Human mesenchymal stem cell exosome preparation loaded with astaxanthin and application thereof

Abstract

The invention discloses a human mesenchymal stem cell exosome preparation loaded with astaxanthin and application of the human mesenchymal stem cell exosome preparation, and relates to the technical field of biological preparations. The preparation is prepared by performing in-vitro amplification on human mesenchymal stem cells, extracting secreted exosomes, then mixing the exosomes with an RVG modification enhancing preparation and loading an astaxanthin medicine. The human mesenchymal stem cell exosome preparation loaded with astaxanthin is obtained, and the therapeutic effect and safety of the human mesenchymal stem cell exosome preparation in cell models and animal experiments are studied. The invention provides a new thought and method for clinical treatment of Parkinson's disease, and has certain innovativeness and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to a human mesenchymal stem cell exosome preparation loaded with astaxanthin and an application thereof. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease, whose main characteristics include movement disorders, tremors, rigidity and bradykinesia. The current treatment methods are mainly drug therapy and surgical treatment, but these methods can only relieve symptoms and cannot cure the disease. Astaxanthin, as a natural antioxidant, plays a neuroprotective role in Parkinson's disease, but its stability and bioavailability in the body are low, which greatly limits its application. In recent years, stem cell therapy has attracted widespread attention due to its potential regenerative ability. Among them, human mesenchymal stem cells have become a hot topic of research due to their good proliferation ability and multidirectional differentiation potential. However, the tumorigenicity, thrombotic potential and ethical issues of stem cells limit their application. Exosomes, as an important medium for intercellular communication, have good biocompatibility and targeting. Studies have shown that human mesenchymal stem cell exosomes can carry a variety of bioactive molecules, such as proteins and RNA, and participate in intercellular signaling. However, the separation and extraction efficiency of exosomes is low, the therapeutic effect in Parkinson's disease has not met expectations, and the brain targeting ability is poor.

[0003] Recently, exosomes have been found to be a new type of natural drug carrier that increases the bioavailability and stability of drugs. Given the unique drug-carrying function of exosomes, studies have been conducted on using exosomes to load drugs with poor stability and low bioavailability, including the astaxanthin loaded in this patent. In the past, researchers used milk exosomes to load astaxanthin, but this study was not aimed at neurological diseases, so the brain targeting was not accurate; and the efficacy of milk-derived exosomes in Parkinson's disease is not as accurate as that of stem cell-derived exosomes. In addition, the output efficiency and encapsulation rate of drug-loaded exosomes are usually less than 30%, which limits their application. Summary of the invention

[0004] The key technical problem that the present invention intends to solve is how to effectively improve the stability and bioavailability of astaxanthin and enhance its brain targeting in the treatment of Parkinson's disease. To this end, the present invention provides a human mesenchymal stem cell exosome preparation loaded with astaxanthin and its application to solve the problems raised in the prior art.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing a human mesenchymal stem cell exosome preparation loaded with astaxanthin, comprising the following preparation steps:

[0007] Step 1: Select human mesenchymal stem cells, expand them in vitro, and culture them at 37°C and 5% CO2. When the cells reach 80%-90% fusion, subculture them and extract the secreted exosomes by ultracentrifugation;

[0008] Step 2: The exosomes extracted in step 1 were mixed with the targeting modifier, incubated overnight at 34-37°C, and then astaxanthin was added and treated with pulsed ultrasound, followed by incubation at 34-37°C for 1 hour to obtain RVG-AST@exo, i.e., human mesenchymal stem cell exosome preparation loaded with astaxanthin.

[0009] Among them, the targeting modifier is DSPE-PEG2000-RVG; the mass ratio of exosomes, targeting modifier and astaxanthin is 10:10:1.

[0010] The specific steps of pulsed ultrasonic treatment are as follows:

[0011] Set the ultrasonic power to 100-120W, ultrasonicate for 15-20s, then cool down for 15-20s, repeat 3 times, take out and place in an ice bath for 2min, repeat the above ultrasonication and ice bath process 3 times in total.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] Astaxanthin has a therapeutic effect on PD, but its stability and bioavailability are low; stem cells have a significant therapeutic effect on PD, but their tumorigenicity and thrombotic properties greatly limit their application; although stem cell-derived exosomes also have a therapeutic effect on PD, the extraction yield is low, the therapeutic effect does not meet expectations, and the brain targeting is poor. Therefore, the present invention simulates the real growth environment of stem cells in vivo through 3D cell culture technology, which not only improves the yield and quality of stem cells and exosomes, but also better simulates the growth environment in vivo, thereby enhancing the therapeutic effect of stem cells and their exosomes, and increasing the encapsulation rate and efficacy of drugs; and then utilizes the modification of RVG to enhance the targeting of the preparation in the treatment of Parkinson's disease and efficiently complete the drug loading of astaxanthin, which significantly improves the therapeutic effect. In the cell model, the RVG-AST@exo preparation can effectively reduce the level of ROS in the cell, increase the proportion of functional mitochondria, increase TH and reduce the expression of α-syn. In animal experiments, the drug can effectively cross the blood-brain barrier and enrich in brain tissue. After RVG-AST@exo treatment, the behavioral and pathological changes of mice are significantly improved, and there are no obvious toxic and side effects.

[0014] The present invention efficiently and high-quality combines the therapeutic effects of astaxanthin, stem cells and their exosomes, the drug-loading function of exosomes and the brain-targeting advantage of RVG, which is expected to improve the therapeutic effects and brain targeting of astaxanthin and exosomes, and achieve effective treatment of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a transmission electron micrograph of the unengineered exosomes in Example 1;

[0016] Figure 2 This is a nanoparticle tracking analysis diagram of the unengineered exosomes in Example 1;

[0017] Figure 3 This is a WB detection image of the unengineered exosomes in Example 1;

[0018] Figure 4 This is a transmission electron microscopy image of RVG-AST@exo prepared in Example 1;

[0019] Figure 5 This is a nanoparticle tracking analysis diagram of RVG-AST@exo prepared in Example 1;

[0020] Figure 6 This is the ultraviolet absorption spectrum of RVG-AST@exo prepared in Example 1;

[0021] Figure 7 This is the HPLC curve of RVG-AST@exo prepared in Example 1;

[0022] Figure 8 This is the uptake capacity performance of RVG-AST@exo in SH-SY5Y cells in Example 1;

[0023] Fig. 9 The Cck-8 method for measuring different MPP in Example 2 + Viability of SH-SY5Y cells after treatment with drug concentrations;

[0024] Fig.10 The Cck-8 method in Example 2 was used to measure the viability of SH-SY5Y cells after treatment with different engineered exosomes concentrations;

[0025] Fig.11 , 12 The expression of TH and α-syn in each group was detected by qRT-PCR in Example 2;

[0026] Fig.13 The expression of TH and α-syn in each group was detected by western blot in Example 2;

[0027] Fig.14 For the control group and MPP in Example 2 + Group, MPP + Fluorescence detection of cells in the +AST group stained with JC-1;

[0028] Fig.15 MPP in Example 2 + +exo group, MPP + +AST@exo group, MPP + Fluorescence detection of cells in the +RVG-AST@exo group stained with JC-1;

[0029] Fig.16 For the control group and MPP in Example 2 + Group, MPP + Fluorescence detection of cells in the +AST group after ROS staining;

[0030] Fig.17 is the MPP in Example 2 + +exo group, MPP + +AST@exo group, MPP + Fluorescence detection of ROS staining in the +RVG-AST@exo group;

[0031] Fig.18 These are the in vivo images of small animals after 4h and 6h of DIR staining in Example 3;

[0032] Fig.19 This is a behavioral test diagram of mice subjected to the tremor experiment in Example 3;

[0033] Fig. 20 This is a behavioral test diagram of mice subjected to the forced swimming test in Example 3;

[0034] Fig.21 This is a graph showing the behavioral test of mice in the pole climbing experiment in Example 3;

[0035] Fig. 22 This is a behavioral test diagram of mice subjected to the hanging experiment in Example 3;

[0036] Fig.23 This is a graph showing the behavioral test of mice in the open field experiment in Example 3;

[0037] Fig.24 This is the TH immunohistochemical staining image in Example 3;

[0038] Fig.25 This is a graph showing the weight changes of mice before and after treatment in Example 4;

[0039] Fig.26 This is the drug hemolysis test detection diagram in Example 4. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1: Preparation and characterization of a human mesenchymal stem cell exosome preparation loaded with astaxanthin, the specific steps are as follows:

[0042] Step 1: Select healthy human mesenchymal stem cells, place them in Hyclone brand α-MEM medium for in vitro expansion, culture them at 37°C and 5% CO2, subculture when the cells reach 80%-90% confluence, and extract the secreted exosomes by ultracentrifugation; use ultracentrifugation to extract exosomes and characterize them;

[0043] from Figure 1 Transmission electron microscopy showed that the exosomes had a saucer-like morphology; Figure 2 Nanoparticle tracking analysis technology was used to detect the particle size of exosomes, and the tested particle size was 78nm; Figure 3 WB was used to detect the positive and negative proteins CD9, CD81, Tsg101 and Calnexin on the surface of exosomes. It can be seen that MSCs-exo express the exosome surface marker proteins CD9, CD81, TSG101, but not Calnexin. In summary, the prepared exosomes were successfully characterized and can be used for subsequent experiments.

[0044] Step 2: Mix the exosomes extracted in step 1 with the targeting modifier DSPE-PEG2000-RVG in a mass ratio of 1:1, and incubate overnight at 37°C to ensure the effective binding of the targeting modifier to the exosomes; then use ultrasound to load astaxanthin into the targeted modified exosomes; wherein, the mass ratio of exosomes to astaxanthin is 10:1, and pulse ultrasound treatment is performed, the ultrasound power is set to 120W, and the ultrasound is continued for 15s and then cooled for 15s, repeated 3 times, and then taken out and placed in an ice bath for 2min, and the above ultrasound and ice bath process is repeated 3 times. Then incubate at 37°C for 1 hour to restore the stability of the exosome membrane. After the preparation, RVG-AST@exo is obtained, and RVG-AST@exo is characterized:

[0045] in Figure 4 Transmission electron microscopy showed that the engineered exosomes had a saucer-like morphology; Figure 5 The particle size of engineered exosomes detected by nanoparticle tracking analysis technology was 86nm, which is similar to Figure 2 The exosomes in the middle were slightly larger than those in the middle, but still within the exosome size range; Figure 6The absorbance of astaxanthin was determined by ultraviolet measurement of the maximum absorption peak, which was 474 nm; Figure 7 Determine the drug standard curve for HPLC y = 1E8x-36153 (R 2 =0.9951), and the drug encapsulation efficiency was determined to be 30.8%; finally, the uptake capacity of SH-SY5Y cells was evaluated, and the exosomes were incubated with DiO (5 μM) dye in the dark for 30 min, and the free dye was removed by centrifugation to obtain labeled exosomes. After the exosomes were co-incubated with SH-SY5Y cells for 24 h, they were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, and incubated with DAPI in the dark for 10 min. Laser scanning confocal microscopy was used to observe whether MSCs-exo could be taken up by SH-SY5Y cells, such as Figure 8 As shown, it is clear that the engineered exosomes can be taken up by SH-SY5Y.

[0046] Example 2: The therapeutic effect of RVG-AST@exo prepared in Example 1 in a Parkinson's disease cell model was studied. The specific scheme is as follows:

[0047] First, SH-SY5Y cells were cultured and subcultured. When the cells grew to 70% to 80%, an appropriate amount of trypsin was added for digestion, and then the digestion was terminated with DMEM / F12 complete medium. The cell suspension was collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and fresh complete medium was added to resuspend and inoculate into a 10 cm dish.

[0048] Reuse MPP + SH-SY5Y cells were treated to construct PD cell model, and 1×10 4 SH-SY5Y cells were incubated overnight, the old medium was discarded, and then 0mM, 1mM, 2mM, 3mM, 4mM, 5mM MPP was used. + After 24 hours of treatment, different MPPs were measured by CCK-8 method. + To evaluate the viability of SH-SY5Y cells after concentration treatment, 10 μL of CCK-8 solution was added to a 96-well plate, and the absorbance of each group at 450 nm was read using an ELISA reader, and the cell activity at each concentration was plotted. The results are shown in Fig. 9 , the optimal dosing concentration of 2 mM was selected for subsequent experiments;

[0049] Then, 1×10 4SH-SY5Y cells were inoculated in a 96-well plate and incubated overnight. The old culture medium was discarded, and engineered exosomes (RVG-AST@exo) loaded with astaxanthin at concentrations of 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM were used. After incubation for 24 hours, the optimal dosing concentration of engineered exosomes with different drug contents was determined by the CCK-8 method. 10 μL of CCK-8 solution was added to each well, incubated at 37°C for 1 hour, and then the absorbance value of the sample at a wavelength of 450 nm was detected by an enzyme marker. The results are shown in Figure 2. Fig.10 , the engineered exosome preparation at 20 μM astaxanthin was selected;

[0050] Then, qRT-PCR and western blot were used to detect the control group and MPP + Group, MPP + +AST group, MPP + +exo group, MPP + +AST@exo group, MPP + The expression of TH and α-syn in SH-SY5Y cells of the +RVG-AST@exo group;

[0051] Among them, the SH-SY5Y cells in the control group were treated with PBS throughout the whole process;

[0052] MPP + SH-SY5Y cells in the group were treated with 2 mM MPP + PBS was added 24 h after drug treatment;

[0053] MPP + SH-SY5Y cells in the +AST group were exposed to 2 mM MPP + After 24 hours of drug treatment, 20 μM astaxanthin was added for 48 hours;

[0054] MPP + SH-SY5Y cells in the +exo group were incubated with 2 mM MPP + After 24 hours of drug treatment, the exosomes extracted in step 1 of Example 1 were added for 48 hours, and the amount of exosomes used was equivalent to 10 times the mass of 20 μM astaxanthin;

[0055] MPP + SH-SY5Y cells in the +AST@exo group were exposed to 2 mM MPP + After 24 hours of drug treatment, AST@exo preparation (AST loading concentration is 20 μM) was added and treated for 48 hours; wherein the AST@exo preparation was prepared by pulsed ultrasonic loading of astaxanthin by the exosomes in Example 1; the mass ratio of exosomes to astaxanthin was 10:1.

[0056] MPP + SH-SY5Y cells in the +RVG-AST@exo group were exposed to 2 mM MPP + After 24 hours of drug treatment, RVG-AST@exo preparation (AST loading concentration is 20 μM) was added and treated for 48 hours; wherein the RVG-AST@exo preparation was prepared by mixing the exosomes in Example 1 with RVG, incubated overnight at 37°C, and then loaded with astaxanthin by pulsed ultrasound; the mass ratio of RVG, exosomes and astaxanthin was 10:10:1;

[0057] The above groups are marked as con, PD, AST / ast, exo, ast@exo, RVG-ast@exo (applicable throughout the text), respectively. Fig.11 , 12 , 13, MPP + After modeling, the expression of TH mRNA and protein decreased, and increased significantly after treatment in the RVG-AST@exo group; + The mRNA and protein expressions of α-syn increased after modeling, and decreased significantly after treatment in the RVG-AST@exo group, which confirmed its therapeutic effect on the PD cell model;

[0058] Finally, cells in each group were stained with JC-1 and ROS to clarify the anti-oxidative stress effect of the drug. Fig.14 , 15 It can be seen that MPP + The number of functional mitochondria decreased after modeling, and the number of functional mitochondria in the RVG-AST@exo group increased significantly after treatment; Fig.16 , 17 It can be seen that MPP + The level of ROS in cells increased after modeling, and the level of ROS in the RVG-AST@exo group was significantly reduced after treatment.

[0059] Example 3: The therapeutic effect of RVG-AST@exo prepared in Example 1 in an animal model of Parkinson's disease was studied. The specific scheme is as follows:

[0060] First, the animal model of Parkinson's disease was prepared: SPF male C57BL / 6J mice were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China), aged 8 to 10 weeks, weighing 22 to 25 g, and were adaptively raised for 3 days. The animals were given behavioral experimental training in climbing pole test, open field test, hanging test and forced swimming test before modeling. After the training, behavioral tests were performed. Mice that passed the behavioral tests were selected for subsequent experiments. The PD group was given an intraperitoneal injection of 30 mg / kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) once a day for 8 consecutive days. No drug treatment or other intervention was given on the 9th day. Behavioral tests were performed on the 10th day to determine the modeling effect.

[0061] Subsequently, small animal in vivo imaging technology was used to determine whether the drug could pass through the mouse blood-brain barrier and be enriched in brain tissue. DiR was co-incubated with engineered exosomes for 30 minutes, and then free dye was removed by centrifugation to obtain labeled engineered exosomes. 200 μl of labeled engineered exosomes were injected into mice through the tail vein, and small animal in vivo imaging technology was used to observe whether the engineered exosome drugs could be enriched in the brain at different time points, such as Fig.18 As shown, compared with the MPTP+AST@exo group, the drug in the MPTP+RVG-AST@exo group can efficiently pass through the blood-brain barrier and be enriched in brain tissue;

[0062] Subsequently, the behavioral changes of mice in the control group, MPTP group, MPTP+AST group, MPTP+exo group, MPTP+AST@exo group, and MPTP+RVG-AST@exo group were determined through tremor test, forced swimming test, pole climbing test, hanging test, and open field test.

[0063] Among them, mice in the control group were treated with normal saline throughout the whole process;

[0064] Mice in the MPTP group were injected intraperitoneally with 30 mg / kg MPTP for 8 days and then injected with normal saline into the tail vein every other day for a total of 4 times;

[0065] The mice in the MPTP+AST group were intraperitoneally injected with 30 mg / kg MPTP for 8 days, and then injected with 10 mg / kg astaxanthin into the tail vein every other day for a total of 4 times;

[0066] Mice in the MPTP+exo group were injected intraperitoneally with 30 mg / kg MPTP for 8 days, and then received a tail vein injection of 100 mg / kg exosomes every other day for a total of 4 times;

[0067] Mice in the MPTP+AST@exo group were intraperitoneally injected with 30 mg / kg MPTP for 8 days, and then injected with 110 mg / kg AST@exo via the tail vein every other day for a total of 4 times; AST@exo contained exosomes and astaxanthin in a mass ratio of 10:1.

[0068] Mice in the MPTP+RVG-AST@exo group were injected intraperitoneally with 30 mg / kg MPTP for 8 days before being injected intravenously with 210 mg / kg RVG-AST@exo every other day for a total of 4 times; RVG-AST@exo included exosomes, RVG, and astaxanthin in a mass ratio of 10:10:1.

[0069] The specific behavioral test methods are as follows:

[0070] Tremor experiment: After the mice were injected with MPTP intraperitoneally, they were immediately observed for 3 hours. The scoring criteria were: 0 points, similar to normal mice, without any symptoms; 1 point, erect hair, arched back, intermittent fine tremors, but free movement; 2 points, frequent swallowing, frequent tremors, hind limbs spread, tail shaking, and gradually limited movement; 3 points, drooling, continuous tremors, stiff limbs, and limited movement; 4 points, death due to general paralysis;

[0071] Suspension test: Place the two front paws of the tested mouse at the midpoint of a horizontal wire 50 cm long, 30 cm high, and 5 mm in diameter, lift the mouse's tail upside down, and then release it, and record the situation of its hind paws grasping the wire upwards. Scoring criteria: 3 points, both hind paws can grasp; 2 points, one hind paw can grasp; 1 point, both hind paws cannot grasp;

[0072] Open field test: a 50cm×50cm×50cm square box was divided into 3×3 squares, and the mouse was placed in the middle square. The number of times the mouse crossed into the adjacent squares with more than three paws within 2 minutes was recorded. The scoring criteria were: 0 points, the number of crossings was >40 times; 1 point, the number of crossings was 31-40 times; 2 points, the number of crossings was 21-30 times; 3 points, the number of crossings was <21 times;

[0073] Climbing pole test: A plastic ball with a diameter of 2.5 cm is fixed to the top of an acrylic pole with a length of 50 cm and a thickness of 1.5 cm, and gauze is wrapped around it to increase friction. Then the mouse to be tested is placed on the ball, and the time required for the mouse's four feet to land on the ground is recorded. The scoring criteria are: 0 points, climbing pole time <4.01s; 1 point, climbing pole time 4.01-8.0s; 2 points, climbing pole time 8.01-12.0s; 3 points, climbing pole time >12.00s;

[0074] Forced swimming test: Place the mouse in a 40cm×16cm×25cm water tank with a water depth of 15cm and a water temperature of (27±2)℃. Record the total immobility time for 3min. The immobility time requires the mouse to float for at least 3s. The scoring criteria are: 0 points, floating for 0-30s; 1 point, floating for 31-90s; 2 points, floating for 91-150s; 3 points, floating for 151-180s.

[0075] Depend on Figure 19-23 The experimental test results showed that the behavior of mice in the disease group was impaired, while that of the MPTP+RVG-AST@exo group improved after treatment. TH immunohistochemical staining was then used to confirm the pathological changes in the mouse brain tissue. Fig.24 It can be seen that the number of DA neurons in the brain tissue of mice in the disease group decreased, while the number of DA neurons in the RVG-AST@exo group increased after treatment.

[0076] Example 4: The safety of RVG-AST@exo prepared in Example 1 in animal and cell models was studied. The specific scheme is as follows:

[0077] The mice tested in Example 3 were taken and the weight changes of mice in each group were recorded. Fig.25 It can be seen that the weight of mice did not decrease significantly after treatment. The hemolysis test of the drug was used to determine whether the drug would cause hemolysis. Fig.26 It can be seen that from left to right, the first tube is the negative control of normal saline, the last tube is the positive control of ddH2O, and the middle tube is different concentrations of drugs, and the drugs did not cause obvious hemolytic reaction.

[0078] Conclusion: The present invention effectively improves the stability of astaxanthin, the yield and encapsulation rate of stem cell exosomes, enhances the targeting of astaxanthin-loaded exosomes in the treatment of Parkinson's disease, significantly improves the therapeutic effect, and provides new ideas and methods for the clinical treatment of Parkinson's disease. It has certain innovation and application value.

[0079] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for preparing a human mesenchymal stem cell exosome preparation loaded with astaxanthin, characterized in that: The method comprises the following preparation steps: Step 1: Select human mesenchymal stem cells, expand them in vitro, and extract the secreted exosomes by ultracentrifugation; Step 2: The exosomes extracted in step 1 were mixed with the targeting modifier, incubated overnight at 34-37°C, and then astaxanthin was added and treated with pulsed ultrasound, followed by incubation at 34-37°C for 1 hour to obtain RVG-AST@exo, i.e., human mesenchymal stem cell exosome preparation loaded with astaxanthin.

2. The method for preparing a human mesenchymal stem cell exosome preparation loaded with astaxanthin according to claim 1, characterized in that: The targeting modifier is DSPE-PEG2000-RVG; the mass ratio of exosomes, targeting modifier and astaxanthin is 10:10:

1.

3. The method for preparing a human mesenchymal stem cell exosome preparation loaded with astaxanthin according to claim 1, characterized in that: During in vitro expansion, cells were cultured at 37°C and 5% CO2, and passaged when the cells reached 80%-90% confluence.

4. The method for preparing a human mesenchymal stem cell exosome preparation loaded with astaxanthin according to claim 1, characterized in that: The specific steps of pulse ultrasonic treatment include: setting the ultrasonic power to 100-120W, ultrasonicating for 15-20s and then cooling for 15-20s, repeating 3 times, taking out and placing in an ice bath for 2 minutes, and repeating the above ultrasonic and ice bath process 3 times in total.

5. A human mesenchymal stem cell exosome preparation loaded with astaxanthin, characterized in that: It is prepared by the method for preparing a human mesenchymal stem cell exosome preparation loaded with astaxanthin as described in any one of claims 1 to 4.

6. The human mesenchymal stem cell exosome preparation loaded with astaxanthin according to claim 5, characterized in that: The preparation can improve the efficacy and stability of astaxanthin.

7. Use of the human mesenchymal stem cell exosome preparation loaded with astaxanthin as claimed in claim 5 in the preparation of a drug for treating Parkinson's disease.

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