Exosome-loaded curcumin nano sustained-release drug delivery system and application thereof

Abstract

The application discloses a curcumin nano-loaded exosome sustained-release drug delivery system and application thereof, and belongs to the technical field of biological medicines. A curcumin solid lipid nano-particle Cur-SLN is transfected into human bronchial epithelial cells BEAS-2B by using an active loading method, and then a modified ultracentrifugation method is used to collect exosomes Cur-SLN-Exo. The curcumin nano-loaded exosome sustained-release drug delivery system has the beneficial effects that after inhalation administration, the Cur-SLN-Exo is deposited in small airways, due to the homology between the exosomes from target cells and the cells in the body, the clearance of the body circulation system can be avoided, the loaded drug is not degraded in the extracellular environment, the special structure of the lipids and proteins on the surface of the exosomes can be combined with the target cells and then rapidly fused with the target cells, the loaded drug can be delivered into the cells, compared with a lipid nano drug delivery system, the drug loading capacity can be improved, the administration frequency and dose can be reduced, and the lung action time can be prolonged, and the curcumin nano-loaded exosome sustained-release drug delivery system provides a treatment scheme for COPD mitochondrial damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a sustained-release drug delivery system for exosomes loaded with curcumin nanoparticles and its application. Background Technology

[0002] Extracellular vesicles (EVs) and other bioparticles, as alternatives to lung nanoparticles (LNPs), have been optimized for mRNA encapsulation, cellular uptake, and delivery. EVs, including exosomes (Exosomes), are nanoscale vesicles secreted by various cell types into almost all biological fluids. Exosomes have known therapeutic properties in various disease applications. Exosomes contain a variety of molecular components, including RNA and proteins, which vary depending on their cellular origin. Exosomes play an important role in maintaining respiratory microenvironment homeostasis and supporting normal lung function. They deliver bioactive components such as genetic material and proteins to recipient cells, reducing the release of inflammatory factors, alleviating inflammatory responses, inhibiting alveolar macrophage apoptosis and promoting proliferation, repairing damaged alveolar capillaries, promoting regeneration, preventing fibrosis, and protecting lung integrity. Mesenchymal stem cells (MSCs) and MSC-derived exosomes are considered potential therapeutic agents for lung inflammatory and fibrotic diseases such as ARDS due to their significant immunomodulatory effects. Exosomes secreted by lung cells may contain molecular components and membrane features recognized in the lung microenvironment. Using lung-derived exosomes as delivery carriers for inhaled drugs can more effectively evade immune clearance while enhancing the targeting ability of lung cells, thereby increasing drug retention and efficacy in the lungs. In addition to drug delivery, lung-derived exosomes themselves have also shown therapeutic benefits.

[0003] Studies have found that, compared with exosomes derived from mesenchymal stem cells, lung-derived exosomes can better restore lung function and reduce the degree of fibrosis in animal models of idiopathic pulmonary fibrosis.

[0004] Exosomes extracted by conventional ultracentrifugation often contain impurities, such as microvesicles.

[0005] In summary, how to provide a sustained-release drug delivery system with minimal impurities loaded with curcumin nanoparticles on exosomes and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a sustained-release drug delivery system for exosome-loaded curcumin nanoparticles and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A sustained-release drug delivery system for exosomes loaded with curcumin nanoparticles is described. Curcumin solid lipid nanoparticles (Cur-SLN) are transfected into human bronchial epithelial cells BEAS-2B using an active loading method. The exosomes are then collected by ultracentrifugation. The exosomes are named Cur-SLN-Exo.

[0009] A method for preparing an exosome-loaded curcumin nanoparticle sustained-release drug delivery system includes the following steps:

[0010] Human bronchial epithelial cells BEAS-2B were cultured until the cell confluence reached 80% or more. The lyophilized powder of curcumin solid lipid nanoparticles Cur-SLN was added to the cell culture flask and incubated for 4 hours. After culturing, centrifugation, filtration, and resuspending, the cells were placed in a VP-M1 nebulizer for inhalation.

[0011] Further steps include the following:

[0012] (1) Culture of human bronchial epithelial cells BEAS-2B;

[0013] (2) Curcumin solid lipid nanoparticles (Cur-SLN) were transfected into human bronchial epithelial cells BEAS-2B:

[0014] (21) When the cell fusion degree is above 80%, the lyophilized powder of curcumin solid lipid nanoparticles Cur-SLN is diluted with fetal bovine serum culture medium to a concentration of 50-200 μg / mL containing Cur-SLN. After preheating to 37°C, the original culture medium is removed, and the diluted fetal bovine serum culture medium containing curcumin solid lipid nanoparticles Cur-SLN is added to the culture flask of human bronchial epithelial cells BEAS-2B. The flask is then placed back into the incubator and incubated for 4 hours. At this time, the human bronchial epithelial cells BEAS-2B have internalized most of the Cur-SLN into the cells.

[0015] (22) Take out the culture flask, rinse it twice with PBS preheated to 37°C, then add exosome-free serum culture medium, put it back in the incubator and continue to culture for 24 hours. At this time, the fetal bovine serum added to the culture medium has removed the exosomes it contains, so the interference of exosomes carried in the culture medium can be eliminated.

[0016] The exosome-free serum culture medium is an exosome-free high glucose culture medium with 10% FBS added;

[0017] (3) Collection of exosomes by ultracentrifugation:

[0018] The supernatant was obtained by centrifuging three times, filtering through a 0.45μm microporous membrane, precipitating by ultracentrifugation, resuspending in PBS, and precipitating by ultracentrifugation again. This yielded Cur-SLN-Exo.

[0019] Further, the specific operation of step (1) is as follows: Select human bronchial epithelial cells BEAS-2B in the logarithmic growth phase, add trypsin to digest for 5 minutes, then scrape off the adherent cells, add fetal bovine serum culture medium preheated to 37°C, and repeatedly pipette to prepare a single-cell suspension, then according to 10.0~20.0×10 5 The cells were seeded at a density of 10 cells / ml in a cell culture flask, and exosome-free serum-free medium was added. The culture flask was gently shaken to disperse the cells evenly. The flask was placed in a 37°C incubator with a 5% CO2 atmosphere and the cells were observed to adhere to the culture vessel after 24 hours of culture.

[0020] The exosome-free serum culture medium is made by adding 10% FBS to an exosome-free high-glucose culture medium.

[0021] Furthermore, in step (21), the number of dead cells is controlled to be less than 5%.

[0022] Further, the specific operation of step (3) is as follows: collect the culture medium in the cell culture flask, filter it through a 0.22μm filter, add it to a centrifuge tube, centrifuge at 4℃ and 800×g for 20min, and aspirate the supernatant; centrifuge at 4℃ and 2000×g for 20min, and aspirate the supernatant; centrifuge at 4℃ and 10000×g for 40min, and aspirate the supernatant; filter through a 0.45μm microporous membrane; after strictly balancing the collected supernatant, centrifuge at 4℃ and 130000×g for 70min, discard the supernatant, add the obtained precipitate to pre-cooled PBS, strictly balance it, continue to centrifuge at 4℃ and 130000×g for 70min, discard the supernatant, and the obtained precipitate is Cur-SLN-Exo.

[0023] Furthermore, the method for preparing the lyophilized powder of curcumin solid lipid nanoparticles (Cur-SLN) includes:

[0024] (1) The weight ratio of curcumin: glyceryl monostearate: PEG-40 stearate: poloxamer 188: Tween-80: lyophilization protectant: water is 1~1.25: 9~11: 19~22: 9~11: 19~22: 36~44: 64~79: 1800~2200;

[0025] (2) Weigh out curcumin, glyceryl monostearate and PEG-40 stearate according to the above mass ratio, dissolve them in anhydrous ethanol, and mix them on a magnetic stirrer at 75°C to obtain an organic phase.

[0026] (3) Weigh out poloxamer 188 and Tween-80 according to the above mass ratio, dissolve them in water, and stir magnetically at 75°C to dissolve and mix them evenly to obtain an aqueous phase.

[0027] (4) The organic phase was slowly injected into the aqueous phase at 75-78℃ and 1000 rpm / min under magnetic stirring, and then the temperature was raised to 85℃ and the organic solvent was removed by continuous stirring to obtain Cur-SLN thermal suspension.

[0028] (5) The obtained Cur-SLN hot suspension was then rapidly injected into the same volume of cold water at 0-4℃ under constant temperature magnetic stirring. The mixture was stirred continuously in an ice-water bath for 3 hours and then filtered through a 0.45μm filter membrane to obtain the Cur-SLN suspension.

[0029] (6) Place the Cur-SLN suspension in a freeze dryer for programmed freeze drying to obtain powder, and pass it through a 200-mesh sieve to obtain the freeze-dried powder of curcumin solid lipid nanoparticles Cur-SLN.

[0030] The application of the above-mentioned sustained-release drug delivery system and the sustained-release drug delivery system prepared by any of the above preparation methods in the preparation of drugs for treating chronic obstructive pulmonary disease.

[0031] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: Because pulmonary exosomes are homologous to body cells, they can avoid clearance from the systemic circulatory system, preventing the loaded drug from being degraded in the extracellular environment; the lipids and proteins with special surface structures can rapidly fuse with target cells after binding, delivering the loaded drug into the cells, significantly increasing the drug loading and local drug concentration of the pulmonary inhalation drug delivery system, reducing the frequency and dosage of administration, and prolonging the duration of action in the lungs. Simultaneously, because Cur-SLN-Exo has suitable particle size and stability, it can be precisely delivered to the small airways after inhalation, achieving dual-targeted delivery of COPD treatment drugs. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0033] Figure 1 The attached figure shows the particle size and concentration results of Exo and Cur-SLN-Exo in Example 2 of the present invention;

[0034] Figure 2 The attached figure shows the morphological characteristics of Exo and Cur-SLN-Exo under a transmission electron microscope in Embodiment 3 of the present invention;

[0035] Figure 3The attached figure shows the protein expression of Cur-SLN-Exo surface markers TSG101, HSP70, and CD63 in Example 4 of this invention;

[0036] Figure 4 The attached figures show the lung and tracheal irritation of rats in Example 6 of this invention after inhaling blank SLN-Exo and Cur-SLN-Exo, where A is the air control group; B is the blank SLN-Exo group; C is the Cur-SLN-Exo group; the top figure shows the trachea, and the bottom figure shows the lung.

[0037] Figure 5 The attached figure shows the pathological changes (HE) of lung inflammation in COPD mice caused by Cur-SLN-Exo in Example 7 of the present invention. The top part is a partial view of the lungs, and the bottom part is a partial view of the trachea. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The reagents required for this invention are conventional laboratory reagents, purchased from commercially available channels; for example:

[0040] Human bronchial epithelial cells BEAS-2B were purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0041] Experimental methods not mentioned are standard experimental methods and will not be described in detail here.

[0042] Example 1

[0043] (1) Culture of human bronchial epithelial cells BEAS-2B:

[0044] Human bronchial epithelial cells (BEAS-2B) in the logarithmic growth phase were selected, digested with trypsin for 5 minutes, and then the adherent cells were scraped off and added to fetal bovine serum culture medium preheated to 37°C. The cells were repeatedly pipetted to prepare a single-cell suspension, which was then cultured at a rate of 10.0–20.0 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of 10 cells / ml in a cell culture flask, and exosome-free serum-free medium was added. The culture flask was gently shaken to disperse the cells evenly. The flask was placed in a 37°C incubator with a 5% CO2 atmosphere and the cells were observed to adhere to the culture vessel after 24 hours of culture.

[0045] The exosome-free serum culture medium is made by adding 10% FBS to an exosome-free high-glucose culture medium.

[0046] (2) Preparation of freeze-dried curcumin solid lipid nanoparticles Cur-SLN:

[0047] (21) The weight ratio of curcumin: glyceryl monostearate: PEG-40 stearate: poloxamer 188: Tween-80: lyophilization protectant: water is 1~1.25: 9~11: 19~22: 9~11: 19~22: 36~44: 64~79: 1800~2200;

[0048] (22) Weigh curcumin, glyceryl monostearate and PEG-40 stearate according to the above mass ratio, dissolve them in anhydrous ethanol, and mix them on a magnetic stirrer at 75°C to obtain an organic phase.

[0049] (23) Poloxamer 188 and Tween-80 were weighed according to the above mass ratio and dissolved in water. The mixture was then magnetically stirred at a constant temperature of 75°C to dissolve and mix the aqueous phase.

[0050] (24) The organic phase was slowly injected into the aqueous phase at 75-78°C and 1000 rpm / min under magnetic stirring, and then the temperature was raised to 85°C and the organic solvent was removed by continuous stirring to obtain Cur-SLN thermal suspension.

[0051] (25) The obtained Cur-SLN hot suspension was then rapidly injected into the same volume of cold water at 0-4℃ under constant temperature magnetic stirring. The mixture was stirred continuously in an ice-water bath for 3 hours and then filtered through a 0.45μm filter membrane to obtain the Cur-SLN suspension.

[0052] (26) The Cur-SLN suspension was placed in a freeze dryer for programmed freeze drying to obtain powder, which was then passed through a 200-mesh sieve to obtain the freeze-dried powder of curcumin solid lipid nanoparticles Cur-SLN.

[0053] (3) Curcumin solid lipid nanoparticles (Cur-SLN) were transfected into human bronchial epithelial cells BEAS-2B:

[0054] (31) When the cell fusion degree is above 80%, the lyophilized powder of curcumin solid lipid nanoparticles Cur-SLN is diluted with fetal bovine serum culture medium to a concentration of 50-200 μg / mL containing Cur-SLN. After preheating to 37°C, the original culture medium is removed, and the diluted fetal bovine serum culture medium containing curcumin solid lipid nanoparticles Cur-SLN is added to the human bronchial epithelial cell BEAS-2B cell culture flask and placed back into the incubator for 4 hours.

[0055] Keep the number of dead cells below 5%;

[0056] (32) Remove the culture flask, rinse it twice with PBS preheated to 37°C, then add exosome-free serum culture medium, and put it back into the incubator to continue culturing for 24 hours;

[0057] The exosome-free serum culture medium is an exosome-free high glucose culture medium with 10% FBS added;

[0058] (4) Collection of exosomes by ultracentrifugation:

[0059] Collect the culture medium from the cell culture flask, filter it through a 0.22 μm filter, add it to a centrifuge tube, centrifuge at 800 × g for 20 min at 4 °C, and collect the supernatant; centrifuge at 2000 × g for 20 min at 4 °C, and collect the supernatant; centrifuge at 10000 × g for 40 min at 4 °C, and collect the supernatant; filter through a 0.45 μm microporous membrane; balance the collected supernatant, centrifuge at 130000 × g for 70 min at 4 °C, discard the supernatant, add the obtained precipitate to pre-cooled PBS, balance it strictly, and continue to centrifuge at 130000 × g for 70 min at 4 °C, discard the supernatant, and the obtained precipitate is Cur-SLN-Exo.

[0060] The extracted Cur-SLN-Exo was resuspended in 100 μL of PBS and then frozen at -80°C for later use.

[0061] Example 2

[0062] Particle size determination of Cur-SLN-Exo

[0063] Take 20 μL of Cur-SLN-Exo prepared by the method in Example 1, dilute it to 1 mL using a 0.45 μm filter membrane, and inject the sample into the Nanosight 300 detection cell using a syringe. Analyze the particle size and concentration of Exo without Cur-SLN loading and Cur-SLN-Exo. Use PBS filtered through a 0.45 μm microporous membrane as a control to determine its particle size. The results are as follows: Figure 1 As shown.

[0064] The average particle sizes of blank Exo and Cur-SLN-Exo were 144.0 nm and 153.8 nm, respectively, and the particle concentrations were 6.8E+9 / mL and 3.3E+10 / mL, respectively.

[0065] Example 3

[0066] Morphology of Cur-SLN-Exo observed by transmission electron microscopy (TEM)

[0067] Take 30 μL of the Cur-SLN-Exo suspension prepared by the method in Example 1 and place it on a copper grid. Let it stand at room temperature for 5 min. After absorbing the floating liquid with filter paper, add 10 μL of pH 7.0, 4% phosphotungstic acid solution to the copper grid and negatively stain at room temperature for 5 min. Blot the negative stain with filter paper, dry at room temperature for 15 min, and observe and photograph the morphology of Cur-SLN-Exo under TEM. The morphology of Exo is detected using the same method. The results are as follows: Figure 2 As shown.

[0068] Example 4

[0069] Western blot was used to verify the protein expression of Cur-SLN-Exo surface markers CD63, TSG101, and HSP70.

[0070] Cur-SLN-Exo is secreted by cells and mainly contains cytoplasmic proteins and membrane proteins, as well as small amounts of nucleic acids and mitochondrial proteins.

[0071] Prepare RIPA (strong) lysis buffer containing 1% protease inhibitor in advance, and add it directly to the Cur-SLN-Exo precipitate and control cell (BEAS-2B cell) precipitate prepared in Example 1. After gently mixing, transfer to pre-cooled EP tubes and centrifuge at 12000×g for 15 min at 4°C. The supernatant is retained, which is the Cur-SLN-Exo protein and control cell protein.

[0072] Protein quantification of Cur-SLN-Exo protein and control cell protein was performed using the BCA protein quantification kit. Protein samples were diluted with 1% RIPA (strong) lysis buffer containing a protease inhibitor at a specific ratio, mixed, and heated to 100℃ for protein denaturation for 5 min. A 10% separating gel and a 5% stacking gel were prepared, and the proteins in the mixture were separated by SDS-PAGE electrophoresis. First, electrophoresis was performed on the separating gel at a constant voltage of 110V for 50 min, followed by electrophoresis on the stacking gel at a constant voltage of 80V for 30 min. A PVDF membrane suitable for the electrophoresis gel was cut, immersed in methanol for 10 s, and then the proteins on the electrophoresis gel were transferred to the PVDF membrane under constant voltage of 100V for 2 h. After transfer, the PVDF membrane was removed, washed three times with TBST for 10 min each time, then blocked with 5% skim milk at room temperature for 2 h, and the primary antibody corresponding to the protein to be detected was added. The membrane was incubated at 4℃ for 12 h. Place the PVDF membranes of each sample on a shaker, remove the primary antibody, and wash three times with TBST within 1 hour. Then, add secondary antibody dilution buffer (1:1000 dilution) to ensure thorough contact with the membrane, and incubate at room temperature for 2 hours. Remove the secondary antibody, and wash three times with TBST on a shaker for decolorization. Remove the PVDF membrane and place it on plastic wrap. Mix the A and B components of the ECL chemiluminescence solution in a 1:1 volume ratio and drop the mixture onto the PVDF membrane. Observe and photograph the results. The results are as follows: Figure 3 As shown.

[0073] The results showed that Western blotting detected the specific expression of CD63, TSG101 and HSP70 proteins in Cur-SLN-Exo.

[0074] Example 5

[0075] Determination of drug loading in Cur-SLN-Exo:

[0076] Cur was measured using a fluorometer.

[0077] The fluorescence excitation conditions for Cur are: excitation at 450 nm and emission at 520 nm.

[0078] Loading rate % = (Cur in Cur - SLN - Exo / Amount of total Cur added) × 100%

[0079] The loading rate of curcumin on Cur-SLN-Exo prepared in Example 1 was determined to be 28.57%.

[0080] Example 6

[0081] Irritation test of lungs and trachea in Cur-SLN-Exo rats after inhalation administration

[0082] Preparation of blank SLN-Exo:

[0083] (1) The weight ratio of glyceryl monostearate: PEG-40 stearate: poloxamer 188: Tween-80: lyophilization protectant: water is 0.9-1.1: 1.9-2.2: 0.9-1.1: 1.9-2.2: 3.6-4.4: 6.4-7.9: 180-220;

[0084] (2) Weigh out glyceryl monostearate and PEG-40 stearate according to the above mass ratio, dissolve them in anhydrous ethanol, and mix them on a magnetic stirrer at 75°C to obtain an organic phase.

[0085] (3) Weigh out poloxamer 188 and Tween-80 according to the above mass ratio, dissolve them in water, and stir magnetically at 75°C to dissolve and mix them evenly to obtain an aqueous phase.

[0086] (4) The organic phase was slowly injected into the aqueous phase at 75-78℃ and 1000 rpm / min under magnetic stirring, and then the temperature was raised to 85℃ and the organic solvent was removed by continuous stirring to obtain a blank SLN thermal suspension.

[0087] (5) The obtained blank SLN hot suspension was then rapidly injected into the same volume of cold water at 0-4℃ under constant temperature magnetic stirring. The mixture was stirred continuously in an ice water bath for 3 hours and then filtered through a 0.45μm filter membrane to obtain the blank SLN suspension.

[0088] (6) Place the blank SLN suspension in a freeze dryer for programmed freeze drying to obtain powder, and pass it through a 200-mesh sieve to obtain the freeze-dried powder of blank SLN.

[0089] (7) Transfect blank SLN into human bronchial epithelial cells BEAS-2B:

[0090] Once the cell confluence reached 80% or higher, the lyophilized blank SLN powder was diluted with fetal bovine serum culture medium to a concentration of 50–200 μg / mL containing SLN. After preheating to 37°C, the original culture medium was removed, and the diluted fetal bovine serum culture medium containing SLN was added to the human bronchial epithelial cell BEAS-2B cell culture flask. The flask was then returned to the incubator and incubated for 4 hours.

[0091] Keep the number of dead cells below 5%; remove the culture flask, rinse twice with PBS preheated to 37°C, then add exosome-free serum medium, and return to the incubator to continue culturing for 24 hours; the exosome-free serum medium is exosome-free high glucose medium with 10% FBS added; the remaining steps are the same as the Cur-SLN-Exo ultracentrifugation collection method.

[0092] The extracted blank SLN-Exo was resuspended in 100 μL of PBS and then frozen at -80°C for later use.

[0093] Eighteen SD rats were randomly divided into three groups of six each using a random number table: an air control group, a blank SLN-Exo group (100 μL / kg), and a Cur-SLN-Exo group (100 μL / kg). Blank SLN-Exo and Cur-SLN-Exo were administered intratracheally once daily using a small animal liquid nebulizer for 14 consecutive days. The air control group received the same volume of air intratracheally using the same device. Twenty-four hours after the last administration, all animals were sacrificed, and tracheal and lung tissues were rapidly removed for hematoxylin-eosin (HE) pathological examination. Results are as follows: Figure 4 As shown.

[0094] The results showed that the air control group had tracheal epithelial loss, a small amount of inflammatory cell infiltration, and tissue edema, but no other obvious lesions. The lung tissue showed a small amount of inflammatory cell infiltration, with intact alveolar walls, no thickening, no inflammatory infiltration, and no congestion. The blank SLN-Exo group showed a small amount of inflammatory cell infiltration and tissue edema, but no other obvious lesions. The alveolar walls were rich in capillaries, with significant local hemorrhage. This indicates that the SLN-Exo sample has good biocompatibility when administered intratracheally, with low airway irritation and no local irritation. The Cur-SLN-Exo group showed a small amount of pseudostratified ciliated columnar epithelial loss and a small amount of inflammatory cell infiltration in the lung tissue. Mild inflammatory cell infiltration was observed in the lung interstitium, but no congestion or edema. This indicates that the Cur-SLN-Exo sample, due to drug encapsulation, has a larger particle size and is slightly less irritating than SLN-Exo, but the irritation is still within the low range, and the biocompatibility is good.

[0095] Example 7

[0096] Study on Cur-SLN-Exo's effect on lung inflammation in COPD model mice

[0097] (1) Preparation and grouping of COPD model mice for drug administration

[0098] The COPD model was established using a combination of fumigation and intratracheal instillation of LPS. Seven days after modeling, surviving animals were randomly divided into four groups according to body weight: a blank control group (n=10), a COPD model group (n=10), a Cur-SLN-Exo group (n=10; inhaled dose 100 μL / kg), and a budesonide group (n=10; nebulized dose 100 μL / kg). The blank control group and COPD model group received the same volume of physiological saline via the airway using a small animal liquid nebulizer, while the Cur-SLN-Exo and budesonide groups received the medication via the trachea using the same small animal liquid nebulizer. All groups received medication once daily for 14 days, starting seven days after modeling.

[0099] (2) Results of inflammatory cell classification and counting in mouse bronchoalveolar perfusion fluid

[0100] Compared with the blank control group, the total number of white blood cells in the bronchoalveolar lavage fluid of COPD model mice was significantly increased (P<0.01). Five-part differential inflammatory cell counting revealed increased numbers of neutrophils, monocytes, and basophils (P<0.01), indicating a large accumulation of inflammatory cells in the bronchi of COPD model mice induced by LPS and smoke exposure, releasing a large amount of inflammatory substances and ultimately altering lung structure. Compared with the COPD model group, administration of exosomes (Cur-SLN-Exo) significantly reduced the number of various inflammatory cells in the bronchoalveolar lavage fluid of mice, particularly the total number of white blood cells and neutrophils (P<0.01), while the budesonide group showed a significant decrease in the total number of white blood cells. These results demonstrate that Cur-SLN-Exo can significantly reduce the number of inflammatory cells in the bronchoalveolar lavage fluid of COPD models and effectively improve the degree of inflammation in the trachea and bronchi of COPD patients.

[0101] Table 1. Results of inflammatory cell classification and counting in BALF perfusion fluid of COPD mouse model using exosomes (Cur-SLN-Exo).

[0102]

[0103] Note: t-test compared to the blank control group. # P < 0.05, ## P < 0.01; ANOVA with the COPD model group, * P < 0.05 ** P < 0.01.

[0104] (3) Pathological changes (HE) of lung inflammation in COPD mice after inhalation of Cur-SLN-Exo

[0105] After aspirating bronchoalveolar lavage fluid, lung tissue was quickly and carefully removed. Residual tissue around the lung tissue was carefully removed with scissors. The tissue was then washed twice with pre-cooled PBS, blotted dry with filter paper, and the left lung was excised and fixed in 4% paraformaldehyde for preservation. The results of hematoxylin-eosin (HE) pathological examination are as follows: Figure 5 As shown.

[0106] The results showed that no obvious lesions were observed in the trachea of ​​the blank control group, but mild inflammatory cell infiltration was observed in the lung tissue. Compared with the blank control group, the COPD model group showed inflammatory cell infiltration, tissue edema, shedding of ciliated columnar epithelium, and hemorrhage in the trachea. Macrophages were abundant in the lung tissue, and mucus accumulation was observed in the cavities. Interstitial inflammatory cell infiltration, alveolar structure disorder, alveolar cavity enlargement, and partial fusion into bullae were observed. Compared with the COPD model group, no obvious lesions were observed in the trachea of ​​the Cur-SLN-Exo group. Mild interstitial inflammatory cell infiltration was observed in the lung tissue. In the budesonide group, edema was occasionally observed in the tracheal tissue, partial shedding of ciliated columnar epithelium in the bronchi, and a small amount of mucus accumulation were observed. The results indicate that Cur-SLN-Exo can effectively inhibit inflammatory changes in the bronchi and lung tissue of the COPD model, reduce the large-scale aggregation of macrophages, improve the degree of remodeling of damaged alveoli, and has a significant therapeutic effect on COPD inflammation and histopathological damage.

[0107] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0108] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sustained-release drug delivery system of exosome loaded curcumin nano, characterized in that, The active loading method is used to transfect curcumin solid lipid nanoparticles Cur-SLN to human bronchial epithelial cells BEAS-2B, and then the ultracentrifugation method is used to collect exosomes, namely the sustained-release drug delivery system, and the exosomes are named Cur-SLN-Exo. The preparation method comprises the following steps: Human bronchial epithelial cells BEAS-2B are cultured, and when the cell confluence is more than 80%, the freeze-dried powder of curcumin solid lipid nanoparticles Cur-SLN is added into the cell culture bottle, and incubated for 4 hours, and then cultured, centrifuged, filtered by a filter membrane, and resuspended.

2. The method of claim 1, wherein the preparation of the exosome loaded curcumin nano-sustained drug delivery system is characterized by, The preparation method comprises the following steps: Human bronchial epithelial cells BEAS-2B are cultured, and when the cell confluence is more than 80%, the freeze-dried powder of curcumin solid lipid nanoparticles Cur-SLN is added into the cell culture bottle, and incubated for 4 hours, and then cultured, centrifuged, filtered by a filter membrane, and resuspended.

3. The production method according to claim 2, wherein The preparation method comprises the following steps: (1) Culturing human bronchial epithelial cells BEAS-2B; (2) Transfecting curcumin solid lipid nanoparticles Cur-SLN to human bronchial epithelial cells BEAS-2B: (21) When the cell confluence is more than 80%, the freeze-dried powder of curcumin solid lipid nanoparticles Cur-SLN is diluted with fetal bovine serum medium to contain Cur-SLN at a concentration of 50-200 μg / mL, and then preheated to 37 DEG C, the original culture medium is removed, the diluted fetal bovine serum medium containing curcumin solid lipid nanoparticles Cur-SLN is added into the human bronchial epithelial cell BEAS-2B culture bottle, and then placed back into the incubator for incubation for 4 hours; (22) The culture bottle is taken out, washed with PBS preheated to 37 DEG C for 2 times, then the exosome-free serum medium is added, and the culture bottle is placed back into the incubator for further culture for 24 hours; The exosome-free serum medium is obtained by adding 10% FBS into the exosome-free high-sugar medium; (3) Collecting exosomes by ultracentrifugation: The supernatant is collected by centrifugation for three times, filtered by a 0.45 μm microporous filter, the precipitate is collected by ultracentrifugation, resuspended by PBS, and the precipitate is collected by ultracentrifugation, thereby obtaining Cur-SLN-Exo.

4. The production method according to claim 3, wherein The specific operation of step (1) is: selecting human bronchial epithelial cells BEAS-2B in logarithmic growth phase, adding trypsin for 5 min, then scraping off the adherent cells, adding preheated fetal bovine serum medium to 37 DEG C, repeatedly blowing to prepare a single cell suspension, and then inoculating in a cell culture bottle at a density of 10.0-20.0 x 10 5 The specific operation of step (1) is: selecting human bronchial epithelial cells BEAS-2B in logarithmic growth phase, adding trypsin for 5 min, then scraping off the adherent cells, adding preheated fetal bovine serum medium to 37 DEG C, repeatedly blowing to prepare a single cell suspension, and then inoculating in a cell culture bottle at a density of 10.0-20.0 x 10 The exosome-free serum medium is obtained by adding 10% FBS into the exosome-free high-sugar medium.

5. The production method according to claim 3, wherein In the step (21), the number of dead cells is controlled to be less than 5%.

6. The production method according to claim 3, wherein The specific operation of the step (3) is that the culture medium in the cell culture bottle is collected, filtered by a 0.22 μm filter, then added into a centrifugal tube, centrifuged at 800xg for 20 min at 4 DEG C, and the supernatant is sucked; centrifuged at 2000xg for 20 min at 4 DEG C, and the supernatant is sucked; centrifuged at 10000xg for 40 min at 4 DEG C, and the supernatant is sucked; filtered by a 0.45 μm microporous filter; the collected supernatant is strictly adjusted to a constant volume, then centrifuged at 130000xg for 70 min at 4 DEG C, the supernatant is discarded, the obtained precipitate is added into pre-cooled PBS, and the PBS is strictly adjusted to a constant volume, then centrifuged at 130000xg for 70 min at 4 DEG C, the supernatant is discarded, and the obtained precipitate is Cur-SLN-Exo.

7. The production method according to claim 2, wherein The preparation method of the freeze-dried powder of curcumin solid lipid nanoparticles Cur-SLN comprises: (1) The mass ratio of curcumin, glyceryl monostearate, PEG-40 stearate, poloxamer 188, Tween-80, lyophilization protectant and water is 1-1.25:9-11:19-22:9-11:19-22:36-44:64-79:1800-2200; (2) The curcumin, glyceryl monostearate and PEG-40 stearate are weighed according to the above mass ratio, dissolved in anhydrous ethanol, and stirred on a constant temperature magnetic stirrer at 75°C to obtain an organic phase; (3) The poloxamer 188 and Tween-80 are weighed according to the above mass ratio, dissolved in water, and stirred on a constant temperature magnetic stirrer at 75°C to obtain an aqueous phase; (4) The organic phase is slowly injected into the aqueous phase under magnetic stirring at 1000 rpm / min at 75-78°C, and then heated to 85°C, and the organic solvent is removed by continuous stirring to obtain a Cur-SLN hot suspension; (5) Then the obtained Cur-SLN hot suspension is quickly injected into the same volume of 0-4°C cold water under constant temperature magnetic stirring, and the ice water bath is continuously stirred for 3h, and then filtered through a 0.45μm filter to obtain a Cur-SLN suspension; (6) The Cur-SLN suspension is placed in a freeze dryer for programmed freeze drying to obtain a powder, which is sieved through a 200 mesh sieve to obtain a lyophilized powder of curcumin solid lipid nanoparticles Cur-SLN.

8. The use of the sustained-release drug delivery system of claim 1 or the sustained-release drug delivery system prepared by the preparation method of any one of claims 2-7 in the preparation of a drug for treating chronic obstructive pulmonary disease.

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