Application of embryonic stem cell exosome ESC-Exos in alopecia treatment

Abstract

The invention relates to the technical field of alopecia treatment and regenerative medicine, in particular to application of embryonic stem cell exosomes (ESC-Exos) in alopecia treatment. Specifically, ESC-Exos is introduced into a scalp dermis layer through a microneedle, and improvement of a hair follicle microenvironment and promotion of hair regeneration are realized by using various growth factors, micro-RNA and immunomodulatory factors carried by the ESC-Exos. According to the method for treating alopecia through the ESC-Exos, the penetrability and absorptivity of exosomes are improved through a microneedle technology, so that the ESC-Exos enter hair follicles and surrounding tissues of the hair follicles, the functions of dermal papilla cells are activated, angiogenesis is promoted, VEGF expression is up-regulated, a Wnt / beta-catenin signal channel is regulated and controlled, long-term hair growth is started, local microinflammation is relieved through an anti-inflammatory mechanism, and the effect of treating alopecia is achieved. Therefore, regeneration treatment of alopecia is realized. The method has the advantages of simplicity and convenience in operation, small trauma, high safety, remarkable curative effect and the like, and is suitable for auxiliary or alternative treatment of various types of male and female alopecia.

Description

Technical Field

[0001] This invention relates to the field of hair loss technology, and more particularly to the application of ESC-Exos in the treatment of hair loss. Background Technology

[0002] Hair loss has a variety of causes, mainly including genetics, endocrine disorders, stress, malnutrition, and medication side effects. Among these, androgenetic alopecia is the most common type, primarily related to genetics and endocrine factors. Hair loss caused by stress is related to excessive stress and insufficient sleep. Hair loss caused by malnutrition is related to an unbalanced diet and a lack of certain essential nutrients. In addition, age is also an important factor affecting hair loss.

[0003] Traditional treatments for hair loss mainly include medication and hair transplantation, but these methods also have many problems. Medication has limited effectiveness and significant side effects. Minoxidil is a commonly used topical medication, but its effectiveness is low, and some patients do not respond well to it, resulting in long treatment cycles and unclear results. Long-term use may also cause adverse reactions such as skin irritation, itching, and erythema, and some patients may even experience allergic reactions. Furthermore, minoxidil may affect a patient's fertility, and patients of childbearing age should use it with extreme caution. After discontinuing the medication, some patients experience hair loss recurrence, further increasing their psychological burden. Hair transplantation has certain limitations. Although hair transplantation is visually appealing to some patients, there are risks of complications such as infection, bleeding, and failure of transplanted hair follicles to survive during the procedure; the cost is relatively high, making it unaffordable for many patients; moreover, hair transplantation is not a permanent solution, and some patients may experience hair loss again within a few years after the procedure, resulting in short-lived treatment effects.

[0004] In recent years, stem cell exosomes (ESC-Exos) have been widely used in various aspects of cell repair and tissue regeneration, showing promising application prospects. For example, exosomes derived from mesenchymal stem cells and their microRNAs have exhibited excellent properties in various tissue repair and regeneration processes, including promoting cell regeneration, regulating inflammatory responses, and inhibiting apoptosis. In hair follicle regeneration, exosomes derived from embryonic stem cells (ESC-Exos) can effectively promote hair regeneration, prolong the hair growth phase, and increase hair density and hair shaft diameter through mechanisms such as activating dermal papilla cells, promoting vascular endothelial growth factor (VEGF) expression, regulating the Wnt / β-catenin signaling pathway, and inhibiting inflammatory responses.

[0005] Based on the above background, the present invention aims to provide a hair loss treatment solution using ESC-Exos, which further improves the effectiveness of exosomes in hair loss treatment by optimizing the preparation and application of exosomes, providing hair loss patients with a safer, more effective and economical treatment option. Summary of the Invention

[0006] Therefore, it is necessary to provide the application of ESC-Exos in hair loss treatment to address the aforementioned technical problems and solve the technical issues raised in the background section.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: Application of ESC-Exos in the treatment of hair loss.

[0008] Furthermore, the hair loss includes at least one of the following types: androgenetic alopecia, alopecia areata, and senile alopecia.

[0009] Furthermore, the application method includes the following steps: 1) Use a 0.5 mm micro-needle to stimulate the scalp locally; 2) The concentration for topical application is 1×10¹ 0 ESC-Exos suspension with particles / ml; 3) The treatment will be performed once a week for a total of 6 weeks.

[0010] Furthermore, the ESC-Exos is derived from human embryonic stem cells, extracted by ultracentrifugation, and purified and identified by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry.

[0011] A microneedle delivery system for treating hair loss, the system comprising microneedles and an ESC-Exos suspension for delivery to the dermal layer of the scalp.

[0012] Furthermore, the depth of the microneedle is 0.5 mm.

[0013] Furthermore, the microneedles are made of biodegradable polymer materials.

[0014] A method for promoting hair regeneration includes the following steps: 1) Obtain ESC-Exos derived from human embryonic stem cells; 2) Inject ESC-Exos locally or introduce it into the hair loss area using a microneedle system.

[0015] Furthermore, the ESC-Exos promotes hair regeneration by activating dermal papillary cells, promoting VEGF expression, regulating the Wnt / β-catenin pathway, and inhibiting inflammatory responses.

[0016] ESC-Exos can be used in any of the following applications: 1) Regulating the microenvironment of hair follicle stem cells; 2) Activate hair follicle stem cells and promote hair follicle regeneration; 3) Inhibits follicular inflammation; 4) Promotes angiogenesis in hair follicles; 5) Increase hair density; 4) Increase hair diameter.

[0017] The technical solution proposed in this invention can significantly improve the limitations of traditional hair loss treatments and effectively realize the precise, safe, and efficient application of ESC-Exos in clinical hair regeneration, thereby solving the technical problems of poor treatment effect, short duration of effect, and strong irritation in existing technologies.

[0018] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects: 1. The application of ESC-Exos in hair loss treatment provided by this invention involves precisely delivering high-purity ESC-Exos to the dermal layer of the scalp via 0.5 mm microneedles. This effectively activates dermal papilla cells, promotes the expression of vascular endothelial growth factor (VEGF), and regulates the Wnt / β-catenin signaling pathway, further stimulating the proliferation and differentiation of hair follicle stem cells, thereby promoting hair growth and prolonging the hair growth phase. Simultaneously, ESC-Exos possesses excellent immunomodulatory and anti-inflammatory properties, which can reduce scalp inflammation and provide a favorable microenvironment for hair follicle regeneration.

[0019] 2. This invention uses a microneedle delivery system to replace traditional injection methods, offering advantages such as non-invasive, precise, and efficient delivery. While improving the bioavailability of ESC-Exos, it significantly reduces discomfort during treatment and enhances patient compliance. The microneedle system is made of biodegradable polymer materials, ensuring high safety. It degrades naturally after treatment, eliminating the need for removal and avoiding secondary trauma, making it suitable for long-term intervention treatment of hair loss. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating the treatment steps; Figure 2 This is a graph showing the changes in average hair density in the treatment group; Figure 3 This is a graph showing the change in the average diameter of hair in the treatment group; Figure 4 Before and after treatment comparison images; Case 1 (male); Note: A: Before treatment; B: 3 months after treatment; C: 6 months after treatment Figure 5 Before and after treatment comparison images; Case 2 (female); Note: A: Before treatment; B: 3 months after treatment; C: 6 months after treatment Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0023] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other.

[0024] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0025] I. Exploration of the ESC-Exos Mechanism 1.1. Mechanism of action of ESC-Exos (embryonic stem cell exosomes) in the treatment of hair loss Hair loss has a variety of causes, mainly including genetics, endocrine disorders, stress, malnutrition, medication side effects, and aging. Androgenetic alopecia (AGA) is the most common type of hair loss, primarily related to genetics and endocrine factors. Traditional treatments such as minoxidil and hair transplantation are effective to some extent, but they have many limitations, such as long treatment cycles, short-lasting effects, high costs, and potential side effects. Therefore, finding a safer and more effective treatment method has become a key research focus.

[0026] In recent years, embryonic stem cell exosomes (ESC-Exos), as a novel type of extracellular vesicle, have attracted attention due to their important role in intercellular communication. Exosomes are small extracellular vesicles (approximately 30-150 nanometers in diameter) with a phospholipid bilayer structure and specific surface markers, enabling them to carry substances that influence cell signaling and gene expression, such as cytokines, growth factors, and regulatory microRNAs (miRNAs). These characteristics make exosomes promising for applications in tissue repair and regenerative medicine.

[0027] 1.1.1 Improvement of the hair follicle microenvironment by ESC-Exos Our in-depth research has revealed that ESC-Exos can improve the hair follicle microenvironment through multiple mechanisms. First, ESC-Exos promotes the activation and proliferation of hair follicle stem cells by regulating cell signaling pathways around the hair follicle, thereby prolonging the hair growth phase. Second, ESC-Exos can improve the hair follicle microenvironment by inhibiting the inflammatory response and reducing the infiltration of inflammatory cells around the hair follicle. Furthermore, ESC-Exos can also increase blood supply to the hair follicle by promoting angiogenesis, providing more nutrients to the hair follicle.

[0028] 1.1.2 The promoting effect of ESC-Exos on hair follicle regeneration ESC-Exos is rich in various bioactive molecules, such as cytokines, growth factors, and microRNAs (miRNAs). These molecules can promote hair follicle regeneration by regulating gene expression and signal transduction in hair follicle cells. For example, some miRNAs in ESC-Exos can promote hair regeneration by targeting specific genes and regulating the proliferation and differentiation of hair follicle cells.

[0029] In summary, embryonic stem cell exosomes can effectively promote hair follicle regeneration and significantly improve the treatment effect of hair loss by regulating the hair follicle microenvironment, activating hair follicle stem cells, inhibiting inflammatory response and promoting angiogenesis. They also have good safety and tolerability, making them a new option for hair loss treatment.

[0030] 1.2 Microneedle Drug Delivery System Biofilm formation around hair follicles is another major obstacle affecting the efficiency of traditional drug treatments in patients with hair loss. Traditional drug delivery methods struggle to penetrate this biofilm, requiring the follicles to be cleaned before application, which causes significant discomfort for patients. Microneedle transdermal drug delivery systems, however, can penetrate this barrier without affecting pain receptors, improving both delivery efficiency and patient compliance. Furthermore, in hair loss treatment, they can effectively penetrate the biofilm to reach the deep hair follicles requiring treatment, achieving targeted therapy, and the relatively closed wound reduces the risk of subsequent infection.

[0031] II. Construction Technology By applying embryonic stem cell exosomes locally, combined with 0.5 mm microneedle technology, the hair loss area was treated to verify its effect in promoting hair regeneration.

[0032] 2.1. Tool Selection Microneedles and embryonic stem cell exosomes (ESC-Exos), currently at the forefront of medical research, were selected as the main tools and research subjects. By combining ESC-Exos with microneedle technology, a novel local delivery system was developed. This system utilizes 0.5 mm microneedles to precisely deliver ESC-Exos to the dermis of the scalp, directly acting on the target area around the hair follicles. This delivery method not only improves drug efficacy but also reduces impact on surrounding normal tissues, exhibiting high targeting and effectiveness.

[0033] Microneedling technology is widely used in skin treatments due to its minimally invasive nature, ease of operation, and high patient compliance. Through precise delivery via microneedles, ESC-Exos can penetrate deep into hair follicles, activate hair follicle stem cells, promote hair follicle regeneration, and improve the hair follicle microenvironment. Furthermore, ESC-Exos itself has antioxidant, anti-inflammatory, and angiogenesis-promoting effects, providing a favorable growth environment for hair follicles and effectively promoting hair regeneration.

[0034] 2.2. Construction Method 2.2.1 Embryonic stem cell exosomes as therapeutic drugs Embryonic stem cell exosomes (ESC-Exos), vesicle-like substances secreted by stem cells, can fuse into damaged hair follicle cells, inducing internal repair of damaged cells. Rich in various active substances, they contribute to improving the hair follicle microenvironment and promoting hair regeneration, avoiding the problem of traditional treatments failing to effectively improve the hair follicle microenvironment. ESC-Exos demonstrates beneficial effects in reducing reactive oxygen species (ROS) and promoting angiogenesis, providing a healthier growth environment for hair follicles.

[0035] In our hair loss treatment, we combined ESC-Exos with microneedling technology. This approach not only promotes repair and regeneration within the hair follicles but also improves local blood circulation, providing the follicles with enhanced nutritional support. This combined treatment method works synergistically to promote both hair follicle health and hair regeneration, with promising preliminary results.

[0036] III. Microneedle Patch Preparation and Treatment Methods 3.1 Preparation of related microneedle patches 3.1.1 Solution Preparation The preparation of solutions for microneedle patches involves the formulation of tip solutions and base layer solutions. The solution components must meet requirements for biocompatibility, biodegradability, and drug loading capacity.

[0037] Needle tip solution preparation: The active ingredient is exosomes derived from human embryonic stem cells (ESC-Exos), and the concentration used is 1×10¹. 0~1×10¹¹ particles / mL. Trehalose (20-60 g / L) and bovine serum albumin (BSA, 2-3 g / L) were added as stabilizers and protectants. The hydrogel matrix material was selected from methacrylamide gelatin (GelMA), hyaluronic acid (HA, 200 mg / mL), or chitosan (5-10 g / L). Phosphate-buffered saline (PBS, 0.01-0.02 M) was used as the solvent. During preparation, trehalose, hyaluronic acid, and other macromolecules were first dissolved in PBS and stirred for 12 hours until fully dissolved. The protein concentration of ESC-Exos was determined using the BCA protein quantification method, and adjusted to the required concentration with PBS. The exosome solution and the matrix solution containing the protectant were slowly mixed at a 1:1 volume ratio, avoiding vigorous stirring. When using GelMA, the photoinitiator was added at a ratio of 100 mg GelMA: 1 mL photoinitiator.

[0038] Preparation of the base layer solution: The main materials are polyvinylpyrrolidone (PVP K30, 1000-3000 g / L), polyvinyl alcohol (PVA), or polycaprolactone (PCL). Polyethylene glycol 400 (PEG400, 10-30 g / L) and squalane (15-25 g / L) are added as plasticizers. The solvent is sterile water or anhydrous ethanol, selected according to the materials. During preparation, the polymer material is dissolved in the appropriate solvent and magnetically stirred for 12 hours until completely dissolved, forming a homogeneous viscous solution.

[0039] 3.1.2 Microneedle fabrication The microneedles of this invention are made of biodegradable polymer materials, specifically PVA / PVP materials.

[0040] Microneedles are fabricated using a micromolding method, ensuring high throughput and reproducible production.

[0041] Mold fabrication: A microneedle array model was designed using CAD software, with parameters set as follows: needle height 800-1200 μm, base diameter 400 μm, and spacing 600-900 μm. A male mold master was fabricated using 3D printing technology. Polydimethylsiloxane (PDMS) was poured onto the male mold, cured at 60°C for 2 hours, and then demolded to obtain the PDMS female mold. Before use, the PDMS mold was subjected to low-power plasma treatment for 30 seconds to enhance its hydrophilicity.

[0042] Solution Infusion and Molding: Using the vacuum filling method, the needle tip solution is added to the PDMS mold and placed in a vacuum desiccator for evacuation (1 psi, 15 minutes) to remove air bubbles and ensure complete solution filling. When using centrifugation, centrifuge at 4000 rpm for 10 minutes to allow the solution to enter the micropores. For photocrosslinked materials, cure by irradiation with ultraviolet light (400-420 nm wavelength) for 1-2 minutes. Remove excess solution from the mold surface with a scraper to ensure a clear needle tip shape.

[0043] Substrate Formation and Demolding: A base layer solution was added to the pre-cured needle tip structure, covering the entire back of the mold. After vacuum degassing, the PVA / PVP substrate was cured in a 60°C drying oven for 2-4 hours. After curing, the microneedle patch was peeled from the PDMS mold. Using ultra-low temperature spray drying technology, the demolded microneedles were rapidly frozen at -70°C. Exosome solution was sprayed onto the needle tip surface under low-temperature vacuum conditions, followed by freezing again and then freeze-drying to remove the solvent. This process was repeated multiple times to increase the loading capacity. The prepared microneedle patch was stored under dry, light-protected conditions.

[0044] 3.2 Research Subjects Fifty patients with hair loss who visited the Fourth Affiliated Hospital of Nanjing Medical University and the First People's Hospital of Hefei between February and June 2023 were selected as the study subjects.

[0045] The study included 30 male and 20 female patients, aged 22 to 50 years, with a mean age of 41.23 years. All patients underwent detailed medical history taking and physical examination, were diagnosed with alopecia, and the degree of hair loss was assessed according to the Hamilton-Norwood classification (males) and Ludwig classification (females). Patients were categorized into 11 with mild alopecia, 31 with moderate alopecia, and 8 with severe alopecia. All patients signed informed consent forms, and the study protocol was approved by the hospital's ethics committee.

[0046] 3.3 Grouping Processing For the main research methods and steps, please refer to [link / reference]. Figure 1 Fifty patients with hair loss were randomly divided into two groups: a treatment group and a control group, with 25 patients in each group. Patients in the treatment group received embryonic stem cell exosomes (ESC-Exos) combined with 0.5 mm microneedles. Specifically, the scalp alopecia areas were clearly identified, mainly including severely alopecia areas such as the forehead, crown, and back of the head. Using a nano-electronic microneedle multi-point injection method, 4 ml of high-purity embryonic stem cell exosomes were directly injected into the dermis of the alopecia area to a depth of 1 mm. Patients in the control group received only 0.5 mm microneedles with saline solution, using the same injection method as the treatment group. Both groups underwent initial efficacy evaluation and intensive treatment one month after injection, followed by re-evaluation and consolidation treatment three months later, and efficacy follow-up at six months.

[0047] 3.4 Observe the relevant results The hair loss status of both groups of patients was assessed at 1, 3, and 6 months after treatment. The main observation indicators included: Hair density: Measured by hair counting to determine the number of hairs per square centimeter of scalp. (See also...) Figure 2 ) Hair diameter: The diameter of newly grown hairs is measured using a microscope. (See also...) Figure 3 ) Patient satisfaction: Patient satisfaction with the treatment outcome was assessed through a questionnaire survey. (See Table 1)

[0048] Adverse reactions: Record any adverse reactions that occur during treatment, such as skin irritation, itching, erythema, etc.

[0049] See Figure 2 , Figure 3 As shown in Table 1, the results indicate that hair density and diameter significantly increased in the treatment group within 1-3 months after injection, and patient satisfaction was significantly higher than in the control group. At 3 months, 41 patients (82%) in the treatment group expressed satisfaction with the treatment effect, while only 10 patients (20%) in the control group expressed satisfaction. (See also...) Figure 4 and Figure 5 Six months later, the hair growth in the treatment group was good, with strong and shiny new hair, while the hair growth improvement in the control group was not significant. No serious adverse reactions occurred in the treatment group, indicating that embryonic stem cell exosome combined with microneedling treatment for hair loss has good safety and tolerability.

[0050] 3.5 Expected Results It has good effects, can significantly improve hair density and diameter in patients with hair loss, promote hair regeneration, and increase patient satisfaction with the treatment results.

[0051] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of ESC-Exos in the treatment of hair loss.

2. The application according to claim 1, characterized in that, The hair loss includes at least one of the following types: androgenetic alopecia, alopecia areata, and senile alopecia.

3. The application according to claim 1, characterized in that, The application method includes the following steps: 1) Use a 0.5 mm micro-needle to stimulate the scalp locally; 2) The concentration for topical application is 1×10¹ 0 ESC-Exos suspension with particles / ml; 3) The treatment will be performed once a week for a total of 6 weeks.

4. In the application according to claim 1, the ESC-Exos is derived from human embryonic stem cells, extracted by ultracentrifugation, and purified and identified by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry.

5. A microneedle delivery system for treating hair loss, characterized in that, The system includes microneedles and an ESC-Exos suspension for delivery to the dermal layer of the scalp.

6. The microneedle delivery system according to claim 4, characterized in that, The depth of the microneedles is 0.5 mm.

7. The microneedle delivery system according to claim 5, characterized in that, The microneedles are made of biodegradable polymer materials.

8. A method for promoting hair regeneration, characterized in that, Includes the following steps: 1) Obtain ESC-Exos derived from human embryonic stem cells; 2) Inject ESC-Exos locally or introduce it into the hair loss area using a microneedle system.

9. The method according to claim 8, characterized in that, The ESC-Exos promotes hair regeneration by activating dermal papilla cells, promoting VEGF expression, regulating the Wnt / β-catenin pathway, and inhibiting inflammatory responses.

10. ESC-Exos can be used in any of the following applications: 1) Regulating the microenvironment of hair follicle stem cells; 2) Activate hair follicle stem cells and promote hair follicle regeneration; 3) Inhibits follicular inflammation; 4) Promotes hair follicle angiogenesis; 5) Increase hair density; 4) Increase hair diameter.

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