Adipose-derived stem cell exosome injection, preparation method and application of adipose-derived stem cell exosome injection in acupoint injection

Abstract

The invention discloses an adipose-derived stem cell exosome injection, a preparation method and application of the adipose-derived stem cell exosome injection to acupoint injection, and belongs to the technical field of biology. A sodium hyaluronate solution; an osmotic pressure regulator; wherein the carrier loaded with the adipose-derived stem cell exosome is prepared by loading the adipose-derived stem cell exosome on the carrier, and the carrier is prepared by reacting hydroxyapatite with sodium alginate; the exosome loading rate of the carrier loaded with the adipose-derived stem cell exosome is greater than or equal to 85%. The preparation method provided by the invention overcomes the key technical problems of low exosome yield, short acupoint residence time, unstable biological activity and the like in the traditional acupoint injection of the adipose-derived stem cell exosome, realizes the efficient, controllable and stable preparation of the adipose-derived stem cell injection, provides a novel choice for the traditional Chinese medicine acupoint injection treatment, and has wide application prospects. The important clinical application value and industrialization prospect are realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an adipose-derived stem cell exosome injection, a preparation method thereof, and its application in acupoint injection. Background Art

[0002] As an important mediator of intercellular communication, stem cell exosomes have shown broad application prospects in the fields of regenerative medicine, tissue repair, and disease treatment. Adipose-derived stem cell exosomes have become a hot topic in biomedical research because of their rich bioactive components, such as growth factors, cytokines, and miRNAs, which can promote angiogenesis, reduce inflammatory responses, and regulate immune functions. Acupoint injection therapy, as a treatment method integrating traditional Chinese and Western medicine, directly injects drugs into specific acupoints, not only exerting the pharmacological effects of the drugs but also utilizing the meridian regulation function of acupuncture at acupoints, with the characteristics of rapid onset and good curative effect.

[0003] In recent years, the application of stem cell exosomes in acupoint injection has gradually attracted attention. However, the existing technology still faces multiple challenges: First, the yield of adipose-derived stem cell exosomes under conventional culture methods is low, making it difficult to meet the needs of clinical applications. For example, the static culture system reported by Wang et al. (2019) can only produce 10 - 15 mg of exosomes per liter of culture medium, severely limiting the possibility of large-scale production. Second, the research by Liu et al. (2021) shows that traditional exosome preparations have a short retention time in the local acupoint environment, usually not exceeding 12 hours, resulting in an unsustained therapeutic effect and the need for frequent injections, increasing the pain of patients and the treatment cost.

[0004] On the other hand, although the exosome delivery system developed by Zhang et al. (2020) improves the stability of exosomes, its carrier material has poor biocompatibility, and the exosome loading rate is only 40% - 50%, resulting in a large loss of exosome active molecules. In addition, exosome preparations in the existing technology are prone to aggregation, and after injection, they are unevenly dispersed, affecting the drug concentration distribution and therapeutic effect at the acupoint. Summary of the Invention

[0005] The present invention discloses an adipose-derived stem cell exosome injection, a preparation method thereof, and its application in acupoint injection, solving the technical problems of low exosome yield, short acupoint retention time, high large-scale production cost, and insufficient exosome delivery efficiency in the existing exosome therapy.

[0006] An adipose-derived stem cell exosome injection disclosed by the present invention comprises the following components:

[0007] A carrier loaded with adipose-derived stem cell exosomes;

[0008] A sodium hyaluronate solution;

[0009] An osmotic pressure regulator;

[0010] Among them, the carrier loaded with adipose-derived stem cell exosomes is prepared by loading adipose-derived stem cell exosomes onto the carrier, and the carrier is prepared by reacting hydroxyapatite with sodium alginate;

[0011] The exosome loading rate of the carrier loaded with adipose-derived stem cell exosomes is ≥85%.

[0012] Preferably, the freeze-drying reconstitution time of the injection solution is ≤3 min.

[0013] Preferably, the molar ratio of the hydroxyapatite to the sodium alginate is 1:(0.6 - 1.0).

[0014] Preferably, the molecular weight of the hyaluronic acid is 1.0 - 2.0 MDa, and the addition amount is 0.03% - 0.07% of the total mass of the injection solution.

[0015] Preferably, the osmotic pressure regulator is selected from sodium chloride or glucose, and the osmotic pressure of the injection solution after adding the osmotic pressure regulator is 275 - 285 mOsm / kg.

[0016] Preferably, the preparation method of the carrier loaded with adipose-derived stem cell exosomes comprises the following steps:

[0017] (1) Prepare a 0.4% - 0.6% hydroxyapatite nano-suspension, and ultrasonically treat it until the particle size is ≤60 nm;

[0018] (2) Mix the hydroxyapatite and the sodium alginate, add 0.08% - 0.12% calcium chloride for crosslinking, control the stirring rate at 200 - 400 rpm at a pH of 6.8 - 7.2, and the reaction time is 30 - 60 min. The crosslinked product is aseptically filtered through a 0.22 μm pore size filter membrane;

[0019] (3) Dry under supercritical CO2 conditions, with a pressure of 7 - 9 MPa, a temperature of 30 - 40 °C, and a time of 1.5 - 2.5 h to obtain a microcarrier;

[0020] (4) Mix the adipose-derived stem cell exosomes and the microcarrier at a drug loading amount of 0.8 - 1.0 mg / mL, and stir at a rotation speed of 100 - 200 rpm at a temperature of 4 - 8 °C for 2 - 4 hours to obtain the carrier loaded with adipose-derived stem cell exosomes.

[0021] Preferably, the preparation method of the adipose-derived stem cell exosomes comprises the following steps:

[0022] Cultivate adipose-derived stem cells in a bioreactor at 35 - 38 °C until the density reaches 1×10 6 / mL, apply a square wave pulse of 1.0 - 1.4 kV / cm, with a pulse width of 40 - 60 μs and a frequency of 8 - 12 Hz. Incubate statically for 12 - 24 hours, centrifuge to collect the supernatant, concentrate it through a 100 kDa ultrafiltration membrane, and use size exclusion chromatography to collect the fraction with a particle size of 30 - 150 nm to obtain adipose stem cell exosomes.

[0023] Preferably, the viscosity of the injection is 1.2 - 1.5 mPa·s.

[0024] The present invention also discloses a preparation method of the above injection, including the following steps: at 2 - 8 °C, mix the carrier loaded with adipose stem cell exosomes with a hyaluronic acid solution having a molecular weight of 1.0 - 2.0 MDa, add an osmotic pressure regulator to adjust the osmotic pressure to 275 - 285 mOsm / kg, and then aseptically dispense to obtain the adipose stem cell exosome injection.

[0025] The present invention also discloses an application of the above injection in acupoint injection therapy, including: selecting acupoints of Zusanli, Guanyuan, and Shenshu, with an acupuncture depth of 1.5 - 3.0 cm, a dosage of 0.5 - 1.0 mL each time, a treatment cycle of once a week for 4 consecutive weeks.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] (1) In the present invention, adipose stem cells are induced to secrete exosomes by applying an electrical stimulation method, which increases the exosome yield by more than 90%, significantly improving the production efficiency; at the same time, the carrier constructed by hydroxyapatite and sodium alginate achieves an exosome loading rate of ≥85%, greatly increasing the drug loading rate.

[0028] (2) In the present invention, by adding sodium hyaluronate and an osmotic pressure regulator, the viscosity of the injection is maintained at 1.2 - 1.5 mPa·s, which not only ensures injectability but also achieves compatibility with the local tissue of the acupoints, extending the residence time of the drug at the acupoints to more than 48 hours, while the residence time of the traditional preparation is only 12 - 16 hours.

[0029] (3) The exosome injection of the present invention is processed under supercritical CO2 conditions through a specific drying process (pressure 7 - 9 MPa, temperature 30 - 40 °C, time 1.5 - 2.5 h), and the freeze-dried reconstitution time ≤3 min, which is significantly faster than the reconstitution time of 10 - 15 minutes generally required in the prior art, improving the convenience of clinical use and the preservation rate of exosome activity.

[0030] (4) The present invention adopts an acupoint injection scheme, making the CD31+ blood vessel density reach 153.6 - 175.4 per mm 2 , compared with 87.5 - 94.8 per mm of the comparative example 2It is increased by more than about 70%, the blood perfusion ratio reaches 0.80 - 0.90, which is increased by more than about 60% compared with 0.48 - 0.52 of the comparative example, significantly enhancing the local microcirculation and angiogenesis effects of the acupoints. Brief Description of the Drawings

[0031] Figure 1 It is a graph showing the change in the amount of exosomes released in vitro of the injection solutions prepared in Examples 1 - 5 and Comparative Examples 1 - 3 at pH 6.5. Detailed Description of the Embodiments

[0032] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] Example 1

[0034] Use a Sartorius Biostat 50L bioreactor, control the dissolved oxygen concentration (DO) at 40.0%, the pH value at 7.20, and the culture temperature at 37.0°C. The culture medium uses modified α-MEM, containing 0.75 μg / mL vitamin C and 5.00 μg / mL VEGF, and the glucose concentration is controlled to decrease from 4.8 g / L to 2.1 g / L by gradient. When the cell density reaches 1×10 6 cells / mL, use a BTX ECM 830 electroporator, set the square wave pulse parameters as the field strength of 1.2 kV / cm, the pulse width of 50 μs, the frequency of 10 Hz, and the treatment time of 2 minutes for pulsed electric field stimulation treatment. Subsequently, immediately detect the CD63+ exosome secretion efficiency by flow cytometry, and the result shows that the secretion efficiency reaches 93.5%.

[0035] Then use a Malvern Zetasizer Nano ZS particle size analyzer to monitor. Prepare a 0.5% hydroxyapatite (HA) nano-suspension with the particle size controlled at 50 and the Zeta potential of -25 mV; mix according to the molar ratio of HA: sodium alginate (SA) = 1:0.83, and add 0.1% calcium chloride solution for cross-linking. Adopt the supercritical CO2 drying process, control the drying pressure at 8 MPa, the temperature at 35°C, and the time at 2 hours. The pore size distribution is measured by the BET method to be 50 - 150 nm.

[0036] Finally, use Thermo Scientific Hybrid system, load microcarriers with exosomes to a drug loading of 0.83 mg / mL, add 0.05% hyaluronic acid (molecular weight 1.5 MDa), and monitor and adjust the osmotic pressure of the final preparation to 280 mOsm / kg using an osmometer.

[0037] Example 2

[0038] Use a Sartorius Biostat 50 L bioreactor, control the dissolved oxygen concentration (DO) at 38.5%, the pH value at 7.15, and the culture temperature at 36.8 °C. The culture medium uses modified α-MEM, containing 0.80 μg / mL vitamin C and 4.75 μg / mL VEGF, and control the glucose concentration to decrease from 5.0 g / L to 2.3 g / L by gradient. When the cell density reaches 1×10 6 cells / mL, perform pulsed electric field stimulation treatment: Use a BTX ECM 830 electroporator, set the square wave pulse parameters as field strength 1.1 kV / cm, pulse width 55 μs, frequency 9 Hz, and treatment time 2.5 minutes. Immediately afterwards, detect the secretion efficiency of CD63+ exosomes by flow cytometry, and the result shows that the secretion efficiency reaches 94.2%.

[0039] Then use a Malvern Zetasizer Nano ZS particle size analyzer to monitor, prepare a 0.55% hydroxyapatite (HA) nano-suspension with a particle size controlled at 48 nm and a Zeta potential of -23 mV; mix according to the molar ratio of HA: sodium alginate (SA) = 1:0.85, and add a 0.12% calcium chloride solution for crosslinking; adopt a supercritical CO2 drying process, control the drying pressure at 7.8 MPa, the temperature at 34 °C, and the time at 2.2 hours, and measure the pore size distribution by the BET method to be 55 - 145 nm.

[0040] Finally use a Thermo Scientific Hybrid system, load microcarriers with exosomes to a drug loading of 0.85 mg / mL, add 0.06% hyaluronic acid (molecular weight 1.6 MDa), and monitor and adjust the osmotic pressure of the final preparation to 275 mOsm / kg using an osmometer.

[0041] Example 3

[0042] Use a Sartorius Biostat 50 L bioreactor, control the dissolved oxygen concentration (DO) at 41.5%, the pH value at 7.25, and the culture temperature at 37.2 °C. The culture medium uses modified α-MEM, containing 0.70 μg / mL vitamin C and 5.25 μg / mL VEGF, and control the glucose concentration to decrease from 4.5 g / L to 2.0 g / L by gradient. When the cell density reaches 1×106 When the cell density reached 1×10 cells / mL, pulsed electric field stimulation treatment was carried out: using a BTX ECM 830 electroporator, the square wave pulse parameters were set as a field strength of 1.3 kV / cm, a pulse width of 45 μs, a frequency of 11 Hz, and a treatment time of 1.8 minutes. Subsequently, the secretion efficiency of CD63+ exosomes was immediately detected by flow cytometry, and the result showed that the secretion efficiency reached 95.1%.

[0043] Then, it was monitored using a Malvern Zetasizer Nano ZS particle size analyzer. A 0.45% hydroxyapatite (HA) nano-suspension was prepared with a particle size controlled at 52 nm and a Zeta potential of -26 mV; it was mixed according to a molar ratio of HA: sodium alginate (SA) = 1:0.80, and a 0.09% calcium chloride solution was added for crosslinking; a supercritical CO2 drying process was adopted, controlling the drying pressure at 8.2 MPa, the temperature at 36 °C, and the time at 1.8 hours. The pore size distribution was measured by the BET method to be 45 - 155 nm.

[0044] Finally, a Thermo Scientific mixing system was used to load exosomes onto microcarriers to a drug loading amount of 0.80 mg / mL, 0.04% hyaluronic acid (molecular weight 1.4 MDa) was added, and the final preparation osmotic pressure was monitored and adjusted to 285 mOsm / kg by an osmometer.

[0045] Example 4

[0046] A Sartorius Biostat 50 L bioreactor was used, controlling the dissolved oxygen concentration (DO) at 39.0%, the pH value at 7.10, and the culture temperature at 36.5 °C. The culture medium used was modified α-MEM, containing 0.85 μg / mL vitamin C and 4.50 μg / mL VEGF, and the glucose concentration was controlled to decrease from 5.2 g / L to 2.4 g / L by gradient. When the cell density reached 1×10 6 cells / mL, pulsed electric field stimulation treatment was carried out: using a BTX ECM 830 electroporator, the square wave pulse parameters were set as a field strength of 1.0 kV / cm, a pulse width of 60 μs, a frequency of 8 Hz, and a treatment time of 3.0 minutes. Subsequently, the secretion efficiency of CD63+ exosomes was immediately detected by flow cytometry, and the result showed that the secretion efficiency reached 92.8%.

[0047] Subsequently, it was monitored using a Malvern Zetasizer Nano ZS particle size analyzer. A 0.60% hydroxyapatite (HA) nano-suspension was prepared with the particle size controlled at 45 nm and the Zeta potential of -22 mV. It was mixed according to the molar ratio of HA: sodium alginate (SA) = 1:0.88, and a 0.13% calcium chloride solution was added for crosslinking. The supercritical CO2 drying process was adopted, with the drying pressure controlled at 7.5 MPa, the temperature at 33 °C, and the time at 2.5 hours. The pore size distribution measured by the BET method was 60 - 140 nm.

[0048] Finally, a Thermo Scientific mixing system was used to load exosomes onto the microcarriers to a drug loading amount of 0.88 mg / mL. 0.07% hyaluronic acid (molecular weight 1.7 MDa) was added, and the osmotic pressure of the final preparation was monitored and adjusted to 270 mOsm / kg by an osmometer.

[0049] Example 5

[0050] A Sartorius Biostat 50 L bioreactor was used, with the dissolved oxygen concentration (DO) controlled at 42.0%, the pH value at 7.30, and the culture temperature at 37.5 °C. The culture medium used was modified α-MEM, containing 0.65 μg / mL vitamin C and 5.50 μg / mL VEGF, and the glucose concentration was controlled to decrease from 4.2 g / L to 1.9 g / L by gradient. When the cell density reached 1×10 6 cells / mL, pulsed electric field stimulation treatment was carried out: using a BTX ECM 830 electroporator, the square wave pulse parameters were set as the field strength of 1.4 kV / cm, the pulse width of 40 μs, the frequency of 12 Hz, and the treatment time of 1.5 minutes. Subsequently, the CD63+ exosome secretion efficiency was immediately detected by flow cytometry, and the result showed that the secretion efficiency reached 96.3%.

[0051] Subsequently, it was monitored using a Malvern Zetasizer Nano ZS particle size analyzer. A 0.40% hydroxyapatite (HA) nano-suspension was prepared with the particle size controlled at 55 nm and the Zeta potential of -28 mV. It was mixed according to the molar ratio of HA: sodium alginate (SA) = 1:0.78, and a 0.08% calcium chloride solution was added for crosslinking. The supercritical CO2 drying process was adopted, with the drying pressure controlled at 8.5 MPa, the temperature at 38 °C, and the time at 1.5 hours. The pore size distribution measured by the BET method was 40 - 160 nm.

[0052] Finally, a Thermo Scientific Hybrid system, loading microcarriers with exosomes to a drug loading amount of 0.78 mg / mL, adding 0.03% hyaluronic acid (molecular weight 1.3 MDa), and monitoring and adjusting the osmotic pressure of the final preparation to 290 mOsm / kg using an osmometer.

[0053] Comparative Example 1

[0054] Using a Sartorius Biostat A 50 L bioreactor was used, controlling the dissolved oxygen concentration (DO) at 40.0%, the pH value at 7.20, and the culture temperature at 37.0 °C. The culture medium was modified α-MEM, containing 0.75 μg / mL vitamin C and 5.00 μg / mL VEGF, and the glucose concentration was controlled to decrease from 4.8 g / L to 2.1 g / L by gradient. Different from Example 1, in this comparative example, pulsed electric field stimulation treatment was not used. Instead, the cultured adipose stem cells were directly harvested, the culture supernatant was separated by conventional centrifugation (300×g, 10 minutes), and the exosomes were collected by filtration using a 0.22 μm filter membrane. Flow cytometry detection showed that the secretion efficiency of CD63+ exosomes was only 52.3%, significantly lower than 93.5% in Example 1.

[0055] Subsequently, it was monitored using a Malvern Zetasizer Nano ZS particle size analyzer. A 0.5% hydroxyapatite (HA) nano-suspension was prepared, with the particle size controlled at 50 nm and the Zeta potential at -25 mV; it was mixed according to the molar ratio of HA: sodium alginate (SA) = 1:0.83, and a 0.1% calcium chloride solution was added for crosslinking; a supercritical CO2 drying process was adopted, controlling the drying pressure at 8 MPa, the temperature at 35 °C, and the time at 2 hours. The pore size distribution was measured by the BET method to be 50 - 150 nm.

[0056] Finally, using Thermo Scientific Hybrid system, loading microcarriers with exosomes. However, due to the low initial exosome yield, the final drug loading amount only reached 0.45 mg / mL, significantly lower than 0.83 mg / mL in Example 1; 0.05% hyaluronic acid (molecular weight 1.5 MDa) was added, and the osmotic pressure of the final preparation was monitored and adjusted to 280 mOsm / kg using an osmometer.

[0057] Compared with Example 1, the exosome yield in this comparative example decreased by 45.8%, the expected acupoint retention time was shortened by 50%, and the pro-angiogenic efficiency decreased by about 42%.

[0058] Comparative Example 2

[0059] Using a Sartorius Biostat A 50L bioreactor was used, with the dissolved oxygen concentration (DO) controlled at 40.0%, the pH value at 7.20, and the culture temperature at 37.0 °C. The culture medium used was modified α-MEM, containing 0.75 μg / mL vitamin C and 5.00 μg / mL VEGF. The glucose concentration was controlled to decrease from 4.8 g / L to 2.1 g / L by gradient. When the cell density reached 1×10 6 cells / mL, pulsed electric field stimulation treatment was carried out: using a BTX ECM 830 electroporator, the square wave pulse parameters were set as the field strength of 1.2 kV / cm, the pulse width of 50 μs, the frequency of 10 Hz, and the treatment time of 2 minutes. Subsequently, the secretion efficiency of CD63+ exosomes was immediately detected by flow cytometry, and the result showed that the secretion efficiency reached 93.5%.

[0060] The main difference between this comparative example and Example 1 was that instead of using the biomimetic mineralization microcarrier technology, a traditional liposome encapsulation method was used:

[0061] The extracted exosomes were mixed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000 in a molar ratio of 5:4:1. A thin film was formed at 40 °C using a rotary evaporator, vacuum dried for 12 hours, hydrated with PBS buffer for 60 minutes, ultrasonicated (power 150 W, 2 minutes), and then filtered through a 0.22 μm filter membrane. Particle size analysis showed that the average diameter of the formed liposomes was 120 nm, the potential was -15 mV, and the exosome encapsulation efficiency was 68.5%, significantly lower than 92.3% of Example 1.

[0062] Finally, 0.05% hyaluronic acid (molecular weight 1.5 MDa) was added, and the osmotic pressure was adjusted to 280 mOsm / kg.

[0063] Compared with Example 1, in this comparative example, with a similar initial exosome yield, due to the low encapsulation efficiency, the final drug loading amount was only 0.57 mg / mL, the acupoint retention time was only 12 hours, and the pro-angiogenesis efficiency decreased by about 35%.

[0064] Comparative Example 3

[0065] Using a Sartorius Biostat A 50L bioreactor was used, with the dissolved oxygen concentration (DO) controlled at 40.0%, the pH value at 7.20, and the culture temperature at 37.0 °C. The culture medium used was modified α-MEM, containing 0.75 μg / mL vitamin C and 5.00 μg / mL VEGF. The glucose concentration was controlled to decrease from 4.8 g / L to 2.1 g / L by gradient. When the cell density reached 1×10 6When the cell density reached 1×10⁶ cells / mL, pulsed electric field stimulation was performed: using a BTX ECM 830 electroporator, the square wave pulse parameters were set as a field strength of 1.2 kV / cm, a pulse width of 50 μs, a frequency of 10 Hz, and a treatment time of 2 minutes. Subsequently, flow cytometry was immediately used to detect the secretion efficiency of CD63+ exosomes, and the result showed that the secretion efficiency reached 93.5%.

[0066] Then, using a Malvern Zetasizer Nano ZS particle size analyzer for monitoring, a 0.5% hydroxyapatite (HA) nano-suspension was prepared with a particle size controlled at 50 nm and a Zeta potential of -25 mV; different from Example 1, in this comparative example, sodium alginate was not added for compounding, but pure HA nanoparticles were directly used; 0.1% calcium chloride solution was added for simple cross-linking; a conventional freeze-drying method (temperature -40 °C, pressure 20 Pa, time 24 hours) was used instead of supercritical CO₂ drying. The pore size distribution of the final material determined by the BET method was uneven, mainly concentrated in 200 - 350 nm, significantly larger than 50 - 150 nm in Example 1; at the same time, the porosity was only 35%, lower than 68% in Example 1. When the exosomes were mixed with the prepared microcarriers, the loading efficiency only reached 70.2%, significantly lower than 92.3% in Example 1.

[0067] Finally, using a Thermo Scientific mixing system, the microcarriers were loaded with exosomes to the carrier-loaded exosomes. Due to uneven pore size and low loading efficiency, the final drug loading amount was only 0.58 mg / mL, significantly lower than 0.83 mg / mL in Example 1. When formulating the preparation, 0.05% hyaluronic acid (molecular weight 1.5 MDa) was added, and the osmometer was used to monitor and adjust the osmotic pressure of the final preparation to 280 mOsm / kg.

[0068] The injection prepared in this comparative example showed an obvious "burst release" phenomenon in the in vitro release test, and the release amount exceeded 85% within 24 hours. Compared with about 50% release in 24 hours in Example 1, it lacked the long-acting controlled release characteristics. The retention time after in vivo acupoint injection was only 14 hours, much lower than 24 - 48 hours in Example 1, and the angiogenesis promotion efficiency decreased by about 38%.

[0069] Performance Detection

[0070] 1. Determination of Exosome Secretion Efficiency

[0071] Test procedure: First, collect the adipose stem cell culture supernatants of each example and comparative example, and precipitate the exosomes by ultracentrifugation (100,000×g, 70 minutes, 4 °C). Resuspend the precipitate in PBS, and use a BCA protein quantification kit (Thermo Scientific TM)Determine the total protein content. Take 10 μL of the exosome suspension and incubate it with the CD63 antibody (Abcam, catalog number ab134045) for 30 minutes (at room temperature, protected from light), and use a BD FACSCalibur TM flow cytometer to detect the percentage of CD63-positive exosomes. At the same time, determine the particle size distribution and concentration of exosomes using a nanoparticle tracking analyzer (Malvern NanoSight NS300).

[0072] Calculate the secretion efficiency according to the formula: Secretion efficiency (%) = (Number of CD63+ exosomes / Theoretical maximum number of exosomes) × 100%.

[0073] 2. Determination of microcarrier loading efficiency

[0074] Test procedure: Mix and incubate the prepared microcarriers with exosomes (37 °C, 4 hours), with an oscillation speed of 60 rpm. After completion, centrifuge and collect (3,000 × g, 10 minutes), and determine the content of unloaded exosomes in the supernatant using a BCA protein quantification kit.

[0075] Loading efficiency calculation formula: Loading efficiency (%) = [(Total exosome protein amount - Exosome protein amount in the supernatant) / Total exosome protein amount] × 100%.

[0076] 3. In vitro release characteristics test

[0077] Test procedure: Take 1 mL of the samples of each example and comparative example respectively, load them into a dialysis bag (molecular weight cut-off value 12 - 14 kDa), immerse them in 50 mL of PBS buffer (two conditions of pH 7.4 and pH 6.5, simulating normal tissue and acupoint microenvironment), and place them in a constant temperature oscillating water bath at 37 °C (oscillation speed 100 rpm). Sample 1 mL at preset time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h) and supplement an equal amount of fresh buffer.

[0078] Determine the content of exosome markers (CD9, CD63, TSG101) in the collected samples by ELISA method (R&D Systems), and calculate the cumulative release percentage: Cumulative release (%) = (Release amount at time t / Total loading amount) × 100%.

[0079] 4. Determination of acupoint retention time

[0080] Test procedure: Healthy adult SD rats (8 weeks old, body weight 250±20 g) were selected, and animal experiments were conducted in accordance with the "Regulations on the Administration of Laboratory Animals". Exosomes were labeled with DiR fluorescent dye (Thermo Fisher, catalog number D12731) to prepare fluorescently labeled injection solutions of each example and comparative example. Twelve animals were in each group, and 0.2 mL of the labeled injection solution was injected into the Zusanli acupoint (3 cun below the lower outer side of the fibular head). Use The Spectrum in vivo imaging system was used to perform in vivo fluorescence imaging at preset time points (0 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h), observe the fluorescence signal intensity and perform quantitative analysis, and calculate the retention time, which was defined as the time required for the fluorescence intensity to decrease to 50% of the initial value.

[0081] 5. Evaluation of angiogenesis efficiency

[0082] Test procedure: A rat hindlimb ischemia model was established, and an ischemic environment was created by ligating the root of the femoral artery. The animals were randomly divided into groups (10 animals in each group), and 0.2 mL of each example and comparative example was injected into the Zusanli acupoint. On the 7th and 14th days after injection, a laser Doppler blood flow imaging instrument (Moor FLPI-2) was used to measure the blood flow perfusion of the lower limbs, and the blood flow perfusion ratio of the ischemic side to the healthy side was calculated. On the 14th day, the animals were sacrificed, and the calf muscle tissue was taken. After fixation with 4% paraformaldehyde and paraffin embedding, sections (thickness 5 μm) were made. Immunohistochemistry was used to detect CD31-positive blood vessels (anti-CD31 antibody, Abcam, catalog number ab28364, diluted 1:200). Five fields of view were randomly selected under a 400× light microscope, and the CD31-positive blood vessel density (number / mm 2 ) and the percentage of CD31-positive area were calculated.

[0083] IV. Test data tables

[0084] Table 1. Record table of exosome secretion efficiency and performance

[0085]

[0086] Table 2. Record table of microcarrier performance and loading efficiency

[0087]

[0088]

[0089] Table 3. Evaluation table of acupoint retention time and biological effects

[0090]

[0091] Data analysis

[0092] As can be seen from Table 1, the CD63+ exosome secretion efficiencies of Examples 1-5 and Comparative Examples 2-3 using pulsed electric field stimulation technology are all above 92%, and the yields are between 84-94 μg / 10 6 cells. In contrast, Comparative Example 1 that did not use this technology had only a secretion efficiency of 52.3% and a yield of 47.2 μg / 10 6 cells, which were reduced by 43.9% and 44.7% respectively, indicating that pulsed electric field can significantly promote exosome secretion.

[0093] As can be seen from Table 2, the microcarriers prepared by the biomimetic mineralization technology in Examples 1-5 have an ideal pore size distribution (40-160 nm) and a relatively high porosity (65.8% - 72.1%), resulting in an exosome loading efficiency of over 91.5% and a final drug loading amount between 0.78 - 0.88 mg / mL. In contrast, Comparative Example 2 using the traditional liposome encapsulation method had a loading efficiency of only 68.5% and the drug loading amount decreased to 0.57 mg / mL; although Comparative Example 3 used hydroxyapatite material, it did not add sodium alginate to form a composite material and used the conventional freeze-drying method, resulting in too large pore size (200-350 nm) and low porosity (35.2%), with a loading efficiency of only 70.2% and a final drug loading amount of 0.58 mg / mL, which was significantly lower than that of the examples.

[0094] From Figure 1 it can be seen that Examples 1-5 showed good sustained-release characteristics under the condition of pH 6.5 (simulating the acupoint microenvironment), with the release amount at 24 hours between 46.5% - 54.8% and the release amount at 48 hours between 81.5% - 87.2%, presenting a stable and continuous release curve. In contrast, Comparative Example 2 and Comparative Example 3 released 38.5% and 42.3% of the drug respectively in the first 2 hours, the release amount at 8 hours exceeded 65%, and the release amount at 24 hours was as high as over 85%, showing an obvious "burst release" phenomenon. This indicates that the biomimetic mineralized microcarriers can effectively control the release rate of exosomes in the acupoint microenvironment and avoid the waste of drug efficacy caused by too fast release.

[0095] As can be seen from Table 3, the acupoint retention times of Examples 1-5 were all above 30 hours, and Example 5 was the longest, reaching 39.4 hours; while the retention times of Comparative Examples 1-3 were only 12.5 - 16.2 hours, less than half of that of the examples. In terms of promoting angiogenesis, the blood perfusion ratio (0.80 - 0.90) and CD31+ blood vessel density (153.6 - 175.4 per / mm 2 ) of the examples were significantly higher than those of the comparative examples (0.48 - 0.52 and 87.5 - 94.8 per / mm 2), with the improvement rates being above 60% and 70% respectively. Example 5 showed the best performance in these indicators, with its CD31+ blood vessel density reaching 175.4 / mm 2 , and the area percentage reaching 40.1%, which was about 90% higher on average than that of the comparative example, fully demonstrating that the adipose stem cell injection prepared by the present invention has significantly enhanced acupoint retention and biological activity.

[0096] In summary, the present invention significantly improves the exosome secretion efficiency and yield of adipose stem cells through the pulsed electric field stimulation technology, greatly enhances the loading efficiency and drug loading capacity of exosomes through the biomimetic mineralized microcarrier technology, realizes the goal of continuously and stably releasing exosomes in the acupoint microenvironment, and finally significantly prolongs the acupoint retention time and enhances the pro-angiogenic effect.

[0097] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Any equivalent changes and improvements made within the scope of the present invention application shall still fall within the scope covered by the patent of the present invention.

Claims

1. An adipose stem cell exosome injection, characterized in that: Includes the following components: A vector loaded with adipose stem cell exosomes; Sodium hyaluronate solution; Osmotic pressure regulators; Wherein, the carrier loaded with adipose stem cell exosomes is prepared by loading a carrier with adipose stem cell exosomes, and the carrier is prepared by reacting hydroxyapatite with sodium alginate; The exosome loading rate of the carrier loaded with adipose stem cell exosomes is ≥85%.

2. The injection according to claim 1, characterized in that: The freeze-dried reconstitution time of the injection is ≤3 min.

3. The injection according to claim 1, characterized in that: The molar ratio of the hydroxyapatite to the sodium alginate is 1:(0.6-1.0).

4. The injection according to claim 1, characterized in that: The molecular weight of the hyaluronic acid is 1.0-2.0 MDa, and the added amount is 0.03% to 0.07% of the total mass of the injection.

5. The injection according to claim 1, characterized in that: The osmotic pressure regulator is selected from sodium chloride or glucose, and the osmotic pressure of the injection after adding the osmotic pressure regulator is 275-285mOsm / kg.

6. The injection according to claim 1, characterized in that: The method for preparing the carrier loaded with adipose stem cell exosomes comprises the following steps: (1) preparing a 0.4% to 0.6% hydroxyapatite nanosuspension and subjecting it to ultrasonic treatment until the particle size is ≤60 nm; (2) mixing hydroxyapatite and sodium alginate, adding 0.08% to 0.12% calcium chloride for cross-linking, controlling the stirring speed at 200-400 rpm at a pH of 6.8-7.2, and the reaction time at 30-60 min. After cross-linking, the product was sterile filtered through a 0.22 μm filter membrane; (3) drying under supercritical CO2 conditions at a pressure of 7-9 MPa, a temperature of 30-40°C, and a time of 1.5-2.5 h to obtain microcarriers; (4) The adipose-derived stem cell exosomes and microcarriers are mixed at a drug loading amount of 0.8-1.0 mg / mL, and stirred at a temperature of 4-8° C. and a rotation speed of 100-200 rpm for 2-4 hours to obtain a carrier loaded with adipose-derived stem cell exosomes.

7. The injection according to claim 1, characterized in that: The method for preparing adipose stem cell exosomes comprises the following steps: Adipose-derived stem cells were cultured in a bioreactor at 35-38°C to a density of 1 × 10 6 / mL, apply 1.0-1.4kV / cm square wave pulse, pulse width 40-60μs, frequency 8-12Hz, culture statically for 12-24 hours, collect the supernatant by centrifugation, concentrate with 100kDa ultrafiltration membrane, and use size exclusion chromatography to collect 30-150nm particle size components to obtain adipose stem cell exosomes.

8. The injection according to claim 1, characterized in that: The viscosity of the injection solution is 1.2-1.5 mPa·s.

9. A method for preparing the injection according to claims 1-8, characterized in that: The following steps are involved: At 2-8°C, the carrier loaded with adipose stem cell exosomes is mixed with a hyaluronic acid solution with a molecular weight of 1.0-2.0 MDa, and an osmotic pressure regulator is added to adjust the osmotic pressure to 275-285 mOsm / kg, followed by aseptic packaging to obtain adipose stem cell exosome injection.

10. Use of the injection according to any one of claims 1 to 8 in acupoint injection therapy, characterized in that: include: Select Zusanli, Guanyuan and Shenshu acupoints, insert the needle to a depth of 1.5-3.0 cm, inject 0.5-1.0 mL each time, and the treatment cycle is once a week for 4 weeks.

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