Menstrual blood stem cell exosome drug delivery composition loaded with artemisinin as well as preparation method and application of menstrual blood stem cell exosome drug delivery composition

Abstract

The invention discloses a preparation method of an artemisinin-loaded menstrual blood stem cell exosome drug delivery composition, which comprises the following steps: 1) diluting a menstrual blood stem cell exosome to 0.1-10 mg / mL to obtain an exosome solution; (2) preparing an artemisinin solution with the concentration of 0.1 to 5 mg / mL; (3) mixing the exosome solution in the step (1) and the artemisinin solution in the step (2), and co-incubating at 26-40 DEG C for 1-5 hours; 4, loading is conducted through an ultrasonic method.The menstrual blood stem cell exosome drug delivery composition loaded with artemisinin is easy to obtain and store, stable in property, free of immunogenicity, good in biocompatibility, high in bioavailability, high in targeting performance and good in treatment effect on spinal cord injuries.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to a menstrual stem cell exosome drug delivery composition loaded with artemisinin, and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) is the leading cause of premature death and long-term disability, and its morbidity and mortality rates are increasing year by year. SCI can lead to sensory and motor dysfunction below the injury level, persistent organ dysfunction and permanent neurological deficits, manifested as paralysis, pain, spasticity, incontinence and intestinal dysfunction and many other complications, which seriously reduce the quality of life of patients and greatly increase the economic burden. Due to its high morbidity, severe disability rate and high treatment costs, it greatly threatens the health of patients. At present, surgery, drugs, rehabilitation and other treatment options are still the main treatment options, but none of them can effectively reverse the recovery of damaged nerves. Nerve repair after spinal cord injury is still a world-class problem. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides an artemisinin-loaded menstrual stem cell exosome drug delivery composition, and a preparation method and application thereof. The artemisinin-loaded menstrual stem cell exosome drug delivery composition has the effect of promoting nerve regeneration and repair after spinal cord injury in vitro and in vivo.

[0004] On the one hand, the present invention provides the following technical solution: a menstrual stem cell exosome drug delivery composition loaded with artemisinin, comprising menstrual stem cell exosomes and artemisinin.

[0005] On the other hand, the present invention also provides the following technical solution: a method for preparing a blood stem cell exosome drug delivery composition loaded with artemisinin, comprising the following steps: 1) Dilute the menstrual stem cell exosomes to 0.1-10 mg / mL to obtain an exosome solution; 2) Prepare artemisinin solution with a concentration of 0.1~5 mg / mL; 3) mixing the exosome solution described in step 1) and the artemisinin solution described in step 2), and incubating them at 26-40° C. for 1-5 hours; 4) Ultrasonic loading is used.

[0006] Preferably, in step 1), the concentration of the exosome solution is 0.5-5 mg / mL.

[0007] Preferably, in step 2), the concentration of the artemisinin solution is 0.2-2 mg / mL.

[0008] Preferably, in step 3), the volume ratio of the exosome solution to the artemisinin solution is 1:(1-5).

[0009] Preferably, in step 3), the mixture is incubated at 35-38° C. for 1-2 h; Preferably, in step 4), the procedure of the ultrasonic method is as follows: (1) 10-30% amplitude, ultrasonication for 5-20 s, interval of 10-30 s, duration of 2-10 min, ice bath for 1-5 min as one cycle, and multiple cycles; (2) incubation for 0.5-2 h at a temperature of 30-40° C. to obtain a mixed solution; (3) ultrafiltration of the mixed solution to obtain exosomes loaded with artemisinin.

[0010] Preferably, the incubation is 1-2 hours at a temperature of 35-37°C.

[0011] Preferably, the ultrafiltration conditions are: 4°C, centrifugation at 4500×g for 15 min.

[0012] On the other hand, the present invention also provides the use of the menstrual stem cell exosome drug delivery composition loaded with artemisinin in the treatment of nerve repair after spinal cord injury.

[0013] In summary, the present invention has the following beneficial effects: The artemisinin-loaded menstrual stem cell exosome drug delivery system of the present invention firstly retains the dual advantages of menstrual stem cell exosomes as both a therapeutic agent and a drug delivery system; secondly, the use of self-sourced exosomes as drug carriers has good non-immunogenicity and biocompatibility, which can improve the effective utilization rate and reduce the clearance rate of the drug; at the same time, the solubility and stability of artemisinin are greatly enhanced, the circulation time in the body is prolonged, the targeting of the drug transport to the injured site is enhanced, and the bioavailability is improved; the combination of the two has a synergistic effect, producing a "1+1≥2" effect; the artemisinin-loaded menstrual stem cell exosome drug delivery system is easy to obtain and store, has stable properties, is non-immunogenic, has good biocompatibility, high bioavailability, strong targeting, and has a good therapeutic effect on spinal cord injury; at the same time, the artemisinin-loaded menstrual stem cell exosome drug delivery system can be factory-standardized and mass-produced, and as a cell derivative preparation combined with effective ingredients of traditional Chinese medicine, it has broad social and economic value and benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For the isolation and identification of exosomes and the identification of exosomes loaded with artemisinin: A: The morphology of exosomes from menstrual stem cells was observed by transmission electron microscopy, with a scale of 100 nm; B: The morphology of exosomes loaded with artemisinin was observed by transmission electron microscopy, with a scale of 100 nm; C: The diameter of the exosomes from menstrual stem cells was detected by a particle size analyzer, with the unit being nm; D: The diameter of the exosomes loaded with artemisinin was detected by a particle size analyzer, with the unit being nm; E: Western blot was used to detect the expression of corresponding markers in cells (Cell), supernatant (Supernatant), exosomes (Exo) and exosomes loaded with artemisinin (Art-Exo); F: The absorbance of the artemisinin solution was measured by an ultra-micro spectrophotometer; G: The absorbance of the exosome solution of menstrual stem cells was measured by an ultra-micro spectrophotometer; H: The absorbance of the exosome solution loaded with artemisinin was measured by an ultra-micro spectrophotometer.

[0015] Figure 2 Characterization of exosomes loaded with artemisinin: A: Drawing of artemisinin standard curve; B: Artemisinin precision test; C: Artemisinin encapsulation efficiency determination.

[0016] Figure 3 Comparison of the precision and encapsulation efficiency of the drug delivery system of menstrual stem cell exosomes and adipose stem cell exosomes loaded with artemisinin: A: Intra-day precision; B: Inter-day precision; C: Encapsulation efficiency determination.

[0017] Figure 4 Study on drug release from artemisinin-loaded menstrual stem cell exosomes: The release of artemisinin from artemisinin-loaded menstrual stem cell exosomes was studied using a dynamic dialysis method in release media at pH 5.5 and pH 7.4.

[0018] Figure 5 Comparison of drug release from menstrual stem cell exosomes and adipose stem cell exosomes loaded with artemisinin: A: Drug release of artemisinin-loaded exosomes from menstrual stem cells and adipose-derived stem cells under normal physiological conditions simulated at pH 7.4; B: Drug release of artemisinin-loaded exosomes from menstrual stem cells and adipose-derived stem cells in lysosomes simulated at pH 5.5.

[0019] Figure 6 Stability assessment of artemisinin-loaded menstrual stem cell exosomes: A: Stability evaluation of artemisinin-loaded exosomes in PBS for 7 days. The figure shows the diameter data of exosomes measured by NTA. B: Stability of artemisinin-loaded exosomes in 10% FBS for 24 hours. The figure shows the diameter data of exosomes measured by NTA.

[0020] Figure 7 Uptake of artemisinin-loaded menstrual stem cell exosomes by nerve cells: A: Uptake of exosomes from menstrual stem cells by SHSY5Y; B: Uptake of exosomes loaded with artemisinin by SHSY5Y; F: Statistical analysis of A and B showed that the uptake of drug-loaded exosomes by SHSY5Y was higher than that of exosomes from menstrual stem cells, and the difference was statistically significant.

[0021] Figure 8 For intracellular drug delivery: After overnight culture, SHSY5Y cells were incubated with common exosomes (MenSCs-Exo) and drug-loaded exosomes (Art-MenSCs-Exo) for 4 h, 8 h, and 12 h; the artemisinin concentration was measured after collection.

[0022] Fig. 9 Comparison of intracellular drug delivery of artemisinin-loaded menstrual stem cell exosomes and adipose stem cell exosomes: Fig.10 To investigate the effects of artemisinin-loaded exosomes on the biological behaviors of neuronal cells; Artemisinin-loaded exosomes and menstrual stem cell exosomes were co-cultured with SHSY5Y cells. SHSY5Y cells are similar in morphology, physiology and biochemical functions to normal nerve cells, and were selected to simulate the effects of artemisinin-loaded exosomes on nerve cells in vivo. SHSY5Y cells were co-cultured with MenSCs-Exo and Art-MenSCs-Exo, and cell morphology was observed using an inverted phase contrast microscope three days later. Figures A1 and A2: Bright field images, cells cultured with Art-MenSCs-Exo have longer axons and more branches. Cells from the MenSCs-Exo and Art-MenSCs-Exo groups were randomly selected for quantitative analysis. The Art-MenSCs-Exo group was superior to the menstrual stem cell exosome group without artemisinin in terms of axon number (Figure A3), total axon length (Figure D), branch number (Figure E), and branching frequency on a single axon (Figure F). This indicates that artemisinin-loaded exosomes have a good ability to promote nerve cell growth, which is necessary for promoting regeneration of spinal cord injury. Figures B1 and B2: Live / dead The results showed that both exosomes could support the survival of neurons, but the number of dead cells in the Art-MenSCs-Exo group was less than that in the MenSCs-Exo group; Figure B3: Live / dead test quantitative statistics.

[0023] Fig.11 Menstrual stem cell exosomes loaded with artemisinin improve local pathological conditions of SCI rats: Nissl staining showed that the number of atrophic and degenerated Nissl bodies in the Art-MenSCs-Exo group was less than that in the other injury groups, and the number of newly formed Nissl bodies was increased; HE staining showed that the cell shrinkage and fragmentation in the injured spinal cord in the Art-MenSCs-Exo group was improved, the number of inflammatory cells was reduced, and the syringomyelia phenomenon was significantly improved compared with other groups; Masson staining showed that the degree of fibrosis in the spinal cord injury area of ​​rats treated with Art-MenSCs-Exo group was lower than that in other injury groups, and the volume of the cavity was smaller.

[0024] Fig.12 To load artemisinin exosomes to improve motor function and axon regeneration after spinal cord injury in SD rats: Figure A: BBB scores were performed on SD rats at different time points after surgery. The BBB scores of rats in the Art-MenSCs-Exo group were significantly higher than those in other injury groups starting from the 7th day. Figure B: Catwalk gait analysis was performed on SD rats 28 days after surgery. The figure shows that the footprint morphology, strength, distance between the two hind paws, and relative footprint distance of rats in the Art-MenSCs-Exo group were significantly improved compared with those in the SCI group, and were better than those in the MenSCs-Exo group. This shows that Art-MenSCs-Exo treatment can improve the motor function of SCI rats. ; Figure C: Spinal cord tissue of rats was taken 28 days after surgery, frozen and sectioned for immunofluorescence examination (green is GFAP, red is NF200, blue is DAPI, 20X); As can be seen from the above figure: in the SCI group, axons were interrupted at the spinal cord injury site, and there was no regenerated axons; although some axons regenerated in the MenSCs-Exo group, the number of NF200-positive neurofilaments was small and arranged in disorder; the NF200 fluorescence intensity of the Art-MenSCs-Exo group was stronger than that of the other injury groups, showing obvious axon regeneration and orderly arrangement. DETAILED DESCRIPTION

[0025] The present invention will be further described with reference to the accompanying drawings.

[0026] In preliminary experiments, the applicant found that artemisinin can have a good repair effect on a variety of animal injury models, but its poor water solubility, low bioavailability, short half-life, unsatisfactory distribution in the body, obstruction of the blood-brain barrier, difficulty in reaching an effective dose in the central nervous system, and problems with targeted and accurate delivery all affect the therapeutic effect, resulting in limited application.

[0027] Menstrual blood-derived mesenchymal stem cells (MenSCs) are a new type of mesenchymal stem cells that are derived from the endometrium shed during menstruation and are discharged from the body through menstrual blood. Compared with other invasive methods of acquisition, MenSCs can be obtained safely and non-invasively from the menstrual blood of women of childbearing age. Previous research results have shown that menstrual blood stem cells have shown good effects in the treatment of SCI; at the same time, previous in vitro research results have found that MenSCs promote the increase in the length of spinal cord neuron axons and the number of branches after injury by releasing exosomes. However, exosomes of menstrual blood stem cells have a short half-life, a limited number of homing to the injury site, and low colonization efficiency. In addition, since most of them are administered intravenously, the peak value is at the beginning and a stable effective concentration cannot be maintained.

[0028] In order to overcome the deficiencies of menstrual stem cell exosomes and artemisinin and enhance their anti-injury and repair effects in SCI, this application uses an ultrasonic method to load artemisinin into menstrual stem cell exosomes. Compared with free artemisinin, the menstrual stem cell exosome drug delivery system loaded with artemisinin can not only pass through the blood-brain barrier and the blood-spinal cord barrier to reach the damaged lesion site, but also deliver the encapsulated artemisinin to the damaged tissue, effectively enriching it at the site of injury, achieving targeted delivery and precise release of the drug; it can also improve the solubility and stability of artemisinin, prolong the in vivo circulation time, and improve bioavailability; at the same time, it can play the dual advantages of menstrual stem cell exosomes as both a therapeutic agent and a drug delivery carrier, improve the survival and proliferation of neuronal cells from multiple angles, improve the microenvironment at the spinal cord injury site, promote nerve regeneration, and improve neurological function recovery. The results of the previous experiments showed that the exosomes loaded with artemisinin showed good stability and drug loading effect, and could also be taken up by nerve cells. At the same time, the research results also showed that the menstrual stem cell exosome drug delivery system loaded with artemisinin showed good neurological function improvement effects on damaged neuronal cells and SCI rats. The details are as follows: Example 1: Construction and characterization of artemisinin-loaded menstrual stem cell exosome drug delivery system Exosomes from menstrual stem cells were obtained by ultracentrifugation and diluted to 1 mg / mL for later use. Cell culture medium was first harvested from MenSCs and centrifuged at 300g and 4°C for 10 min using an ultracentrifuge to remove detached cells. The supernatant was collected and filtered through a 0.22µm filter to remove contaminating apoptotic bodies, microvesicles, and cell debris. The clarified cell culture medium was then centrifuged at 100,000g for 90 min at 4°C in an ultracentrifuge using a 50.2 Ti rotor (k factor: 157.7) to precipitate exosomes. The supernatant was carefully removed and the pellet containing crude exosomes was resuspended in 1 mL of 4°C PBS. A second round of ultracentrifugation was performed (50.2 Ti rotor (k factor: 157.7), 4°C, 100,000g for 90 min), and the resulting exosome pellet was resuspended in 4°C PBS and adjusted to a concentration of 1 mg / mL to obtain exosomes from menstrual stem cells for later use.

[0029] Artemisinin was weighed and dissolved with dimethyl sulfoxide (DMSO) to prepare a stock solution with a final concentration of 1 mg / mL for later use. After mixing equal volumes of the exosome solution and artemisinin solution, they were incubated at 37°C for 2 hours, and then loaded using a gentle ultrasonic method (Sonic Dismembrator, Fisher Scientific, USA). The procedure was as follows: 20% amplitude, ultrasonic for 10 seconds, intermittent for 10 seconds, for 3 minutes, and an ice bath for 2 minutes as one cycle, and repeated 6 cycles. Incubate at 37°C for 1 hour to stabilize the exosome membrane; transfer the mixed solution into a 100KD ultrafiltration tube, and ultrafilter at 4°C, 4500×g for 15 minutes to remove unbound artemisinin to obtain exosomes loaded with artemisinin. Figure 1 and Figure 2 As shown, the absorbance of the artemisinin-loaded exosome solution was then determined by an ultra-micro spectrophotometer; at the same time, the drug-loaded exosome morphology was detected by TEM, the drug-loaded exosome particle size was detected by NTA, and the drug-loaded exosome protein markers were identified by Western Blot. Transmission electron microscopy results showed that both artemisinin-loaded exosomes and ordinary exosomes had a double-layer membrane structure, and the morphology of the exosomes did not change much after drug loading. NTA results showed that the particle size of artemisinin-loaded exosomes increased slightly, with a diameter of 30-150nm. Western blot detected that the corresponding protein markers CD9, CD63, and TSG101 were expressed in ordinary exosomes and artemisinin-loaded exosomes. The absorbance of the artemisinin-loaded exosome solution measured by ultra-micro spectrophotometer has a double peak.

[0030] Example 2: Detection of the precision and encapsulation efficiency of the blood stem cell exosome drug delivery system loaded with artemisinin like Figure 2As shown in the figure, the absorbance of 5 artemisinin solutions with different concentrations (0.2 mg / mL, 0.6 mg / mL, 1.0 mg / mL, 1.5 mg / mL and 2.0 mg / mL) was measured at the maximum absorption wavelength of 292 nm using an ultra-micro spectrophotometer, and the PBS solution was used as the blank control group. The absorbance of the 5 groups of samples was measured at 9:00, 11:00 and 13:00 every day for 3 consecutive days. The values ​​within the same day and at the same time point were statistically analyzed to calculate the intra-day precision, inter-day precision and relative standard deviation (RSD).

[0031] The absorbance of the ultrafiltered artemisinin-loaded exosome solution at 292 nm was measured using an ultra-micro spectrophotometer, and the mass concentration of the corresponding artemisinin was calculated by substituting it into the standard curve equation. The encapsulation efficiency of exosomes loaded with artemisinin was calculated according to the encapsulation efficiency calculation formula.

[0032] Encapsulation efficiency (EE%) = (mass of Art in Exo+Art / total mass of Art added) × 100%.

[0033] The concentration of artemisinin in exosomes was determined using a high performance liquid chromatography (HPLC) system. The method was briefly described as follows: methanol was used to lyse the exosomes to release the encapsulated artemisinin. After centrifugation to remove the protein contained in the exosomes, 20 μL of the supernatant was injected into the HPLC system and the DAD probe was used to collect the absorbance at 292 nm. The concentration of artemisinin in the exosomes was then calculated based on the standard curve.

[0034] Comparative Example 1: Comparison of the precision and encapsulation efficiency of artemisinin-loaded menstrual stem cell exosomes and adipose stem cell exosomes delivery systems Menstrual stem cell exosomes (i.e., Art-MenSC-Exo) and adipose stem cell exosomes (i.e., Art-ADSC-Exo) were loaded with artemisinin under the same conditions, and then the precision and encapsulation efficiency were tested and compared according to the methods and steps in Example 2. Figure 3 As shown, the results showed that menstrual stem cell exosomes were significantly superior to adipose stem cell exosomes in intra-day precision, inter-day precision and encapsulation efficiency.

[0035] Example 3: Study on drug release from artemisinin-loaded menstrual stem cell exosome delivery system like Figure 4As shown in the figure, the release of artemisinin from menstrual stem cell exosomes loaded with artemisinin was studied using a dynamic dialysis method in release media at pH 5.5 and pH 7.4. The exosome samples loaded with artemisinin and the free artemisinin solution were placed in a dialysis bag (MWCO = 100 kDa), and then the dialysis bag was immersed in PBS and stirred at 37 ° C. At 0h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h, the dialysis bag was taken out and placed in a new dish of PBS prepared in advance. At the same time, the PBS solution in the original dish was collected and the artemisinin concentration was determined by an ultra-micro spectrophotometer, and the cumulative release percentage of artemisinin was calculated.

[0036] Comparative Example 2: Comparison of drug release from adipose stem cell exosomes and menstrual stem cell exosomes like Figure 5 As shown, the menstrual stem cell exosomes and adipose stem cell exosomes loaded with artemisinin were simultaneously tested for drug release according to the method in Example 3, and the former was significantly better than the latter.

[0037] Example 4: Stability Assessment like Figure 6 As shown, Art-MenSCs-Exo and MenSCs-Exo dissolved in PBS were stored at 4°C for 7 days. The changes in exosome particle size and concentration were detected by nanoparticle tracking analyzer (NTA) before and after storage to evaluate the stability of the exosomes under storage conditions; Art-MenSCs-Exo and MenSCs-Exo were added to complete culture medium without exosomes (containing 10% FBS) and retained at 37°C for 24 hours, and then NTA was used to detect the changes in exosome particle size and concentration to evaluate the stability of the exosomes under culture conditions.

[0038] Example 5: Observation of Cellular Uptake of Exosomes like Figure 7 As shown, PKH67 fluorescent dye was used to label artemisinin-loaded menstrual stem cell exosomes to study the uptake of these drug-loaded exosomes by SHSY5Y cells. The labeled exosomes were co-incubated with SHSY5Y cells, and the intracellular fluorescence signals were analyzed using confocal laser scanning microscopy and flow cytometry.

[0039] Example 6: Intracellular drug delivery like Figure 8As shown, SHSY5Y cells were cultured overnight and incubated with common exosomes (MenSCs-Exo) and drug-loaded exosomes (Art-MenSCs-Exo) for 4h, 8h, and 12h. The surviving cells at each time point were collected and counted, and 250μL of pure water (DDW) was added. Repeated freezing and thawing released the artemisinin contained in the cells, and 4 times the volume of acetonitrile was added to extract artemisinin and precipitate the protein. After centrifugation, 10μL of the supernatant was taken and the artemisinin concentration was determined by HPLC-MS / MS. The intracellular drug concentration = artemisinin assay content (ng) / number of viable cells.

[0040] Comparative Example 3: Comparison of intracellular drug delivery between adipose stem cell exosomes and menstrual stem cell exosomes like Fig. 9 As shown, the menstrual stem cell exosomes and adipose stem cell exosomes loaded with artemisinin were simultaneously tested for intracellular drug delivery according to the method in Example 6, and the former was significantly better than the latter.

[0041] Application Example 1: Effect of artemisinin-loaded menstrual stem cell exosome drug delivery system on axon regeneration of injured neurons In vitro, a co-culture system of artemisinin-loaded menstrual stem cell exosomes and spinal cord neuron injury models was constructed to observe the morphology of spinal cord neurons, the length, number and branch number of axon regeneration, and the morphology of growth cones; the effect of the artemisinin-loaded menstrual stem cell-derived exosome delivery system on promoting neuronal axon regeneration was clarified. Fig.10 As shown, the results showed that neuronal cells cultured with artemisinin-loaded exosomes had longer axons and more branches, which indicated that artemisinin-loaded exosomes had a good ability to promote the growth of nerve cells.

[0042] Application Example 2: Artemisinin-loaded menstrual stem cell exosomes improve local pathological conditions in SCI rats Eight-week-old SD female rats were used to establish a T10 hemisection model of spinal cord injury. Exosomes of menstrual stem cells loaded with artemisinin were injected into the tail vein. Routine postoperative care, such as warming, urination assistance, wound disinfection, and penicillin injection, were performed. Spinal cord tissues of rats were obtained 28 days after surgery, and frozen sections were made and stained for pathological analysis. Fig.11 As shown, Nissl staining showed that the number of atrophic and degenerated Nissl bodies in the Art-MenSCs-Exo group was less than that in the other injury groups, and the number of newly formed Nissl bodies was increased; HE staining showed that the cell shrinkage and fragmentation in the injured spinal cord in the Art-MenSCs-Exo group was improved, the number of inflammatory cells was reduced, and the syringomyelia phenomenon was significantly improved compared with other groups; Masson staining showed that the degree of fibrosis in the spinal cord injury area of ​​rats treated with Art-MenSCs-Exo group was lower than that in other injury groups, and the volume of the cavity was smaller.

[0043] Application Example 3: Effect of artemisinin-loaded menstrual stem cell exosome drug delivery system on neural regeneration after spinal cord injury in rats Eight-week-old SD female rats were used to make a T10 hemisection model of spinal cord injury. The exosomes of menstrual stem cells loaded with artemisinin were injected into the tail vein. Routine postoperative care, such as warming, urination assistance, wound disinfection, and penicillin injection, were performed. BBB score and Catwalk gait analysis were used to observe the recovery of motor function 28 days after surgery, and immunofluorescence was used to detect nerve regeneration. Fig.12 As shown, the results showed that exosomes loaded with artemisinin can effectively promote the recovery of motor function and regeneration of nerve axons after spinal cord injury in SD rats.

[0044] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A drug delivery composition of menstrual stem cell exosomes loaded with artemisinin, characterized by: Including menstrual stem cell exosomes and artemisinin.

2. A method for preparing a drug delivery composition of menstrual stem cell exosomes loaded with artemisinin, characterized in that: The steps include: 1) Dilute the menstrual stem cell exosomes to 0.1-10 mg / mL to obtain an exosome solution; 2) Prepare artemisinin solution with a concentration of 0.1~5 mg / mL; 3) mixing the exosome solution described in step 1) and the artemisinin solution described in step 2), and incubating them at 26-40° C. for 1-5 hours; 4) Ultrasonic loading is used.

3. The method for preparing the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to claim 2, characterized in that: In step 1), the concentration of the exosome solution is 0.5-5 mg / mL.

4. The method for preparing the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to claim 3, characterized in that: In step 2), the concentration of the artemisinin solution is 0.2-2 mg / mL.

5. The method for preparing the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to claim 4, characterized in that: In step 3), the volume ratio of the exosome solution to the artemisinin solution is 1:(1-5).

6. The method for preparing the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to claim 5, characterized in that: In step 3), incubate at 35-38°C for 1-2 hours.

7. The method for preparing the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to claim 6, characterized in that: In step 4), the procedure of the ultrasonic method is as follows: (1) 10-30% amplitude, ultrasonication for 5-20 seconds, interval of 10-30 seconds, duration of 2-10 minutes, ice bath for 1-5 minutes as one cycle, and multiple cycles; (2) incubation for 0.5-2 hours at a temperature of 30-40°C to obtain a mixed solution; (3) ultrafiltration of the mixed solution to obtain exosomes loaded with artemisinin.

8. The method for preparing the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to claim 7, characterized in that: Incubate for 1 to 2 hours at 35 to 37°C.

9. The method for preparing the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to claim 8, characterized in that: The ultrafiltration conditions are: 4°C, centrifugation at 4500×g for 15 min.

10. Use of the artemisinin-loaded menstrual stem cell exosome drug delivery composition according to any one of claims 1 to 9 in the treatment of neural repair after spinal cord injury.

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