A rapamycin-loaded exosome composition and a preparation method and application thereof

Abstract

This invention relates to an exosome composition loaded with rapamycin, its preparation method, and its application. The invention uses exosomes loaded with rapamycin to prepare the exosome composition, wherein the exosomes are lemon exosomes. Rapamycin is loaded onto lemon exosomes via electroporation to obtain the exosome composition loaded with rapamycin. The rapamycin in this composition can be used to treat androgenetic alopecia. Lemon exosomes can improve the local stability and delivery efficiency of rapamycin, and can also scavenge or reduce intracellular reactive oxygen species levels. The combination of these two factors can provide efficient and targeted treatment for androgenetic alopecia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an exosome composition loaded with rapamycin, its preparation method, and its application. Background Technology

[0002] Androgenetic alopecia (AGA) is a common hair loss disorder that primarily affects post-pubertal individuals. Its pathological features include follicle miniaturization, shortened anagen (growth) phase, and prolonged telogen (resting) phase. Epidemiological data shows that the prevalence of AGA in Chinese men is 21%, while the prevalence in men and women over 70 years of age is 41.4% and 11.8%, respectively, and is increasing annually. AGA patients often experience psychological problems such as anxiety and social phobia. The pathogenesis involves the binding of dihydrotestosterone (DHT) to the androgen receptor (AR), leading to premature aging of hair follicle cells (DPCs), increased reactive oxygen species (ROS), and abnormalities in autophagy-related signaling pathways (such as PI3K / Akt / mTOR). As key cells in the hair follicle, DPCs are responsible for regulating the hair follicle cycle. Influenced by DHT and ROS, they are prone to aging, preventing hair follicles from transitioning from the telogen phase to the anagen phase. The core pathophysiological process can be summarized as follows: after circulating testosterone enters the dermal papilla cells, it is converted into highly active dihydrotestosterone (DHT) under the catalysis of 5α-reductase. Under continuous stimulation by DHT, the anagen phase of the hair follicle is significantly shortened, prematurely entering the telogen phase. The hair follicle volume progressively atrophies, causing the growing hair to gradually change from coarse terminal hair to fine, light-colored vellus hair. As the disease progresses, the hair follicle gradually loses its ability to functionally transition from the telogen phase to the anagen phase. The activity of melanocytes in the hair bulb decreases, melanin synthesis ceases, and ultimately, the hair follicle completely closes, forming a visible area of ​​hair loss. This invention aims to solve the problems of low drug delivery efficiency, poor bioavailability, and inability to simultaneously target oxidative stress and autophagy mechanisms in existing AGA treatments. By constructing a novel nanocarrier system, it achieves efficient and safe subcutaneous delivery, improves drug uptake and release in DPCs, synergistically inhibits premature hair follicle aging, and promotes hair growth.

[0003] Rapamycin itself lacks targeting, which causes it to indiscriminately inhibit mTOR in all cells. This type of specific inhibition can lead to side effects on normal cells. Patent document CN120346178A mentions a biomimetic nanoparticle loaded with rapamycin and its preparation method and application. The substrate on which it is loaded is RZ NPs. Loading rapamycin onto this substrate does not significantly improve drug delivery efficiency in the treatment of androgenetic alopecia.

[0004] Exosomes are nanoscale vesicle structures actively secreted by cells, and they have significant research value in areas such as intercellular communication, disease diagnosis, and treatment. Exosomes have functions such as intercellular information transmission, removal of metabolic waste, and participation in immune regulation. Patent document CN119564765B mentions the application of lemon exosomes in the preparation of drugs for the prevention, relief, or treatment of skin ulcers, but does not mention their application in rapamycin delivery systems.

[0005] In summary, the treatment of androgenetic alopecia with rapamycin requires addressing the targeted delivery of rapamycin by combining it with lemon exosomes. Therefore, it is crucial to research a rapamycin-loaded exosome composition, its preparation method, and its application. Summary of the Invention

[0006] One object of the present invention is to provide an exosome composition loaded with rapamycin for the treatment of androgenetic alopecia. Rapamycin is a specific inhibitor of mTOR and can treat androgenetic alopecia. Exosomes can improve the local stability and delivery efficiency of rapamycin. The combination of the two results in an exosome composition loaded with rapamycin for the treatment of androgenetic alopecia.

[0007] Another objective of this invention is to provide lemon exosomes as a loading substrate for rapamycin. Lemon exosomes are a stable source, have antioxidant properties, and synergistically work with rapamycin.

[0008] Another objective of this invention is to provide a method for preparing lemon exosomes, which can produce lemon exosomes simply and effectively.

[0009] Another object of the present invention is to provide a rapamycin-loaded exosome composition, which uses electroporation loading technology to prepare a rapamycin-loaded lemon exosome.

[0010] To achieve the above objectives, this invention provides an exosome composition loaded with rapamycin, wherein the composition is a rapamycin-loaded nanocarrier, and the nanocarrier is lemon exosomes. This invention selects lemon as the exosome-like nanovesicle, as lemon is rich in vitamin C, polyphenolic compounds, organic acids, and various antioxidant active ingredients, giving the derived exosome-like nanovesicles significant antioxidant capacity in their natural state. This allows the composition of this invention to effectively scavenge or reduce intracellular reactive oxygen species levels. Furthermore, because lemon exosomes have good biocompatibility and low immunogenicity, and their lipid bilayer structure is similar to that of mammalian cell membranes, the exosome membrane and cell membrane fuse upon passing through animal cell membranes, releasing rapamycin. Rapamycin can inhibit the mTOR pathway, enabling cells to resume autophagy, reducing dermal papilla cell aging, thereby combating androgenetic alopecia caused by premature aging of dermal papilla cells, oxidative stress, and autophagy damage. Rapamycin binds to FK506-binding protein 12, inhibiting mTORC1 activity, restoring autophagy, and promoting the functional recovery of dermal papilla cells and hair follicle regeneration. Lemon vesicles themselves are rich in plant miRNAs and metabolites with anti-inflammatory and antioxidant activities. When rapamycin treats dermal papilla cells, it synergistically enhances the effects of drugs by inhibiting the NF-κB pathway or scavenging free radicals.

[0011] Preferably, the composition comprises rapamycin loaded onto a nanocarrier. Loading rapamycin onto a nanocarrier allows for localized administration of the drug. The carrier locks the drug in the skin and hair follicle areas, significantly reducing the amount of drug entering the bloodstream, mitigating the side effects of rapamycin, and maintaining the therapeutic efficacy of the invention while greatly reducing side effects. The loading method of the composition allows the nanocarrier to protect rapamycin from premature consumption. After the carrier enters the cells, the drug is not released instantaneously but slowly as the carrier degrades, achieving a long-lasting effect during treatment and avoiding frequent administration.

[0012] As a preferred nanocarrier, lemon exosomes are used. Lemon exosomes are a safe, inexpensive nanocarrier with natural anti-inflammatory and antioxidant activities, suitable for transdermal drug delivery, and are suitable for long-term local drug delivery in this invention. The lemon exosome nanovesicles do not contain potential animal-derived pathogens; they can avoid the complement activation effect like synthetic liposomes. The lipid composition of lemon exosome vesicles is mainly glycolipids and phosphatidic acids, without cationic lipids, and has good blood compatibility. Rapamycin is a highly hydrophobic macrolide that tends to embed into the hydrophobic core of the lipid bilayer. The lemon vesicle membrane is rich in phytosterols and glycolipids, and its acyl chain arrangement is more rigid and ordered than the phospholipid bilayer of animal cell membranes, which can provide a more stable hydrophobic anchoring environment for rapamycin, resulting in a high encapsulation rate.

[0013] Preferably, the loading method is electroporation. Electroporation is a highly efficient and controllable active drug delivery technology. Rapamycin can enter the interior of lemon exosomes through an electric field. After the electric field is removed, the lipid molecules of lemon exosomes rearrange, the gaps close, and rapamycin is encapsulated in the lumen.

[0014] A method for preparing lemon exosomes includes the following steps: S1. Pre-treat lemons by juicing them to obtain lemon juice; S2. Centrifuge the lemon juice three times consecutively, and remove the supernatant after each centrifugation to obtain crude lemon juice; S3. Add phosphate buffer to crude lemon juice for dilution, filter to obtain nanovesicle fluid, and purify the nanovesicle fluid to obtain lemon exosome dispersion.

[0015] Preferably, the pre-treated lemons in S1 are subjected to mechanical juicing after cleaning surface impurities; the first centrifugation condition in S2 is centrifugation at 5000g for 15 minutes, and the second and third centrifugation conditions are centrifugation at 8000g for 20 minutes.

[0016] Preferably, the ratio of crude lemon juice to phosphate buffer in S3 is 1:5; the filtration in S3 is performed using a 0.22μm microporous membrane; and the purification of S3 is performed by filtering the nanovesicle fluid through an ultrafiltration device.

[0017] A method for preparing an exosome composition loaded with rapamycin includes the following steps: S1. Pre-treat lemon exosome dispersion and rapamycin for later use; S2. Take the treated lemon exosome dispersion and rapamycin, mix them well to obtain the pre-electroporation mixture; S3. The above mixture is pre-incubated at 37°C for 1 hour, then electroporated. An equal volume of buffer A-2 is added, and the mixture is incubated at 4°C for 1 hour.

[0018] Preferably, the pretreatment in S1 involves resuspending the lemon exosome dispersion in electroporation buffer A-1 to obtain an exosome particle concentration of 1.5 × 10⁻⁶. 10The buffer-1 reagent consists of 1000 parts water, 85 parts sucrose, 19 parts trehalose, 3 parts N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), and 0.1 parts anhydrous magnesium chloride, with a pH of 7.4. The buffer-2 reagent consists of 1000 parts pure water, 38 parts trehalose, 8 parts sodium chloride, 2.5 parts N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), 1 part human serum albumin, 0.5 parts potassium chloride, and 0.2 parts anhydrous magnesium chloride. The volume ratio of rapamycin to lemon exosome dispersion in S2 is 0.3-0.5:1.

[0019] The application of an exosome composition loaded with rapamycin, characterized in that the composition comprises rapamycin and a nanocarrier, for the treatment of androgenetic alopecia.

[0020] The beneficial effects of this invention are: (1) The lemon exosome composition loaded with rapamycin of the present invention can effectively scavenge or reduce the level of intracellular reactive oxygen species (ROS), thereby reducing the damage of oxidative stress to cell structure and function. (2) The lemon exosome composition loaded with rapamycin of the present invention utilizes lemon-derived exosome-like nanovesicles as natural nanocarriers to load rapamycin, constructing a synergistic therapeutic system to achieve efficient delivery and multiple biological regulation. Lemon exosomes exert antioxidant effects by regulating intracellular reactive oxygen species levels, and rapamycin induces protective autophagy in cells by regulating the mTOR signaling pathway. The two work together to improve the functional state of dermal papilla cells, thereby promoting the improvement of the hair follicle microenvironment and hair growth. (3) The lemon exosome composition loaded with rapamycin of the present invention can significantly reduce the level of cellular oxidative damage and enhance cell activity under in vitro conditions. It has good biocompatibility and safety, can improve drug delivery efficiency, reduce production costs, and promote hair follicle regeneration through multi-target synergistic effects, and has good application prospects.

[0021] (4) The lemon exosomes in the lemon exosome loaded with rapamycin composition of the present invention are widely available and low in cost; moreover, the quality of the preparation process is highly controllable. Attached Figure Description

[0022] Figure 1 Image showing morphological changes of lemon exosomes during isolation; Figure 2 Ultrastructure of lemon exosomes; Figure 3 Ultrastructure of rapamycin loaded on lemon exosomes; Figure 4 Lemon exosome size distribution diagram; Figure 5Particle size distribution of lemon exosomes loaded with rapamycin; Figure 6 Lemon exosomes and potential diagrams of lemon exosomes loaded with rapamycin; Figure 7 Chromatographic peaks of rapamycin in lemon exosomes loaded with rapamycin; Figure 8 Changes in the fur on the back of a mouse; Figure 9 Relative number of hair follicles in mice; Figure 10 Relative dermal thickness of the skin on the back of a mouse; Figure 11 Observational image of mouse skin tissue; Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0025] Example 1: Extraction of lemon exosomes; Fresh lemons were selected, and after washing away surface impurities, they were mechanically juiced to collect the original juice. The juice was then filtered through four layers of medical gauze to remove coarse fibers and pulp. The juice was then centrifuged at 5000g for 15 minutes to further remove pulp fibers and small particulate impurities. The supernatant was collected and centrifuged at 8000g for 20 minutes to further remove cell debris and residual tissue. This was followed by another centrifugation at 8000g for 20 minutes to obtain a clear lemon juice supernatant. The treated lemon juice was diluted with phosphate-buffered saline (PBS) at a volume ratio of 1:5 to reduce sample viscosity and facilitate subsequent filtration. The diluted sample was filtered through a 0.22μm microporous membrane to remove residual large particles and microbial contamination. The filtered sample was used for subsequent separation and purification of exosome-like nanovesicles. The sample loading volume (the volume of lemon juice along with exosome primordia) was controlled according to the ultrafiltration device capacity, with a single loading volume not exceeding 64ml. Add PBS to another ultrafiltration flask (equilibration flask) to 3 / 2 of the container volume. The ultrafiltration temperature is 4°C, using a 100kDa ultrafiltration membrane, and centrifuge at 4000-5000g to obtain lemon exosomes.

[0026] Figure 1The study demonstrates the changes in liquid appearance during the stepwise separation and enrichment of LEVs from raw lemon juice. The raw freshly squeezed lemon juice was obviously turbid. After centrifugation, a relatively clear supernatant was obtained. After further machine purification, a pale white precipitate could be seen at the bottom of the centrifuge tube. Multiple differential centrifugations can significantly remove large particulate impurities and cell debris.

[0027] Example 2: Lemon exosomes loaded with rapamycin; Lemon-derived exosome nanovesicles loaded with rapamycin were prepared by electroporation. The specific steps are as follows: First, the prepared lemon exosomes were resuspended in electroporation buffer (buffer A-1) (redispersed into a homogeneous suspension) and the exosome particle concentration was adjusted to 1.5 × 10⁻⁶. 10 particles / mL; rapamycin was pre-dissolved in DMSO (dimethyl sulfoxide) to prepare a 10 mM (10 -3 The stock solution (mol / L) was stored at -20℃ in the dark; 279 μL of buffer A-1 was placed in an electroporation cup, and 15 μL of exosome suspension (1.5 × 10⁻⁶ mol / L) was added sequentially. 10 Add 6 μL of rapamycin solution (containing 2.7428 μg of rapamycin) to the particle / mL solution, and gently mix to bring the total volume of the system to 300 μL. This is the pre-electroporation mixture, where the volume ratio of rapamycin solution to lemon exosome suspension is approximately 0.4:1. Incubate the mixture at 37°C for 1 h, followed by electroporation. Transfer the pre-incubated electroporation system to an electrode cup and perform electroporation using an electroporation apparatus. Set the electroporation parameters as follows: pulse electric field strength of 2000 V / cm, single pulse width of 1500 μs, number of pulses of 3, and pulse interval of 1000 ms. Then incubate at 4°C for 1 h. After electroporation, add an equal volume of buffer A-2 (containing the same concentration of rapamycin as before electroporation) to the system to promote exosome membrane structure recovery and improve rapamycin loading stability, adjusting the total volume of the system to 600 μL.

[0028] Example 3: Morphological observation of lemon exosomes and lemon exosome-rapamycin; Ten μL of lemon exosome samples and lemon exosomes loaded with rapamycin were used for transmission electron microscopy (TEM) observation. A sealing film was laid flat on a glass slide, and a copper mesh was placed on the film using tweezers. The exosome sample was then dropped onto the copper mesh surface and allowed to stand for about 10 minutes to absorb the adsorption. Excess liquid was then gently blotted away with filter paper. After adsorption, 10 μL of 2% (w / v) uranium acetate was dropped onto the copper mesh surface for negative staining. After staining for 2 minutes, excess staining solution was again blotted away with filter paper. The stained copper mesh was then allowed to air dry at room temperature for about 10 minutes. After drying, the samples were placed in a TEM microscope and observed and images were acquired under an accelerating voltage of 100 kV to obtain the morphological characteristics of the exosomes.

[0029] The ultrastructure of lemon exosomes was observed using TEM. Figure 2 As shown, the vesicles are mostly round or typical "cup-shaped" structures. The cup-shaped structure is a typical structure formed due to water loss during the negative staining drying process. The vesicle boundaries are clear, the membrane structure is intact, and no obvious protein aggregates are seen in the background. The ultrastructure of lemon exosomes-rapamycin was observed using TEM. Figure 3 As shown, rapamycin is embedded in the vesicle lipid membrane or adsorbed onto the surface. The images show that lemon exosomes-rapamycin maintain a round or "teacup-shaped" structure with clear membrane boundaries, a gentle drug loading process, and intact vesicle structure.

[0030] Example 4: Nanoparticle tracking analysis of lemon exosomes and lemon exosome-rapamycin; 1) Turn on the NTA instrument, start the laser module, and preheat for 30 minutes; rinse the sample inlet channel 2-3 times with ultrapure water, then add polystyrene standard microspheres of known particle size and concentration (1:250000 dilution), with a calibration error within ±5nm; then wash with 0.01mol / L phosphate buffered saline.

[0031] 2) Dilute the preservation solution of lemon exosomes loaded with rapamycin and lemon exosomes with phosphate-buffered saline filtered through a 0.02 μm filter membrane at a dilution ratio of 1:2500, and prepare three sets of test samples for each. 3) Use a 1mL needleless syringe to evenly inject the sample into the sample cell, and aim the laser focus at a position slightly below the center of the field of view; record 5 60s videos for each sample group, and between each video segment, use a syringe pump to very slowly advance the sample (20-50μL / min), and the test temperature is 25℃; 4) Analyze and process to obtain a statistical chart of particle size distribution.

[0032] The nanoscale distribution characteristics of lemon exosomes were analyzed using NTA technology. Figure 4As shown, its particle size distribution curve exhibits a single peak, demonstrating good uniformity; the particle size of LEVs is 143.23±3.45 nm, and the particle concentration of the sample reaches 2.4×10⁻⁶. 6 The particle size distribution (particles / mL) indicates that the LEVs purified by this invention have a concentrated size distribution, consistent with the typical size characteristics of exosome nanovesicles, and exhibit a high recovery concentration. The nanoscale distribution characteristics of lemon exosomes-rapamycin, analyzed using NTA technology, are... Figure 5 As shown, particle size analysis revealed that LEVs-Rapa were concentrated, with a hydrated particle size of 174.05±6.20 nm, slightly higher than that of blank LEVs. This indicates that rapamycin has been embedded in the vesicle lipid membrane or adsorbed on the surface, but the overall size is still less than 200 nm, which is conducive to local penetration and cellular uptake.

[0033] Example 5: Potential testing of lemon exosomes loaded with rapamycin; Turn on the analyzer and let the laser preheat for at least 30 minutes. Then, set up a zeta potential measurement in the software, select liposomes as the material, with a refractive index of 1.45, an absorption of 0.001, water as the dispersion medium, set the temperature to 25℃ and equilibrate for 120 seconds, set the number of measurements to 3, set the voltage to automatic, and use an Omega capillary cell as the sample cell. Next, prepare the sample. Dilute the empty lemon exosome stock solution with 1mM sodium chloride solution and observe the real-time count rate, adjusting it to approximately 100 to 500 kcps. First, wash the rapamycin-loaded sample twice with 1mM sodium chloride at 4°C using a 100kDa ultrafiltration tube. Then, dilute to a suitable count rate and gently mix the sample by flicking the bottom of the tube with your finger and rubbing it between your palms. Use a 1ml needleless syringe to draw up the diluted sample and slowly push it into the Omega capillary cell. After the liquid fills both electrode arms, carefully check that there are no residual air bubbles at the U-shaped electrode contact points. Seal the injection port with the matching stopper, wipe the outer wall of the sample cell clean with lint-free paper, and place it into the sample cell with the gold electrode side facing the contact spring inside the instrument. Close the cap. Click "Start Measurement" and observe the phase diagram on the screen. Confirm that there are no violent sawtooth jumps and the frequency shift is symmetrical. After three measurements are completed, the potential distribution should show a single, clean peak. Record the results.

[0034] Zeta potential measurements showed that, as Figure 6 As shown, the blank LEVs were -6.56±1.29mV, while the lemon exosome-rapamycin LEVs decreased to -3.89±0.87mV, still maintaining a negative charge, demonstrating the dispersion stability.

[0035] Example 6: High-performance liquid chromatography (HPLC) determination of rapamycin loaded on lemon exosomes; A standard curve for rapamycin was established using high-performance liquid chromatography (HPLC) to quantitatively analyze its loading in lemon exosomes. The procedure was as follows: First, a standard curve for rapamycin was prepared. 2.50 mg of rapamycin was accurately weighed and placed in a 10 mL volumetric flask, then diluted to the mark with methanol to obtain a stock solution (250 μg / mL). Subsequently, 800, 400, 200, 100, 40, 20, 10, and 4 μL of the stock solution were respectively added to 5 mL volumetric flasks and diluted to the mark with methanol to obtain standard solutions with concentrations of 40, 20, 10, 5, 2, 1, 0.5, and 0.2 μg / mL. The peak area of ​​each standard crystal was detected using HPLC, and a linear regression equation between peak area (S) and concentration (C) was established. The standard curve was plotted for quantitative analysis of LEVs-RAPA samples. Mobile phase and chromatographic conditions: Column: Agilent TC-C18; Mobile phase: methanol-water (84:16); Flow rate: 1.0 mL / min; Detection wavelength: 278 nm; Column temperature: 50℃; Injection volume: 200 μL; LEVs-Rapa samples were subjected to ultrafiltration centrifugation (100 kDa ultrafiltration tube, 4500 rpm, 20 min) to separate exosomes from free drug. After centrifugation, the lower filtrate was collected and the volume Vfree was recorded. The concentration of free drug Cfree was determined by HPLC. Triple replicates were performed, and the encapsulation efficiency EE% and LC were calculated using the following formulas: (1) EE% = (C_total - C_travel) / C_total × 100%; (2) LC (ug / 109 particles) = (Ctotal - Ctraffic) × Vtotal / Ntotal × 109; Wherein C total - the initial total concentration of RAPA in the preparation system (unit: μg / mL). The concentration of free drug in the filtrate collected in the bottom collection tube after centrifugation by ultrafiltration (unit: μg / mL). V is the total volume of the drug-loaded reaction system (unit: mL). N_total - The total number of exosome particles in the reaction system as measured by NTA (unit: particles). according to Figure 7 Calculate the drug loading and encapsulation efficiency.

[0036] The following sets of tests were conducted: The first group consists of buffer 1 and buffer 2, with lemon exosome particle concentrations of 1.1×10¹⁰ particles / mL and 3.3×10⁹ particles / mL, respectively. The amount of RAPA used is 0.702 μg, and the total volume is 300 μL. The second group is buffer 1, with lemon exosome particle concentrations of 1.1×10¹⁰ particles / mL and 3.3×10⁹ particles / mL, RAPA dosage of 0.702 μg, and a total volume of 300 μL; The third group is buffer 2, with lemon exosome particle concentrations of 1.1×10¹⁰ particles / mL and 3.3×10⁹ particles / mL, RAPA dosage of 0.702 μg, and a total volume of 300 μL; The fourth group consisted of buffer 1 and buffer 2, with lemon exosome particle concentrations of 2.75×10¹⁰ particles / mL and 4.12×10⁹ particles / mL, respectively. The amount of RAPA used was 0.702 μg, and the total volume was 300 μL. The fifth group consisted of buffer 1 and buffer 2, with lemon exosome particle concentrations of 2.75×10¹⁰ particles / mL and 4.12×10⁹ particles / mL, respectively. The amount of RAPA used was 0.585 μg, and the total volume was 300 μL.

[0037] Table 1. Relevant data for groups 1-5.

[0038]

[0039] Table 2 shows the test results for groups 1-5.

[0040] Free RAPA in LEVs-Rapa was removed by ultrafiltration and centrifugation, and the total drug content and free drug content were determined using HPLC standard curves. This study optimized the LEVs-RAPA feed ratio and the electroporation buffer system during the electroporation drug loading process. Electroporation uses a transient high-voltage electric field to form nanopores in the LEVs lipid bilayer, achieving efficient drug loading.

[0041] Experimental example: Rapamycin-loaded lemon exosomes in mice; Laboratory animals: 6-week-old healthy male C57BL / 6 mice, weighing 16-18g, obtained from the Experimental Animal Center of Hangzhou Medical College; and acclimatized to the environment at 18-25℃, 60-70% humidity, and a 12-hour light-dark cycle. All animal experiments were conducted in accordance with the Animal Care and Use Guidelines of the Chinese Physiological Society and with the permission of the Animal Breeding and Use Committee of Zhejiang Provincial Animal Medical Center.

[0042] Adaptive feeding of laboratory animals: Forty-eight healthy male C57BL / 6 mice aged 6 weeks were used in this experiment. After being transferred to a standard animal facility, the mice underwent 3-5 days of acclimatization feeding to reduce the impact of environmental stress on the experiment.

[0043] The preparation of the drugs and reagents used in the experiment is as follows: The drug used in the AGA model was DHT, prepared as a 10 mg / mL stock solution using corn oil as a solvent. DHT powder was ground and accurately weighed, and an appropriate volume of corn oil was added. The solution was sonicated for approximately 5 minutes in the dark until clear and free of visible particles. It was then prepared in batches and stored at 4°C in the dark. The preparation process for rapamycin injection was as follows: 9.1417 mg of rapamycin powder was weighed and dissolved in 1 mL of DMSO to obtain a 10 mM stock solution; subsequently, it was diluted with 0.9% sodium chloride solution to a working concentration of 1.6 μM and aliquoted and stored at 4°C for later use.

[0044] AGA model construction: A DHT-induced AGA model was established using intraperitoneal injection. Except for the blank control group, mice in each group received a fixed dose of 2 mg / mouse, with 200 μL of DHT solution injected intraperitoneally every two days. Two pre-injections of DHT were performed before the formal experiment to ensure stable androgen levels. For back hair removal: after anesthesia with isoflurane, the back hair (approximately 2.0 cm × 3.0 cm) was removed using a razor. Depilatory cream was then evenly applied and left for 5 minutes. Residual hair and cream were then wiped away with a warm, damp gauze until the skin was exposed, indicating that the hair follicles were in the resting phase, providing a consistent basis for subsequent experiments. This study used intraperitoneal injection for DHT stimulation. Except for the blank control group, all mice received a fixed dose of 2 mg / mouse, with 200 μL of DHT solution injected intraperitoneally every two days. Two consecutive pre-injections of DHT were required before the back hair removal procedure.

[0045] Experimental grouping: After determining the administration method and concentration to the mice and completing the hair removal treatment on their backs, 48 ​​mice were randomly divided into 6 groups of 8 mice each, and each group received an intervention. 1) Blank control group (Control): No DHT injection was performed, and no treatment was applied to the hair removal area. This group served as a reference for healthy growth. 2) Model control group (DHT): Received only intraperitoneal injections of DHT every 2 days, with no drug intervention in the hair removal area; 3) Lemon exosome group (LEVs / DHT): Under the background of continuous DHT injection, an appropriate amount of LEVs was injected subcutaneously into the hair removal area; 4) Rapamycin group (RAPA / DHT): 1.6 μM free RAPA solution was subcutaneously injected into the hair removal area under the background of continuous DHT injection; 5) Experimental group of this invention (LEVs-RAPA / DHT): Under continuous DHT injection, LEVs loaded with an equivalent of 1.6 μM RAPA were subcutaneously injected into the hair removal area (LEVs-Rapa); 6) Other drug control group (Minoxidil / DHT): Under the background of continuous DHT injection, 200 μL of 3% Minoxidil solution was evenly applied to the hair removal area daily.

[0046] Throughout the experimental period, the growth of hair on the back of mice in each group was continuously observed, and histological examinations were conducted according to the experimental design to systematically evaluate the therapeutic effects and potential mechanisms of action of each intervention strategy. H&E staining of mouse dorsal tissue: Immediately after euthanizing the mice, dorsal skin tissue was harvested and fixed in 10% neutral buffered formalin for 24 hours to maintain tissue integrity. After fixation, the tissue underwent a gradient alcohol dehydration process (70% ethanol for 1 hour → 80% ethanol for 1 hour → 95% ethanol twice for 1 hour each → anhydrous ethanol twice for 30 minutes each), followed by two clearing treatments in xylene for 15 minutes each. After dehydration and clearing, the tissue was immersed in paraffin at 58-60°C for 3 hours to prepare paraffin blocks. These blocks were then continuously cut into thin slices of approximately 4 μm using a Leica RM2235 rotary cutter and attached to glass slides for later use.

[0047] After hydration, the sections are first dewaxed in xylene, then rehydrated to a hydrated state using a gradient of alcohols, followed by modified H&E staining. Hematoxylin is added to the sections for staining for 5-10 minutes, followed by differentiation with 1% hydrochloric acid alcohol for 30 seconds and then blued under running water for 10 minutes. Eosin solution is added for staining for 2-5 minutes. After staining, the sections are dehydrated using a gradient of alcohols (80%, 90%, 95%, anhydrous ethanol I, and anhydrous ethanol II, 5 minutes each), and then cleared twice in xylene for 5 minutes each time. Finally, neutral resin is added for mounting, and a coverslip is gently placed on top to avoid air bubbles, ensuring the sections are fixed and structurally stable. The processed sections can be observed under an Olympus BX53 optical microscope at 200x magnification.

[0048] The condition of the mice was observed sequentially from day 1, day 7, day 14, and day 21, examining the condition of the fur on their backs. Figure 8-10As shown, in both the blank control group and the model control group, mice treated with DHT showed no significant hair growth. Therefore, the DHT-induced androgenetic alopecia in mice in this experiment is as expected. Based on the results from the lemon exosome group, rapamycin group, and experimental group, it can be seen that the therapeutic effect of lemon exosomes loaded with rapamycin is significantly stronger than that of rapamycin alone or lemon exosomes. Rapamycin is strongly hydrophobic and has a large molecular weight, resulting in extremely low penetration of conventional topical formulations. Lemon exosomes have antioxidant and anti-inflammatory effects, but do not possess significant therapeutic factors and cannot directly intervene in hair follicle cells, nor can they directly induce autophagy in hair follicle stem cells. Lemon exosomes have extremely weak direct intervention capabilities for fibrosis and androgen signaling; the combination of the two improves the transdermal efficiency and tissue retention of rapamycin, achieving effective reversal of hair follicle condition. Utilizing the natural biocompatibility and suitable small particle size of lemon exosomes, the penetration rate of rapamycin can be increased, achieving targeted drug delivery deep within the hair follicle; the composition of this invention can prolong local retention time, and the lemon exosome system can deeply integrate with skin tissue, achieving rapid initial release to reach effective concentration, followed by stable sustained release, ensuring a sustained effective drug concentration in the hair follicle target area and overcoming the defect of easy local drug clearance. From a biological perspective, lemon exosome-rapamycin intervention significantly shortens the alopecia period in AGA mice; from Figure 8 Observations showed that the skin changed from grayish-white in the resting phase to black in the growth phase, and hair grew rapidly and gradually covered the bald area. From the experimental group and other drug control groups, it can be seen that the efficacy of minoxidil, the first-line drug for treating androgenetic alopecia, is slightly different from that of the experimental group, indicating that the efficacy of the present invention is sufficient to treat androgenetic alopecia.

[0049] according to Figure 11 It can be seen that, compared with the normal group and the DHT group, after the onset of the disease, the dermis thinned, the hair follicle volume decreased, the hair bulb became smaller, the hair follicles became shallower, and some hair follicles degenerated, the number of hair follicles decreased, and the arrangement of hair follicles became disordered. In the lemon exosome group, the dermal thickness increased, the number of hair follicles recovered to some extent, the diameter of hair follicles increased slightly, and the number of deep hair follicles increased, but small hair follicles still existed, and the arrangement was not fully restored. Lemon exosomes alone can partially reverse DHT loss, but the recovery is limited. In the rapamycin group, the degree of hair follicle activation increased, the number of deep hair follicles increased, and the rapamycin group showed improved hair follicle activation. While rapamycin showed more significant effects on the recovery of hair follicles during the anagen phase, the recovery effect was significantly different from that of the normal group. The recovery in the lemon exosome-rapamycin group was very obvious, with dermal thickness returning to near normal values, a significant increase in the number of hair follicles, and an increase in the proportion of large hair follicles and deep hair follicles, indicating that the hair follicles re-entered the anagen phase, the hair bulb structure recovered, the proliferation of surface hair matrix cells recovered, the hair growth capacity recovered, and the arrangement of hair follicles became more regular again, proving that the synchronization of the hair follicle cycle was improved and the miniaturization of hair follicles was reversed. The therapeutic effect of this invention is significantly better than that of minoxidil.

[0050] This application constructs a novel drug delivery system for lemon-derived exosome-like nanovesicles (LEVs) loaded with rapamycin (RAPA), achieving effective reversal of hair follicle state by improving drug transdermal efficiency and tissue retention. RAPA is strongly hydrophobic and has a large molecular weight, resulting in extremely low penetration efficiency in conventional topical formulations. This application utilizes the natural biocompatibility and suitable particle size (approximately 143 nm) of LEVs nanocarriers to achieve targeted drug delivery deep within the hair follicle. Addressing the deficiency of free drugs being easily and rapidly washed away locally, the LEVs system can deeply integrate with skin tissue, and its biphasic release characteristics (rapid initial release followed by stable sustained release) ensure a sustained effective drug concentration in the hair follicle target area. LEVs-Rapa intervention significantly shortened the alopecia period in AGA mice. Gross phenotypic observation showed that the skin changed from grayish-white in the resting phase to black in the anagen phase, with hair rapidly germinating and gradually covering the bald area. LEVs-Rapa intervention significantly shortened the alopecia period in AGA mice. Gross phenotypic observation showed that the skin changed from grayish-white in the resting phase to black in the anagen phase, and hair rapidly sprouted and gradually covered the balding area. LEVs-Rapa significantly promoted the activation of melanocytes and melanin deposition in the hair bulb, which is an important biological marker of successful hair follicle return to the anagen phase.

[0051] It should be noted that the above embodiments and comparative examples are only used to illustrate the technical solutions of the present invention and not 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 technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the present invention.

Claims

1. An exosome composition loaded with rapamycin, characterized in that, The composition includes rapamycin and nanocarriers.

2. The exosome composition loaded with rapamycin according to claim 1, characterized in that, Rapamycin is loaded onto a nanocarrier.

3. The exosome composition loaded with rapamycin according to claim 1 or 2, characterized in that, The nanocarrier is a lemon exosome, and the particle size of the lemon exosome is 143.23±3.45 mm.

4. The exosome composition loaded with rapamycin according to claim 2, characterized in that, The load method is electroporation load.

5. A method for preparing lemon exosomes, characterized in that, The method for preparing the lemon exosomes, according to claims 1-4, comprises the following steps: S1. Pre-treat lemons to obtain lemon juice; S2. Centrifuge the lemon juice three times consecutively, and remove the supernatant after each centrifugation to obtain crude lemon juice; S3. Add phosphate buffer to crude lemon juice for dilution, filter to obtain nanovesicle fluid, and purify the nanovesicle fluid to obtain lemon exosome dispersion.

6. The method for preparing lemon exosomes according to claim 5, characterized in that, The pre-treated lemons in S1 are mechanically juiced after cleaning surface impurities; the first centrifugation in S2 is centrifugation at 5000g for 15 minutes, and the second and third centrifugation are centrifugation at 8000g for 20 minutes.

7. The method for preparing lemon exosomes according to claim 5, characterized in that, The ratio of crude lemon juice to phosphate buffer in S3 is 1:5; the filtration in S3 is performed using a 0.22μm microporous membrane; the purification of S3 is performed by filtering the nanovesicle fluid through an ultrafiltration device.

8. A method for preparing an exosome composition loaded with rapamycin, characterized in that, The preparation method for the exosome composition loaded with rapamycin according to claims 1-4 comprises the following steps: S1. Pre-treat lemon exosome dispersion and rapamycin for later use; S2. Take the treated lemon exosome dispersion and rapamycin, mix them well to obtain the pre-electroporation mixture; S3. The above mixture is pre-incubated at 37°C for 1 hour, then electroporated. An equal volume of bufferA-2 reagent is added, and the mixture is incubated at 4°C for 1 hour.

9. The method for preparing an exosome composition loaded with rapamycin according to claim 8, characterized in that, The pretreatment in S1 involves resuspending the lemon exosome dispersion in electroporation buffer A-1 to obtain an exosome particle concentration of 1.5 × 10⁻⁶. 10 The buffer-1 reagent consists of 1000 parts water, 85 parts sucrose, 19 parts trehalose, 3 parts N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), and 0.1 parts anhydrous magnesium chloride, with a pH of 7.

4. The buffer-2 reagent consists of 1000 parts pure water, 38 parts trehalose, 8 parts sodium chloride, 2.5 parts N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), 1 part human serum albumin, 0.5 parts potassium chloride, and 0.2 parts anhydrous magnesium chloride. The volume ratio of the dispersion of rapamycin and lemon exosomes in S2 is 0.3-0.5:

1.

10. The application of an exosome composition loaded with rapamycin, characterized in that, The composition includes rapamycin and a nanocarrier for the treatment of androgenetic alopecia.

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