Pancreas targeted drug delivery system based on bitter gourd extracellular vesicles and application thereof
Through bitter melon extracellular vesicles as carriers, the efficient targeted delivery of drugs in the pancreas is achieved, and the problem of insufficient accumulation of drugs in the pancreas is solved, which improves the efficacy and reduces systemic toxic side effects.
Patent Information
- Application Number
- CN202510306152.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-07-11
AI Technical Summary
Existing treatment methods are difficult to achieve efficient accumulation of drugs in the pancreatic area, resulting in reduced efficacy of drugs and may cause systemic toxic side effects. There is a lack of efficient targeted delivery methods for pancreatic area.
Bitter melon extracellular vesicles are used as carriers, and the targeted drug delivery system is prepared by intraperitoneal injection or oral administration, using its unique lipids and protein composition to achieve efficient targeted delivery of the pancreas.
It increases the accumulation of drugs in the pancreas, enhances biosafety, reduces systemic toxic side effects, and has broad application prospects.
Smart Images
Figure CN120290452A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pharmaceutical preparations, and particularly relates to a pancreatic-targeted drug delivery system based on bitter gourd extracellular vesicles and its applications. Background Art
[0002] In recent years, the incidence of pancreatic-related diseases has been increasing year by year and has received more and more attention. On the one hand, pancreatic cancer is widely regarded as the malignant tumor with the worst prognosis. On the other hand, more and more evidence shows that the lesions of the pancreatic microenvironment are one of the important reasons for the occurrence and development of diabetes. However, due to the complexity of the diagnosis and treatment of pancreatic diseases, the treatment and management of pancreatic diseases are still very difficult. In particular, the existing treatment methods are difficult to achieve the efficient accumulation of drugs at the pancreatic target site, reducing the drug efficacy and possibly causing serious systemic toxic side effects. Therefore, the lack of an efficient targeted delivery and treatment method for the pancreas is still a key scientific problem and medical need to be solved urgently in the current field of pancreatic diseases.
[0003] Eukaryotic organisms such as animals and plants can all carry out cross-cell, cross-tissue and even cross-species information transmission and function regulation by secreting extracellular vesicles. Extracellular vesicles generally refer to membrane vesicles with a lipid bilayer structure, ranging in size from nanometers to micrometers, actively secreted by somatic cells. Extracellular vesicles are usually mainly divided into three categories according to the particle size range: exosomes, microvesicles, and apoptotic bodies. Among them, exosomes are a type of nanoscale extracellular vesicles secreted through the multivesicular body pathway, with a diameter usually between 50 - 150 nm in mammals, while in plants, due to differences in cell structure and secretion mechanisms, they often have a larger particle size range. Extracellular vesicles represented by exosomes usually contain various active components of the source cells, including nucleic acids, proteins, and small molecule metabolites, and can regulate the physiological functions of the body by transmitting these functional molecules between cells. Due to its function of information transmission in the body, as well as its source-specific lipid, protein composition and physicochemical properties, extracellular vesicles usually have unique in vivo distribution characteristics. The natural phospholipid bilayer structure of extracellular vesicles makes them excellent drug carriers, which can efficiently load cargo molecules with different properties and molecular weights to avoid the influence of the external environment, thereby improving the drug stability. At the same time, extracellular vesicles such as exosomes also have high heterogeneity according to their sources, carry different nucleic acids and proteins according to different source cells, and have different lipid compositions, showing natural specific targeting effects and pharmacological activities. Among them, extracellular vesicles such as phytogenic exosomes avoid zoonotic diseases compared with animal-derived biological products, greatly reducing the costs of breeding, extraction, testing and quarantine; compared with biological products extracted by cell culture, the production efficiency is greatly improved, avoiding the high costs of aseptic breeding. Generally speaking, plant-derived vesicles have both excellent drug-targeted delivery performance and significant advantages such as wide sources and easy industrial application.
[0004] At present, existing studies have pointed out that extracellular vesicles derived from some animals and plants can maintain structural stability in the gastrointestinal environment and transfer internal payloads across species to human intestinal epithelial cells. By distinguishing the protein and lipid compositions of extracellular vesicles such as extracellular vesicles from different sources, analyzing their in vivo distribution characteristics and pharmacological activities, and using their own properties to construct a new type of nano-drug targeted delivery system has great application prospects and clinical transformation value. However, there are still very few reports on the in vivo distribution characteristics of extracellular vesicles at present.
[0005] Momordica charantia, as a typical representative of "medicinal and edible homology" in traditional Chinese medicine, has been widely used in the prevention and treatment of diabetes in traditional medicine. However, so far, no relevant research on using extracellular vesicles of Momordica charantia to prepare a drug delivery system targeting the pancreas has been found. Summary of the Invention
[0006] To solve the problems that currently available pancreatic treatment drugs are difficult to accumulate efficiently in the pancreas, have poor targeting, low bioavailability, and are prone to cause systemic toxic side effects, etc., in order to meet the treatment needs of pancreatic diseases and improve the survival rate of patients with pancreatic-related diseases such as pancreatitis, diabetes, and pancreatic cancer, the object of the present invention is to provide a pancreatic-targeted drug delivery system based on extracellular vesicles of Momordica charantia and its application. The inventors surprisingly found that due to their unique lipid and protein compositions, extracellular vesicles of Momordica charantia have a significant enrichment effect in the pancreas of mice after intraperitoneal injection and oral administration. Therefore, the present invention creatively links the extracellular vesicles of traditional Chinese medicine Momordica charantia with pancreatic targeting function, and constructs a targeted drug delivery system with extracellular vesicles of Momordica charantia as the carrier. Compared with the existing drug delivery methods, it can target the pancreatic organ more efficiently and achieve the efficient accumulation of drugs or detection markers in the pancreas. At the same time, it has strong biological safety, is conducive to large-scale production, and has application transformation value.
[0007] To achieve the above object, the present invention provides the following technical solutions:
[0008] In the first aspect, the present invention provides a method for preparing extracellular vesicles of Momordica charantia, comprising the following steps:
[0009] (1) Wash and cut fresh Momordica charantia into pieces, then homogenize, filter, centrifuge the filtrate at 300 - 500×g for 10 - 60 min, centrifuge the obtained supernatant at 1500 - 2500×g for 10 - 60 min, take the supernatant, centrifuge at 14000 - 16000×g for 10 - 60 min, take the supernatant, and concentrate to obtain crude extracellular vesicles of Momordica charantia;
[0010] (2) Load the crude extracellular vesicles of bitter gourd obtained in step (1) onto a Sepharose 4B size exclusion chromatography, and elute with a PBS buffer solution at pH 7.4 containing 0.5 - 2 mM EDTA, and collect the eluate containing bitter gourd extracellular vesicles;
[0011] (3) Prepare sucrose aqueous solutions with concentrations of 0.60 g / mL, 0.30 g / mL, and 0.15 g / mL respectively, and successively take equal volumes and add them into an ultracentrifuge tube to form a sucrose density gradient layer with increasing concentration from top to bottom. Add the eluate containing bitter gourd extracellular vesicles obtained in step (2) to the top layer, and ultracentrifuge at 100,000×g for 0.5 - 1.5 h. Collect the extracellular vesicles between the sucrose concentrations of 0.30 and 0.60 g / mL, add PBS buffer solution to disperse evenly, and dialyze to remove sucrose to obtain bitter gourd extracellular vesicles.
[0012] Based on the above technical solution, further, the centrifugation in step (1) is carried out at 1 - 5 °C; the concentration is carried out by using an ultrafiltration tube with a cut-off molecular weight of 100 kDa.
[0013] Based on the above technical solution, further, the Sepharose 4B size exclusion chromatography in step (2) is pre-equilibrated with a PBS buffer solution containing 1 mM EDTA at pH 7.4 before use; the elution flow rate is 0.3 - 1 mL / min.
[0014] Based on the above technical solution, further, the centrifugation and dialysis in step (3) are carried out at 1 - 5 °C, and the cut-off molecular weight of the dialysis bag used for dialysis is 40 kDa.
[0015] In the second aspect, the present invention provides bitter gourd extracellular vesicles prepared by the above preparation method.
[0016] In the third aspect, the present invention provides a pancreatic-targeted drug delivery system based on bitter gourd extracellular vesicles. The pancreatic-targeted drug delivery system includes the above bitter gourd extracellular vesicles and a drug active substance loaded in the bitter gourd extracellular vesicles or a drug-loaded carrier loaded with a drug active substance.
[0017] Based on the above technical solution, further, the method for loading the drug active substance or the drug-loaded carrier loaded with a drug active substance into the bitter gourd extracellular vesicles includes co-incubation method, ultrasonic method, extrusion method, electroporation method, chemical perforation method, repeated freezing and thawing method.
[0018] Based on the above technical solution, further, the drug-loaded carrier loaded with a drug active substance loads the drug active substance into the carrier by adsorption method, chemical binding method, embedding method, co-precipitation method, solvent evaporation method, supercritical fluid method, spray drying method.
[0019] Based on the above technical solution, further, the pharmaceutically active substance includes anti-inflammatory drugs, anti-cancer drugs, anti-diabetic drugs, and immunomodulators.
[0020] Based on the above technical solution, further, the anti-inflammatory drugs include cortisone, hydrocortisone, methylprednisolone, dexamethasone, aspirin, sodium salicylate, paracetamol, phenylbutazone, oxyphenbutazone, indomethacin, sulindac, diclofenac, ibuprofen, naproxen, piroxicam, rofecoxib, triamcinolone acetonide acetate, clopidogrel bisulfate, rituximab, infliximab, adalimumab;
[0021] The anti-cancer drugs include cisplatin, paclitaxel, fluorouracil, cyclophosphamide, capecitabine, gemcitabine, vinorelbine, raltitrexed, doxorubicin, bleomycin, anastrozole, letrozole, tamoxifen, imatinib, gefitinib, interleukin, interferon, trastuzumab, rituximab, bevacizumab, iodine-131, radium-223, thallium-201, arsenic-211;
[0022] The anti-diabetic drugs include metformin, pioglitazone, rosiglitazone, sodium glucose cotransporter 2 inhibitor, glibenclamide, gliclazide, glimepiride, repaglinide, nateglinide, sitagliptin phosphate, empagliflozin, dorzagliatin, insulin, insulin glargine, insulin detemir, pramlintide, exenatide, liraglutide, semaglutide, tirzepatide;
[0023] The immunomodulators include but are not limited to interferon, interleukin, cyclosporine, tacrolimus, methotrexate, azathioprine.
[0024] In a fourth aspect, the present invention provides the use of the above-mentioned pancreatic-targeted drug delivery system in the preparation of a drug for treating pancreatitis, diabetes, or pancreatic cancer.
[0025] Based on the above technical solution, further, the drug further includes pharmaceutically acceptable excipients.
[0026] Based on the above technical solution, further, the pharmaceutically acceptable excipients include fillers, antioxidants, pH regulators, osmotic pressure regulators, solubilizers, cosolvents, antioxidants, bacteriostatic agents, lyoprotectants, suspending agents, and flavoring agents.
[0027] Based on the above technical solutions, further, the filler includes one or more of starch, sucrose, dextrin, lactose, microcrystalline cellulose, mannitol, bovine serum albumin; the antioxidant includes vitamin C, vitamin E, butylated hydroxyanisole, dibutylhydroxytoluene, propyl gallate, sodium sulfite, sodium metabisulfite, sodium thiosulfate, disodium edetate; the pH regulator includes phosphoric acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate, citric acid, sodium citrate, acetic acid, sodium acetate, tartaric acid, sodium tartrate, citric acid, sodium citrate, histidine; the bacteriostatic agent includes sodium benzoate, p-hydroxybenzoate, potassium sorbate, sorbitol; the lyoprotectant includes sucrose, trehalose, mannitol, sorbitol, sodium glutamate, proline, casein, skim milk; the flavoring agent includes sweeteners, aromatic agents, mucilage agents, effervescent agents.
[0028] Based on the above technical solutions, further, the dosage forms of the drug include tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.
[0029] The beneficial effects of the present invention compared with the prior art are as follows:
[0030] The present invention discovers that the drug-loaded Momordica charantia extracellular vesicles can be targeted and delivered to pancreatic tissue after intraperitoneal injection and oral administration. Compared with free drugs, the enrichment degree of the drug-loaded system in the pancreas is significantly increased after loading the drugs. Momordica charantia-derived extracellular vesicles, as natural drug carriers, have significant advantages such as rich sources, stable yields, low production costs, and high use safety. The vesicles can be efficiently targeted and delivered to the pancreas via the lymphatic circulation and surface ligand mediation without modification or engineering; loading drug active ingredients into the vesicles can not only improve drug stability, overcome the gastrointestinal barrier, but also significantly improve drug absorption efficiency and pancreatic accumulation. The present invention opens up new ideas for the research and treatment of pancreatic-related diseases and has broad application prospects. Description of the Drawings
[0031] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments will be briefly introduced below.
[0032] Figure 1 It is a transmission electron microscope morphology characterization diagram (scale: 100 nm) of Momordica charantia extracellular vesicles in Example 4.
[0033] Figure 2 It is a transmission electron microscope morphology characterization diagram (scale: 50 nm) of Momordica charantia extracellular vesicles loaded with mesoporous silica nanoparticles in Example 6.
[0034] Figure 3Drug leakage curves of the drug-loaded mesoporous silica nanoparticles of Example 7 and the drug-loaded mesoporous silica nanoparticles coated with Momordica charantia extracellular vesicles in simulated gastrointestinal fluids.
[0035] Figure 4 Comparison chart of the targeting effect of Momordica charantia extracellular vesicles at the cellular level in Example 8.
[0036] Figure 5 Fluorescence distribution diagrams of various organs after intraperitoneal injection of Momordica charantia extracellular vesicles examined by fluorescence imaging method in Example 9.
[0037] Figure 6 Statistical chart of the fluorescence intensity of various organs after intraperitoneal injection of Momordica charantia extracellular vesicles in Example 9.
[0038] Figure 7 Fluorescence distribution of various organs after oral administration of Momordica charantia extracellular vesicles examined by fluorescence imaging method in Example 10.
[0039] Figure 8 Statistical chart of the fluorescence intensity of various organs after oral administration of Momordica charantia extracellular vesicles in Example 10. Detailed implementation manners
[0040] The present invention will be described in detail below in conjunction with the embodiments. However, the implementation manners of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods and techniques well-known to those skilled in the art, and the reagents or materials are all obtained through commercial channels.
[0041] Example 1
[0042] Extraction of Momordica charantia extracellular vesicles by ultracentrifugation
[0043] Wash fresh Momordica charantia (purchased from the local farmers' market), cut it into small pieces, homogenize it with a squeezing juicer, and filter it through a silk cloth. Take the filtered Momordica charantia juice, centrifuge it at 400×g for 30 min at 4°C to remove tissue debris and residual cells, and take the supernatant. Centrifuge the supernatant at 2000×g for 30 min at 4°C to remove cell debris, and take the supernatant. Centrifuge the supernatant at 15000×g for 30 min at 4°C to remove residual cell debris, and take the supernatant. Concentrate the obtained supernatant using an ultrafiltration tube (100 kDa) to obtain crude Momordica charantia extracellular vesicles.
[0044] The extracellular vesicles of bitter gourd were extracted by ultracentrifugation, and the specific operation was as follows: The crude extracellular vesicles of bitter gourd were added into an ultracentrifugation tube and ultracentrifuged at 4°C and 100,000×g for 1 h. The precipitate was evenly dispersed with pre-cooled 1 mM PBS to obtain the extracellular vesicles of bitter gourd.
[0045] After diluting PBS 10-fold, the particle size distribution of the extracellular vesicles of bitter gourd was detected by the DLS method. The protein content of the extracellular vesicle solution was determined using a BCA protein quantification kit. Bovine serum albumin (BSA) was used as a standard to draw a working curve of protein concentration - absorbance. The proteins of the extracellular vesicles of bitter gourd were extracted using a whole protein extraction kit, diluted to the concentration range of the working curve, and co-incubated with the BCA working solution at 37°C for 30 min. The absorbance was measured at a wavelength of 562 nm, and the protein content was calculated according to the working curve.
[0046] The results showed that the particle size distribution of the extracellular vesicles of bitter gourd extracted by this method was 489.0 ± 111.1 nm, the PDI was 0.395 ± 0.046, and the total protein concentration was 3.01 mg / mL. The extracellular vesicles obtained by this method were deformed by the centrifugal force during ultracentrifugation and were difficult to be evenly dispersed again after stacking with each other.
[0047] Example 2
[0048] Extraction of extracellular vesicles of bitter gourd by sucrose density gradient method
[0049] The crude extracellular vesicles of bitter gourd were prepared by the method in Example 1. The extracellular vesicles of bitter gourd were extracted by the sucrose density gradient method, and the specific operation was as follows: Sucrose aqueous solutions with concentrations of 0.60 g / mL, 0.30 g / mL, and 0.15 g / mL were respectively prepared and 1 mL of each was taken into an ultracentrifugation tube in turn to form a sucrose density gradient layer with increasing concentration from top to bottom. The concentrated crude extracellular vesicle sample of bitter gourd was gently added to the top layer and ultracentrifuged at 4°C and 100,000×g for 1 h. The precipitate between the 0.30 - 0.60 g / mL sucrose solution layers was collected. After the precipitate was evenly dispersed with pre-cooled 1 mM PBS, it was loaded into a dialysis bag (MW = 40 kDa) and dialyzed overnight at 4°C to remove sucrose, obtaining the extracellular vesicles of bitter gourd.
[0050] The particle size distribution and total protein content of the extracellular vesicles of bitter gourd were detected by the method in Example 1. The results showed that the particle size distribution of the extracellular vesicles of bitter gourd extracted by this method was 189.8 ± 18.6 nm, the PDI was 0.417 ± 0.042, and the total protein concentration was 2.85 mg / mL. The extracellular vesicles obtained by this method had a wide particle size distribution and poor monodispersity.
[0051] Example 3
[0052] Extraction of extracellular vesicles of bitter gourd by size exclusion chromatography
[0053] The crude extracellular vesicles of bitter gourd cells were prepared by the method in Example 1. The extracellular vesicles of bitter gourd were purified by size exclusion chromatography. The specific operation was as follows: The crude extracellular vesicle sample of bitter gourd was slowly loaded onto a Sepharose 4B size exclusion chromatography pre-equilibrated with PBS containing 1 mM EDTA pH 7.4. Elution was carried out at a constant speed of 0.5 mL / min with PBS containing 1 mM EDTA at pH 7.4, and one tube of eluate was collected every 1 mL. The eluate containing the required sample was determined by the DLS method to obtain the extracellular vesicles of bitter gourd cells.
[0054] The particle size distribution and total protein content of the extracellular vesicles of bitter gourd were detected by the method in Example 1. The results showed that the particle size distribution of the extracellular vesicles of bitter gourd extracted by this method was 190.1 ± 9.1 nm, the PDI was 0.208 ± 0.085, and the total protein concentration was 0.71 mg / mL. The extracellular vesicles obtained by this method had a relatively uniform particle size but a low concentration.
[0055] Example 4
[0056] Extraction of extracellular vesicles of bitter gourd by size exclusion chromatography combined with sucrose density gradient method
[0057] The extracellular vesicles of bitter gourd were collected by the method in Example 3, and the sample was subjected to sucrose density gradient centrifugation by the method in Example 2 to obtain the extracellular vesicles of bitter gourd.
[0058] The particle size distribution and total protein content of the extracellular vesicles of bitter gourd were detected by the method in Example 1. The results showed that the particle size distribution of the extracellular vesicles of bitter gourd extracted by this method was 189.5 ± 10.0 nm, the PDI was 0.088 ± 0.034, and the total protein concentration was 3.22 mg / mL. The extracellular vesicles obtained by this method had a uniform particle size and a high protein concentration. The extracellular vesicle suspension of bitter gourd was dropped on the surface of a 200-mesh copper grid and stained with 1% uranium staining solution for 30 s. After discarding the staining solution, the copper grid was dried and observed with a transmission electron microscope at a voltage of 80 kV, and the morphology was as Figure 1 shown.
[0059] Example 5
[0060] Direct loading of active substances into extracellular vesicles of bitter gourd by ultrasonic method:
[0061] Taking the anti-inflammatory active compound curcumin as an example, the specific method is as follows: Dissolve curcumin in DMSO and dilute it with physiological saline to 0.5 mg / mL. Add the Momordica charantia extracellular vesicles of Example 4 so that the mass ratio of curcumin to Momordica charantia extracellular vesicles is 1:15. Under the condition of an ice-water bath, sonicate continuously at a power of 100 W for 1 min and then cool for 2 min, repeating 6 cycles. Centrifuge at 4°C and 12,000×g for 10 min to remove any potentially precipitated drugs. Centrifuge at 4°C and 100,000×g for 30 minutes, discard the supernatant, add pre-cooled PBS to wash the precipitate, centrifuge again under the same conditions, and collect the precipitate suspended in PBS at pH 7.4 to obtain drug-loaded Momordica charantia extracellular vesicles.
[0062] The drug loading of the obtained drug-loaded extracellular vesicles was measured to be 4.97 ± 0.37%.
[0063] Example 6
[0064] Loading active substances in Momordica charantia extracellular vesicles using inorganic nanoparticles:
[0065] Taking mesoporous silica, which has been widely used in various nano-drug delivery systems, as an example, the drug is loaded into mesoporous silica by the adsorption equilibrium method, and the mesoporous silica is loaded into the Momordica charantia extracellular vesicles of Example 4 by the extrusion method. The specific operation is as follows: Taking tirzepatide as a model protein drug, the drug is loaded by the adsorption method. Mix an aqueous solution of 2 mg / mL tirzepatide and a suspension of 1 mg / mL mesoporous silica nanoparticles evenly dispersed in equal volumes, stir at 4°C for 4 h, centrifuge at 4°C and 12,000×g for 5 min, and collect the precipitate, which is mesoporous silica nanoparticles loaded with tirzepatide. The drug-loaded mesoporous silica nanoparticles are re-dispersed evenly in PBS at pH 7.4 at a concentration of 1.0 mg / mL, mixed evenly with an equal volume of a suspension of 1.5 mg / mL Momordica charantia extracellular vesicles, and extruded repeatedly through a 0.4 μm pore size micro-extruder at least 11 times. Centrifuge at 4°C and 12,000×g for 10 min to remove free Momordica charantia extracellular vesicles and leaked tirzepatide, and collect the precipitate as Momordica charantia extracellular vesicles coated with mesoporous silica nanoparticles loaded with tirzepatide. Wash with pre-cooled PBS and re-suspend. Store at -80°C for later use.
[0066] The morphology of Momordica charantia extracellular vesicles after loading mesoporous silica is as Figure 2 shown; after detection, the drug loading of the obtained drug-loaded Momordica charantia extracellular vesicles was 24.95 ± 0.83%.
[0067] Example 7
[0068] In this example, the release behavior of drug-loaded mesoporous silica and drug-loaded mesoporous silica coated with Momordica charantia extracellular vesicles in simulated gastrointestinal fluids was evaluated. Momordica charantia extracellular vesicles were uniformly dispersed in an acidic solution (0.1 mol / L hydrochloric acid solution) simulating the gastric environment to 0.5 mg / mL and incubated at 37 °C for 2 h. After centrifugation, the precipitate was taken and redispersed in simulated intestinal fluid (6.8 g of potassium dihydrogen phosphate, dissolved in 500 mL of water, adjusted to pH 6.8 with 0.1 mol / L sodium hydroxide, and diluted to 1000 mL with water) and incubated for another 10 h. Supernatants were taken at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h respectively, and the drug leakage amount of the drug-loading system was detected by high performance liquid chromatography. The specific method was as follows: chromatographic column ZORBAX 300SB-C8 (Agilent, C18, 4.6×150 mm, 5 μm), mobile phase: 0.1M potassium dihydrogen phosphate solution with pH 2.5 - acetonitrile (70:30), constant elution, flow rate: 1 mL / min, column temperature: 50 °C, detection wavelength: 220 nm, injection volume: 10 μL. The first 2 h was simulated gastric fluid, and the subsequent 10 h was simulated intestinal fluid. The results are as Figure 3 , and the results showed that in simulated gastrointestinal fluids, the drug-loaded mesoporous silica nanoparticles coated with Momordica charantia extracellular vesicles had a significantly lower drug release amount compared to the uncoated drug-loaded particles, which could protect the loaded drugs and prevent premature drug leakage.
[0069] Example 8
[0070] Targeting effect of Momordica charantia extracellular vesicles at the cellular level
[0071] MIN6 cells were digested and counted, then inoculated into 24-well plates and cultured overnight at 37 °C with 5% CO2. When the cells entered the logarithmic growth phase, fluorescein isothiocyanate (FITC)-labeled mesoporous silica nanoparticles and FITC-labeled mesoporous silica nanoparticles coated with Momordica charantia extracellular vesicles were suspended in pH 7.4 PBS to make the final concentration of mesoporous silica 20 μg / mL. After replacing the original medium, they were cultured in the dark under the same conditions for 4 h. After washing repeatedly with pre-cooled PBS 3 times, the cells were digested with 1 mL of trypsin for 2 min, centrifuged at 1000×g for 3 min to collect the cells, and then redispersed evenly with 1 mL of PBS. The fluorescence intensity of the cells was detected using an Agilent NovoCyte flow cytometer. The results are as Figure 4 shown, and it can be seen that mesoporous silica nanoparticles coated with Momordica charantia extracellular vesicles can aggregate more effectively into the cells.
[0072] Example 9
[0073] Pancreatic targeting effect after intraperitoneal injection of Momordica charantia extracellular vesicles
[0074] The fluorescence labeling method was used to investigate the in vivo distribution of Momordica charantia extracellular vesicles after intraperitoneal injection. The specific method was as follows: Healthy KM mice (SPF grade, 4 - 5 weeks old, half male and half female) were randomly divided into 2 groups, with 12 mice in each group. They were intraperitoneally injected with free cyanine (Cy7) and Cy7-labeled Momordica charantia extracellular vesicles of Example 4 with the same fluorescence intensity. Among them, Cy7 was covalently bound to the amino groups on the surface of extracellular vesicles by the EDC / NHS chemical coupling method, and free Cy7 was removed using an ultrafiltration tube (100 kDa). After administration, 3 mice were sacrificed at 2 h, 4 h, 8 h, and 16 h respectively, and the heart, liver, spleen, lung, kidney, pancreas, and intestinal lymph nodes were taken, and the fluorescence intensity of the ex vivo organs was observed under a small animal imaging system.
[0075] The results were as Figure 5 , the total fluorescence intensity values of each organ were recorded and compared, and the results were as Figure 6 . The results showed that Momordica charantia extracellular vesicles could accumulate significantly in pancreatic tissue after intraperitoneal injection, having a specific targeting effect.
[0076] Example 10
[0077] Pancreatic targeting effect of Momordica charantia extracellular vesicles after oral administration
[0078] The fluorescence labeling method was used to investigate the in vivo distribution of Momordica charantia extracellular vesicles after oral administration. The specific method was as follows: Healthy KM mice (SPF grade, 4 - 5 weeks old, half male and half female) were randomly divided into 2 groups, with 12 mice in each group. They were orally administered with free Cy7 and Cy7-labeled Momordica charantia extracellular vesicles of Example 4 with the same fluorescence intensity. After administration, 3 mice were sacrificed at 2 h, 4 h, 8 h, and 16 h respectively, and the heart, liver, spleen, lung, kidney, pancreas, and intestinal lymph nodes were taken, and the fluorescence intensity of the ex vivo organs was observed under a small animal imaging system.
[0079] The results were as Figure 7 , the total fluorescence intensity values of each organ were recorded and compared, and the results were as Figure 8 . The results showed that Momordica charantia extracellular vesicles could accumulate significantly in pancreatic tissue after oral administration, having a specific targeting effect.
[0080] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: They can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing extracellular vesicles of Momordica charantia, characterized in that, It includes the following steps: (1) Wash fresh bitter gourds, cut them into pieces, homogenize, filter, centrifuge the filtrate at 300 - 500×g for 10 - 60 min, centrifuge the obtained supernatant at 1500 - 2500×g for 10 - 60 min, take the supernatant, centrifuge at 14000 - 16000×g for 10 - 60 min, take the supernatant, and concentrate to obtain crude extracellular vesicles of bitter gourd; (2) Load the crude extracellular vesicles of bitter gourd obtained in step (1) onto a Sepharose 4B size - exclusion chromatography, elute with a PBS buffer solution at pH 7.4 containing 0.5 - 2 mM EDTA, and collect the eluate containing extracellular vesicles of bitter gourd; (3) Prepare sucrose aqueous solutions with concentrations of 0.60 g / mL, 0.30 g / mL, and 0.15 g / mL respectively, and successively take equal volumes and add them into an ultra - centrifuge tube to form a sucrose density gradient layer with increasing concentration from top to bottom. Add the eluate containing extracellular vesicles of bitter gourd obtained in step (2) to the top layer, ultra - centrifuge at 100000×g for 0.5 - 1.5 h, collect the extracellular vesicles between the sucrose concentrations of 0.30 and 0.60 g / mL, add PBS buffer solution to disperse evenly, and dialyze to remove sucrose to obtain extracellular vesicles of bitter gourd.
2. The preparation method according to claim 1, characterized in that, The centrifugation in step (1) is carried out at 1 - 5°C; the concentration is carried out by using an ultrafiltration tube with a molecular weight cut - off of 100 kDa; Before use, the Sepharose 4B size - exclusion chromatography in step (2) is pre - equilibrated with a PBS buffer solution containing 1 mM EDTA at pH 7.4; The elution flow rate is 0.3 - 1 mL / min; The centrifugation and dialysis in step (3) are carried out at 1 - 5°C, and the molecular weight cut - off of the dialysis bag used for dialysis is 40 kDa.
3. The extracellular vesicles of bitter gourd prepared by the preparation method according to claim 1 or 2.
4. A pancreatic-targeted drug delivery system based on bitter gourd extracellular vesicles, characterized in that, The pancreatic - targeting drug delivery system described above includes the extracellular vesicles of bitter gourd of claim 3 and a drug active substance loaded in the extracellular vesicles of bitter gourd or a drug - loaded carrier loaded with a drug active substance.
5. The pancreatic targeting drug delivery system according to claim 4, wherein The method for loading the drug active substance or the drug - loaded carrier loaded with a drug active substance into the extracellular vesicles of bitter gourd includes co - incubation method, ultrasonic method, extrusion method, electroporation method, chemical punching method, and repeated freezing - thawing method.
6. The pancreatic-targeted drug delivery system according to claim 4, wherein The drug - loaded carrier loaded with a drug active substance loads the drug active substance into the carrier by adsorption method, chemical binding method, embedding method, co - precipitation method, solvent evaporation method, supercritical fluid method, and spray drying method.
7. The pancreatic targeting drug delivery system according to claim 4, wherein The drug active substances include anti - inflammatory drugs, anti - cancer drugs, drugs for treating diabetes, and immunomodulators.
8. Use of the pancreatic - targeting drug delivery system according to any one of claims 4 - 7 in the preparation of a drug for treating pancreatitis, diabetes, or pancreatic cancer.
9. The application according to claim 8, wherein The drug also includes pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients include fillers, antioxidants, pH regulators, osmotic pressure regulators, solubilizers, cosolvents, antioxidants, bacteriostatic agents, lyophilization protectants, suspending agents, and flavoring agents.
10. The application according to claim 8, wherein, The dosage forms of the drug include tablets, granules, oral liquid preparations, drops, injection preparations, and capsule preparations.