Exosome compound containing NMN-ergothioneine as well as preparation method and application of exosome compound

Abstract

The invention discloses an exosome compound containing NMN-ergothioneine as well as a preparation method and application thereof, and relates to the technical field of exosome preparations, the exosome compound comprises an exosome matrix and an amphiphilic active intermediate loaded on the exosome matrix; the amphiphilic active intermediate is formed by assembling or physically embedding a lipophilic carrier material and an active composition through an intermolecular force; the active composition comprises nicotinamide mononucleotide and ergothioneine; the amphiphilic active intermediate is embedded into a lipid bilayer of the exosome matrix or entrapped in an inner cavity of the exosome matrix to form a compound with a core-shell structure or a membrane fusion structure. By constructing a unique membrane embedding structure, the entrapment efficiency and stability of hydrophilic components are improved, medicine leakage is effectively prevented, meanwhile, the biological activity of exosomes is reserved, the cellular uptake ability and intracellular delivery efficiency are enhanced, and effective repair and lasting anti-oxidation effects on mitochondrial functions are achieved.

Description

Technical Field

[0001] This invention relates to the field of exosome formulation technology, specifically to an exosome complex containing NMN-ergothioneine, its preparation method, and its application. Background Technology

[0002] Nicotinamide mononucleotide and ergothioneine are key bioactive substances in organisms, widely involved in the regulation of cellular energy metabolism and antioxidant processes. Exosomes, as nanoscale vesicles secreted by cells, contain abundant bioactive components and possess excellent biocompatibility and intercellular communication functions, often considered ideal natural drug delivery carriers. In the current research and development of anti-aging cosmetics and tissue repair drugs, utilizing exosomes to carry various active factors to achieve deep penetration and cellular function regulation has become an important research and application direction in the industry.

[0003] To load hydrophilic small-molecule active substances into exosomes, existing technologies typically employ physical mixing or mechanical methods. However, in practical applications, due to the extremely high water solubility of nicotinamide mononucleotide and ergothioneine, these methods struggle to stably confine them within the lipid bilayer structure or lumen of exosomes, easily leading to rapid leakage of active ingredients and low encapsulation efficiency. Furthermore, conventional loading processes often damage the membrane structure of exosomes, resulting in insufficient stability during blood circulation or skin application. These substances may degrade or be released before reaching target cells, resulting in a low effective concentration that ultimately enters the cell and exerts its effects, particularly in the mitochondria, thus failing to achieve the desired repair and anti-aging effects. Summary of the Invention

[0004] The purpose of this invention is to provide an exosome complex containing NMN-ergothionein, its preparation method and application, thereby solving the problems existing in the background art.

[0005] To address the aforementioned technical problems, this invention provides an exosome complex containing NMN-ergothioneine, comprising an exosome matrix and an amphiphilic active intermediate loaded on the exosome matrix; Amphiphilic active intermediates are formed by the assembly or physical encapsulation of lipophilic carrier materials and active compositions through intermolecular forces; The active composition includes nicotinamide mononucleotide and ergothioneine; Amphiphilic active intermediates are embedded in the lipid bilayer of the exosome matrix or encapsulated in the lumen of the exosome matrix, forming complexes with core-shell or membrane fusion structures.

[0006] A method for preparing an exosome complex containing NMN-ergothioneine is also provided, comprising the following steps: Step S1: Cell culture and multi-stage centrifugation purification to prepare high-purity exosome suspension; Step S2: Prepare an amphiphilic active intermediate loaded with nicotinamide mononucleotide and ergothionein by thin-film hydration or solvent evaporation; the amphiphilic active intermediate is formed by assembling or physically embedding a lipophilic carrier material and an active composition through intermolecular forces. Step S3: Mix the exosome suspension with the amphiphilic active intermediate at a mass ratio, and process them through a membrane fusion induction process to obtain an exosome complex containing NMN-ergothionein.

[0007] Preferably, step S1 specifically includes the following sub-steps: Step S1.1: Select mesenchymal stem cells for adherent culture. When the cell confluence reaches 70%-90%, replace with serum-free culture medium and continue culture. Collect cell supernatant. Step S1.2: Differential centrifugation pretreatment: The collected cell supernatant is subjected to multi-step differential centrifugation at a low temperature of 4°C to remove dead cells, cell debris and large molecular proteins, and the clear supernatant is collected. Step S1.3: Ultrafiltration concentration: Place the clarified supernatant in a tangential flow ultrafiltration device or centrifugal ultrafiltration tube with a molecular weight cutoff of 10kDa-100kDa, and concentrate it at 4°C until the volume is concentrated to 1 / 10-1 / 5 of the original volume to obtain the concentrate. Step S1.4: Ultracentrifugation: Centrifuge the concentrate at 4°C, discard the supernatant, and resuspend the precipitate in phosphate buffer to obtain the exosome suspension.

[0008] Preferably, the differential centrifugation operation in step S1.2 is specifically as follows: Centrifuge at 300g-500g for 5min-15min and collect the supernatant; Centrifuge at 2000g-3000g for 15-30 minutes and collect the supernatant; Finally, centrifuge at 10000g-12000g for 30min-60min and collect the supernatant.

[0009] Preferably, the ultra-high speed centrifugation conditions in step S1.4 are: centrifugal force 100000g-120000g, centrifugation time 60min-120min.

[0010] Preferably, step S2 specifically includes the following sub-steps: Step S2.1: Weigh out phospholipid compounds and cholesterol as lipophilic carrier materials, dissolve them in an organic solvent, and obtain a clear lipid organic solution; Step S2.2: Dissolve nicotinamide mononucleotide and ergothionein in a polar solvent that is miscible with organic solvents to obtain an active ingredient solution; Step S2.3: Mix the lipid organic solution and the active ingredient solution in a predetermined ratio, and carry out a constant temperature stirring reaction at 25℃-45℃ and 300rpm-800rpm for 2h-6h under inert gas protection to obtain a mixed reaction solution. Step S2.4: Place the mixed reaction solution in a rotary evaporator and perform rotary evaporation at 35℃-50℃ and under reduced pressure to remove all solvents and form a uniform drug-containing lipid film on the inner wall of the container. Step S2.5: Add an aqueous buffer solution to the container, hydrate the drug-containing lipid film under water bath conditions, and then disperse it by ultrasonication or high-pressure homogenization to obtain a dispersion of amphiphilic active intermediate with uniform particle size.

[0011] Preferably, in step S2.1, the phospholipid compound is selected from one or more of dipalmitoylphosphatidylcholine or soybean lecithin; the organic solvent is one or more of chloroform, dichloromethane or ethanol; In step S2.2, the polar solvent is methanol; In step S2.3, the conditions for the constant temperature stirring reaction are: temperature 25℃-45℃, rotation speed 300rpm-800rpm, and reaction time 2h-6h.

[0012] Preferably, in step S2.5, the conditions for ultrasonic dispersion of the probe are: power 100W-300W, working time 3s-5s, interval time 3s-5s, ice bath cooling throughout, and total processing time 5min-15min.

[0013] Preferably, step S3 specifically involves the following steps: The exosome suspension prepared in step S1 and the amphiphilic active intermediate dispersion prepared in step S2 were mixed evenly at a protein to lipid mass ratio of 1:5 to 1:20. The mixture is placed in a polycarbonate membrane extruder and, under constant temperature conditions of 37℃-45℃, the mixture is reciprocated through a polycarbonate filter membrane with a pore size of 100nm-200nm 10-30 times using high-pressure gas to induce the fusion or recombination of the amphiphilic active intermediate with the exosome membrane. The extruded mixture was then incubated at 4°C for 2-6 hours to obtain the complex.

[0014] It also provides the application of an exosome complex containing NMN-ergothionein in anti-skin aging cosmetics or drugs for repairing mitochondrial function.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Through specific intermolecular assembly and membrane fusion processes, high-density loading of hydrophilic active ingredients in hydrophobic membrane structures has been successfully achieved. This effectively overcomes the defect of easy leakage of water-soluble small molecules in lipid carriers, improves the encapsulation level of active ingredients, and exhibits excellent structural stability during storage and transportation. It can lock in the internal active substances for a long time, ensuring that the formulation can maintain a high effective drug loading concentration during use, thereby ensuring the consistency and reliability of product quality.

[0016] It fully preserves the key bioactive proteins on the surface of the carrier matrix, maintaining natural cell homing ability and biocompatibility. With the help of optimized membrane fluidity and flexibility design, it can pass through the dense intercellular matrix more efficiently and be recognized and taken up by target cells, increasing the flux of active substances into the cell. This allows more effective ingredients to break through the cell membrane barrier and accurately accumulate in the cell to exert their biological functions, reducing the ineffective loss of active substances in non-target areas.

[0017] Once inside the cell, it enables the continuous release of active ingredients, prolonging the intracellular window of action. By improving the biodistribution of active substances within the cell, it can more effectively act on core organelles such as mitochondria, enhancing the efficiency of scavenging reactive oxygen species and repairing damaged cell functions. As a result, the complex exhibits good biological effects in improving skin condition, delaying the aging process, and promoting tissue repair, and has broad application prospects. Detailed Implementation

[0018] Example 1 This embodiment provides an exosome complex containing NMN-ergothioneine, wherein the exosome matrix is ​​derived from human umbilical cord mesenchymal stem cells, and the amphiphilic active intermediate is constructed through a specific lipid self-assembly technology. By utilizing the natural affinity of the exosome matrix and the lipid anchoring effect of the amphiphilic active intermediate, the aim is to effectively improve the penetration efficiency of NMN and ergothioneine in the stratum corneum of the skin. Specifically, in step S1, human umbilical cord mesenchymal stem cells are selected for adherent culture. When the cell confluence reaches 70%, the serum-free culture medium is replaced. In the differential centrifugation pretreatment in step S1.2, the collected cell supernatant is first centrifuged at 300g for 5 min and the supernatant is collected; then centrifuged at 2000g for 15 min and the supernatant is collected; finally, centrifuged at 10000g for 30 min and the supernatant is collected. Subsequently, in step S1.3, the clarified supernatant is concentrated to 1 / 10 of its original volume using a tangential flow ultrafiltration device with a molecular weight cutoff of 10 kDa. Then, in step S1.4, the concentrate is centrifuged at 100000g for 60 min to obtain a high-purity exosome suspension. This stage uses gradient differential centrifugation combined with gentle ultra-high-speed centrifugation and a specific molecular weight cutoff to maximize the preservation of the bioactive structure of surface marker proteins such as CD9 and CD63 in the exosome matrix and reduce physical damage caused by mechanical shearing. In step S2, during the preparation of the amphiphilic active intermediate, step S2.1 involves weighing dipalmitoylphosphatidylcholine and cholesterol at a mass ratio of 4:1 as lipophilic carrier materials and dissolving them in chloroform to obtain a clear lipid organic solution; this ratio helps to form a lipid membrane with suitable rigidity. Step S2.2 involves adding nicotinamide mononucleotide (NMN) and ergothioneine at a mass ratio of 1:1 to a polar solvent, methanol, and sonicating at 40 kHz for 10-15 min until the solution is clear and transparent, obtaining the active ingredient solution; here, the ultrasonic cavitation effect is utilized to promote the dispersion and dissolution of highly polar active molecules in the organic phase. Step S2.3 involves mixing the lipid organic solution and the active ingredient solution at a volume ratio of 2:1 and incubating at 25°C. The reaction was carried out under constant temperature stirring at 300 rpm for 2 hours. This step utilizes the miscibility of polar and organic solvents to promote electrostatic adsorption or hydrogen bonding between the hydrophilic active ingredient and the phospholipid head group at the molecular level, forming a stable intermolecular assembly. In step S2.4, the solvent was removed by rotary evaporation at 35°C and reduced pressure to form a drug-containing lipid film. In step S2.5, an aqueous buffer solution was added, and the mixture was ultrasonically dispersed using a probe at 100W power, with a 3-second working time followed by a 3-second interval, for a total time of 5 minutes, to obtain a uniform amphiphilic active intermediate dispersion. This step, by strictly controlling the low temperature and ultrasonic power, prevented the thermal degradation of NMN and ergothioneine, ensuring the chemical stability of the active composition. Finally, in step S3, after detecting the concentration of the exosome suspension using a BCA protein concentration assay kit, the exosome suspension and the amphiphilic active intermediate dispersion were mixed at a protein to lipid mass ratio of 1:5. The mixture was placed in a polycarbonate membrane extruder and, under constant temperature of 37°C, the mixture was reciprocated through a polycarbonate filter membrane with a pore size of 100 nm 10 times using high-pressure gas. Subsequently, it was incubated at 4°C for 2 hours to obtain the exosome complex containing NMN-ergothioneine. Under these conditions, through the synergistic effect of the instantaneous shear force generated by mechanical extrusion and the lipid phase transition temperature, the amphiphilic active intermediate was embedded in the lipid bilayer of the exosome matrix, forming a complex with a core-shell structure or a membrane fusion structure, thus achieving in-situ loading of the active ingredient.

[0019] Example 2 An exosome complex containing NMN-ergothionein and its preparation method are described in Example 1. This embodiment provides a method for preparing an exosome complex containing NMN-ergothioneine, wherein the process parameters are selected within a limited range to balance the preparation efficiency and loading. In step S1, the supernatant is collected when the mesenchymal stem cell confluence reaches 80%. The differential centrifugation in step S1.2 is as follows: first centrifuge at 400g for 10 min, then at 2500g for 20 min, and finally at 11000g for 45 min. In step S1.3, a centrifugal ultrafiltration tube with a molecular weight cutoff of 50 kDa is used to concentrate the solution to 1 / 8 of its original volume. The ultracentrifugation conditions in step S1.4 are set as follows: centrifugal force 110000g, centrifugation time 90 min. These parameters help to remove more small molecule proteins and improve the purity of the exosome matrix while ensuring exosome recovery rate. In step S2, step S2.1 involves dissolving soybean lecithin and cholesterol in dichloromethane at a mass ratio of 3:1. The lower cholesterol ratio provides the intermediate with better flexibility. In step S2.2, NMN and ergothioneine are added to methanol at a mass ratio of 2:1 and dissolved using ultrasound at 40kHz for 15 minutes until the solution is completely clear, yielding the active ingredient solution. In step S2.3, the lipid organic solution and the active ingredient solution are mixed at a volume ratio of 3:1, and the constant temperature stirring reaction conditions are adjusted to: temperature 35℃, rotation speed 500rpm, and reaction time 4h, to ensure sufficient contact between the active ingredient and the lipophilic carrier material and complete assembly. In step S2.4, rotary evaporation is performed at 42℃. In step S2.5, the probe ultrasonic dispersion conditions are: power 200W, 4s working, 4s intermittent, and a total processing time of 10min. The moderately increased energy input helps overcome the van der Waals forces of lipid molecules, forming smaller-particle-size amphiphilic active intermediates, which is beneficial for subsequent fusion. In step S3, the exosome suspension and the amphiphilic active intermediate dispersion are mixed at a protein to lipid mass ratio of 1:12. Under constant temperature of 41°C, the mixture is extruded 20 times through a polycarbonate filter membrane with a pore size of 150 nm, followed by incubation at 4°C for 4 hours. Under this process, the amphiphilic active intermediate is not only embedded in the lipid bilayer of the exosome matrix, but also partially encapsulated in the lumen of the exosome matrix. The resulting complex exhibits good structural stability and prolonged blood circulation time in the application of drugs for repairing mitochondrial function, which helps the active composition to be continuously released into target cells.

[0020] Example 3 An exosome complex containing NMN-ergothionein and its preparation method are described in Example 1. This embodiment provides a method for preparing an exosome complex containing NMN-ergothioneine, wherein the process parameters are selected at high values ​​within a limited range to maximize loading efficiency; In step S1, the cells were processed when the confluence reached 90%; in step S1.2, differential centrifugation was performed: first at 500g for 15 min, then at 3000g for 30 min, and finally at 12000g for 60 min; in step S1.3, the cells were concentrated to 1 / 5 of their original volume using a device with a molecular weight cutoff of 100kDa; in step S1.4, the ultracentrifugation conditions were 120000g for 120 min; this high-intensity centrifugation ensured a high recovery rate of the exosome matrix. In step S2, step S2.1 involves dissolving dipalmitoylphosphatidylcholine and cholesterol in ethanol at a mass ratio of 6:1. The high proportion of phospholipids makes the membrane structure more compact, which is beneficial to improving the encapsulation efficiency. In step S2.2, NMN and ergothioneine are added to methanol at a mass ratio of 1:2. To ensure complete dissolution of the high-concentration solute, the mixture is ultrasonically treated for 20 minutes at a frequency of 40 kHz and a water bath at 35°C to obtain the active ingredient solution. In step S2.3, the lipid organic solution and the active ingredient solution are mixed at a volume ratio of 1:1. The reaction conditions are: temperature 45°C, rotation speed 800 rpm, and reaction time 6 hours. The higher reaction temperature and rotation speed are beneficial to overcoming the intermolecular barrier and improving the assembly yield of the amphiphilic active intermediate. In step S2.4, rotary evaporation is performed at 50°C. In step S2.5, ultrasonic power is 300W, with a 5-second working time followed by a 5-second interval, for a total time of 15 minutes. In step S3, the exosome suspension and the amphiphilic active intermediate dispersion are mixed at a protein to lipid mass ratio of 1:20; under constant temperature of 45°C, the mixture is extruded 30 times through a filter membrane with a pore size of 200 nm, followed by incubation for 6 hours; the high proportion of lipid input and high temperature extrusion process induce a violent fusion or recombination between the amphiphilic active intermediate and the exosome membrane, resulting in a high drug loading capacity of the complex; this complex, as a carrier of high-concentration active ingredients, is suitable for aging skin care scenarios that urgently need to replenish NAD+ and clear ROS.

[0021] Example 4 An exosome complex containing NMN-ergothionein and its preparation method are described in Example 1. This embodiment adjusts the parameter combination within a limited range, focusing on examining the effects of different lipid materials and solvents; Step S1 is the same as in Example 2; in step S2, step S2.1 uses dipalmitoylphosphatidylcholine and soybean lecithin in a mass ratio of 1:1, and adds cholesterol, with a total phospholipid to cholesterol mass ratio of 5:1, as a lipophilic carrier material, dissolved in a mixed solvent of chloroform and ethanol in a volume ratio of 2:1; in step S2.2, NMN and ergothioneine are added to methanol in a mass ratio of 1:1, and ultrasonically treated at a frequency of 40kHz for 10min to obtain an active ingredient solution; in step S2.3, the lipid organic solution is mixed with the active ingredient solution, and the reaction temperature is controlled at 30℃, the rotation speed at 400rpm, and the time is 3h; in step S2.5, the ultrasonic power is 150W, and the total time is 8min; in step S3, the mixing ratio is 1:8, the extrusion temperature is 39℃, and it is extruded 15 times through a 100nm filter membrane, and incubated for 3h; In this embodiment, the compounded lipophilic carrier material endows the amphiphilic active intermediate with fluidity closer to the cell membrane, making it easier for the amphiphilic active intermediate to embed into the lipid bilayer of the exosome matrix; the resulting complex maintains structural integrity while improving the flexibility of the complex, which is beneficial for it to pass through the dense intercellular matrix.

[0022] Example 5 An exosome complex containing NMN-ergothionein and its preparation method are described in Example 1. This embodiment adjusts the parameter combination within a limited range, focusing on examining the fusion effect at low mixing ratios; Step S1 is the same as in Example 2; in step S2, the preparation parameters of the amphiphilic active intermediate are consistent with those in Example 2; in step S3, the exosome suspension and the amphiphilic active intermediate dispersion are mixed at a protein to lipid mass ratio of 1:6; the extrusion operation is carried out at 43°C, and the mixture is extruded 25 times through a 150nm filter membrane and incubated for 5 hours. Despite the low lipid content, the effect of the membrane fusion induction process was enhanced by increasing the extrusion temperature and number of extrusions, ensuring that the limited number of amphiphilic active intermediates could be efficiently loaded onto the exosome matrix. The complex prepared in this example has a high exosome protein abundance and retains more of the biological signal transduction function of the exosome itself, making it suitable as a carrier for repair drugs with high bioactivity requirements.

[0023] Comparative Example 1 This comparative example uses a physical mixing method and does not prepare amphiphilic active intermediates. The specific operation is as follows: exosome suspension is prepared according to the method of Example 2; nicotinamide mononucleotide and ergothioneine are directly dissolved in PBS buffer; the exosome suspension is directly mixed with the drug solution, the amount of drug is the same as in Example 2, and incubated at 37°C for 4 hours without lipid film preparation or extrusion fusion. In this method, the active composition does not bind to the lipophilic carrier material and does not form amphiphilic active intermediates, which aims to verify the necessity of constructing amphiphilic intermediates for loading efficiency.

[0024] Comparative Example 2 This comparative example prepared liposome drug-loaded particles without exosome matrix. Specifically, the amphiphilic active intermediate dispersion, i.e., conventional liposomes, was prepared according to step S2 of Example 2. The mixing and extrusion operations with exosomes in steps S1 and S3 were not performed, and the dispersion was directly used as the final product. This complex lacks the exosome matrix as a bioactive carrier, which aims to verify the key role of the exosome matrix in cellular uptake and targeted delivery.

[0025] Comparative Example 3 This comparative example uses electroporation for drug loading, rather than membrane fusion extrusion. Specifically, an exosome suspension was prepared according to Example 2; NMN and ergothioneine were directly added to the exosome suspension, and an electroporator was used at 300V and 150μF for electroporation, followed by recovery culture. This method did not construct amphiphilic active intermediates but instead attempted to forcibly introduce hydrophilic drugs into exosomes through physical perforation, aiming to verify the advantages of membrane fusion induction technology over traditional electroporation in maintaining membrane integrity and encapsulation efficiency.

[0026] Verification test To verify the performance of the exosome complex containing NMN-ergothionein of the present invention, the physicochemical properties and biological effects of the samples prepared in Examples 1-5 and Comparative Examples 1-3 were characterized.

[0027] Test Standards Average particle size (nm) and PDI: The dynamic light scattering particle size analyzer (DLS, model Malvern Zetasizer NanoZS) was used to measure the particle size at 25°C. The test was repeated 3 times and the average value was taken. Encapsulation efficiency %: Free drug was separated by ultrafiltration centrifuge tubes with a molecular weight cutoff of 3 kDa. The contents of NMN and ergothioneine in the filtrate were determined by high performance liquid chromatography (HPLC), and the encapsulation efficiency was calculated. Cell uptake percentage: Using DiO / DiI fluorescent labeling, the proportion of positive cells was detected by flow cytometry after the sample was co-incubated with human skin fibroblast HDF for 4 h. Mitochondrial ROS clearance rate %: An H2O2-induced oxidative damage model was established, and DCFH-DA fluorescent probes were used for labeling. The fluorescence intensity was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the ROS clearance rate was calculated.

[0028] Specific testing process All samples were stored at 4°C before testing, and physicochemical indicators were tested within 24 hours after preparation. In cell experiments, HDF cells were seeded in 96-well plates at a density of 5×10^3 cells / well. After the cells adhered, drug-containing culture medium was added. Each group was set up with 3 replicates, and the experiment was independently repeated 3 times.

[0029] Data table Table 1

[0030] Results Analysis Composition The data in Table 1 and the comparative analysis show that: The complexes prepared in Examples 1-5 all showed stable encapsulation rates of NMN and ergothioneine exceeding 79%, significantly higher than Comparative Example 1 (physical mixing, less than 5%) and Comparative Example 3 (electroporation, approximately 25%). This indicates that constructing amphiphilic active intermediates and utilizing membrane fusion-induced processes can effectively solve the problem of the difficulty in efficiently loading hydrophilic small molecules onto exosome matrices. In particular, Example 3, under high lipid ratio and high-temperature extrusion conditions, achieved the highest encapsulation rate, greater than 91%, confirming that the lipid anchoring strategy can effectively prevent drug leakage. Regarding cellular uptake, the values ​​in the Example group were 88%-93%, which were significantly better than those in Comparative Example 2, i.e., the simple liposomes, which were 55.6%. This indicates that retaining the exosome matrix is ​​crucial for achieving targeted delivery and cell affinity. The amphiphilic active intermediate embedded in the lipid bilayer of the exosome matrix did not disrupt the homing proteins on the exosome surface. On the contrary, Example 4 optimized membrane fluidity by compounding lipids and showed the highest cellular uptake, which was 93.5%. This suggests that the construction of a biomimetic membrane structure helps the complex to overcome the cell membrane barrier. Regarding mitochondrial ROS clearance, the Example Groups exhibited a significant synergistic effect; Comparative Example 1 showed the worst clearance effect because the drug was mainly free outside the cell and could not enter the mitochondrial target site; Comparative Example 2, although with a high drug loading, had limited intracellular antioxidant effect due to the lack of exosome matrix to assist in cell entry; In contrast, the Example Groups combined high drug loading with high uptake, with the amphiphilic active intermediate precisely delivering the active composition into the cell, effectively repairing damaged mitochondrial function; Although Example 3 had a larger particle size, it achieved the best ROS clearance effect of 83.4% due to its extremely high drug loading, verifying the potential of this complex as a highly efficient anti-aging carrier.

[0031] 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 method of preparing an NMN- ergothioneine containing exosome complex, characterized by, Comprising the following steps: Step S1: cell culture and multi-stage centrifugal purification to prepare high-purity exosome suspension; Step S2: preparing the amphiphilic active intermediate loaded with nicotinamide mononucleotide and ergothioneine by thin film hydration method or solvent evaporation method; the amphiphilic active intermediate is formed by assembling or physically embedding the lipophilic carrier material and the active composition through intermolecular forces; Step S3: mixing the exosome suspension and the amphiphilic active intermediate according to the mass ratio, and processing by membrane fusion induction process to obtain the exosome complex containing NMN-ergothioneine; The specific operation of step S3 is: The exosome suspension prepared in step S1 is mixed with the dispersion liquid of the amphiphilic active intermediate prepared in step S2 according to the mass ratio of protein to lipid 1:5 to 1:20; The mixed solution is placed in a polycarbonate membrane extruder, and under the condition of constant temperature at 37-45℃, the mixed solution is reciprocally extruded through a polycarbonate filter membrane with a pore size of 100-200nm by using high-pressure gas driving 10-30 times, to induce the fusion or recombination of the amphiphilic active intermediate and the exosome membrane; Then the extruded mixed solution is incubated at 4℃ for 2-6h to obtain the complex.

2. The method of claim 1, wherein the NMN-ergothioneine containing exosome complex is prepared by, Step S1 specifically comprises the following substeps: Step S1.1: selecting mesenchymal stem cells for adherent culture, and when the cell confluence reaches 70-90%, replacing the serum-free medium for continuous culture, and collecting the cell supernatant; Step S1.2: differential centrifugation pretreatment: the collected cell supernatant is subjected to multi-step differential centrifugation under low temperature conditions at 4℃ to remove dead cells, cell debris and macromolecular impurities, and the clear supernatant is collected; Step S1.3: ultrafiltration concentration: the clear supernatant is placed in a tangential flow ultrafiltration device or a centrifugal ultrafiltration tube with a molecular weight cutoff of 10-100kDa, and filtered and concentrated under the condition of 4℃, until the volume is concentrated to 1 / 10-1 / 5 of the original volume, to obtain a concentrated solution; Step S1.4: ultracentrifugation: the concentrated solution is subjected to ultracentrifugation under the condition of 4℃, the supernatant is discarded, and the precipitate is resuspended with phosphate buffer to obtain the exosome suspension.

3. The method of claim 2, wherein the NMN-ergothioneine containing exosome complex is prepared by, The differential centrifugation operation of step S1.2 is specifically as follows: Firstly, centrifuge at 300-500g for 5-15min, and collect the supernatant; Then centrifuge at 2000-3000g for 15-30min, and collect the supernatant; Finally, centrifuge at 10000-12000g for 30-60min, and collect the supernatant.

4. The method of claim 2, wherein the NMN-ergothioneine-containing exosome complex is prepared by, The ultracentrifugation condition of step S1.4 is: centrifugal force 100000-120000g, centrifugation time 60-120min.

5. The method of claim 1, wherein the NMN-ergothioneine containing exosome complex is prepared by, Step S2 specifically comprises the following substeps: Step S2.1: weigh the phospholipid compound and cholesterol as the lipophilic carrier material, dissolve in the organic solvent to obtain a clear lipid organic solution; Step S2.2: dissolve nicotinamide mononucleotide and ergothioneine in a polar solvent that can be miscible with the organic solvent to obtain an active ingredient solution; Step S2.3: The lipid organic solution and the active ingredient solution are mixed in a predetermined ratio, and a constant temperature stirring reaction is carried out under inert gas protection, with a temperature control of 25-45°C and a rotation speed of 300-800 rpm, for 2-6 hours, to obtain a mixed reaction liquid; Step S2.4: The mixed reaction liquid is placed in a rotary evaporator and rotary evaporation is carried out at 35-50°C under reduced pressure, to remove all solvents and form a uniform drug-containing lipid film on the inner wall of the container; Step S2.5: The water phase buffer is added to the container, and the drug-containing lipid film is hydrated under water bath conditions, and a uniform particle size of the amphiphilic active intermediate dispersion liquid is obtained by probe ultrasonic dispersion or high pressure homogenization treatment.

6. The preparation method of the NMN-nerve growth factor-containing exosome complex according to claim 5, characterized in that, In step S2.1, the phospholipid compound is selected from one or more of dipalmitoyl phosphatidylcholine or soybean lecithin; and the organic solvent is one or more of chloroform, dichloromethane or ethanol; In step S2.2, the polar solvent is methanol; In step S2.3, the constant temperature stirring reaction conditions are: temperature 25-45°C, rotation speed 300-800 rpm, and reaction time 2-6 hours.

7. The method of claim 5, wherein the NMN-ergothioneine containing exosome complex is prepared by, In step S2.5, the probe ultrasonic dispersion conditions are: power 100-300 W, working time 3-5 s, intermittent time 3-5 s, ice bath cooling throughout the process, and total processing time 5-15 min.

8. An NMN-ergothioneine-containing exosome complex produced by the method of any one of claims 1-7, wherein, The exosome matrix and the amphiphilic active intermediate loaded on the exosome matrix; The amphiphilic active intermediate is formed by assembling or physically embedding the lipophilic carrier material and the active composition through intermolecular forces; the lipophilic carrier material includes phospholipid compounds and cholesterol; The active composition includes nicotinamide mononucleotide and ergothioneine; The amphiphilic active intermediate is embedded in the lipid bilayer of the exosome matrix or loaded in the inner cavity of the exosome matrix, forming a complex with a core-shell structure or a membrane fusion structure.

9. Use of the NMN-ergothioneine containing exosome complex according to claim 8, characterized in that, Specifically, the application is used in anti-skin aging cosmetics or drugs for repairing mitochondrial function. Specifically, the application is used in anti-skin aging cosmetics or drugs for repairing mitochondrial function.

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