Buffer for electroporation-based exosome drug loading and electroporation-based exosome drug loading method

Abstract

Provided are an electroporation buffer and an electroporation-based exosome drug loading method, wherein the electroporation buffer is a liquid medium used in a method for loading an exogenous substance into an exosome on the basis of electroporation, and has an osmotic pressure of 50-400 mOsm / kg and a pH value of 2.0-8.3. By means of the cooperation of osmotic pressure and pH value, the electroporation buffer greatly improves the loading efficiency of exogenous substances into exosomes without causing substantial damage to exosomes and nucleic-acid-based exogenous substances or any damage caused thereto is controllable, thereby meeting the requirements for commercial applications. In addition, the electroporation buffer and the drug loading method also have the advantage of being low cost.

Description

A buffer solution for drug delivery via exosome electroporation and a method for drug delivery via exosomes based on electroporation. Technical Field

[0001] This invention specifically relates to a buffer solution for drug delivery via exosome electroporation and a method for drug delivery via exosomes based on electroporation. Background Technology

[0002] Exosomes, as specialized carriers of intercellular communication, play a crucial role in various physiological processes, including immune responses, antigen presentation, and signal transduction. Almost all eukaryotic cells can secrete exosomes, including adipocytes, epithelial cells, fibroblasts, neurons, and astrocytes. Exosomes are present in almost all body fluids, such as cerebrospinal fluid, urine, saliva, blood, vitreous humor, and breast milk. Exosomes can not only penetrate tissues but also diffuse into the bloodstream and even cross the blood-brain barrier (BBB). Exosomes are rich in nucleic acids, proteins, lipids, and metabolites. They possess a unique ability to induce biological responses and provide diagnostic and prognostic information for some diseases, such as metabolic diseases, cardiovascular diseases, neurodegenerative diseases, and tumors. Furthermore, as nanoparticle-sized endogenous vesicles, their high biocompatibility, natural homing properties, and the ability to achieve more specific enrichment in tissues, organs, and lesions after functionalization make exosomes a valuable novel drug delivery carrier for research. Therefore, exosomes have many promising clinical applications—they can be used as a drug delivery tool, a therapeutic agent for diseases, a novel biomarker for disease diagnosis, or for regenerative medicine-related damage repair and aesthetic treatment.

[0003] Exosome drug delivery is mainly divided into two categories: endogenous loading and exogenous loading. Endogenous loading is an engineered loading method based on parental cells. First, the source cells are modified, such as through direct transfection or co-incubation, to introduce the target molecule. During the production of exosomes, the source cells load the target molecule into intracellular multivesicular bodies, which then travel through the lumen to the extracellular vesicles. Exogenous loading, on the other hand, involves directly loading exogenous substances into isolated exosomes using membrane permeation or other loading strategies, such as electroporation. Exogenous loading can load various therapeutic substances, including small molecule drugs, nucleic acid drugs, proteins, and even nanomaterials.

[0004] To load drugs of various sizes into exosomes, researchers have developed a variety of techniques, including chemical and physical methods such as incubation, sonication, electroporation and chemical transfection (e.g., transmembrane peptides, saponins, Triton), extrusion, freeze-thaw cycles, and dialysis. Chemical methods require the introduction of transfection reagents, most of which are cytotoxic, posing a significant obstacle to the application of exosome-based drugs. Currently, most physical methods suffer from low drug loading efficiency or severe exosome damage, hindering progress in exosome-based drug delivery.

[0005] Compared to endogenous loading technologies based on parental cell engineering and other exogenous loading technologies, exogenous loading technologies based on electroporation are relatively convenient and offer more stable and controllable loading effects, making them a promising candidate for becoming the primary technology for exosome drug delivery. Electroporation utilizes electrical pulses to generate a potential difference across an exosome, creating openings in the exosome membrane. Exogenous molecules then pass through these openings to enter the exosome, completing the loading process. Electroporation is a safe, efficient, and cost-effective technology for exosome drug loading.

[0006] In the development of electroporation buffers, existing technologies almost exclusively focus on cell electroporation buffers. Currently, there are no commercially available electroporation buffers specifically designed for drug loading onto exosomes. Due to the differences in size and characteristics between cells and exosomes, cell electroporation buffers cannot be universally used for exosome electroporation. There are some reports on exosome electroporation buffers, such as CN114181974B, which discloses an electroporation buffer composed of 3-9 mg / ml NaCl and 10-40 mg / ml sucrose, or 0.1-1 mg / ml KCl and 10-40 mg / ml sucrose, with a pH of 1-2. While this electroporation buffer can achieve good loading results, studies have found that exosomes and nucleic acids are easily damaged at this pH value, thus affecting the application efficacy of drug-loaded exosomes.

[0007] In addition, data shows that when the pH value is 1.6, the glycosidic bond between purine bases and ribonucleotides (deoxyribonucleotides) will break, causing damage to nucleic acid substances.

[0008] Therefore, there is still a need to develop an electroporation buffer that can achieve high loading efficiency while causing no substantial damage or controllable damage to exosomes and nucleic acid-like exogenous substances, in order to meet the requirements of commercial applications. Summary of the Invention

[0009] One objective of this invention is to provide an exosome electroporation buffer that, while causing no substantial damage or only controllable damage to exosomes and nucleic acid-like exogenous substances, has high loading efficiency to meet the requirements of commercial applications.

[0010] A second objective of this invention is to provide a method for exosome drug delivery by electroporating exosomes using the above-mentioned electroporation buffer to introduce exogenous substances.

[0011] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0012] The first aspect of this invention provides an electroporation buffer solution, which is a liquid medium used in a method for loading exogenous substances into exosomes based on electroporation. The osmotic pressure of the electroporation buffer solution is 50–400 mOsm / kg, for example, 50 mOsm / kg, 55 mOsm / kg, 60 mOsm / kg, 65 mOsm / kg, 70 mOsm / kg, 75 mOsm / kg, 80 mOsm / kg, 85 mOsm / kg, 90 mOsm / kg, 95 mOsm / kg, 100 mOsm / kg, 105 mOsm / kg, 110 mOsm / kg, 115 mOsm / kg, 120 mOsm / kg, and 125 mOsm / kg. / kg, 130mOsm / kg, 135mOsm / kg, 140mOsm / kg, 145mOsm / kg, 150mOsm / kg, 155mOsm / kg, 160mOsm / kg, 165mOsm / kg, 170mOsm / kg, 175mOsm / kg, 180mOsm / kg, 185mOsm / kg、190mOsm / kg、195mOsm / kg、200mOsm / kg、205mOsm / kg、210mOsm / kg、215mOsm / kg、220mOsm / kg、225mOsm / kg、230mOsm / kg、235mOsm / kg、240m Osm / kg, 245mOsm / kg, 250mOsm / kg, 255mOsm / kg, 260mOsm / kg, 265mOsm / kg, 270mOsm / kg, 275mOsm / kg, 280mOsm / kg, 285mOsm / kg, 290mOsm / kg, 295mOsm / kg, 300mOsm / kg, 305mOsm / kg, 310mOsm / kg, 315mOsm / kg, 320mOsm / kg, 325mOsm / kg, 330mOsm / kg, 335mOsm / kg, 340mOsm / kg, 345mOsm / kg, 350mOsm / kg, 3 55 mOsm / kg, 360 mOsm / kg, 365 mOsm / kg, 370 mOsm / kg, 375 mOsm / kg, 380 mOsm / kg, 385 mOsm / kg, 390 mOsm / kg, 395 mOsm / kg, or 400 mOsm / kg; pH value 2.0–8.3, for example, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, or 8.3.

[0013] According to the first embodiment, the pH value of the electroporation buffer solution is 2.1 to 4.3, for example, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, or 4.3; the osmotic pressure is 50 to 400 mOsm / kg, for example, 50 mOsm / kg, 55 mOsm / kg, 60 mOsm / kg, 65 mOsm / kg, 70 mOsm / kg, 75 mOsm / kg, 80 mOsm / kg, 85 mOsm / kg, 90 mOsm / kg, etc. mOsm / kg, 95mOsm / kg, 100mOsm / kg, 105mOsm / kg, 110mOsm / kg, 115mOsm / kg, 120mOsm / kg, 125mOsm / kg, 130mOsm / kg, 135mOsm / kg, 140mOsm / k g, 145mOsm / kg, 150mOsm / kg, 155mOsm / kg, 160mOsm / kg, 165mOsm / kg, 170mOsm / kg, 175mOsm / kg, 180mOsm / kg, 185mOsm / kg, 190mOsm / kg, 195 mOsm / kg, 200mOsm / kg, 205mOsm / kg, 210mOsm / kg, 215mOsm / kg, 220mOsm / kg, 225mOsm / kg, 230mOsm / kg, 235mOsm / kg, 240mOsm / kg, 245mOsm 3 00mOsm / kg, 305mOsm / kg, 310mOsm / kg, 315mOsm / kg, 320mOsm / kg, 325mOsm / kg, 330mOsm / kg, 335mOsm / kg, 340mOsm / kg, 345mOsm / kg, 350mOs m / kg, 355mOsm / kg, 360mOsm / kg, 365mOsm / kg, 370mOsm / kg, 375mOsm / kg, 380mOsm / kg, 385mOsm / kg, 390mOsm / kg, 395mOsm / kg or 400mOsm / kg.

[0014] In this first embodiment, preferably, the pH value of the electroporation buffer is 2.1 to 4.3, and the osmotic pressure is 50 to 350 mOsm / kg.

[0015] In this first embodiment, more preferably, the electroporation buffer solution has a pH value of 2.1 to 4.3 and an osmotic pressure of 50 to 200 mOsm / kg.

[0016] In this first embodiment, more preferably, the electroporation buffer has a pH of 2.1 to 4.3 and an osmotic pressure of 50 to 120 mOsm / kg.

[0017] According to the second embodiment, the pH value of the electroporation buffer solution is 2.1–4.3, for example, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, or 4.3; the osmotic pressure is 200–350 mOsm / kg, for example, 200 mOsm / kg, 205 mOsm / kg, 210 mOsm / kg, 215 mOsm / kg, 220 mOsm / kg, 225 mOsm / kg, 230 mOsm / kg, 235 mOsm / kg, 240 mOsm / kg. kg、245mOsm / kg、250mOsm / kg、255mOsm / kg、260mOsm / kg、265mOsm / kg、270mOsm / kg、275mOsm / kg、280mOsm / kg、285mOsm / kg、290mOsm / kg、295mOsm / k g, 300mOsm / kg, 305mOsm / kg, 310mOsm / kg, 315mOsm / kg, 320mOsm / kg, 325mOsm / kg, 330mOsm / kg, 335mOsm / kg, 340mOsm / kg, 345mOsm / kg or 350mOsm / kg.

[0018] According to the third embodiment, the pH value of the electroporation buffer solution is 2.1 to 8.3, for example, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5 2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, or 8.3; osmotic pressure is 50–200 mOsm / kg, for example, 50 mOsm / kg, 55 mOsm / kg, 60mOsm / kg, 65mOsm / kg, 70mOsm / kg, 75mOsm / kg, 80mOsm / kg, 85mOsm / kg, 90mOsm / kg, 9 5mOsm / kg, 100mOsm / kg, 105mOsm / kg, 110mOsm / kg, 115mOsm / kg, 120mOsm / kg, 125mOsm / kg, 130mO sm / kg, 135mOsm / kg, 140mOsm / kg, 145mOsm / kg, 150mOsm / kg, 155mOsm / kg, 160mOsm / kg, 165mOsm / kg, 170mOsm / kg, 175mOsm / kg, 180mOsm / kg, 185mOsm / kg, 190mOsm / kg, 195mOsm / kg or 200mOsm / kg.

[0019] In this third embodiment, preferably, the electroporation buffer has a pH of 2.1 to 8.3 and an osmotic pressure of 50 to 150 mOsm / kg.

[0020] In this third embodiment, more preferably, the electroporation buffer has a pH of 2.1 to 8.3 and an osmotic pressure of 50 to 120 mOsm / kg.

[0021] According to the fourth embodiment, the pH value of the electroporation buffer solution is 4.5–8.3, for example, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7. 0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, or 8.3; osmotic pressure of 50–200 mOsm / kg, for example, 50 mOsm / kg, 55 mOsm / kg, 60 mOsm / kg, 65 mOsm / kg, 70 mOsm / kg, 75 mOsm / kg, 80mOsm / kg, 85mOsm / kg, 90mOsm / kg, 95mOsm / kg, 100mOsm / kg, 105mOsm / kg, 110mO sm / kg, 115mOsm / kg, 120mOsm / kg, 125mOsm / kg, 130mOsm / kg, 135mOsm / kg, 140mOsm / kg、145mOsm / kg、150mOsm / kg、155mOsm / kg、160mOsm / kg、165mOsm / kg、170mOsm / k g, 175mOsm / kg, 180mOsm / kg, 185mOsm / kg, 190mOsm / kg, 195mOsm / kg or 200mOsm / kg.

[0022] In this fourth embodiment, preferably, the electroporation buffer solution has a pH value of 4.5 to 8.3 and an osmotic pressure of 50 to 150 mOsm / kg.

[0023] In this fourth embodiment, more preferably, the electroporation buffer solution has a pH value of 4.5 to 8.3 and an osmotic pressure of 50 to 120 mOsm / kg.

[0024] According to the fifth embodiment, the pH value of the electroporation buffer solution is 4.5–8.3, for example, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7. 7.8, 7.9, 8.0, 8.1, 8.2, or 8.3; osmotic pressure of 200–400 mOsm / kg, for example, 200 mOsm / kg, 205 mOsm / kg, 210 mOsm / kg, 215 mOsm / kg, 220 mOsm / kg, 225 mOsm / kg, 230 mOsm / kg, 235 mOsm / kg, 240 mOsm / kg, 245 mOsm / kg, 250 mOsm / kg. g, 255mOsm / kg, 260mOsm / kg, 265mOsm / kg, 270mOsm / kg, 275mOsm / kg, 280mOsm / kg, 285mOsm / kg, 290m Osm / kg, 295mOsm / kg, 300mOsm / kg, 305mOsm / kg, 310mOsm / kg, 315mOsm / kg, 320mOsm / kg, 325mOsm / kg , 330mOsm / kg, 335mOsm / kg, 340mOsm / kg, 345mOsm / kg, 350mOsm / kg, 355mOsm / kg, 360mOsm / kg, 365mO sm / kg, 370mOsm / kg, 375mOsm / kg, 380mOsm / kg, 385mOsm / kg, 390mOsm / kg, 395mOsm / kg or 400mOsm / kg.

[0025] In this fifth embodiment, preferably, the electroporation buffer solution has a pH value of 4.5 to 8.3 and an osmotic pressure of 200 to 350 mOsm / kg.

[0026] In this fifth embodiment, more preferably, the electroporation buffer solution has a pH value of 4.5 to 8.3 and an osmotic pressure of 200 to 300 mOsm / kg.

[0027] According to the first to fifth embodiments, the exogenous substance to be delivered is mRNA or siRNA.

[0028] According to some specific embodiments, the conductivity of the electroporation buffer is 3 to 17 mS / cm, for example, 3 mS / cm, 4 mS / cm, 5 mS / cm, 6 mS / cm, 7 mS / cm, 8 mS / cm, 9 mS / cm, 10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 mS / cm or 17 mS / cm.

[0029] According to some specific embodiments, the electroporation buffer includes cations, anions, sugars, and solvents.

[0030] Further, the cation is selected from one or more of sodium ions, potassium ions, calcium ions, and magnesium ions. Even further, the cation is selected from two, three, or four of sodium ions, potassium ions, calcium ions, and magnesium ions.

[0031] In this embodiment, the concentration of sodium ions is 5 to 175 mM, for example, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM or 175 mM.

[0032] In this embodiment, the concentration of potassium ions is 2 to 175 mM, for example, 2 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM or 175 mM.

[0033] Further, the concentration of potassium ions is 2–100 mM. Even further, the concentration of potassium ions is 2–50 mM. Still further, the concentration of potassium ions is 2–30 mM, for example, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM.

[0034] In this embodiment, the concentration of calcium ions is 4 to 50 mM, for example, 4 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM.

[0035] In this embodiment, the concentration of magnesium ions is 5 to 50 mM, for example, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM.

[0036] Furthermore, the cations include sodium ions and potassium ions, and selectively include one or more of calcium ions and magnesium ions.

[0037] Further, the anion is selected from one or more of chloride ions, hydrogen phosphate ions, dihydrogen phosphate ions, sulfate ions, and citrate ions. Even further, the anion is selected from one, two, three, four, or five of chloride ions, hydrogen phosphate ions, dihydrogen phosphate ions, sulfate ions, and citrate ions.

[0038] In this embodiment, the concentration of chloride ions is 2 to 175 mM, for example, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM or 175 mM.

[0039] In this embodiment, the concentration of the hydrogen phosphate ion or the dihydrogen phosphate ion is 8 to 150 mM, for example, 8 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM or 150 mM.

[0040] In this embodiment, the concentration of sulfate ions is 3 to 100 mM, for example, 3 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM.

[0041] In this embodiment, the concentration of citrate ions is 3 to 100 mM, for example, 3 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM.

[0042] Furthermore, the sugar is selected from one or more of trehalose, sucrose, glucose, fructose, and mannose. Even further, the sugar is selected from one, two, three, four, or five of trehalose, sucrose, glucose, fructose, and mannose.

[0043] Furthermore, the solvent is water.

[0044] According to some specific embodiments, the total concentration of the cation is 10-200 mM, for example, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM.

[0045] According to some specific embodiments, the total concentration of the anions is 10-200 mM, for example, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM.

[0046] According to some specific embodiments, the total sugar concentration is 10-200 mM, for example, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM.

[0047] According to this first embodiment, the electroporation buffer comprises 15–200 mM of cations (e.g., 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM). M, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM or 200mM), and anions of 15–200mM (e.g., 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM). 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, or 200mM), sugars of 20-200mM (e.g., 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM), and 20-200mM sugars. mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130 mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM or 200mM).

[0048] According to this second embodiment, the electroporation buffer comprises 50–175 mM of cations (e.g., 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 1...). 45mM, 150mM, 155mM, 160mM, 165mM, 170mM or 175mM), and anions of 50–175mM (e.g. 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM). M, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM or 175mM), sugars of 20-200mM (e.g., 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM), 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, or 200mM).

[0049] According to this third embodiment, the electroporation buffer comprises 15-100 mM of cations (e.g., 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM) and 15-100 mM of anions (e.g., 15 mM, 20 mM, 25 mM, 30 mM, 35 mM). 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM), and sugars of 20 to 100mM (e.g., 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM).

[0050] According to this fourth embodiment, the electroporation buffer comprises 15-100 mM of cations (e.g., 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM) and 15-100 mM of anions (e.g., 15 mM, 20 mM, 25 mM, 30 mM, 35 mM). 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM), and sugars of 20 to 100mM (e.g., 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM).

[0051] According to this fifth embodiment, the electroporation buffer comprises 50-200 mM of cations (e.g., 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM) and 50-200 mM of anions (e.g., 50 mM, 55 mM, 60 mM, 65 mM, 70 mM). ,75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 1 30mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM 185mM, 190mM, 195mM or 200mM), 20-100mM sugars (e.g. 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM).

[0052] Furthermore, the electroporation buffer solution selectively contains a buffer system.

[0053] Furthermore, the cations and anions are derived from inorganic salts and pH adjusters. The pH adjuster is selected from one or more of hydrochloric acid, sulfuric acid, citric acid, sodium hydroxide, and potassium hydroxide. The inorganic salt is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0054] According to some specific implementation methods, the exogenous substances include compounds, nucleic acids, peptides, and proteins, to be prepared into drugs or vaccines for treating and / or preventing various diseases in humans or animals, test kits, and medical aesthetic products, etc.

[0055] Furthermore, the nucleic acid includes siRNA, miRNA, mRNA, antisense oligonucleotide, or DNA.

[0056] A second aspect of the present invention provides a method for drug delivery to exosomes based on electroporation, wherein the method uses the above-mentioned electroporation buffer to load exogenous substances into exosomes.

[0057] According to some specific implementations, the method uses a static electroporation apparatus or a flow cytometry electroporation apparatus, along with corresponding consumables, for electroporation. The flow cytometry apparatus, in particular, can achieve simultaneous electroporation while the fluid is flowing, enabling continuous electroporation and offering the advantage of high throughput.

[0058] According to some specific embodiments, the electric field strength of the electroporation is controlled to be 250–5000 V / cm, for example, 250 V / cm, 300 V / cm, 350 V / cm, 400 V / cm, 450 V / cm, 500 V / cm, 600 V / cm, 700 V / cm, 800 V / cm, 900 V / cm, 1000 V / cm, 1100 V / cm, 1200 V / cm, 1300 V / cm, 1400 V / cm, 1500 V / cm, 1600 V / cm, 1700 V / cm, 1800 V / cm, 1900 V / cm, 2000 V / cm, 2100 V / cm, 220 V / cm, etc. 0V / cm, 2300V / cm, 2400V / cm, 2500V / cm, 2600V / cm, 2700V / cm, 2800V / cm, 2900V / cm, 3000V / cm, 3100V / cm , 3200V / cm, 3300V / cm, 3400V / cm, 3500V / cm, 3600V / cm, 3700V / cm, 3800V / cm, 3900V / cm, 4000V / cm, 4100V / cm, 4200V / cm, 4300V / cm, 4400V / cm, 4500V / cm, 4600V / cm, 4700V / cm, 4800V / cm, 4900V / cm or 5000V / cm; control the total pulse width of the electroporation to be 0.05 to 50 ms, for example, 0.05 ms, 0.5 ms, 1 ms, 5 ms, 15 ms, 20 ms, 25 ms, 30 ms, 35 ms, 40 ms, 45 ms or 50 ms.

[0059] According to some specific implementations, the number of pulses for electroporation is controlled to be 1 to 10 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0060] According to some specific embodiments, the pulse interval of the electroporation is controlled to be 0.1 to 6000 ms, for example, 0.1 ms, 1 ms, 10 ms, 50 ms, 100 ms, 200 ms, 300 ms, 400 ms, 500 ms, 600 ms, 700 ms, 800 ms, 900 ms, 1000 ms, 1100 ms, 1200 ms, 1300 ms, 1400 ms, 1500 ms, 1600 ms, 1700 ms, 1800 ms, 1900 ms, 2000 ms, 2100 ms, 2200 ms, 2300 ms, 2400 ms, 2500 ms, 2600 ms, 2700 ms. ms, 2800ms, 2900ms, 3000ms, 3100ms, 3200ms, 3300ms, 3400ms, 3500ms, 3600ms, 3700ms, 3800ms, 3900ms, 4000ms, 4100ms, 4200ms, 4300ms, 44 00ms, 4500ms, 4600ms, 4700ms, 4800ms, 4900ms, 5000ms, 5100ms, 5200ms, 5300ms, 5400ms, 5500ms, 5600ms, 5700ms, 5800ms, 5900ms or 6000ms.

[0061] According to some specific embodiments, the particle number ratio of the exosomes to the exogenous substance is 1:100 to 1:100000, for example 1:100, 1:500, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:5500, 1:6000, 1:6500, 1:7000, 1:7500, 1:8000, 1:8500, 1:9000, 1:9500, 1:10000, 1:10500, 1:11000, 1:11500, 1:12000, 1:12500, 1:13000, 1:13500, 1... 1:14000, 1:14500, 1:15000, 1:15500, 1:16000, 1:16500, 1:17000, 1:17500, 1:18000, 1:18500, 1:19000, 1:19500, 1:20000, 1:20500, 1:21000, 1:2150 0, 1:22000, 1:22500, 1:23000, 1:23500, 1:24000, 1:24500, 1:25000, 1:25500, 1:26000, 1:26500, 1:27000, 1:27500, 1:28000, 1:28500, 1:29000, 1:2 9500, 1:30000, 1:30500, 1:31000, 1:31500, 1:32000, 1:32500, 1:33000, 1:33500, 1:34000, 1:34500, 1:35000, 1:35500, 1:36000, 1:36500, 1:37000 1:37500, 1:38000, 1:38500, 1:39000, 1:39500, 1:40000, 1:40500, 1:41000, 1:41500, 1:42000, 1:42500, 1:43000, 1:43500, 1:44000, 1:44500, 1:450 00, 1:45500, 1:46000, 1:46500, 1:47000, 1:47500, 1:48000, 1:48500, 1:49000, 1:49500, 1:50000, 1:50500, 1:51000, 1:51500, 1:52000, 1:52500, 1: 53000, 1:53500, 1:54000, 1:54500, 1:55000, 1:55500, 1:56000, 1:56500, 1:57000, 1:57500, 1:58000, 1:58500, 1:59000, 1:59500, 1:60000, 1:605001:61000, 1:61500, 1:62000, 1:62500, 1:63000, 1:63500, 1:64000, 1:64500, 1:65000, 1:65500, 1:66000, 1:66500, 1:67000, 1:67500, 1:68000, 1:68500, 1:69000, 1:69500, 1:70000, 1:7050 0, 1:71000, 1:71500, 1:72000, 1:72500, 1:73000, 1:73500, 1:74000, 1:74500, 1:75000, 1:75500, 1:76000, 1:76500, 1:77000, 1:77500, 1:78000, 1:78500, 1:79000, 1:79500, 1:80000, 1:80 500, 1:81000, 1:81500, 1:82000, 1:82500, 1:83000, 1:83500, 1:84000, 1:84500, 1:85000, 1:85500, 1:86000, 1:86500, 1:87000, 1:87500, 1:88000, 1:88500, 1:89000, 1:89500, 1:90000, 1: 90500, 1:91000, 1:91500, 1:92000, 1:92500, 1:93000, 1:93500, 1:94000, 1:94500, 1:95000, 1:95500, 1:96000, 1:96500, 1:97000, 1:97500, 1:98000, 1:98500, 1:99000, 1:99500, or 1:100000.

[0062] According to some specific embodiments, the ratio of the number of exosome particles to the mass of the exogenous substance is 1E+10p:(1~1000)μg.

[0063] According to some specific implementations, the exogenous substance is added in excess compared to the exosomes.

[0064] According to some specific embodiments, the exosomes are fed in the form of storage in a buffer solution, wherein the density of the exosomes in the buffer solution is controlled to be above 1E+9p / mL.

[0065] According to some specific embodiments, the osmotic pressure of the electroporation system is controlled to be 50-400 mOsm / kg, and the pH value is 2.5-8.3.

[0066] According to the first embodiment, the osmotic pressure of the electroporation system is controlled to be 50-400 mOsm / kg, and the pH value is 2.1-4.3.

[0067] Furthermore, the osmotic pressure of the electroporation system is controlled to be 50–300 mOsm / kg, and the pH value is 2.1–4.3.

[0068] According to the second embodiment, the osmotic pressure of the electroporation system is controlled to be 200-350 mOsm / kg, and the pH value is 2.1-4.3.

[0069] According to the third embodiment, the osmotic pressure of the electroporation system is 50-200 mOsm / kg, and the pH value is 2.1-8.3.

[0070] According to the fourth embodiment, the osmotic pressure of the electroporation system is 50-200 mOsm / kg, and the pH value is 4.5-8.3.

[0071] According to the fifth embodiment, the osmotic pressure of the electroporation system is 200-400 mOsm / kg, and the pH value is 4.5-8.3.

[0072] According to some specific implementation methods, before electroporation of the prepared electroporation system, it is incubated in an incubator at a temperature of 35°C to 38°C for 1 to 2 hours.

[0073] Compared with the prior art, the present invention has the following advantages:

[0074] This invention, taking advantage of the characteristics of exosomes, develops an electroporation buffer solution suitable for loading exogenous substances onto exosomes via electroporation. Compared to chemical loading methods, this solution does not introduce harmful chemicals, meeting the requirements of drug clinical trial application regulations. Furthermore, by adjusting the osmotic pressure and pH value, this electroporation buffer solution significantly improves the loading efficiency of exosomes with exogenous substances without causing substantial damage or with controllable damage to exosomes and nucleic acid-based exogenous substances, meeting the requirements for commercial applications. In addition, the electroporation buffer solution and drug loading method of this invention have the advantage of low cost. Attached Figure Description

[0075] Figure 1 shows the detection results of exosome particle integrity in Example 8;

[0076] Figure 2 shows the detection results of siRNA loading efficiency in Example 9;

[0077] Figure 3 shows the detection results of exosome particle integrity in Example 9;

[0078] Figure 4 shows the detection results of siRNA loading efficiency in Example 10;

[0079] Figure 5 shows the results of the peptide loading efficiency test in Example 11;

[0080] Figure 6 shows the results of mRNA loading efficiency in Example 12. Detailed Implementation

[0081] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0082] Because current electroporation-based exosome loading methods suffer from the problem of not being able to simultaneously address exosome loading efficiency and exosome / nucleic acid damage, the applicant, after extensive research and summarization, firstly developed an electroporation buffer specifically for loading exosomes into exosomes. Secondly, based on this electroporation buffer, the applicant further designed suitable electroporation conditions and achieved a high loading rate of exosomes into exosomes by precisely controlling the electric field.

[0083] Specifically, because exosomes are very small in size and have poor sensitivity to electric fields, it is difficult to effectively open pores using only an electric field. Therefore, the applicant uses relatively mild pH conditions to ensure that the pH value will not cause substantial damage to exosomes and nucleic acid drugs. Furthermore, by using appropriate osmotic pressure, it is easier to form electroporation under a suitable electric field, thereby allowing exogenous substances such as nucleic acid molecules, small molecule compounds, peptides, and proteins to enter the exosomes, achieving the purpose of loading exogenous substances such as nucleic acids, small molecule compounds, peptides, and proteins onto the exosomes.

[0084] This application further improves the loading efficiency and loading stability of exosomes by modifying the formulation of the electroporation buffer under suitable pH and osmotic pressure.

[0085] Furthermore, extensive research has demonstrated that the electroporation buffer solution and exosome drug loading method of this application not only have high loading efficiency and stability, but also exhibit minimal exosome fragmentation after electroporation, thus reducing the likelihood of exosome fragmentation and aggregation.

[0086] The exosomes used in this application can be exosomes from various sources, including but not limited to exosomes from human, animal, and plant sources. For example, animal-derived exosomes include, but are not limited to, exosomes derived from cells, milk, blood, and saliva of animals such as humans, mice, rats, pigs, cattle, sheep, and chickens, such as HEK293 cell exosomes, MSC cell exosomes, epithelial cell exosomes, macrophage exosomes, dendritic cell exosomes, mast cell exosomes, neuronal cell exosomes, tumor cell exosomes, oligodendrocyte exosomes, B cell exosomes, T cell exosomes, and milk exosomes. Plant-derived exosomes include, but are not limited to, lemon exosomes, ginger exosomes, carrot exosomes, watermelon exosomes, grape exosomes, olive exosomes, sunflower seed exosomes, rose exosomes, and ginseng exosomes.

[0087] The technical solutions and effects of the present invention will be further illustrated below with reference to embodiments and comparative examples.

[0088] In the following examples and comparative examples, all raw materials and reagents used were commercially available or prepared using existing methods. Unless otherwise specified, "%" in the following examples and comparative examples refers to mass percentage. The exosomes used in Examples 1-10 and Comparative Example 1 were exosomes derived from HEK293 cells, and the siRNA was siRNA targeting the human Gapdh gene. The osmotic pressure and pH values ​​used in this application were measured under standard conditions (temperature 25°C, atmospheric pressure 1 atmosphere).

[0089] Example 1: Preparation of buffer-1

[0090] Add 0.035g NaCl, 0.02g KCl, 0.06g NaH2PO4, 0.06g Na2HPO4, 0.15g CaCl2, 0.85g sucrose, and 0.85g trehalose to 100mL of sterile water. Adjust the pH to 6.5 using HCl and NaOH to prepare an electroporation buffer with an osmotic pressure of 120mOsm / kg, named buffer-1.

[0091] Example 2: Preparation of buffer-2

[0092] Add 0.05g NaCl, 0.05g KCl, 0.05g CaCl2, 0.05g MgCl2, 0.5g sucrose, and 1.0g trehalose to 100mL of sterile water, adjust the pH to 2.3 with HCl, and prepare an electroporation buffer with an osmotic pressure of 112mOsm / kg, named buffer-2.

[0093] Example 3: Preparation of buffer-3

[0094] Add 0.60g NaCl, 0.02g KCl, 0.12g MgSO4, 0.10g CaCl2, and 1.5g sucrose to 100mL of sterile water, adjust the pH to 2.2 with HCl, and prepare an electroporation buffer with an osmotic pressure of 280mOsm / kg, named buffer-3.

[0095] Example 4: Preparation of buffer-4

[0096] Add 0.07g NaCl, 0.05g KCl, 0.05g MgCl2, 0.5g sucrose, and 1.2g trehalose to 100mL of sterile water, adjust the pH to 2.2 with HCl, and prepare an electroporation buffer with an osmotic pressure of 119mOsm / kg, named buffer-4.

[0097] Example 5: Preparation of buffer-5

[0098] Add 0.07g NaCl, 0.05g KCl, 0.05g MgCl2, 0.5g sucrose, and 1.2g trehalose to 100mL of sterile water, adjust the pH to 2.4 with HCl, and prepare an electroporation buffer with an osmotic pressure of 116mOsm / kg, named buffer-5.

[0099] Example 6: Preparation of buffer-6

[0100] Add 0.07g NaCl, 0.05g KCl, 0.05g MgCl2, 0.5g sucrose, and 1.2g trehalose to 100mL of sterile water, adjust the pH to 2.7 with HCl, and prepare an electroporation buffer with an osmotic pressure of 112mOsm / kg, named buffer-6.

[0101] Example 7: Preparation of buffer-7

[0102] Add 0.60g NaCl, 0.12g KCl, 0.12g Na2HPO4, 0.05g MgCl2, 0.75g sucrose, and 0.75g trehalose to 100mL of sterile water. Adjust the pH to 6.6 using HCl and NaOH to prepare an electroporation buffer with an osmotic pressure of 275mOsm / kg, named buffer-7.

[0103] Preparation of Buffer-8 (Comparative Example 1)

[0104] Add 0.60g NaCl, 0.02g KCl, 0.12g MgSO4, 0.1g CaCl2, and 1.5g sucrose to 100mL of sterile water, adjust the pH to 1.6 with HCl, and prepare an electroporation buffer with an osmotic pressure of 282mOsm / kg, named buffer-8.

[0105] Example 8: Exosome electroporation loading of siRNA

[0106] (1) Preparation of exosomes

[0107] Exosomes were stored in PBS at a density of 1.5E+11p / mL.

[0108] (2) Preparation of exogenous substances

[0109] Dissolve the siRNA in water or PBS to a concentration of 428 ng / μL.

[0110] (3) Preparation of electroporation system

[0111] The exosomes prepared in step (1) and the siRNA prepared in step (2) were mixed evenly with the buffers prepared in the above examples and comparative examples to prepare 100 μL of electroporation system. The particle ratio of exosomes to siRNA was 1:1E+4. The osmotic pressure, pH value and amount of each component of the prepared electroporation system are shown in Table 1 below.

[0112] (4) Incubate the prepared electroporation system at 37°C for 1 hour.

[0113] (5) Transfer the incubated electroporation system to an electrode cup and perform electroporation using an electroporation instrument. Set the electroporation parameters as follows: pulse electric field strength is 2000V / cm, single pulse width is 1500μs, number of pulses is 3, and pulse interval is 1000ms.

[0114] (6) After electroporation, the system was diluted 100 times with PBS and subjected to nanoflow cytometry (by Deobio) to detect the integrity of exosomes, i.e., the proportion of intact exosome particles in the mixture. The detection results are shown in Figure 1. Figure 1 shows the detection results of buffer-7, buffer-3 and buffer-8 from left to right. The particle integrity of buffer-7 was 80.8%, that of buffer-3 was 77%, and that of buffer-8 was 35.5%.

[0115] Table 1

[0116] Example 9: Exosome electroporation loading of siRNA

[0117] Following the method in Example 8, siRNA was loaded into exosomes. After electroporation, the system was diluted 100-fold with PBS and subjected to nanoflow cytometry (by Dubio) to test the siRNA loading efficiency and exosome integrity. The loading efficiency results are shown in Figure 2, and the exosome particle integrity percentage is shown in Figure 3. Figures 2 and 3, from left to right, show the detection results for buffer-7, buffer-1, and buffer-2, respectively. The loading efficiency of buffer-7 was 8.7%, buffer-1 was 36.3%, and buffer-2 was 80.1%. The particle integrity of buffer-7 was 81.4%, buffer-1 was 78.8%, and buffer-2 was 76.1%.

[0118] Example 10: Exosome electroporation loading of siRNA

[0119] Following the method in Example 8, siRNA was loaded into exosomes. After electroporation, the system was diluted 100-fold with PBS and subjected to nanoflow cytometry (by Dubio) to test the siRNA loading efficiency. The results are shown in Figure 4. Figure 4 shows the detection results for buffer-4, buffer-5, and buffer-6 from left to right. The loading efficiency of buffer-4 was 56.2%, buffer-5 was 51.2%, and buffer-6 was 47.4%. Furthermore, using these three buffers for exosome electroporation resulted in relatively good preservation of exosome particle integrity.

[0120] Example 11: Exosome Electroporation Loading of Peptides

[0121] (1) Preparation of experimental materials

[0122] Exosomes derived from MSC cell supernatant were prepared and purified in-house and stored in PBS at a particle concentration of 2E+11p / mL. Ultrapure water was used to prepare peptide solutions at a concentration of 0.2μg / μL. The peptides were FITC-labeled nonapeptides purchased from Nanjing Jietai Biotechnology Co., Ltd.

[0123] (2) Preparation of electroporation system

[0124] Based on the properties of the buffers and the results of siRNA loading via exosome electroporation described above, we selected buffer-1, buffer-2, and buffer-6 to test the effectiveness of peptide loading via exosome electroporation. The exosomes and peptides prepared in step (1) were mixed thoroughly with buffer-1, buffer-2, and buffer-6 from the above examples to prepare a 100 μL electroporation system. The osmotic pressure, pH value, and amount of each component in the prepared electroporation system are shown in Table 2 below.

[0125] (3) Incubate the prepared electroporation system at 37°C for 1 hour.

[0126] (4) Transfer the incubated electroporation system to an electrode cup and perform electroporation using an electroporation instrument. Set the electroporation parameters as follows: pulse electric field strength is 2000V / cm, single pulse width is 1500μs, number of pulses is 3, and pulse interval is 1000ms.

[0127] (5) After electroporation, the system was diluted 100 times with PBS and used as a blank control for Beckman nanocomputing. The FITC positivity rate of exosomes after electroporation was detected, and exosomes with FITC fluorescence were those loaded with peptides. The detection results are shown in Figure 5, where, from left to right, the detection results are for the blank control, buffer-1, buffer-2, and buffer-6. The loading efficiency of buffer-1 was 59.5%, that of buffer-2 was 87.6%, and that of buffer-6 was 75.9%, indicating that buffer-2 had the best electroporation loading effect on exosomes.

[0128] Table 2

[0129] Example 12: Exosome electroporation loading of mRNA

[0130] (1) Material preparation

[0131] Milk exosomes were purchased and stored in PBS with a particle concentration of 2E+11p / mL; ultrapure aqueous solution of mRNA with a concentration of 2μg / μL and a full length of 980nt was purchased from Kaituo Life Science (Suzhou) Co., Ltd.

[0132] (2) Preparation of electroporation system

[0133] Based on the properties of the buffer and the results of the above exosome electroporation loading of siRNA / peptide, we selected buffer-2 to test the effect of milk exosome electroporation loading of mRNA. The exosomes and mRNA prepared in step (1) were mixed evenly with buffer-2 in the above examples to prepare 100 μL of electroporation system. The osmotic pressure, pH value and amount of each component of the prepared electroporation system are shown in Table 3 below.

[0134] (3) Incubate the prepared electroporation system at 37°C for 1 hour.

[0135] (4) Transfer the incubated electroporation system to an electrode cup and perform electroporation using an electroporation instrument. Set the electroporation parameters as follows: pulse electric field strength is 2000V / cm, single pulse width is 1500μs, number of pulses is 3, and pulse interval is 1000ms.

[0136] (5) After electroporation, the electroporated exosomes were stained using Thermo's SYTO™ 9 green fluorescent nucleic acid staining agent, diluted 1:10000. Staining was performed at room temperature in the dark for 15 minutes. After staining, the exosomes were diluted 100-fold and detected by Beckman nanocytography. Exosomes that had not undergone electroporation were used as a blank control. The positive rate of green fluorescence in the exosomes after electroporation was measured; exosomes with green fluorescence were those loaded with mRNA. The results are shown in Figure 6, where, from left to right, the results are for the blank control and buffer-2. The loading efficiency of buffer-2 was 12.5%.

[0137] Table 3

[0138] Example 13: Exosome electroporation loading of small molecule compounds

[0139] (1) Preparation of experimental materials

[0140] Lemon exosomes were purchased and stored in PBS at a particle concentration of 2E+11 p / mL, with a particle size of approximately 80 nm. The small molecule compound to be loaded was doxorubicin, purchased from Selleck, which was dissolved in ultrapure water to a final concentration of 2 μg / μL.

[0141] (2) Electroporation system preparation

[0142] Based on the properties of the buffer and experience in loading other substances, we selected buffer-2 and buffer-3 to test the effect of exosome electroporation loading of doxorubicin. The exosomes and doxorubicin prepared in step (1) were mixed evenly with buffer-2 and buffer-3 in the above examples to prepare 100 μL of electroporation system. The osmotic pressure, pH value and the amount of each component of the prepared electroporation system are shown in Table 4 below.

[0143] (3) Incubate the prepared electroporation system at 37°C for 1 hour.

[0144] (4) During incubation at 37°C, take 190 μL of buffer-7 and add 10 μL of doxorubicin for later use.

[0145] (5) Transfer the incubated electroporation system to an electrode cup and perform electroporation using an electroporation instrument. Set the electroporation parameters as follows: pulse electric field strength is 2000V / cm, single pulse width is 1500μs, number of pulses is 3, and pulse interval is 1000ms.

[0146] (6) Transfer the exosomes obtained after electroporation in step (5) to an EP tube, add the solution prepared in step (4), and mix the solution obtained in step (5) with the solution in step (4) in a 1:1 ratio. Incubate at 4°C for 1 hour.

[0147] (7) The mixture from step (6) is purified and separated into exosomes using a 10 kDa ultrafiltration tube to obtain pure exosomes.

[0148] (8) The purified exosomes were lysed using exosome lysis buffer.

[0149] (9) The lysis products obtained in step (8) were subjected to HPLC analysis to determine the amount of doxorubicin loaded in the exosomes. Finally, the HPLC results showed that in the first group of experiments, the exosomes were loaded with 4.65 μg of doxorubicin; in the second group of experiments, the exosomes were loaded with 3.37 μg of doxorubicin. Detailed results are shown in Table 5.

[0150] Table 4

[0151] Table 5

[0152] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. An electroporation buffer, characterized by: The electroporation buffer is a liquid medium used in a method of loading an exogenous substance into an exosome by electroporation, and has an osmotic pressure of 50 to 400 mOsm / kg and a pH of 2.0 to 8.

3.

2. The electroporation buffer of claim 1, wherein: The electroporation buffer has a pH of 2.1 to 4.3 and an osmotic pressure of 50 to 400 mOsm / kg, preferably an osmotic pressure of 50 to 350 mOsm / kg, further preferably an osmotic pressure of 50 to 200 mOsm / kg, and more preferably an osmotic pressure of 50 to 120 mOsm / kg.

3. The electroporation buffer of claim 1, wherein: The electroporation buffer has a pH of 2.1 to 4.3 and an osmotic pressure of 200 to 350 mOsm / kg.

4. The electroporation buffer of claim 1, wherein: The electroporation buffer has a pH of 2.1 to 8.3 and an osmotic pressure of 50 to 200 mOsm / kg, preferably an osmotic pressure of 50 to 150 mOsm / kg, further preferably an osmotic pressure of 50 to 120 mOsm / kg.

5. The electroporation buffer of claim 1, wherein: The electroporation buffer has a pH of 4.5 to 8.3 and an osmotic pressure of 50 to 200 mOsm / kg, preferably an osmotic pressure of 50 to 150 mOsm / kg, further preferably an osmotic pressure of 50 to 120 mOsm / kg.

6. The electroporation buffer of claim 1, wherein: The electroporation buffer has a pH of 4.5 to 8.3 and an osmotic pressure of 200 to 400 mOsm / kg, preferably an osmotic pressure of 200 to 350 mOsm / kg, further preferably an osmotic pressure of 200 to 300 mOsm / kg.

7. The electroporation buffer of any one of claims 2 to 6, wherein: The exogenous substance to be delivered is mRNA or siRNA.

8. The electroporation buffer according to any one of claims 1 to 6, characterized in that: The electroporation buffer has a conductivity of 3 to 17 mS / cm.

9. The electroporation buffer according to any one of claims 1 to 6, characterized in that: The electroporation buffer includes a cation, an anion, a sugar, and a solvent.

10. The electroporation buffer of claim 9, wherein: The cation is selected from one or more of sodium ion, potassium ion, calcium ion, and magnesium ion; The anion is selected from one or more of chloride ion, hydrogen phosphate ion, dihydrogen phosphate ion, sulfate ion, and citrate ion; The sugar is selected from one or more of trehalose, sucrose, glucose, fructose, and mannose; The solvent is water.

11. The electroporation buffer of claim 9, wherein: The total concentration of the cation is 10 to 200 mM, the total concentration of the anion is 10 to 200 mM, and the total concentration of the sugar is 10 to 200 mM.

12. The electroporation buffer of claim 2, wherein: The electroporation buffer includes 15 to 200 mM of the cation, 15 to 200 mM of the anion, and 20 to 200 mM of the sugar.

13. The electroporation buffer of claim 3, wherein: The electroporation buffer includes 50 to 175 mM of the cation, 50 to 175 mM of the anion, and 20 to 200 mM of the sugar.

14. The electroporation buffer of claim 4 or 5, wherein: The electroporation buffer includes 15 to 100 mM of the cation, 15 to 100 mM of the anion, and 20 to 100 mM of the sugar.

15. The electroporation buffer of claim 6, wherein: The electroporation buffer includes 50 to 200 mM of the cation, 50 to 200 mM of the anion, and 20 to 200 mM of the sugar.

16. The electroporation buffer of claim 9, wherein: The electroporation buffer selectively contains a buffer system.

17. The electroporation buffer of claim 9, wherein: The cation and the anion are derived from an inorganic salt and a pH adjuster selected from one or more of hydrochloric acid, sulfuric acid, citric acid, sodium hydroxide, and potassium hydroxide.

18. The electroporation buffer of any one of claims 1 to 17, wherein: The exogenous substance includes compounds, nucleic acids, peptides and proteins.

19. The electroporation buffer of claim 18, wherein: The nucleic acid includes siRNA, miRNA, mRNA, antisense oligonucleotide or DNA.

20. An electroporation-based exosome drug loading method, comprising: The method uses the electroporation buffer in any one of claims 1 to 19 to load the exogenous substance into the exosome.

21. The electroporation-based exosome drug loading method of claim 20, wherein: The method uses a static or flow electroporator and the matching consumables.

22. The electroporation-based exosome drug loading method of claim 20, wherein: The electric field intensity of the electroporation is controlled to be 250-5000 V / cm, and the total pulse width is 0.05-50 ms.

23. The electroporation-based exosome drug loading method of claim 20, wherein: The particle number ratio of the exosome to the exogenous substance is 1:100-1:100000.

24. The electroporation-based exosome drug loading method of claim 20, wherein: The particle number ratio of the exosome to the exogenous substance is 1E+10 p:(1-1000) μg.

25. The electroporation-based exosome drug loading method of claim 20, wherein: The osmotic pressure of the system of the electroporation is controlled to be 50-400 mOsm / kg, and the pH value is 2.5-8.3.

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