A minoxidil-loaded hair follicle exosome complex, and a preparation method and application thereof
By introducing a cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate into the hair follicle exosome complex to form a core-shell structure, the problem of stable loading and release of minoxidil in vivo was solved, achieving efficient and intelligent drug delivery and improving therapeutic efficacy and safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XIAMEN HONGYI TESTING CO LTD
- Filing Date
- 2026-01-23
- Publication Date
- 2026-06-02
Smart Images
Figure CN121550175B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a follicular exosome complex loaded with minoxidil, its preparation method, and its application. Background Technology
[0002] Androgenetic alopecia is a common hair loss condition. Minoxidil is one of the main drugs used in clinical practice to treat this type of hair loss. Its mechanism of action is mainly to stimulate hair follicle growth. With the development of biotechnology, stem cell-derived exosomes, as an endogenous nanovesicle, are considered as potential drug delivery carriers due to their good biocompatibility and low immunogenicity.
[0003] In practical applications, exosomes are often used as carriers to load various drugs, aiming to cross biological barriers and reach deep lesions for synergistic therapeutic effects. To ensure therapeutic efficacy, sufficient drug must be loaded around or inside the carrier using physical or chemical means, ensuring stable delivery. However, minoxidil is a poorly soluble drug with poor solubility and dispersion stability in aqueous media. Existing carrier construction methods often fail to guarantee efficient drug loading, easily leading to drug leakage, precipitation, or crystallization during preparation or storage. Furthermore, natural exosomes lack structural stability during in vivo circulation or local application, easily degrading or being rapidly cleared by the complex enzymatic environment or immune system, preventing prolonged retention in the hair follicle. Non-specific drug release before reaching the target area, or failure to release the drug at the lesion site according to changes in the microenvironment caused by inflammation, results in low drug utilization, difficulty maintaining effective therapeutic concentrations, safety concerns, and poor therapeutic effects. Summary of the Invention
[0004] The purpose of this invention is to provide a hair follicle exosome complex loaded with minoxidil, its preparation method and application, thereby solving the problems existing in the background art.
[0005] To address the aforementioned technical problems, this invention provides a hair follicle exosome complex loaded with minoxidil, having a core-shell structure, comprising: a hair follicle mesenchymal stem cell exosome as the core; a cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate with its hydrophobic end inserted into the lipid bilayer of the hair follicle mesenchymal stem cell exosome; and minoxidil contained within the hydrophilic β-cyclodextrin cavity of the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate; wherein the ROS-sensitive linker in the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate contains a thioketal bond or a disulfide bond, and the complex undergoes responsive cleavage in a reactive oxygen species microenvironment.
[0006] Preferably, the method includes the following steps: Step S1: Separate and purify the hair follicle mesenchymal stem cell exosome suspension; Step S2: Synthesize the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate; Step S3: Perform membrane intercalation assembly of the conjugate obtained in Step S2 and the exosome suspension obtained in Step S1 to prepare surface-modified exosomes; Step S4: Mix the minoxidil solution with the surface-modified exosomes obtained in Step S3, perform drug loading and separation and purification to obtain the hair follicle exosome complex loaded with minoxidil.
[0007] Preferably, step S1 specifically includes the following sub-steps: Step S1.1: Take hair follicle tissue, cut it into small pieces, add a type II collagenase solution with a mass fraction of 0.1%-0.25%, and digest it enzymatically in a 37℃ constant temperature shaker at a speed of 60rpm-100rpm for 30min-60min. Isolate and culture hair follicle mesenchymal stem cells. When the cell confluence reaches 70%-80%, wash the cells with phosphate buffer 2-3 times, replace the exosome-free serum medium, and continue culturing for 24h-48h. Collect the cell supernatant; Step S1.2: Incubate the collected cell supernatant at 4℃... Using differential centrifugation, centrifuge at 300g for 10 min, 2000g for 20 min, and 10000g for 30 min sequentially to remove cell debris, apoptotic bodies, and large molecular proteins, and collect the supernatant; Step S1.3: After filtering the supernatant collected in step S1.2 through a polyethersulfone membrane with a pore size of 0.22 μm, perform ultracentrifugation at 4℃ with a centrifugal force of 100000g-120000g and a centrifugation time of 60 min-90 min, discard the supernatant, and resuspend the precipitate with pre-cooled phosphate buffer to obtain the hair follicle mesenchymal stem cell exosome suspension.
[0008] Preferably, step S2 specifically includes the following sub-steps: Step S2.1: Preparation of ROS-sensitive linker: If preparing a linker containing a thioketal bond: Under nitrogen protection, 3-mercaptopropionic acid and anhydrous acetone are mixed in a molar ratio of 2:(1-2), and dry hydrogen chloride gas is introduced until saturation. The reaction is magnetically stirred at 20℃-30℃ for 4-6 hours. After the reaction, the mixture is cooled to 0℃-4℃ in an ice bath, allowed to stand for crystallization, filtered, and the filter cake is washed 3-5 times with cold hexane and vacuum dried. A dicarboxylic acid linker containing a thioketal bond is obtained. To prepare a linker containing a disulfide bond: 3,3'-dithiodipropionic acid is directly selected as the dicarboxylic acid linker containing a disulfide bond. Step S2.2: Preparation of the cholesterol activation intermediate: The dicarboxylic acid linker obtained in step S2.1 is dissolved in anhydrous dichloromethane, and cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine are added, controlling the molar ratio of the dicarboxylic acid linker to cholesterol to be... 1:(0.8-1.2), reacted at room temperature in the dark with stirring for 12-24 h. The reaction solution was purified by silica gel column chromatography, using a mixture of dichloromethane and methanol in a volume ratio of 50:1-20:1 as the eluent. The fraction containing one free carboxyl group was collected, and the solvent was removed by rotary evaporation to obtain the cholesterol-ROS-sensitive linker monoester; Step S2.3: Synthesis of the conjugate: Mono-6-amino-β-cyclodextrin was dissolved in anhydrous dimethyl sulfoxide, and the product obtained in step S2.2 was added. Cholesterol-ROS-sensitive linker monoester and condensing agent were prepared. The molar ratio of monoester to mono-6-amino-β-cyclodextrin was controlled at 1:(1.0-1.2). The reaction was stirred at 30℃-40℃ for 24h-48h. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 500Da-1000Da and dialyzed in deionized water for 48h-72h to remove unreacted small molecules and solvent. After freeze-drying, cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate was obtained.
[0009] Preferably, in step S2.2, the molar ratio of the dicarboxylic linker arm, cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:(0.8-1.2):(1.5-2.0):(0.1-0.3); in step S2.3, the condensing agent is 1,3-dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
[0010] Preferably, step S3 specifically involves: dissolving the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate prepared in step S2 in ethanol or dimethyl sulfoxide to prepare a stock solution with a concentration of 5 mg / mL-10 mg / mL; adding the stock solution dropwise to the exosome suspension prepared in step S1 at a rate of 10 μL / min-50 μL / min, according to a mass ratio of exosome protein to conjugate of 1:(0.5-2.0); placing the mixture in a 37°C constant temperature shaker and incubating it in the dark at a speed of 60 rpm-100 rpm for 1 h-2 h to allow the cholesterol groups to insert into the exosome membrane structure; after incubation, placing the mixture into a dialysis bag with a molecular weight cutoff of 10 kDa-14 kDa and dialyzing it in phosphate buffer for 12 h-24 h, changing the dialysis solution every 4 h-6 h to remove unbound conjugates and organic solvents, and obtaining surface-modified exosomes.
[0011] Preferably, step S4 specifically involves: dissolving minoxidil in acetic acid buffer or ethanol aqueous solution to prepare a minoxidil solution with a concentration of 20 mg / mL-40 mg / mL; mixing the surface-modified exosomes obtained in step S3 with the minoxidil solution at a volume ratio of 1:(2-4), and magnetically stirring at 200 rpm-400 rpm for 4 h-8 h at 25℃-30℃; after the reaction is completed, centrifuging the mixture at 100000g for 60 min, discarding the supernatant, resuspending the precipitate in phosphate buffer, and then ultrasonically dispersing it for 1 min-2 min with a power of 30W-50W to obtain the hair follicle exosome complex loaded with minoxidil.
[0012] Also provided is the use of a follicular exosome complex loaded with minoxidil in the preparation of a medicament for treating androgenetic alopecia or alopecia areata.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] By introducing specific conjugates onto the surface of biological vesicles, a protective hydration shell is formed. Anchoring groups are inserted into the lipid bilayer using hydrophobic interactions, while the hydrophilic ends face outwards. This effectively shields the vesicle surface, reducing the recognition and clearance of the vesicles by biological systems. As a result, the circulation and residence time of the carrier in the biological environment are prolonged, the membrane structure is stabilized, premature degradation of the carrier during transportation is prevented, and the integrity of the carrier's own biological activity is ensured, enabling it to reach the site of action more effectively.
[0015] By utilizing a specific macrocyclic cavity structure to encapsulate drug molecules, and through the recognition interaction between the host cavity and the guest molecule, hydrophobic drugs are contained within the cavity, effectively dispersing the drug in an aqueous medium. This solves the problem of poor solubility of the active ingredient. The assembly of the host and guest molecules on the carrier surface provides a high density of loading sites, enhancing the drug loading capacity of the system. It also avoids competition for space between the drug and endogenous bioactive factors within the carrier, ensuring that the exogenous drug and the carrier's own active ingredients can work together.
[0016] The connecting portion of the conjugate is designed as a chemical bond responsive to specific microenvironmental stimuli. In pathological environments with elevated oxidative stress levels, this bond breaks, acting as a molecular switch. Under normal physiological conditions, the drug is stably encapsulated. Upon reaching a target tissue with high oxidative levels, the protective shell dissociates, triggering the release of the encapsulated drug and the exposure of the therapeutic carrier, achieving on-demand delivery. This intelligent release behavior matches pathological characteristics and improves drug utilization at the target site. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 This is a balance diagram of drug loading and particle uniformity in this invention. Detailed Implementation
[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0020] Example 1
[0021] This embodiment provides a hair follicle exosome complex loaded with minoxidil. Its core design concept is to solve the dual problems of poor exosome stability and weak drug targeting by using a core-shell heterostructure. In particular, the ROS-sensitive linker in the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate is specifically designed to contain a thioketal bond. This chemical selection enables the complex to undergo responsive cleavage in the high concentration of reactive oxygen species microenvironment in the hair loss area, realizing a smart delivery mechanism of sensing-response-release.
[0022] In step S1 of this embodiment, to obtain a highly active carrier core, hair follicle tissue is minced and mixed with a 0.1% (w / w) type II collagenase solution. The mixture is then digested at 60 rpm for 60 min in a 37°C constant-temperature shaker. This low-concentration digestion condition aims to reduce damage to cell membrane surface proteins such as CD9 and CD63, preserving the natural homing ability of exosomes. Hair follicle mesenchymal stem cells are isolated and cultured. When the cell confluence reaches 70%, the cells are washed twice with phosphate-buffered saline, replaced with exosome-free serum culture medium, and cultured for another 24 h. The cell supernatant is then collected. The collected cell supernatant was then centrifuged at 4°C using differential centrifugation at 300g for 10 min, 2000g for 20 min, and 10000g for 30 min to remove cell debris, apoptotic bodies, and large molecular proteins. The supernatant was then collected. After filtering the supernatant through a polyethersulfone membrane with a pore size of 0.22 μm, it was centrifuged at 4°C at 100000g for 90 min. The supernatant was discarded, and the precipitate was resuspended in pre-cooled phosphate buffer to obtain the hair follicle mesenchymal stem cell exosome suspension.
[0023] In step S2 of this embodiment, an environmentally responsive molecular switch is constructed. First, in step S2.1, under nitrogen protection, 3-mercaptopropionic acid and anhydrous acetone are mixed at a molar ratio of 2:1, and dry hydrogen chloride gas is introduced until saturation. The reaction is magnetically stirred at 20°C for 6 hours. After the reaction, the mixture is cooled to 0°C in an ice bath, allowed to stand for crystallization, filtered, and the filter cake is washed three times with cold hexane and vacuum dried to obtain a dicarboxyl linker containing a thioketal bond. Then, in step S2.2, the obtained dicarboxyl linker is dissolved in anhydrous dichloromethane, and cholesterol and 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; at this time, the molar ratio of the dicarboxylic linker, cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine was set to 1:0.8:1.5:0.1, and the reaction was stirred at room temperature in the dark for 24 h; the reaction solution was purified by silica gel column chromatography, the eluent was a mixture of dichloromethane and methanol in a volume ratio of 50:1, and the solvent was removed by rotary evaporation to obtain cholesterol-ROS-sensitive linker monoester; finally, in step S2.3, the mono- 6-Amino-β-cyclodextrin was dissolved in anhydrous dimethyl sulfoxide, and a cholesterol-ROS-sensitive linker monoester and the condensing agent 1,3-dicyclohexylcarbodiimide were added. The molar ratio of the monoester to mono-6-amino-β-cyclodextrin was controlled at 1:1.0, and the reaction was stirred at 30°C for 48 h. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in deionized water for 48 h to remove unreacted small molecules and solvent. After freeze-drying, the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate was obtained. The conjugate was then analyzed by DMSO-d6 at 400 MHz. The product structure was confirmed by 1H-NMR as follows: δ 5.68 (brs, cyclodextrin-OH), 5.31 (m, cholesterol C6-H), 4.82 (s, cyclodextrin C1-H), 4.45 (m, cyclodextrin-OH), 3.50-3.80 (m, cyclodextrin ring proton), 2.65-2.85 (m, linker arm -CH2-), 0.65-2.00 (m, cholesterol backbone proton). The overall yield of the reaction was calculated to be 68%. In this step, the introduction of the thioketal bond provides a chemical basis for the subsequent site-directed release under inflammatory conditions.
[0024] In step S3 of this embodiment, the fluidity of the biological membrane is utilized for non-destructive modification. The cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate prepared in step S2 is dissolved in ethanol to prepare a stock solution with a concentration of 5 mg / mL. The stock solution is added dropwise to the exosome suspension prepared in step S1 at a rate of 10 μL / min, according to the mass ratio of exosome protein to conjugate of 1:0.5. The mixture is placed in a 37°C constant temperature shaker and incubated at 60 rpm in the dark for 2 h. This process utilizes the hydrophobicity of the cholesterol group to allow it to spontaneously insert into the exosome membrane structure, while the hydrophilic cyclodextrin end faces outward, forming a protective hydration layer. After incubation, the mixture is placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in phosphate buffer for 24 h. The dialysate is changed every 6 h to remove unbound conjugates and organic solvents, resulting in surface-modified exosomes.
[0025] In step S4 of this embodiment, the drug is loaded via host-guest recognition. Minoxidil is dissolved in acetic acid buffer to prepare a minoxidil solution with a concentration of 20 mg / mL. The surface-modified exosomes obtained in step S3 are mixed with the minoxidil solution at a volume ratio of 1:2 and magnetically stirred at 200 rpm for 8 hours at 25°C. Minoxidil is then loaded into the cyclodextrin cavity using host-guest recognition. After the reaction, the mixture is centrifuged at 100,000 g for 60 min, the supernatant is discarded, the precipitate is resuspended in phosphate buffer, and then ultrasonically dispersed at 30 W for 2 min to obtain the hair follicle exosome complex loaded with minoxidil. This loading method avoids minoxidil directly entering the exosome and crowding out the space of bioactive factors, while also solving the problem of poor water solubility of minoxidil.
[0026] Example 2
[0027] This embodiment provides a follicular exosome complex loaded with minoxidil and its preparation method; this embodiment aims to balance the recovery rate of exosomes and the loading efficiency of drugs by adjusting the preparation parameters, especially for the process adaptation to the characteristics of disulfide linker arms;
[0028] In this embodiment, the ROS-sensitive linker uses a disulfide bond. This chemical bond has excellent responsiveness to intracellular high glutathione environments or specific oxidative stress environments, providing release kinetics characteristics different from those of thioketal bonds.
[0029] In step S1 of this embodiment, hair follicle tissue is minced and mixed with a 0.25% (w / w) type II collagenase solution. The mixture is then digested at 100 rpm for 30 min in a 37°C constant temperature shaker. The higher enzyme concentration and speed shorten the digestion time, which helps to quickly obtain cells. Hair follicle mesenchymal stem cells are isolated and cultured. When the cell confluence reaches 80%, the cells are washed three times with phosphate buffer, and the culture medium is replaced with exosome-free serum for 48 h. The cell supernatant is collected. The subsequent differential centrifugation steps are the same as in Example 1. In the ultra-high speed centrifugation step, the centrifugation force is adjusted to 120,000 g and the centrifugation time is 60 min. The supernatant is discarded, and the precipitate is resuspended in pre-cooled phosphate buffer to obtain the hair follicle mesenchymal stem cell exosome suspension.
[0030] In step S2 of this embodiment, a conjugate containing a disulfide linker is synthesized. Step S2.1: 3,3'-dithiodipropionic acid is directly selected as the dicarboxylic acid linker containing a disulfide bond, omitting the synthesis step of the thioketal bond. Step S2.2: The dicarboxylic acid linker containing a disulfide bond is dissolved in anhydrous dichloromethane, and cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine are added. The molar ratio of the dicarboxylic acid linker, cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is controlled at 1:1.0:2.0:0.3. The reaction is stirred at room temperature in the dark for 12 hours. The reaction solution is purified by silica gel column chromatography, with dichloromethane and methanol as the eluent in a volume ratio of 2:1. A 0:1 mixture was analyzed by thin-layer chromatography to merge fractions containing a single spot. The solvent was removed by rotary evaporation to obtain a cholesterol-ROS-sensitive linker monoester. Step S2.3: Mono-6-amino-β-cyclodextrin was dissolved in anhydrous dimethyl sulfoxide, and the above monoester and condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added. The molar ratio of monoester to mono-6-amino-β-cyclodextrin was controlled at 1:1.2, and the mixture was stirred at 40°C for 24 h. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in deionized water for 72 h. After freeze-drying, the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate was obtained. 1H-NMR confirmed that the product structure was as expected, and the overall yield was approximately 65%.
[0031] In step S3 of this embodiment, the conjugate prepared in step S2 is dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 10 mg / mL. The stock solution is added dropwise to the exosome suspension prepared in step S1 at a rate of 50 μL / min, according to the mass ratio of exosome protein to conjugate of 1:2.0. The higher proportion of conjugate is intended to increase the modification density on the surface of exosomes and enhance their carrying capacity for minoxidil. The mixture is placed in a constant temperature shaker at 37°C and incubated at 100 rpm in the dark for 1 h. After incubation, the mixture is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed in phosphate buffer for 12 h, with the dialysate being changed every 4 h to obtain surface-modified exosomes.
[0032] In step S4 of this embodiment, minoxidil is dissolved in an ethanol-water solution to prepare a minoxidil solution with a concentration of 40 mg / mL. The surface-modified exosomes obtained in step S3 are mixed with the minoxidil solution at a volume ratio of 1:4 and magnetically stirred at 400 rpm for 4 hours at 30°C. The higher minoxidil concentration and stirring speed help to improve the saturation of host-guest inclusion. After the reaction is completed, the mixture is centrifuged at 100,000 g for 60 min, the supernatant is discarded, the precipitate is resuspended in phosphate buffer, and then ultrasonically dispersed at 50 W for 1 min to obtain the hair follicle exosome complex loaded with minoxidil. This embodiment significantly improves the drug loading of the product through high-intensity physical parameter settings, which is suitable for treatment scenarios with high dosage requirements.
[0033] Example 3
[0034] This embodiment provides a hair follicle exosome complex loaded with minoxidil and its preparation method; this embodiment focuses on optimizing intermediate parameters to obtain a complex with uniform particle size and good stability, especially by finely controlling the reaction temperature and dialysis conditions;
[0035] In step S1 of this embodiment, hair follicle tissue is minced and mixed with a 0.15% (w / w) type II collagenase solution. The mixture is then digested by enzymatic digestion at 80 rpm for 45 min in a constant temperature shaker at 37°C. Hair follicle mesenchymal stem cells are isolated and cultured. When the cell confluence reaches 75%, the cells are washed and the culture medium is replaced. The culture is then cultured for 36 h, and the cell supernatant is collected. After removing impurities by differential centrifugation, the cells are subjected to ultra-high speed centrifugation at a centrifugal force of 110,000 g for 75 min. The precipitate is then resuspended to obtain a suspension of hair follicle mesenchymal stem cell exosomes.
[0036] In step S2 of this embodiment, step S2.1 is the same as in Example 1, preparing a dicarboxyl linker containing a thioketal bond; in step S2.2, the molar ratio of the dicarboxyl linker, cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:0.9:1.8:0.2, and the reaction is carried out at room temperature with stirring in the dark for 18 hours; the eluent is a mixture of dichloromethane and methanol in a volume ratio of 30:1, to obtain the cholesterol-ROS-sensitive linker intermediate; in step S2.3, The condensing agent used was 1,3-dicyclohexylcarbodiimide, and the molar ratio of the intermediate to mono-6-amino-β-cyclodextrin was controlled at 1:1.1. The reaction was carried out at 35°C with stirring for 36 h. The molecular weight cutoff of the dialysis bag was 1500 Da, and dialysis was performed for 60 h to obtain the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate. The yield of the conjugate synthesized under these conditions reached 70%, and the structure was confirmed by NMR. This combination of parameters aims to ensure the reaction conversion rate while reducing the formation of by-products and improving the purity of the conjugate.
[0037] In step S3 of this embodiment, the conjugate is prepared into a stock solution of 8 mg / mL, wherein the solvent is ethanol; the stock solution is added to the exosome suspension at a rate of 30 μL / min according to the mass ratio of exosome protein to conjugate of 1:1.0; the mixture is incubated at 37°C and 80 rpm for 1.5 h; the molecular weight cutoff of the dialysis bag is 12 kDa, and dialysis is performed for 18 h, with the solution changed every 5 h; this ratio ensures that the surface coverage of the exosomes is moderate, which can effectively load drugs while maintaining the flexibility of the exosomes and prevents the increase in rigidity caused by excessive modification;
[0038] In step S4 of this embodiment, minoxidil was prepared into a 30 mg / mL solution using acetate buffer as the solvent; the surface-modified exosomes were mixed with the minoxidil solution at a volume ratio of 1:3 and stirred at 28°C and 300 rpm for 6 h; after the reaction was completed, the precipitate was collected by ultracentrifugation and dispersed by ultrasonication at 65 W for 1.5 min to obtain the hair follicle exosome complex loaded with minoxidil; this embodiment obtained the complex with the narrowest particle size distribution, i.e. the smallest PDI, by setting moderate parameters, demonstrating the best colloidal stability.
[0039] Example 4
[0040] This embodiment provides a follicular exosome complex loaded with minoxidil and its preparation method; this embodiment pays special attention to the influence of solvent ratio and reaction temperature on product structure during the synthesis of the linker arm, in order to obtain a complex that is more sensitive to ROS response;
[0041] In step S1 of this embodiment, a type II collagenase solution with a mass fraction of 0.2% was used for digestion at 37°C and 90 rpm for 40 min; cell confluence was controlled at 75% and cultured in serum-free medium for 30 h; ultracentrifugation was performed at 115,000 g for 80 min; this step ensured the stability of exosome production and provided sufficient raw materials for subsequent processes.
[0042] In step S2 of this embodiment, during step S2.1, when preparing the ROS-sensitive linker, the molar ratio of 3-mercaptopropionic acid to anhydrous acetone was adjusted to 2:1.5, the reaction temperature was controlled at 25°C, and the reaction time was 5 hours. Appropriately increasing the acetone ratio helps to improve the uniformity of the ketal reaction and reduce the formation of oligomers. In step S2.2, the molar ratio of the dicarboxylic acid linker, cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine was 1:0.95:1.7:0.25, and the reaction time was 20 hours. In step S2.3, the molar ratio of the monoester to mono-6-amino-β-cyclodextrin was 1:1.15, the reaction temperature was 38°C, and the reaction time was 30 hours. The molecular weight cutoff of the dialysis bag was selected as 800 Da, and dialysis was performed for 50 hours. The final product was identified by 1H-NMR, and the yield was 66%.
[0043] In step S3 of this embodiment, the concentration of the conjugate mother liquor is 6 mg / mL, and the solvent is anhydrous dimethyl sulfoxide; the mass ratio of exosome protein to conjugate is 1:1.5, the dropping rate is 20 μL / min; the incubation conditions are 37°C, 70 rpm, and 1.5 h; the dialysis conditions are the same as in Example 3; the choice of solvent is crucial to the dispersion state of the conjugate in the aqueous phase, and the use of anhydrous dimethyl sulfoxide promotes the extension of the hydrophobic cholesterol end and improves the intercalation efficiency;
[0044] In step S4 of this embodiment, the concentration of minoxidil solution was 25 mg / mL; the mixing volume ratio was 1:2.5; the stirring conditions were 26°C, 250 rpm, and 5 h; the ultrasonic power was 60 W; and the treatment time was 1.5 min to obtain the hair follicle exosome complex loaded with minoxidil. This embodiment demonstrates the positive impact of solvent system fine-tuning on the performance of the final product and obtains a complex with balanced overall performance.
[0045] Example 5
[0046] This embodiment provides a hair follicle exosome complex loaded with minoxidil and its preparation method; this embodiment aims to verify the feasibility of preparing the complex by extending the physical assembly time with a lower amount of chemical reagents, so as to reduce the potential risk of reagent residue.
[0047] In step S2 of this embodiment, in step S2.1, the molar ratio of 3-mercaptopropionic acid to anhydrous acetone is 2:2, the reaction temperature is 30°C, and the reaction time is 4 h; in step S2.2, the molar ratio of the dicarboxyl linker, cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:0.85:1.6:0.15, and the reaction time is 16 h; in step S2.3, the molar ratio of the intermediate to mono-6-amino-β-cyclodextrin is 1:1.05, the reaction temperature is 32°C, and the reaction time is 40 h. The mild reaction conditions reduce the risk of deformation of the cyclodextrin cavity; the target conjugate was successfully synthesized by 1H-NMR, with a yield of 69%.
[0048] In step S3 of this embodiment, the concentration of the conjugate mother liquor is 7 mg / mL; the mass ratio of exosome protein to conjugate is 1:0.8, the dropping rate is 40 μL / min; the incubation conditions are 37°C and 90 rpm, but the time is extended to 2 h to ensure that cholesterol groups can be fully inserted into the membrane under a lower concentration gradient; the dialysis time is 20 h; long-term low-speed incubation is conducive to the formation of a more thermodynamically stable membrane structure.
[0049] In step S4 of this embodiment, the minoxidil solution concentration was 35 mg / mL; the mixing volume ratio was 1:3.5; the stirring conditions were 28°C, 350 rpm, and 7 h; the ultrasonic power was 70 W; and the treatment time was 1 min to obtain the hair follicle exosome complex loaded with minoxidil. This embodiment demonstrates a time-for-space strategy, proving that a qualified complex can still be prepared with low reagent consumption, and has good industrialization prospects.
[0050] Comparative Example 1
[0051] This comparative example provides a physical mixture with the same components as in Example 3, but without chemical conjugation and membrane intercalation assembly. Specifically, the extracted hair follicle mesenchymal stem cell exosomes, minoxidil, β-cyclodextrin, and cholesterol are directly mixed and used after simple stirring. This comparative example lacks the key structure of the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate, and cannot form a stable core-shell structure. It aims to verify the necessity of covalent coupling and membrane intercalation assembly for the stability of the complex.
[0052] Comparative Example 2
[0053] This comparative example provides a non-ROS-responsive complex; its preparation method is basically the same as that of Example 3, except that in step S2.1, adipic acid is used instead of the dicarboxyl linker containing the thioketal bond, thereby synthesizing a cholesterol-adipic acid-β-cyclodextrin conjugate without the ROS-sensitive bond; although the complex has a core-shell structure, the linker does not have the ability to break in response to ROS, and the shell cannot detach under inflammatory conditions, which aims to verify the key role of the ROS-sensitive bond in achieving on-demand drug release.
[0054] Comparative Example 3
[0055] This comparative example provides a simple minoxidil-cyclodextrin inclusion complex without exosomes. The preparation method is as follows: minoxidil and β-cyclodextrin are mixed and stirred in the proportions in Example 3 to form an inclusion complex by utilizing host-guest interaction, but it does not contain the core bioactive component, hair follicle mesenchymal stem cell exosomes. This comparative example aims to verify the irreplaceable role of exosomes as the core bioactive component and carrier in the system.
[0056] Comparative Example 4
[0057] This comparative example provides a direct drug-loaded exosome without connector arm modification. The preparation method is as follows: extracted hair follicle mesenchymal stem cell exosomes are mixed with minoxidil solution, and the drug is forcibly loaded into the exosomes using an electroporation method with a voltage of 200V and a capacitance of 100μF, without any surface modification. This comparative example aims to verify the advantages of the surface host-guest loading strategy of the present invention over traditional internal drug loading methods in maintaining the integrity of exosomes and the amount of drug loaded.
[0058] Verification test
[0059] The physicochemical properties and in vitro release tests of the minoxidil-loaded follicular exosome complexes or control samples prepared in Examples 1-5 and Comparative Examples 1-4 are shown below:
[0060] Particle size and potential stability test methods: The average hydrated particle size and polydispersity index (PDI) of each sample in PBS buffer were determined by dynamic light scattering (DLS); the samples were stored at 4°C for 7 days and then measured again to evaluate the colloidal stability.
[0061] Drug loading and encapsulation efficiency testing methods: The content of minoxidil was determined by high performance liquid chromatography (HPLC); the free drug was separated by ultrafiltration centrifugation, and the encapsulation efficiency was calculated, which is the ratio of the encapsulated drug amount to the total drug amount multiplied by 100%.
[0062] ROS responsive release assay method: The dialysis method was used to simulate the microinflammatory environment of the hair loss area, i.e., a PBS solution containing 100 μM H2O2 and pH 7.4, and the normal physiological environment, i.e., a PBS solution with pH 7.4; dialysis was performed at 37℃ for 24 h, and the drug concentration in the release medium was measured at regular intervals.
[0063] Data table:
[0064] Table 1 Summary of performance tests for each embodiment and comparative example
[0065]
[0066] Result analysis components:
[0067] Based on Table 1 Figure 1 It can be seen that the core-shell structured complex constructed in this invention exhibits significant advantages in terms of stability, drug loading capacity, and smart responsiveness.
[0068] First, comparing Examples 1-5 with Comparative Example 1, it can be seen that the complexes formed by covalent bonding and membrane intercalation have uniform particle size, i.e., PDI is less than 0.2, while simple physical mixing leads to severe aggregation, i.e., particle size is greater than 1 μm. This confirms the key role of cholesterol anchoring groups in maintaining the colloidal stability of exosomes, and the hydration layer they form provides the necessary steric hindrance. At the same time, the encapsulation efficiency of the Example group is about 70%, which is much higher than the 15.2% of the physical mixing group and the 42.6% of Comparative Example 4. This is attributed to the high density of external drug loading sites provided by the cyclodextrin cavity, which effectively avoids the shortcoming of limited internal volume of exosomes.
[0069] Secondly, regarding environmental responsiveness, with the introduction of H2O2 into the release medium, the drug release rate of Examples 1-5 showed an explosive increase, jumping from about 12% to about 80%, while the release behavior of Comparative Example 2, which used a non-responsive linker, showed no significant difference in the two media; this mechanistically confirms the effectiveness of thioketal bonds or disulfide bonds as molecular switches: under ROS stimulation, the linker breaks, causing the cyclodextrin shell to detach, thereby simultaneously releasing the drug and exposing the exosomes; this design perfectly matches the pathological characteristics of elevated oxidative stress levels in androgenetic alopecia areas;
[0070] Finally, comparing Example 3 with Comparative Example 4, it was found that the surface modification strategy not only improved the drug loading capacity but also avoided the potential damage to the membrane structure caused by electroporation. In summary, this invention has successfully realized a nanomedicine delivery system with high drug loading capacity, high stability, and lesion-targeting activation function by finely controlling the chemical structure and assembly process.
[0071] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.
Claims
1. A minoxidil-loaded hair follicle exosome complex, characterized in that, It has a core-shell structure, comprising: a hair follicle mesenchymal stem cell exosome as the core; a cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate inserted into the lipid bilayer of the hair follicle mesenchymal stem cell exosome; and minoxidil contained in the hydrophilic β-cyclodextrin cavity of the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate; wherein the ROS-sensitive linker in the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate contains a thioketal bond or a disulfide bond, and the complex undergoes responsive cleavage in a reactive oxygen microenvironment; The method for preparing the hair follicle exosome complex loaded with minoxidil includes the following steps: Step S1: Separate and purify the hair follicle mesenchymal stem cell exosome suspension; Step S2: Synthesize the cholesterol-ROS-sensitive linker arm-β-cyclodextrin conjugate; Step S3: Perform membrane intercalation assembly of the conjugate obtained in Step S2 and the exosome suspension obtained in Step S1 to prepare surface-modified exosomes; Step S4: Mix the minoxidil solution with the surface-modified exosomes obtained in Step S3, perform drug loading and separation and purification to obtain the hair follicle exosome complex loaded with minoxidil. Step S2 specifically includes the following sub-steps: Step S2.1: Preparation of ROS-sensitive linker: To prepare a linker containing a thioketal bond: Under nitrogen protection, 3-mercaptopropionic acid and anhydrous acetone are mixed in a molar ratio of 2:(1-2), and dry hydrogen chloride gas is introduced until saturation. The reaction is magnetically stirred at 20℃-30℃ for 4-6 hours. After the reaction, the mixture is cooled to 0℃-4℃ in an ice bath, allowed to stand for crystallization, filtered, and the filter cake is washed 3-5 times with cold hexane and dried under vacuum to obtain the desired product. Dicarboxylic acid linker containing a thioketal bond; if preparing a linker containing a disulfide bond: directly select 3,3'-dithiodipropionic acid as the dicarboxylic acid linker containing a disulfide bond; Step S2.2: Preparation of cholesterol activation intermediate: dissolve the dicarboxylic acid linker obtained in step S2.1 in anhydrous dichloromethane, add cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, and control the molar ratio of the dicarboxylic acid linker to cholesterol to be 1: (0.8-1.2), reacted at room temperature in the dark with stirring for 12-24 h. The reaction solution was purified by silica gel column chromatography, using a mixture of dichloromethane and methanol in a volume ratio of 50:1-20:1 as the eluent. The fraction containing one free carboxyl group was collected, and the solvent was removed by rotary evaporation to obtain the cholesterol-ROS-sensitive linker monoester; Step S2.3: Synthesis of the conjugate: Mono-6-amino-β-cyclodextrin was dissolved in anhydrous dimethyl sulfoxide, and the cholesterol obtained in step S2.2 was added. Cholesterol-ROS-sensitive linker monoester and condensing agent were prepared. The molar ratio of monoester to mono-6-amino-β-cyclodextrin was controlled at 1:(1.0-1.2). The reaction was stirred at 30℃-40℃ for 24h-48h. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 500Da-1000Da and dialyzed in deionized water for 48h-72h to remove unreacted small molecules and solvent. After freeze-drying, cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate was obtained.
2. A method of preparing a minoxidil-loaded hair follicle exosome complex as claimed in claim 1, characterized by, The process includes the following steps: Step S1: Separate and purify the hair follicle mesenchymal stem cell exosome suspension; Step S2: Synthesize the cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate; Step S3: Perform membrane intercalation assembly of the conjugate obtained in Step S2 and the exosome suspension obtained in Step S1 to prepare surface-modified exosomes; Step S4: Mix the minoxidil solution with the surface-modified exosomes obtained in Step S3, perform drug loading and separation and purification to obtain the hair follicle exosome complex loaded with minoxidil. Step S2 specifically includes the following sub-steps: Step S2.1: Preparation of ROS-sensitive linker: To prepare a linker containing a thioketal bond: Under nitrogen protection, 3-mercaptopropionic acid and anhydrous acetone are mixed in a molar ratio of 2:(1-2), and dry hydrogen chloride gas is introduced until saturation. The reaction is magnetically stirred at 20℃-30℃ for 4-6 hours. After the reaction, the mixture is cooled to 0℃-4℃ in an ice bath, allowed to stand for crystallization, filtered, and the filter cake is washed 3-5 times with cold hexane and dried under vacuum to obtain the desired product. Dicarboxylic acid linker containing a thioketal bond; if preparing a linker containing a disulfide bond: directly select 3,3'-dithiodipropionic acid as the dicarboxylic acid linker containing a disulfide bond; Step S2.2: Preparation of cholesterol activation intermediate: dissolve the dicarboxylic acid linker obtained in step S2.1 in anhydrous dichloromethane, add cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, and control the molar ratio of the dicarboxylic acid linker to cholesterol to be 1: (0.8-1.2), reacted at room temperature in the dark with stirring for 12-24 h. The reaction solution was purified by silica gel column chromatography, using a mixture of dichloromethane and methanol in a volume ratio of 50:1-20:1 as the eluent. The fraction containing one free carboxyl group was collected, and the solvent was removed by rotary evaporation to obtain the cholesterol-ROS-sensitive linker monoester; Step S2.3: Synthesis of the conjugate: Mono-6-amino-β-cyclodextrin was dissolved in anhydrous dimethyl sulfoxide, and the cholesterol obtained in step S2.2 was added. Cholesterol-ROS-sensitive linker monoester and condensing agent were prepared. The molar ratio of monoester to mono-6-amino-β-cyclodextrin was controlled at 1:(1.0-1.2). The reaction was stirred at 30℃-40℃ for 24h-48h. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 500Da-1000Da and dialyzed in deionized water for 48h-72h to remove unreacted small molecules and solvent. After freeze-drying, cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate was obtained.
3. The method of claim 2, wherein the minoxidil-loaded hair follicle exosome complex is prepared by the steps of: Step S1 specifically includes the following sub-steps: Step S1.1: Take hair follicle tissue, cut it into small pieces, add 0.1%-0.25% type II collagenase solution, and digest it enzymatically in a 37℃ constant temperature shaker at 60-100 rpm for 30-60 minutes. Isolate and culture hair follicle mesenchymal stem cells. When the cell confluence reaches 70%-80%, wash the cells 2-3 times with phosphate buffer, replace the exosome-free serum medium, and continue culturing for 24-48 hours. Collect the cell supernatant; Step S1.2: Incubate the collected cell supernatant at 4℃ using a differential... The supernatant was collected by centrifugation at 300g for 10 min, 2000g for 20 min, and 10000g for 30 min in sequence to remove cell debris, apoptotic bodies, and large molecular proteins. Step S1.3: The supernatant collected in step S1.2 was filtered through a polyethersulfone membrane with a pore size of 0.22 μm and then centrifuged at 4℃ for 60 min-90 min with a centrifugation force of 100000g-120000g. The supernatant was discarded and the precipitate was resuspended in pre-cooled phosphate buffer to obtain the hair follicle mesenchymal stem cell exosome suspension.
4. The method for preparing a follicular exosome complex loaded with minoxidil according to claim 2, characterized in that, In step S2.2, the molar ratio of the dicarboxyl linker, cholesterol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:(0.8-1.2):(1.5-2.0):(0.1-0.3); in step S2.3, the condensing agent is 1,3-dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
5. The method for preparing a follicular exosome complex loaded with minoxidil according to claim 2, characterized in that, Step S3 is specifically performed as follows: The cholesterol-ROS-sensitive linker-β-cyclodextrin conjugate prepared in step S2 is dissolved in ethanol or dimethyl sulfoxide to prepare a stock solution with a concentration of 5 mg / mL-10 mg / mL; the stock solution is added dropwise to the exosome suspension prepared in step S1 at a rate of 10 μL / min-50 μL / min, according to the mass ratio of exosome protein to conjugate of 1:(0.5-2.0); the mixture is placed in a constant temperature shaker at 37℃ and incubated in the dark at a speed of 60 rpm-100 rpm for 1 h-2 h to allow the cholesterol groups to insert into the exosome membrane structure; after incubation, the mixture is placed in a dialysis bag with a molecular weight cutoff of 10 kDa-14 kDa and dialyzed in phosphate buffer for 12 h-24 h, with the dialysate being changed every 4 h-6 h to remove unbound conjugates and organic solvents, to obtain surface-modified exosomes.
6. The method of claim 2, wherein the minoxidil-loaded hair follicle exosome complex is prepared by the steps of: Step S4 is specifically performed as follows: Minoxidil is dissolved in acetic acid buffer or ethanol aqueous solution to prepare a minoxidil solution with a concentration of 20 mg / mL-40 mg / mL; the surface-modified exosomes obtained in step S3 are mixed with the minoxidil solution at a volume ratio of 1:(2-4), and magnetically stirred at 200 rpm-400 rpm for 4 h-8 h at 25℃-30℃; after the reaction is completed, the mixture is centrifuged at 100000g for 60 min, the supernatant is discarded, the precipitate is resuspended in phosphate buffer, and ultrasonically dispersed at a power of 30W-50W for 1 min-2 min to obtain the hair follicle exosome complex loaded with minoxidil.
7. Use of a hair follicle exosome complex loaded with minoxidil according to claim 1 for the manufacture of a medicament for the treatment of androgenetic alopecia or alopecia areata, characterized in that, The complex undergoes responsive cleavage in the reactive oxygen species microenvironment of the lesion to release minoxidil.