Preparation method of novel ginseng exosome nano drug delivery system

Abstract

The invention discloses a preparation method of a novel ginseng exosome nano drug delivery system, and mainly relates to the field of functional medical materials. Comprising the following steps: S1, preparing a ginseng exosome crude extract; s2, centrifuging the crude extract and taking supernate; s3, centrifuging the supernate, and taking the precipitate to obtain an exosome resuspension; s4, preparing a purified ginseng exosome; s5, preparing kaempferol nano-enzyme freeze-dried powder; s6, mixing the ginseng exosome solution with the kaempferol nano-enzyme freeze-dried powder to obtain a ginseng exosome kaempferol nano-enzyme mixed solution; and S7, separating the ginseng exosome loaded with the kaempferol nano enzyme to obtain the ginseng exosome nano delivery system. The preparation method has the beneficial effects that the plant active ingredient ginseng exosome is taken as a delivery carrier to carry the water-soluble kaempferol nano-enzyme, and the water-soluble kaempferol nano-enzyme and the ginseng exosome play a synergistic role, so that the effective loading capacity of the exosome on the medicine is improved, the concentration of the medicine is improved, and the preparation method plays a role in various diseases such as wounds and inflammation; the traditional Chinese medicine composition can be better applied to treatment of acute lung injury.

Description

Technical Field

[0001] The present invention relates to the field of functional medical materials, and in particular to a method for preparing a novel ginseng exosome nano drug delivery system. Background Art

[0002] Ginseng is a traditional herb widely used in traditional Chinese medicine and is believed to have a variety of health benefits. Ginseng health benefits include boosting the immune system, anti-fatigue, anti-aging, antioxidant, anti-inflammatory, blood sugar regulation, etc.

[0003] Exosomes are a type of small vesicles secreted by cells, usually about 100nm in diameter, carrying proteins, lipids, RNA and other molecules in the cells, and can transmit signals between cells. Ginseng exosomes refer to small vesicles secreted from ginseng cells. Studies have found that they contain a variety of ingredients related to the medicinal effects of ginseng, including molecules that contribute to immunomodulation, anti-inflammatory and antioxidant effects. Due to the advantages of improving drug targeting, enhancing drug efficacy, and reducing side effects, the application of ginseng exosomes in disease treatment, especially in the fields of cancer, immune-related diseases, neurodegenerative diseases, etc., may bring new treatment approaches.

[0004] Nanozymes are a class of nanomaterials with enzyme-like activity, catalytic properties, and the ability to mimic the biological functions of traditional enzymes. Unlike natural enzymes, nanozymes are usually made of inorganic or organic nanomaterials and can achieve enzyme-like catalytic reactions without the structure and amino acid sequence of natural enzymes. Nanozymes made from traditional Chinese medicine ingredients are made by combining the active ingredients of traditional Chinese medicine, such as flavonoids, with nanomaterials. They are an emerging field of research in recent years, combining the traditional pharmacological effects of traditional Chinese medicine with the advantages of modern nanotechnology, opening up new drug development and treatment strategies. By combining traditional Chinese medicine ingredients with nanozymes, it is possible to enhance their bioavailability, improve their stability, and improve their therapeutic effects while retaining the active ingredients of traditional Chinese medicine.

[0005] Acute lung injury is an acute, diffuse pulmonary inflammatory syndrome caused by infection, trauma or systemic inflammatory response, characterized by alveolar-capillary barrier destruction, pulmonary edema and hypoxemia. In severe cases, it can develop into acute respiratory distress syndrome, which is life-threatening. Ginseng and kaempferol show significant potential in the treatment of ALI. Ginseng is rich in ginsenosides and polysaccharides, and has powerful anti-inflammatory, antioxidant and immunomodulatory effects. It reduces lung inflammation by inhibiting inflammatory signaling pathways and reducing the release of proinflammatory factors; at the same time, its antioxidant components remove excess free radicals, reduce oxidative stress, protect lung tissue and promote repair. Kaempferol, as a natural flavonoid compound, significantly inhibits lung inflammation by inhibiting inflammatory signaling pathways and reducing the release of inflammatory mediators. In addition, kaempferol can also enhance the activity of antioxidant enzymes such as superoxide dismutase and catalase, effectively reducing oxidative stress, regulating immune responses and preventing lung tissue damage. The synergistic effect of the two can not only relieve inflammation and oxidative stress, but also promote lung tissue repair, providing new ideas and prospects for the treatment of acute lung injury. Therefore, it is particularly important to develop a ginseng exosome carrying kaempferol nanozyme as a nano drug delivery system for the treatment of wounds, inflammation and other diseases, especially lung injury. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a new type of ginseng exosome nano drug delivery system, which uses ginseng exosomes, a plant active ingredient, as a delivery carrier, carries water-soluble kaempferol nanozymes, and works synergistically with ginseng exosomes, thereby increasing the effective loading capacity of exosomes for drugs and increasing drug concentration. It plays a role in various diseases such as wounds and inflammation, and can be better applied to the treatment of acute lung injury.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A method for preparing a novel ginseng exosome nano drug delivery system comprises the following steps:

[0009] S1. Wash and cut fresh ginseng into small pieces, transfer to a blender to extract juice and filter to obtain a crude filtrate of ginseng exosomes, centrifuge the crude filtrate of ginseng exosomes at 1000-2500g for 5-20min to remove macromolecular precipitates, and take the supernatant as the crude ginseng exosome extract;

[0010] S2. Transfer the crude ginseng exosome extract in S1 to a centrifuge tube, centrifuge at 1200-3000g for 5-30min and 3000-6000g for 2-30min at 4°C using differential centrifugation, discard the bottom precipitate, and take the supernatant;

[0011] S3. The supernatant obtained in S2 was centrifuged again at 8000-180000g for 0.5-2h, the precipitate was taken, and resuspended with PBS to obtain a heavy suspension of ginseng exosomes;

[0012] S4. Prepare sucrose solutions of different concentrations and add them to ultracentrifuge tubes in sequence, then add the ginseng exosomes resuspension in S3 and centrifuge to obtain a band containing ginseng exosomes, mix the band with PBS and centrifuge again to remove sucrose, collect the precipitate and resuspend it with PBS to obtain purified ginseng exosomes;

[0013] S5. Polyvinyl pyrrolidone, FeCl3·6H2O solution and kaempferol were dissolved in methanol respectively, and then the FeCl3·6H2O solution was slowly added to the polyvinyl pyrrolidone solution at a rate of 0.5 mL / s, and the mixture was fully stirred at room temperature, and then the kaempferol solution was slowly added to the above mixture at a rate of 0.5 mL / s. After stirring for 4 hours, the methanol in the system was removed by dialysis, and the kaempferol nanozyme freeze-dried powder was obtained by freeze drying for 24 hours;

[0014] S6. The purified ginseng exosomes in S4 are diluted with PBS to form an exosome solution, and the kaempferol nanozyme freeze-dried powder in S5 is added to the exosome solution, and the mixture is fully mixed on a shaker, and the mixture is incubated at room temperature for 6 hours to allow the kaempferol nanozyme to be loaded onto the ginseng exosomes to obtain a ginseng exosome kaempferol nanozyme mixed solution;

[0015] S7. Add the prepared sucrose solutions of different gradients to the ginseng exosomes-kaempferol nanozyme mixture in S6 to separate the ginseng exosomes carrying kaempferol nanozyme to obtain the ginseng exosome nanodelivery system.

[0016] Furthermore, in the step S1, the weight ratio of the PBS solution to the fresh ginseng is 5:1-5, the crushing time is 1-5 minutes, and the mesh number of the gauze used for filtering is 100-300 meshes.

[0017] Furthermore, the step S4 is specifically as follows:

[0018] Prepare 8%, 30%, 45% and 60% sucrose PBS solutions respectively, add the ginseng exosome resuspension in S3 into an ultracentrifuge tube, and slowly add 8%, 30%, 45% and 60% sucrose PBS solutions from the bottom in turn. Finally, add the resuspension to 2 mm from the heart tube orifice. After centrifugation at 100000-300000g for 2-5 hours, collect the bands between 30% and 45% sucrose density, add an equal amount of PBS to the bands and resuspend them. Centrifuge at 50000-150000g for 1-3 hours to remove the sucrose. Collect the precipitate and resuspend it with PBS to obtain the ginseng exosome solution.

[0019] Furthermore, the concentration range of the ginseng exosome heavy suspension obtained in step S3 is 150-400 mg / mL.

[0020] Furthermore, in step S5, the mass ratio of polyvinyl pyrrolidone, FeCl3·6H2O and kaempferol is 10:4:1.

[0021] Furthermore, the mass ratio of the ginseng exosomes to the kaempferol nanozyme is 120-260:1.

[0022] Furthermore, the particle size of the ginseng exosome nano-delivery system is 81.3±0.76 nm.

[0023] Furthermore, the prepared new ginseng exosome nano-drug delivery system can be used in the treatment of acute lung injury.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The present invention can combine ginseng exosomes with kaempferol nanozymes to prepare ginseng exosomes carrying kaempferol nanozymes as ginseng exosome nanodelivery systems, so that ginseng exosomes can be successfully extracted and used as drug carriers, inheriting the natural pharmacological properties of ginseng, filling the application gap of ginseng exosomes in the field of drug delivery, and can also prepare the poorly soluble drug kaempferol into water-soluble kaempferol nanozymes, which not only improves the binding efficiency and loading capacity of the drug and the exosome carrier, but also enhances the drug delivery effect, so that the ginseng exosome nanodelivery system of the present invention can utilize the enzyme catalytic properties of kaempferol nanozymes themselves and synergize with ginseng exosomes, and show excellent antioxidant and anti-inflammatory effects in diseases such as acute lung injury;

[0026] 2. The ginseng exosome nano-delivery system of the present invention has uniform particle size distribution, strong free radical scavenging ability, low cytotoxicity, significant anti-inflammatory and antioxidant effects, good therapeutic potential for diseases such as acute lung injury, and shows broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Attached Figure 1 This is a preparation diagram of the ginseng exosome nanodelivery system carrying kaempferol nanozyme of the present invention.

[0028] Attached Figure 2 This is a diagram of the ginseng exosome preparation of the present invention.

[0029] Attached Figure 3 It is a synthetic route map of the kaempferol nanozyme material of the present invention.

[0030] Attached Figure 4 This is a diagram of the freeze-dried kaempferol nanozyme preparation of the present invention.

[0031] Attached Figure 5 This is an electron microscope image of the ginseng exosomes of the present invention.

[0032] Attached Figure 6 This is a diagram of the particle size of ginseng exosomes of the present invention.

[0033] Attached Figure 7 It is a particle size and potential diagram of the ginseng exosomes, kaempferol nanozyme, and ginseng exosome nanodelivery system of the present invention.

[0034] Attached Figure 8 This is a graph showing the results of a DPPH free radical scavenging test using the ginseng exosome nano-delivery system of the present invention.

[0035] Attached Fig. 9 This is a graph showing the results of the ABTS free radical scavenging test of the ginseng exosome nano-delivery system of the present invention.

[0036] Attached Fig.10 It is the ultraviolet absorption spectrum of the kaempferol nanozyme of the present invention.

[0037] Attached Fig.11 This is a diagram showing the cytotoxicity evaluation results of the ginseng exosome nanodelivery system of the present invention.

[0038] Attached Fig.12 This is a diagram showing the experimental results of cell uptake of the ginseng exosome nano-delivery system of the present invention.

[0039] Attached Fig.13 It is a diagram showing the results of a hemolysis experiment of the ginseng exosome delivery system of the present invention.

[0040] Attached Fig.14 This is a diagram of the ginseng exosome delivery system of the present invention regulating mitochondrial membrane potential.

[0041] Attached Fig.15 Schematic diagram of the active oxygen scavenging ability of the ginseng exosome delivery system of the present invention.

[0042] Numbers shown in the accompanying drawings: DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the application equally.

[0044] The present invention discloses a method for preparing a novel nano drug delivery system of ginseng exosomes, comprising the following steps:

[0045] S1. Wash and cut fresh ginseng into small pieces, transfer to a blender to extract juice and filter to obtain a crude filtrate of ginseng exosomes, centrifuge the crude filtrate of ginseng exosomes at 1000-2500g for 5-20min to remove macromolecular precipitation, and the centrifugation temperature is 4°C. Take the supernatant as the crude ginseng exosome extract, wherein the centrifugal speed is more preferably 1500-2000g, most preferably 1800g, and the centrifugation time is preferably 8-15min, more preferably 10min;

[0046] S2. Transfer the crude ginseng exosome extract in S1 to a centrifuge tube, centrifuge at 1200-3000g for 5-30min and 3000-6000g for 2-30min at 4°C using differential centrifugation, discard the bottom precipitate, and take the supernatant;

[0047] S3. The supernatant obtained in S2 is centrifuged at 8000-180000g for 0.5-2h, the precipitate is taken, and resuspended with PBS to obtain a ginseng exosome resuspension, wherein the centrifugal speed is preferably in the range of 100000-150000g, and the best is 120000g, and the centrifugal time is preferably 1-1.5 hours, and the best is 1 hour;

[0048] S4. Prepare sucrose solutions of different concentrations and add them to ultracentrifuge tubes in sequence, then add the ginseng exosomes resuspension in S3 and centrifuge to obtain a band containing ginseng exosomes, mix the band with PBS and centrifuge again to remove sucrose, collect the precipitate and resuspend it with PBS to obtain purified ginseng exosomes;

[0049] S5. Polyvinyl pyrrolidone, FeCl3·6H2O solution and kaempferol were dissolved in methanol respectively, and then the FeCl3·6H2O solution was slowly added to the polyvinyl pyrrolidone solution at a rate of 0.5 mL / s, and the mixture was fully stirred at room temperature, and then the kaempferol solution was slowly added to the above mixture at a rate of 0.5 mL / s. After stirring for 4 hours, the methanol in the system was removed by dialysis, and the kaempferol nanozyme freeze-dried powder was obtained by freeze drying for 24 hours;

[0050] S6. The purified ginseng exosomes in S4 are diluted with PBS to form an exosome solution, and the kaempferol nanozyme freeze-dried powder in S5 is added to the exosome solution, and the mixture is fully mixed on a shaker, and the mixture is incubated at room temperature for 6 hours to allow the kaempferol nanozyme to be loaded onto the ginseng exosomes to obtain a ginseng exosome kaempferol nanozyme mixed solution;

[0051] S7. Add the prepared sucrose solutions of different gradients to the ginseng exosomes-kaempferol nanozyme mixture in S6 to separate the ginseng exosomes carrying kaempferol nanozyme to obtain the ginseng exosome nanodelivery system.

[0052] Preferably, in step S1, the weight ratio of PBS solution to fresh ginseng is 5:1-5, more preferably 5:2-3, most preferably 5:2.5, the crushing time is 1-5 min, more preferably 2-4 min, most preferably 3 min, and the mesh number of gauze used for filtering is 100-300 mesh, more preferably 200 mesh.

[0053] Preferably, the step S4 is specifically:

[0054] Prepare 8%, 30%, 45% and 60% sucrose PBS solutions respectively, add the ginseng exosome resuspension in S3 into an ultracentrifuge tube, and slowly add 8%, 30%, 45% and 60% sucrose PBS solutions from the bottom in turn. Finally, add the resuspension to 2 mm from the heart tube orifice. After centrifugation at 100000-300000g for 2-5 hours, collect the bands between 30% and 45% sucrose density, add an equal amount of PBS to the bands and resuspend them. Centrifuge at 50000-150000g for 1-3 hours to remove the sucrose. Collect the precipitate and resuspend it with PBS to obtain the ginseng exosome solution.

[0055] Preferably, the concentration range of the ginseng exosome heavy suspension obtained in step S3 is 150-400 mg / mL, preferably 200-300 mg / mL, and the optimal concentration is 250 mg / mL.

[0056] Preferably, in step S5, the mass ratio of polyvinyl pyrrolidone, FeCl3·6H2O and kaempferol is 10:4:1.

[0057] Preferably, the mass ratio of the ginseng exosomes to the kaempferol nanozyme is 120-260:1, more preferably 140-220:1, and most preferably 180:1.

[0058] Preferably, the particle size of the ginseng exosome nano-delivery system is 88.5±1.42 nm, more preferably 82.1±1.88 nm, and most preferably 81.3±0.76 nm.

[0059] Preferably, the prepared novel ginseng exosome nano-drug delivery system can be used in the treatment of acute lung injury.

[0060] Example 1: A method for preparing a novel nano drug delivery system of ginseng exosomes, comprising the following steps:

[0061] S1. Wash fresh ginseng with deionized water, wipe dry, weigh 1.5 kg and cut into small pieces, transfer to a crusher, add 1 L of PBS solution, and crush at medium-high speed for 2 minutes to obtain ginseng homogenate. Filter the ginseng homogenate with 200 mesh gauze to obtain ginseng exosome crude filtrate, transfer the ginseng exosome crude filtrate to a centrifuge tube, centrifuge at 4°C and 1800g for 10 minutes to remove macromolecular precipitates, and take the supernatant as ginseng exosome crude extract;

[0062] S2. The crude extract of ginseng exosomes in S1 was divided into 50mL centrifuge tubes and accurately balanced to ensure the smooth progress of the centrifugation process. The tubes were centrifuged at 1800g for 10 minutes at 4°C using differential centrifugation. This step aims to effectively remove large particle impurities contained in the crude extract. After the first centrifugation, the centrifuge tubes were removed and the upper supernatant was transferred to a new 50mL centrifuge tube and accurately balanced again. Then, under the same 4°C temperature condition, the speed of the centrifuge was adjusted to 4000g, and the sample was centrifuged for 20 minutes. The main purpose of this centrifugation was to completely remove the broken cell fragments in the solution. Finally, the upper pure supernatant was carefully retained for subsequent use or analysis.

[0063] S3. The supernatant obtained in S2 was centrifuged at 120,000 g in an ultra-high speed centrifuge at 4°C for 1 hour; this step is to remove macromolecular proteins and soluble impurities in the supernatant; after centrifugation, the supernatant was discarded, and only the crude ginseng exosome extract in the precipitate was retained. Subsequently, a 0.1M PBS solution was aspirated using a 1000μL pipette, and the PBS solution was carefully dripped into the container containing the crude ginseng exosome extract. By gently and repeatedly aspirating and releasing, it was ensured that the solution reached a uniform state without agglomeration. Finally, the concentration of ginseng exosomes was adjusted to 200 mg / mL using PBS to obtain a heavy suspension of ginseng exosomes;

[0064] S4. Prepare sucrose PBS solutions of different concentrations, 8%, 30%, 45% and 60%, respectively, add the ginseng exosome heavy suspension in S3 to an ultracentrifuge tube, slowly add 8%, 30%, 45% and 60% sucrose PBS solutions from the bottom, and finally add 1 mL of ginseng exosome heavy suspension. After balancing the centrifuge tube, centrifuge at 180000g for 4 hours, collect the bands containing ginseng exosomes between 30% and 45% sucrose density, take a new ultracentrifuge tube, add 1 mL of purified ginseng exosome bands and 10 mL of PBS solution, centrifuge at 100000g for 1 hour to remove the residual sucrose solution in the bands, collect the precipitate and resuspend it with 1 mL of PBS to obtain the purified ginseng exosome solution. The protein concentration was determined to be 100 μg / mL.

[0065] S5. Figure 3 As shown, according to the preset ratio, that is, the mass ratio of polyvinyl pyrrolidone (PVP), FeCl3·6H2O and kaempferol is 10:4:1, firstly, the three substances are dissolved in methanol solvent respectively to prepare 15 mg / mL PVP solution, 20 mg / mL FeCl3·6H2O solution and 10 mg / mL kaempferol solution, then, 0.8 mL of FeCl3·6H2O solution is slowly added to the PVP solution at a rate of 0.5 mL / s, and stirred for 5 min to ensure uniform mixing, then, 1 mL of kaempferol solution is added to the above mixture at a dropping rate of 0.5 mL / s, stirred again for 4 h to fully react, and the product is dialyzed using a dialysis bag with a molecular weight cutoff of 2000 Da, dialyzed for 24 h to remove methanol in the system, and then the dialyzed product is freeze-dried at -50 ° C for 24 h to finally obtain a freeze-dried powder of kaempferol nanozyme, as shown in Figure 4 As shown;

[0066] S6. The purified ginseng exosomes in S4 were diluted with 0.1M PBS to form a 200 mg / mL exosome solution, and the kaempferol nanozyme freeze-dried powder in S5 was added according to the mass ratio of ginseng exosomes to kaempferol nanozyme of 150:1, and the solution was shaken on a constant temperature shaker at 37°C for 6 hours to fully mix the solution and load the kaempferol nanozyme onto the ginseng exosomes to obtain a ginseng exosome kaempferol nanozyme mixed solution;

[0067] S7. Add the prepared sucrose solutions of different gradients to the ginseng exosome kaempferol nanozyme mixture in S6 to separate the ginseng exosomes loaded with kaempferol nanozyme to obtain a ginseng exosome nanodelivery system with a particle size of 66.34±1.88nm, as shown in the following figure: Figure 1 shown.

[0068] In the present invention, the resuspension operation is specifically to use a 1000 μL pipette to absorb an appropriate amount of PBS solution, slowly inject the PBS into the substance to be resuspended, and gently and repeatedly absorb several times below the liquid surface until the solution is evenly distributed. In addition, in the present invention, all centrifugation steps need to be balanced. The present invention does not limit the specific conditions for balancing, and technicians can use conventional operating methods known in the art to process.

[0069] Example 2: Electron microscopic characterization of ginseng exosomes:

[0070] The ginseng exosome solution was fixed on a copper grid and stained, and then photographed using a transmission electron microscope. The resulting appearance is as follows Figure 5 As shown in the figure, all ginseng exosomes are regular spherical, which indicates that exosomes have a relatively consistent morphological structure.

[0071] Example 3: Particle size and potential characterization of ginseng exosomes:

[0072] The prepared ginseng exosomes, kaempferol nanozymes and ginseng exosome nanodelivery system were filtered through a 0.8 μm microporous filter membrane, and then the particle size and potential were detected by a particle size analyzer. The experimental results are as follows: Figure 6 , 7 As shown, the results showed that after passing through a 0.8μm microporous filter membrane, the particle size of ginseng exosomes was 63.3±0.21nm, the particle size distribution was uniform, and the potential was -8.74±0.14mV; the particle size of kaempferol nanozyme was 22.5±0.75nm, the particle size distribution was uniform, and the potential was -17.36±0.49mV; the particle size of ginseng exosome nanodelivery system was 81.3nm±0.76, the particle size was uniform, and the potential was -12.4±0.36mV.

[0073] Example 4: Kaempferol nanozyme DPPH free radical scavenging antioxidant experiment:

[0074] Different concentrations of kaempferol nanozymes were used as experimental objects, and the antioxidant properties were tested by using DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) standard. In the experiment, the antioxidant capacity of the samples was accurately quantified using an ELISA instrument, and the ultraviolet absorption spectrum of the solution was scanned and analyzed using an ultraviolet spectrophotometer. The results are as follows: Figure 8 As shown. It was observed that with the gradual increase in the concentration of kaempferol nanozyme, the colors of the two color reaction solutions showed a trend of gradually fading. Further, according to the quantitative data provided by the microplate reader, it can be clearly seen that the increase in the concentration of kaempferol nanozyme is accompanied by a significant increase in the DPPH inhibition rate, that is, the total antioxidant capacity has been correspondingly enhanced. Therefore, it can be scientifically inferred that kaempferol nanozyme has the ability to scavenge free radicals and exhibit antioxidant activity.

[0075] Example 5: Kaempferol nanozyme ABTS total antioxidant experiment:

[0076] Different concentrations of the nanozyme were used for quantitative analysis using the total antioxidant capacity test kit (ABTS). During the experiment, we accurately determined the antioxidant capacity of kaempferol nanozyme at various concentrations using an ELISA instrument. Fig. 9 As shown, the experimental results show that as the concentration of kaempferol nanozyme increases, its antioxidant capacity shows an increasing trend. Therefore, based on the above scientific data and graph analysis, it can be concluded that kaempferol nanozyme has significant antioxidant capacity.

[0077] Example 6: UV absorption spectrum of ginseng exosomes and their preparations:

[0078] After the exosome solution loaded with kaempferol nanozyme was separated and purified by sucrose gradient centrifugation, the obtained product was resuspended in PBS. Subsequently, the resuspended solution was subjected to UV spectral scanning analysis using a Shimadzu UV-visible spectrophotometer, and the scanning wavelength range was set to 200nm-800nm. Fig.10 As shown, after the kaempferol nanozyme was loaded onto the ginseng exosomes, the maximum peak of ultraviolet absorption attributed to the kaempferol nanozyme was observed at a wavelength of about 380 nm.

[0079] Example 7: Cytotoxicity evaluation of ginseng exosomes and their preparations:

[0080] The CCK-8 cell viability assay was used to analyze the cytotoxicity of ginseng exosomes and ginseng exosome nanodelivery system. RAW264.7 macrophage cell line was selected as the test object, and the cell viability was tested for 24 hours. Fig.11 As shown, it is specifically shown that within the 24-hour culture period, ginseng exosomes and ginseng exosome nanodelivery system have little toxic effect on RAW264.7 cells, and the cell survival rate of each test group is maintained above 90%.

[0081] Experimental Example 8: Study on Cellular Uptake and Distribution of Ginseng Exosome Delivery System:

[0082] Laser confocal microscopy was used to observe the distribution of nanoformulations in HUVEC cells after endocytosis. FITC was used as a fluorescent dye, and the prepared ginseng exosomes, kaempferol nanozymes and ginseng exosome nanodelivery systems were loaded. As the concentration increased, the phagocytosis of HUVEC cells was observed. The experimental results showed that as the uptake time of different preparations increased, the cell fluorescence intensity increased accordingly, indicating that the cellular uptake behavior of HUVEC cells for the ginseng exosome nanodelivery system was time-dependent, proving that the ginseng exosome nanocarriers have better bioavailability and can achieve better therapeutic purposes. The experimental results are as follows Fig.12 shown.

[0083] Experimental Example 9: Hemolysis experiment of ginseng exosome delivery system:

[0084] Hemolysis experiments were used to evaluate the blood safety of different formulations of carriers. First, red blood cell suspensions were prepared by collecting blood from SD rats. Subsequently, physiological saline solutions of different preparation groups were prepared to the same concentration (1.0 mg / mL), 300 μL of red blood cell suspension was added to each group, and then 900 μL of solution was added to each group. After standing at room temperature for 6 hours, the mixture was centrifuged at 4°C and 800 g for 10 minutes to observe hemolysis. The hemolysis rate was analyzed by measuring the absorbance at 540 nm using an enzyme reader. The experimental results showed that no obvious hemolysis occurred in the different preparations, proving that the ginseng exosome nanodelivery system has good biosafety. The experimental results are as follows: Fig.13 shown.

[0085] Experimental Example 10: Ginseng exosome delivery system regulates mitochondrial membrane potential:

[0086] The JC-1 mitochondrial membrane potential detection kit was used to detect the changes in mitochondrial membrane potential of HUVEC cells induced by LPS. In brief, HUVEC cells were seeded into culture dishes at a density of 5×104 cells / well and cultured overnight. Before LPS induction, the cells were treated with the same concentration of ginseng exosomes, kaempferol nanozymes, and ginseng exosome nanodelivery system for 2 hours, and then LPS (1ug / mL) was added. After the treatment, the cells were rinsed three times with PBS, and then incubated with 20μM JC-1 at 37°C for 20 minutes. After removing the JC-1 staining solution, the cells were washed three times with JC-1 staining buffer, and then 2mL PBS was added for CLSM imaging. The results showed that LPS induction led to an increase in the J-monomer content and the cells were in an apoptotic state. After treatment with different preparations, the J-monomer content decreased and the J-polymer content increased. The ginseng exosome nanodelivery system can effectively reverse the mitochondrial membrane potential and inhibit cell apoptosis. The results are shown in Fig.14 shown.

[0087] Experimental Example 11: Active oxygen scavenging ability of ginseng exosome delivery system:

[0088] The DCFH-DA fluorescent probe was used to evaluate the changes in the level of reactive oxygen species in HUVEC cells. After LPS (1 μg / mL) activated HUVEC cells, different preparations of the drug were added at equal doses and incubated for another 12 hours. The culture medium was then replaced with fresh culture medium containing 5 μM DCFH-DA, cultured at 37°C in the dark for 0.5 hours, and the changes in intracellular ROS were observed under a fluorescence microscope. The experimental results are shown in Figure 2. Fig.15 As shown, LPS induction caused a significant increase in the reactive oxygen content of HUVEC cells, and the reactive oxygen content of different preparation groups decreased significantly after administration, among which the ginseng exosome nanodelivery system had the best antioxidant effect.

[0089] In summary, the present invention can combine ginseng exosomes with kaempferol nanozymes to prepare ginseng exosomes carrying kaempferol nanozymes as ginseng exosome nanodelivery systems, so that it can successfully extract and use ginseng exosomes as drug carriers, inherit the natural pharmacological properties of ginseng, fill the application gap of ginseng exosomes in the field of drug delivery, and can also prepare the poorly soluble drug kaempferol into water-soluble kaempferol nanozymes, which not only improves the binding efficiency and loading capacity of the drug and the exosome carrier, but also enhances the drug delivery effect, so that the ginseng exosome nanodelivery system of the present invention can utilize the enzyme catalytic properties of kaempferol nanozymes themselves and synergize with ginseng exosomes, and show excellent antioxidant and anti-inflammatory effects in diseases such as acute lung injury; and the ginseng exosome nanodelivery system of the present invention has uniform particle size distribution, strong free radical scavenging ability, low cytotoxicity, significant anti-inflammatory and antioxidant effects, good therapeutic potential for diseases such as acute lung injury, and shows broad application prospects.

Claims

1. A method for preparing a novel nano drug delivery system of ginseng exosomes, characterized in that: The following steps are involved: S1. Wash and cut fresh ginseng into small pieces, transfer to a blender to extract juice and filter to obtain a crude filtrate of ginseng exosomes, centrifuge the crude filtrate of ginseng exosomes at 1000-2500g for 5-20min to remove macromolecular precipitates, and take the supernatant as the crude ginseng exosome extract; S2. Transfer the crude ginseng exosome extract in S1 to a centrifuge tube, centrifuge at 1200-3000g for 5-30min and 3000-6000g for 2-30min at 4°C using differential centrifugation, discard the bottom precipitate, and take the supernatant; S3. The supernatant obtained in S2 was centrifuged again at 8000-180000g for 0.5-2h, the precipitate was taken, and resuspended with PBS to obtain a heavy suspension of ginseng exosomes; S4. Prepare sucrose solutions of different concentrations and add them to ultracentrifuge tubes in sequence, then add the ginseng exosomes resuspension in S3 and centrifuge to obtain a band containing ginseng exosomes, mix the band with PBS and centrifuge again to remove sucrose, collect the precipitate and resuspend it with PBS to obtain purified ginseng exosomes; S5. Polyvinyl pyrrolidone, FeCl3·6H2O solution and kaempferol were dissolved in methanol respectively, and then the FeCl3·6H2O solution was slowly added to the polyvinyl pyrrolidone solution at a rate of 0.5 mL / s, and the mixture was fully stirred at room temperature, and then the kaempferol solution was slowly added to the above mixture at a rate of 0.5 mL / s. After stirring for 4 hours, the methanol in the system was removed by dialysis, and the kaempferol nanozyme freeze-dried powder was obtained by freeze drying for 24 hours; S6. The purified ginseng exosomes in S4 are diluted with PBS to form an exosome solution, and the kaempferol nanozyme freeze-dried powder in S5 is added to the exosome solution, and the mixture is fully mixed on a shaker, and the mixture is incubated at room temperature for 6 hours to allow the kaempferol nanozyme to be loaded onto the ginseng exosomes to obtain a ginseng exosome kaempferol nanozyme mixed solution; S7. Add the prepared sucrose solutions of different gradients to the ginseng exosomes-kaempferol nanozyme mixture in S6 to separate the ginseng exosomes carrying kaempferol nanozyme to obtain the ginseng exosome nanodelivery system.

2. The method for preparing a novel ginseng exosome nano drug delivery system according to claim 1, characterized in that: In the step S1, the weight ratio of PBS solution to fresh ginseng is 5:1-5, the crushing time is 1-5 minutes, and the mesh number of the gauze used for filtering is 100-300 meshes.

3. The method for preparing a novel ginseng exosome nano drug delivery system according to claim 1, characterized in that: The step S4 is specifically as follows: Prepare 8%, 30%, 45% and 60% sucrose PBS solutions respectively, add the ginseng exosome resuspension in S3 into an ultracentrifuge tube, and slowly add 8%, 30%, 45% and 60% sucrose PBS solutions from the bottom in turn. Finally, add the resuspension to 2 mm from the heart tube orifice. After centrifugation at 100000-300000g for 2-5 hours, collect the bands between 30% and 45% sucrose density, add an equal amount of PBS to the bands and resuspend them. Centrifuge at 50000-150000g for 1-3 hours to remove the sucrose. Collect the precipitate and resuspend it with PBS to obtain the ginseng exosome solution.

4. The method for preparing a novel ginseng exosome nano drug delivery system according to claim 1, characterized in that: The concentration range of the ginseng exosome heavy suspension obtained in step S3 is 150-400 mg / mL.

5. The method for preparing a novel ginseng exosome nano drug delivery system according to claim 1, characterized in that: In the step S5, the mass ratio of polyvinyl pyrrolidone, FeCl3·6H2O and kaempferol is 10:4:

1.

6. The method for preparing a novel ginseng exosome nano drug delivery system according to claim 1, characterized in that: The mass ratio of the ginseng exosomes to the kaempferol nanozyme is 120-260:

1.

7. The method for preparing a novel ginseng exosome nano drug delivery system according to claim 1, characterized in that: The particle size of the ginseng exosome nanodelivery system is 81.3±0.76 nm.

8. The method for preparing a novel ginseng exosome nano drug delivery system according to any one of claims 1 to 7, characterized in that: The prepared new ginseng exosome nano drug delivery system can be used in the treatment of acute lung injury.

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