 (25%) gel and N-acetylcysteine ​​(1%) was added, and the transdermal efficiency was increased to 65% (Franz diffusion cell test).

[0175] Example 3: Highly effective transdermal delivery formulation

[0176] Exosome surface modification:

[0177] Before mineralization, transmembrane peptide (TAT, density 10^4 molecules / vesicle) is coupled to the exosome membrane to enhance hair follicle penetration ability.

[0178] Optimization of mineralized layer parameters:

[0179] Mineralization solution formula: outer layer (SeO3 2- 3mM, Ca 2+ 15mM)→Inner layer (SeO3 2- 1mM, Ca 2+ 5mM), alternating electric field frequency 5Hz, 10V / cm, mineralized layer thickness 30nm (CV<4%).

[0180] Thermosensitive gel enhances delivery:

[0181] Poloxamer 407 (22%) and chitosan (0.5%) were compounded, with a gel phase transition temperature of 32° C., a skin retention time of >8 h, and a transdermal efficiency increased by 40% compared with Example 1.

[0182] Quality Control:

[0183] The particle size distribution of the freeze-dried powder is (D10=85 nm, D50=110 nm, D90=135 nm), the drug loading of SeNPs is 8 μg / mg, and the Wnt3a activity is ≥95%.

[0184] Comparative Example 1:

[0185] Compared with Example 1, the difference is that: no gene editing is performed (miR-25-5p / miR-205 is not overexpressed and Wnt3a gene is not integrated), and the rest are the same.

[0186] Comparative Example 2:

[0187] Compared with Example 1, the difference is that the alternating electric field input is cancelled in the mineralization process (only static mixing reaction is performed), and the rest are the same.

[0188] Comparative Example 3:

[0189] Compared with Example 2, the difference is that the hair follicle progenitor cells were not expanded under hypoxic conditions (5% O2) but cultured under normoxia (21% O2). The rest are the same.

[0190] Comparative Example 4:

[0191] Compared with Example 2, the difference is that glutathione is not added in the blue light catalysis step (only ascorbic acid is used), and the rest are the same.

[0192] Comparative Example 5:

[0193] Compared with Example 3, the difference is that the transmembrane peptide (TAT) was not coupled to the surface of the exosomes before mineralization, and the rest were the same.

[0194] Comparative Example 6:

[0195] Compared with Example 3, the difference is that the concentration of poloxamer 407 in the transdermal carrier is 15%, and the rest are the same.

[0196] Experiment 1: Verification of key parameters of the preparation process

[0197] Gene editing effectiveness testing:

[0198] Sample processing: Example 1 (gene-edited exosomes) and Comparative Example 1 (unedited exosomes) were taken, and the exosomes were lysed to extract total RNA and membrane proteins.

[0199] miRNA detection: qPCR was performed (primer sequences: miR-25-5p-F: 5′-GCCCTGAGACCTTAATT GCC-3′, R: universal; miR-205-F: 5′-TCCTTCATTCCACCGGAGTCT-3′, R: universal), using U6 as the internal control, and the ΔΔCt value was calculated.

[0200] Wnt3a activity detection: Exosomes were co-incubated with HEK293T cells (containing the TOPFlash luciferase reporter system) for 24 hours, and the fluorescence intensity (Ex / Em=485 / 535 nm) was detected.

[0201] Mineralized layer structure analysis:

[0202] TEM sample preparation: The exosome suspensions of Example 1 (electric field mineralization) and Comparative Example 2 (static mineralization) were added dropwise to a copper grid, negatively stained with 2% phosphotungstic acid, and observed using a JEM-2100 transmission electron microscope (accelerating voltage 200 kV).

[0203] EDS energy spectrum scanning: Select the mineralized layer area (50nm×50nm), scan the distribution of selenium elements, and calculate the concentration ratio of the inside and outside.

[0204] Hypoxia amplification efficiency test:

[0205] Exosome yield: The culture supernatants of HFPCs from Example 2 (hypoxia) and Comparative Example 3 (normoxia) were subjected to ultracentrifugation (100,000 × g for 2 h), and the particle number was determined by NTA (ZetaView PMX-120).

[0206] Wnt3a loading: Western blot (primary antibody: Anti-Wnt3a 1:1000, secondary antibody: HRP-Goat-anti-Rabbit 1:5000), ImageJ grayscale value quantification.

[0207] Experimental data table:

[0208] Table 1 - Test results of key parameters of preparation process

[0209]

[0210] This experiment verified the irreplaceable role of gene editing, alternating electric field and hypoxia culture in the preparation process. Gene editing precisely regulates the expression of miR-25-5p / miR-205 and Wnt3a through CRISPR / Cas9, directly activates the Wnt / β-catenin pathway and inhibits hair follicle apoptosis signals (such as the Wnt3a activity in comparative example 1 dropped to 7%). The alternating electric field drives Ca 2+ / SeO3 2-Ions migrate to form an asymmetric mineralized layer (gradient ratio of 2.1:1 in Example 1), while static mineralization (Comparative Example 2) results in random ion deposition (gradient ratio of 1.2:1) due to the lack of electric field regulation, confirming the active intervention mechanism of the electric field on the structure and function of the mineralized layer.

[0211] Hypoxic conditions (5% O₂) mimic the physiological microenvironment of the hair follicle stem cell niche, upregulating the HIF-1α signaling pathway and significantly improving the exosome secretion efficiency of HFPCs (the yield in Example 2 increased by 60% compared to Comparative Example 3). This also enhances the stability of Wnt3a (Western blot loading difference of 66%), consistent with the mechanism by which hypoxia inhibits lysosomal degradation pathways. These data demonstrate that the process parameter design of the present invention is not an empirical selection, but rather a deep coupling of molecular mechanisms and physicochemical laws, ensuring the synergistic optimization of the exosome complex in terms of activity, structure, and yield.

[0212] Experiment 2: Delivery system performance verification

[0213] Transdermal targeting efficiency test:

[0214] Sample preparation: Example 3 (TAT-modified exosomes) and Comparative Example 5 (unmodified exosomes) were labeled with DiR fluorescent dye (1 μM, incubated at 37°C for 30 min), and unbound dye was purified by centrifugation.

[0215] In vitro skin model: Pig ear skin (thickness 0.8-1.2 mm) was fixed in a Franz diffusion cell and poloxamer gel containing exosomes (25 mg / cm 2 ), constant temperature at 37℃ for 6 hours.

[0216] Fluorescence quantification: Skin sections (20 μm thickness) were stained with DAPI, and the fluorescence intensity of the hair follicle region was observed under a confocal microscope (Zeiss LSM 880). The targeting ratio (fluorescence ratio of the hair follicle region to the epidermis region) was analyzed using ImageJ.

[0217] LC-MS / MS verification: Skin tissue was homogenized, exosomes were extracted with methanol, and SeNPs concentration was quantified (m / z 78.96→82.92).

[0218] Gel controlled release ability test:

[0219] Viscosity-temperature response test: The gels of Example 3 (Poloxamer 22% + Chitosan 0.5%) and Comparative Example 6 (Poloxamer 15%) were tested by rheometer (TA Instruments DHR-2) during the temperature increase from 25°C to 35°C (shear rate 10s -1 ).

[0220] Release kinetics: Franz diffusion cells were loaded with gel (donor cell) and receiving solution (PBS + 0.1% Tween 80), and the cumulative release of exosomes within 0-6 hours was detected by HPLC (detection wavelength 280 nm).

[0221] Experimental data table:

[0222] Table 2 - Delivery system performance test results

[0223]

[0224] The data from Experiment 2 revealed the dual regulatory mechanism of the transdermal delivery system. The transmembrane peptide TAT guides the exosomes to penetrate along the hair follicle duct (the transdermal depth of Example 3 reaches 320 μm) through electrostatic adsorption of the positive charge (+8) and the negative charge of the hair follicle stratum corneum (zeta potential -20 mV at pH 5.5), while the exosomes of Comparative Example 5 are retained in the epidermis (150 μm) due to the lack of targeted modification. The concentration gradient design of Poloxamer 407 (22% in Example 3) regulates the phase transition temperature through the density of intermolecular micelle entanglement. At 35°C, the viscosity increases sharply (11,200 Pa·s) to form a physical reservoir. However, the low concentration (Comparative Example 6, 15%) causes a burst release (82% release in 3 hours) due to the loose micelle structure. The drug diffuses to non-target areas prematurely, and the actual uptake of the hair follicles is only 38%.

[0225] The microenvironmental response of the mineralized layer further optimizes the release kinetics. The weakly acidic environment of the hair follicle (pH 5.5) preferentially dissolves SeO3 on the outside of the mineralized layer. 2- , releasing SeNPs to block the early apoptosis signal, and after penetrating deep into the hair follicle stem cell niche, the high GSH concentration decomposes the inner calcium phosphate, and the exosomes are slowly released to achieve long-term regeneration. This spatiotemporal sequential release synergizes with the viscosity gradient of the poloxamer gel, so that the drug concentration in the hair follicle area of ​​Example 3 reaches the therapeutic threshold (≥10μg / cm 2 ), which is 3-5 times higher than the traditional delivery system (comparative example 5 / 6), confirming the design advantage of "physical guidance + chemical response" dual active targeting.

[0226] Experiment 3: Formulation stability verification

[0227] HSP70 expression and activity protection test:

[0228] Sample treatment: The exosome suspensions of Example 2 (photocatalysis + glutathione) and Comparative Example 4 (ascorbic acid only) were lysed and centrifuged to obtain the supernatant.

[0229] HSP70 detection: Flow cytometry (CytoFLEX S) was used to detect the HSP70-Alexa Fluor 488 labeling intensity on the exosome surface (Ex / Em=488 / 519 nm).

[0230] Freeze-dried activity retention: Exosomes were co-cultured with hair follicle stem cells (HFSCs) for 48 hours, and the proliferation rate was assessed by CCK-8 assay (absorbance at 450 nm) (with fresh samples as 100%).

[0231] Room temperature storage stability test:

[0232] Oxidation rate detection: After the freeze-dried powder (Example 2 and Comparative Example 4) was stored at room temperature for 6 months, the oxidation products of SeNPs (Se 4+ / Se 6+ , retention time 3.2 / 4.8min).

[0233] Particle size and activity verification: After reconstitution, the exosome particle size distribution was detected by NTA, and the activity retention rate was evaluated by CCK-8 method.

[0234] Experimental data table:

[0235] Table 3 - Formulation stability test results

[0236]

[0237]

[0238] The data from Experiment 3 confirmed the key role of photocatalysis-HSP70 combination in the stability of the formulation. 2- SeNPs are generated by electron transitions in the sample, while glutathione (GSH) acts as an electron donor, not only accelerating SeNP synthesis but also forming a molecular chaperone protection network by activating the heat shock protein HSP70 (expression increased 3.9-fold in Example 2), significantly reducing the risk of denaturation of exosome membrane proteins during freeze-drying (freeze-dried activity retention rate >90%). In contrast, in Comparative Example 4, due to the lack of GSH-mediated upregulation of HSP70, exosomes aggregated after freeze-drying (D50 increased to 135nm), resulting in a nearly 40% loss of activity.

[0239] The oxidative stability of SeNPs is directly related to the protective effect of HSP70. In Example 2, HSP70 inhibits the contact of SeNPs with oxygen free radicals by binding to the hydrophobic domain exposed on the surface of exosomes. After 6 months of storage at room temperature, the oxidation rate is only 11.4%, while in Comparative Example 4, the SeNPs are oxidized due to oxidative stress. 6+ Toxic products accumulate (38.7%), further impairing exosome function. This synergistic protective mechanism enables the preparation of the present invention to maintain particle size uniformity (D50 = 108 nm) and biological activity (>86%) during long-term storage, overcoming the drawback of traditional exosome preparations that are prone to inactivation.

[0240] The coupling of hypoxic amplification and photocatalytic processes enhances exosome stability from the source. Hypoxic conditions (5% O₂) inhibit ROS production, reducing oxidative damage during exosome secretion, while the photocatalytic-HSP70 combination creates a double barrier during subsequent purification and storage. This cross-stage stability design (production-purification-storage) ensures the reliability of the exosome complex from preparation to clinical application, providing key technical support for industrialization.

[0241] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An exosome mixture for treating hair loss, characterized in that: include: (a) Exosomes derived from gene-edited hair follicle progenitor cells; (b) Selenium nanoparticles; (c) A pH and glutathione dual-responsive mineral layer coated on the surface of the exosomes.

2. The exosome mixture for treating hair loss according to claim 1, characterized in that: The gene-edited hair follicle progenitor cells overexpress miR-25-5p and miR-205 through CRISPR / Cas9 technology, and integrate the Wnt3a gene into the regulatory region of the exosomal membrane protein CD63.

3. The exosome mixture for treating hair loss according to claim 1, characterized in that: The mineralized layer consists of calcium phosphate and selenite, and the concentration of selenite in the outer mineralized liquid is 1.5-3 times that of the inner layer.

4. The exosome mixture for treating hair loss according to claim 1, characterized in that The thickness of the mineralized layer is 20-50 nm, and the pH-responsive triggering condition is pH ≤ 5.5 and GSH concentration is 1-10 mM.

5. A method for preparing the exosome mixture according to claim 1, characterized in that: The following steps are involved: (1) Differentiating induced pluripotent stem cells into hair follicle progenitor cells and performing gene editing to overexpress hair growth-promoting miRNA and Wnt3a protein; (2) isolating exosomes secreted by the hair follicle progenitor cells; (3) forming a selenium-containing mineralized layer on the surface of the exosomes through a biomimetic mineralization process; (4) in situ reduction of selenite in the mineralized layer into nano-selenium particles; (5) Purification and preparation of freeze-dried preparations.

6. The preparation method according to claim 5, characterized in that The biomimetic mineralization process is completed in a microfluidic chip, and a pulsed alternating electric field with a frequency of 5-15 Hz and an intensity of 3-10 V / cm is applied to form an asymmetric mineralized layer structure.

7. The preparation method according to claim 5, characterized in that In the method (4), selenite is catalytically reduced by blue light with a wavelength of 440-460 nm, thereby synchronously activating the expression of heat shock protein HSP70 in exosomes.

8. The preparation method according to claim 5, characterized in that The purification adopts multi-stage microfluidic screening, including a ceramic membrane with a pore size of 30-100 nm and charge screening at pH 5.8-6.

2.

9. The preparation method according to claim 5, characterized in that The freeze-dried preparation contains 3-8% trehalose and 2-5% mannitol, and is mixed with poloxamer 407 thermosensitive gel after reconstitution to form a transdermal drug delivery carrier.

10. A use of the exosome mixture according to claim 1, characterized in that: The invention is used for preparing a pharmaceutical composition for treating hair loss, wherein the drug is delivered to the hair follicle area through a transdermal drug delivery system.

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