Buffer solution and method for loading drug into extracellular vesicle

Abstract

Disclosed in the present invention are a buffer solution and method for loading a drug into an extracellular vesicle. Provided is a buffer solution for loading a drug into an extracellular vesicle. The buffer solution is a liquid medium used in a method for loading RNA into an extracellular vesicle, and the buffer solution comprises an anion. The anion comprises an acid radical ion, and the acid radical ion comprises oxygen and at least one multivalent element other than oxygen and is at a concentration of 2-80 mM. The buffer solution causes no substantial damage or causes controllable damage to the extracellular vesicle and RNA while having high loading efficiency, thus meeting the requirements for commercial applications.

Description

Buffer solution for drug delivery to extracellular vesicles and methods for drug delivery to extracellular vesicles

[0001] This application claims priority to Chinese patent application No. CN2025101264128, filed on January 27, 2025. Technical Field

[0002] This invention specifically relates to a buffer solution for drug delivery to extracellular vesicles and a method for drug delivery to extracellular vesicles, particularly a buffer solution used in an electroporation method for loading mRNA into extracellular vesicles. Background Technology

[0003] Extracellular vesicles (EVs) are heterogeneous groups of membrane structures secreted by cells and enclosed in a lipid bilayer. They contain tiny, non-replicating particles secreted by the cell. EVs vary in biological origin and size: exosomes are smaller vesicles, approximately 30–200 nm in diameter, formed within endosomes, organelles responsible for endocytosis and breakdown of intracellular substances; microvesicles are larger vesicles, 50–1000 nm in diameter, formed through cell membrane shedding and maturing and separating via protrusions on the cell membrane; apoptotic bodies are larger vesicles, greater than 1000 nm in diameter, formed during cell death. These vesicles are released when cells enter apoptosis (programmed cell death).

[0004] Extracellular vesicles (EVs) have been extensively studied as a potential alternative to cell therapy. EVs can avoid some of the risks associated with cell therapy applications. Cell therapy uses human cells to treat diseases, but there are some risks in practical applications, such as cells from different sources and the potential for immune responses after implantation. Exosomes, due to their widespread presence and simple content and structure, are considered the most suitable alternative to cell therapy. Exosomes offer several advantages as a viable alternative to cell therapy. As small membrane vesicles, exosomes can carry intracellular biomolecules, such as proteins and nucleic acids, and transmit this information between cells.

[0005] 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 bodily fluids, such as cerebrospinal fluid, urine, saliva, blood, vitreous humor, and breast milk. They can penetrate tissues, diffuse into the bloodstream, and even cross the blood-brain barrier (BBB). Exosomes are rich in nucleic acids, proteins, lipids, and metabolites. The substances carried by exosomes vary greatly depending on the type of source cell and its state (e.g., transformation, differentiation, stimulation, and stress), making them a highly heterogeneous group with a unique ability to induce biological responses. They also provide diagnostic and prognostic information for some diseases, such as metabolic diseases, cardiovascular diseases, neurodegenerative diseases, and tumors. Furthermore, as endogenous vesicles with nanoparticle size, exosomes possess high biocompatibility, natural homing properties, and the ability to achieve more specific enrichment in tissues, organs, and lesions after functional modification, making them a valuable research candidate for novel drug delivery carriers. Therefore, exosomes hold promising clinical applications—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 restoration.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] Nucleic acid molecular drugs are currently recognized in the industry as a class of drugs or drug raw materials that can be used in various disease models. The scientific and industrial communities currently hope to deliver nucleic acid drug molecules to lesions using exosomes, which are highly biocompatible and non-immunogenic, for disease treatment. However, there are still many problems with loading nucleic acid molecular drugs, especially large nucleic acid molecules (mRNA), onto exosomes, such as the stability of mRNA during loading and the low loading efficiency of exosomes.

[0010] Existing technologies also include some reports on exosome electroporation buffers. For example, CN114181974B 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 achieves good loading results, it is only effective for siRNA; its effectiveness for mRNA loading has not been reported. Furthermore, studies have shown that exosomes and mRNA are easily damaged at this pH value, thus affecting the application efficacy of drug-loaded exosomes.

[0011] Therefore, there is still a need to develop a buffer solution that does not cause substantial damage or causes controllable damage to extracellular vesicles and RNA, while maintaining high loading efficiency to meet the requirements of commercial applications. Summary of the Invention

[0012] One object of the present invention is to provide a buffer solution that does not cause substantial damage or causes controllable damage to extracellular vesicles and RNA (especially mRNA) while having high loading efficiency to meet the requirements of commercial applications.

[0013] The second objective of this invention is to provide a method for drug delivery of extracellular vesicles to RNA using the aforementioned buffer solution.

[0014] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0015] A buffer solution for drug delivery via extracellular vesicle electroporation, the buffer solution being a liquid medium used in a method of loading RNA into extracellular vesicles, the buffer solution comprising anions, the anions comprising acid radicals, the acid radicals comprising oxygen and at least one multivalent element other than oxygen, the concentration of the acid radicals being 2-80 mM, for example 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM or 80 mM.

[0016] In this article, "multivalent element" refers to an element with more than one valence, or an element with the ability to form multiple chemical bonds with other atoms. "Extracellular vesicles" are a type of small membrane-bound vesicle secreted by cells outside the cell, with diameters typically ranging from 30 nanometers to 1000 nanometers. Extracellular vesicles with a diameter of 30-200 nanometers are also called exosomes.

[0017] Preferably, the concentration of the anion is 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

[0018] In one embodiment, the anions are added in the form of salts, such as phosphates, hydrogen phosphates, sulfates, sulfites, etc. The concentration of the anions specifically refers to the concentration of anions added in the form of salts.

[0019] Preferably, the at least one multivalent element other than oxygen includes at least one of carbon, silicon, phosphorus, sulfur, and arsenic.

[0020] Preferably, the anion comprises one or more combinations selected from inorganic anions and / or organic anions. Examples of inorganic and organic anions (excluding those containing heavy metals or colored ions) include: arsenate ion (AsO4). 3- Arsenite ions (AsO3) 3- ), borate ion (BO3) 3- ), carbonate ions (CO3) 2- ), phosphate ions (PO4) 3-), phosphate ions (HPO3) 2- ), hydrogen phosphate ion (HPO4) 2- ), dihydrogen phosphate ion (H2PO4) 1- ), thiosulfate ions (S2O3) 2- sulfate ions (SO4) 2- ), sulfite ions (SO3) 2- persulfate ions (S2O8) 2- ), silicate ions (SiO4) 4- ), metasilicate ions (SiO3) 2- ), oxalate ion (C2O4) 2- ), citrate (C6H5O7) 3- )wait.

[0021] In a preferred embodiment, the acid radical ion includes one or more combinations selected from arsenate ion, arsenite ion, borate ion, carbonate ion, phosphate ion, phosphite ion, hydrogen phosphate ion, dihydrogen phosphate ion, thiosulfate ion, sulfate ion, sulfite ion, persulfate ion, silicate ion, metasilicate ion, oxalate ion, and citrate ion.

[0022] In one specific embodiment, the anions include phosphate ions containing oxygen and phosphorus, and the concentration of the phosphate ions is 2-80 mM, for example: 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM or 80 mM. The concentration of the phosphate ions is preferably 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

[0023] Further or optionally, the anions include sulfate ions containing oxygen and sulfur. Even further, the concentration of the sulfate ions is 2-80 mM, for example: 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM, or 80 mM. The concentration of sulfate ions is preferably 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

[0024] Further or alternatively, the anions include carbonate ions containing oxygen and carbon. Even further, the concentration of said carbonate ions is 2-80 mM, for example: 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM, or 80 mM. The concentration of the carbonate ions is preferably 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

[0025] Further or optionally, the anion includes citrate ions. Even further, the concentration of the citrate ions is 2-80 mM, for example: 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM, or 80 mM. The concentration of citrate ions is preferably 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

[0026] Further or optionally, the anion includes oxalate ions. Even further, the concentration of the oxalate ions is 2-80 mM, for example: 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM, or 80 mM. The concentration of oxalate ions is preferably 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

[0027] In one embodiment, the anion further includes a monovalent anion that does not contain any polyvalent elements other than oxygen. The concentration of the monovalent anion is 2-150 mM, for example, 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM, 80 mM, 65 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM, or 25-60 mM, or 25-50 mM.

[0028] Preferably, the concentration of the monovalent anion is 10-120 mM, more preferably 20-100 mM, and even more preferably 30-80 mM.

[0029] Furthermore, the monovalent anion includes chloride ions.

[0030] In one embodiment, the buffer solution comprises cations, the cations comprising one or more combinations selected from sodium ions, potassium ions, magnesium ions, and calcium ions.

[0031] In a first embodiment, the cation comprises sodium ions, and the concentration of sodium ions is 10-150 mM, for example 10 mM, 12 mM, 14 mM, 15 mM, 17 mM, 18 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, or 150 mM. The concentration of sodium ions is more preferably 10-50 mM.

[0032] In the second embodiment, the cation includes potassium ions, and the concentration of potassium ions is 2-20 mM, for example: 2 mM, 4 mM, 5 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM or 20 mM.

[0033] In the third embodiment, the cation includes magnesium ions, and the concentration of magnesium ions is 1-20 mM, for example: 1 mM, 2 mM, 4 mM, 5 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, 18 mM or 20 mM.

[0034] In the fourth embodiment, the cation includes calcium ions, and the concentration of calcium ions is 1-20 mM, for example: 1 mM, 2 mM, 4 mM, 5 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, 18 mM or 20 mM.

[0035] In one embodiment, the buffer solution comprises a sugar, the sugar comprising one or more combinations selected from trehalose, sucrose, mannose, glucose and fructose, the concentration of the sugar being 20-100 mM, for example: 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 76 mM, 80 mM, 65 mM, 90 mM, 95 mM or 100 mM.

[0036] In another embodiment, the buffer solution comprises iodixanol. Preferably, the iodixanol content is 1-25% (W / V)%, for example: 1% (W / V)%, 2% (W / V)%, 4% (W / V)%, 6% (W / V)%, 8% (W / V)%, 10% (W / V)%, 12% (W / V)%, 14% (W / V)%, 15% (W / V)%, 16% (W / V)%, 18% (W / V)%, 20% (W / V)%, 22% (W / V)%, 24% (W / V)%, or 25% (W / V)%.

[0037] In a specific and preferred embodiment, the buffer solution comprises sugar and iodixanol, wherein the sugar concentration is 20-100 mM and the iodixanol content is 1-25 (w / v)%.

[0038] In one embodiment, the solvent of the buffer solution includes water.

[0039] In one embodiment, the pH value of the buffer solution is 2.6-8.2, for example: 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, or 8.2.

[0040] In a preferred embodiment, the pH value of the buffer solution is 2.6-7.0, more preferably 2.6-6.5, and even more preferably 2.6-5.0 or 3.0-6.5.

[0041] In one embodiment, the osmotic pressure of the buffer solution is 30-350 mOsm / kg, for example: 30 mOsm / kg, 35 mOsm / kg, 40 mOsm / kg, 45 mOsm / kg, 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. g, 105mOsm / kg, 110mOsm / kg, 115mOsm / kg, 120mOsm / kg, 125mOsm / kg, 130mOsm / kg, 135mOsm / kg, 140mOsm / kg, 14 5mOsm / kg, 150mOsm / kg, 155mOsm / kg, 160mOsm / kg, 165mOsm / kg, 170mOsm / kg, 175mOsm / kg, 180mOsm / kg, 185mOs m / kg, 190mOsm / kg, 195mOsm / kg, 200mOsm / kg, 205mOsm / kg, 210mOsm / kg, 215mOsm / kg, 220mOsm / kg, 225mOsm / kg , 230mOsm / kg, 235mOsm / kg, 240mOsm / kg, 245mOsm / kg, 250mOsm / kg, 255mOsm / kg, 260mOsm / kg, 265mOsm / kg, 270 mOsm / 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 or 350mOsm / kg.

[0042] In a preferred embodiment, the osmotic pressure of the buffer solution is 30-300 mOsm / kg, more preferably 100-250 mOsm / kg.

[0043] In one embodiment, the conductivity of the buffer solution is 3-17 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.

[0044] In one embodiment, the buffer solution includes a pH adjuster selected from one or more of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, sodium hydroxide, and potassium hydroxide.

[0045] In one embodiment, the raw materials for the buffer solution include:

[0046] NaCl 10-150mM;

[0047] KCl 2-10mM;

[0048] MgCl2 1-20mM;

[0049] Selected from one or more combinations of phosphates, sulfates, carbonates, citrates, and oxalates, 2-50 mM;

[0050] Sugar 20-100mM;

[0051] pH adjuster 0.001-0.0000001M;

[0052] The solvent is water; wherein, the phosphates include one or more combinations selected from phosphates, hydrogen phosphates and dihydrogen phosphates, and the pH adjuster includes one or more selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, sodium hydroxide and potassium hydroxide.

[0053] In another embodiment, the raw materials for the buffer solution include:

[0054] NaCl 10-150mM;

[0055] KCl 2-20mM;

[0056] MgCl2 1-20mM;

[0057] Selected from one or more combinations of phosphates, sulfates, carbonates, citrates, and oxalates, 2-80 mM;

[0058] Iodixanol 1-25 (W / V)%;

[0059] pH adjuster 0.001-0.0000001M;

[0060] The solvent is water; wherein, the phosphates include one or more combinations selected from phosphates, hydrogen phosphates and dihydrogen phosphates, and the pH adjuster includes one or more selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, sodium hydroxide and potassium hydroxide.

[0061] Furthermore, the buffer solution also includes 20-100 mM of sugar.

[0062] In a preferred embodiment, the buffer solution is a liquid medium used in a method for loading mRNA into extracellular vesicles based on electroporation.

[0063] Optionally, the RNA includes mRNA, siRNA, miRNA, shRNA, or circular RNA.

[0064] Another technical solution adopted in this invention is as follows:

[0065] A method for drug delivery into extracellular vesicles, wherein the method uses the aforementioned buffer solution to load RNA into extracellular vesicles.

[0066] In one embodiment, mRNA is loaded into extracellular vesicles using an electroporation method.

[0067] In one embodiment, the method uses the X-Porator H1 electroporation instrument from Suzhou Yida Biotechnology Co., Ltd., and the matching electrode cups for electroporation.

[0068] In one embodiment, the electric field strength of the electroporation is controlled to be 250-1000 V / cm, and the total pulse width is 1000-10000 μs.

[0069] In one specific embodiment, the electroporation parameters are as follows: voltage 400V, pulse width 1500µs, pulse count 3, pulse interval 1000ms. In another specific embodiment, the electroporation parameters are as follows: voltage 300V, pulse width 2000µs, pulse count 3, pulse interval 1000ms.

[0070] In one embodiment, the buffer solution includes cations, anions, sugars, and solvents.

[0071] 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.

[0072] Furthermore, the anion is selected from two, three, four, or five of the following: chloride ion, hydrogen phosphate ion, dihydrogen phosphate ion, sulfate ion, and citrate ion.

[0073] 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.

[0074] Furthermore, the solvent is water.

[0075] Furthermore, the cations and anions are derived from inorganic salts and pH adjusters. The pH adjusters include one or more selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, sodium hydroxide, and potassium hydroxide. The inorganic salts are selected from one or more selected from 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.

[0076] Compared with the prior art, the present invention has the following advantages:

[0077] This invention addresses the characteristics of extracellular vesicles by developing a buffer solution suitable for electroporation loading of RNA (especially mRNA) into extracellular vesicles. Compared to chemical loading methods for exogenous substances, this solution does not introduce harmful chemicals, complying with drug clinical trial regulations. The buffer solution contains acid radicals, which, while causing no substantial damage or only controllable damage to extracellular vesicles and nucleic acid-like exogenous substances, significantly improves the loading efficiency of mRNA onto extracellular vesicles, meeting the requirements for commercial applications. Furthermore, the buffer solution and drug loading method of this invention have the advantage of low cost. Detailed Implementation

[0078] 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.

[0079] Because current electroporation-based extracellular vesicle RNA loading methods suffer from the inability to simultaneously address extracellular vesicle loading efficiency and damage to extracellular vesicles and nucleic acids, the applicant, after extensive research and summarization, firstly developed a buffer solution specifically for loading RNA into extracellular vesicles. Secondly, based on this buffer solution, the applicant further designed suitable electroporation conditions and achieved a high loading rate of mRNA into extracellular vesicles by precisely controlling the electric field.

[0080] Specifically, the applicant uses suitable acidic conditions and appropriate osmotic pressure to make it easier to form electroporation under a suitable electric field, thereby allowing nucleic acid molecules to enter extracellular vesicles and achieve the purpose of loading nucleic acid substances into extracellular vesicles.

[0081] This application further improves the RNA loading efficiency and stability of extracellular vesicles by modifying the buffer formulation components under suitable pH and osmotic pressure, especially for mRNA, exhibiting higher loading efficiency and stability.

[0082] Furthermore, extensive research has demonstrated that the buffer solution and extracellular vesicle drug loading method of this application not only have high and stable RNA loading efficiency, but also result in less extracellular vesicle breakage after electroporation, thus reducing the likelihood of extracellular vesicle breakage and aggregation.

[0083] The present invention will be further illustrated below through specific embodiments.

[0084] Example 1

[0085] Prepare Buffer-1 according to the following components:

[0086] NaCl: 10mM;

[0087] KCl: 4mM;

[0088] Na2HPO4: 2mM;

[0089] Solvent: RNase-free H2O.

[0090] Example 2

[0091] Prepare buffer-2 according to the following components:

[0092] NaCl: 10mM;

[0093] KCl: 2mM;

[0094] MgCl2: 1mM;

[0095] Na2HPO4: 25mM;

[0096] K2HPO4: 5mM;

[0097] Trehalose: 70 mM;

[0098] Solvent: RNase-free H2O.

[0099] Example 3

[0100] Prepare buffer-3 according to the following components:

[0101] NaCl: 10mM;

[0102] KCl: 2mM;

[0103] NaH2PO4: 40mM;

[0104] KH2PO4: 8mM;

[0105] Iodixanol: 22% (w / v);

[0106] Solvent: RNase-free H2O.

[0107] Example 4

[0108] Prepare buffer-4 according to the following components:

[0109] NaCl: 25mM;

[0110] KCl: 4mM;

[0111] MgCl2: 2mM;

[0112] NaH2PO4: 25mM;

[0113] K2HPO4: 5mM;

[0114] Trehalose: 50 mM;

[0115] Solvent: RNase-free H2O.

[0116] Example 5

[0117] Prepare Buffer-5 according to the following components:

[0118] NaCl: 15mM;

[0119] KCl: 4mM;

[0120] MgCl2: 2mM;

[0121] NaH2PO4: 25mM;

[0122] K2HPO4: 5mM;

[0123] Trehalose: 50 mM;

[0124] Solvent: RNase-free H2O.

[0125] Example 6

[0126] Prepare Buffer-6 according to the following components:

[0127] NaCl: 40mM;

[0128] KCl: 2mM;

[0129] MgCl2: 2mM;

[0130] NaH2PO4: 32mM;

[0131] K2HPO4: 5mM;

[0132] Trehalose: 20 mM;

[0133] Iodixanol: 5% (w / v);

[0134] Solvent: RNase-free H2O.

[0135] Example 7

[0136] Prepare Buffer-7 according to the following components:

[0137] NaCl: 10mM;

[0138] KCl: 2mM;

[0139] MgCl2: 1mM;

[0140] Na2SO4: 25mM;

[0141] K2SO4: 5mM;

[0142] Trehalose: 40 mM;

[0143] Solvent: RNase-free H2O.

[0144] Example 8

[0145] Prepare Buffer-8 according to the following components:

[0146] NaCl: 20mM;

[0147] KCl: 2mM;

[0148] MgCl2: 2mM;

[0149] Na2HPO4: 2mM;

[0150] Trehalose: 50 mM;

[0151] Solvent: RNase-free H2O.

[0152] The obtained buffer-8 had an osmotic pressure of 108 mOsm / kg and a conductivity of 3.88 mS / cm. The pH of the buffer was adjusted to 3.50 using hydrochloric acid solution.

[0153] Example 9

[0154] Prepare Buffer-9 according to the following components:

[0155] NaCl: 20mM;

[0156] KCl: 2mM;

[0157] MgCl2: 2mM;

[0158] Na2HPO4: 60mM;

[0159] K2HPO4: 20mM;

[0160] Trehalose: 20 mM;

[0161] Solvent: RNase-free H2O.

[0162] The obtained buffer-9 had an osmotic pressure of 348 mOsm / kg and a conductivity of 16.2 mS / cm. The pH of the buffer was adjusted to 3.65 using hydrochloric acid solution.

[0163] The properties of the buffer solutions obtained in Examples 1 to 9 are as follows:

[0164] pH value is 2.6-8.2;

[0165] The osmotic pressure is 30-350 mosm / kg;

[0166] The electrical conductivity is 3-17 mS / cm.

[0167] The buffer solutions obtained in Examples 1 to 9 can be used as buffer solutions for loading mRNA into exosomes. Depending on the pH of the buffer solution, anions containing multivalent elements other than oxygen can exhibit different valence states, such as -1, -2, -3, and -4.

[0168] Experiment 1:

[0169] 1. Preparation of extracellular vesicles:

[0170] Extracellular vesicles with a density of at least 2E+11 p / mL were stored in PBS for subsequent electroporation.

[0171] 2. Preparation of exogenous substances:

[0172] mRNA was dissolved using RNase-free H2O. The mRNA length was 980nt, and the nucleic acid concentration was prepared to be above 2ug / uL.

[0173] 3. Electroporation equipment:

[0174] Suzhou Yida Biotechnology Co., Ltd. - H1 electroporator and matching electrode cups.

[0175] 4. Experimental protocol for loading nucleic acid drugs via extracellular vesicle electroporation:

[0176] (4-1) Preparation of electroporation system: Buffer (buffer from Examples 1 and 2, and commonly used buffer PBS), extracellular vesicles and mRNA, prepared in appropriate proportions (using the corresponding electrode cup of the Yida H1 electroporator, prepare 100uL of electroporation system = 90uL of electroporation buffer + 5uL of extracellular vesicles + 5uL of nucleic acid);

[0177] (4-2) Electroporation: Transfer the electroporation system to an electrode cup, set the electroporation parameters, and perform electroporation. The electroporation parameters are: voltage 200V, pulse width 1500µs, pulse count 3, pulse interval 1000ms. Perform electroporation on the experimental groups in Table 1 below; do not perform electroporation on the con group.

[0178] Table 1 Comparison of Buffer-1 and Buffer-2

[0179] 5. Purification of extracellular vesicles:

[0180] After the electroporation experiment, the electroporation system was removed from the electroporation cup and transferred to a new EP tube. An appropriate amount of Oligo dT magnetic beads was added to purify the extracellular vesicles. The tube was placed on a magnetic rack and left to stand for 1 minute to remove free nucleic acids. The supernatant was the purified extracellular vesicles.

[0181] Note: 1 mg of Oligo dT magnetic beads corresponds to 2 μg of mRNA.

[0182] 6. Extraction and reverse transcription of mRNA loaded in extracellular vesicles:

[0183] (1) Total RNA was extracted from extracellular vesicles using the Trizol method.

[0184] (2) Using the PrimeScript reverse transcription kit TM The RT reagent kit (TaRaKa, RR037A) is used for reverse transcription of extracted RNA.

[0185] 7. qPCR detection:

[0186] (1) Primer customization: primers were synthesized at Suzhou Anshengda Biotechnology Co., Ltd.

[0187] (2) Use ChamQ Universal SYBR qPCR Master Mix (vazyme, Q711-02) for qPCR testing.

[0188] 8. The results of qPCR show the amplification of the target gene mRNA fragment in the electroporation group relative to the con group. The results show that buffer 2 has the best effect. See Figure 1 for details.

[0189] Experiment 2:

[0190] Based on buffer-2, we compared the effect of changing pH on loading performance when the osmotic pressure of the buffer solution remained approximately constant.

[0191] 1. Using the experimental scheme of Experiment 1 above, compare the effects of the three buffer solutions of Examples 2, 4 and 5 on the electroporation loading of mRNA onto exosomes.

[0192] 2. The experimental grouping is shown in Table 2 below. The con group does not undergo electroporation.

[0193] Table 2 Comparison of Buffer-2, Buffer-4 and Buffer-5

[0194] 3. The experimental results are shown in Figure 2. The results show that buffer-2 has the best loading effect.

[0195] Experiment 3:

[0196] Based on buffer-2, we compared the effect of changing the osmotic pressure on the loading effect when the pH of the buffer solution remained approximately constant.

[0197] 1. Using the experimental scheme of Experiment 1 above, compare the effects of the three buffer solutions of Examples 2, 3 and 6 on the electroporation loading of mRNA onto exosomes.

[0198] 2. The experimental grouping is shown in Table 3 below. The con group does not undergo electroporation.

[0199] Table 3 Comparison of Buffer-2, Buffer-3 and Buffer-6

[0200] 3. The experimental results are shown in Figure 3. The results show that buffer-2 has the best loading effect.

[0201] Experiment 4:

[0202] Based on Buffer-2, the phosphate in the raw material was replaced with sulfate, and it was observed whether this would affect the loading effect of exosomes.

[0203] 1. Using the experimental scheme of Experiment 1 above, compare the effects of the two buffer solutions in Example 2 and Example 7 on the electroporation loading of mRNA onto exosomes.

[0204] 2. The experimental groups are shown in Table 4 below. The con group does not undergo electroporation.

[0205] Table 4 Comparison of Buffer-2 and Buffer-7

[0206] 3. The experimental results are shown in Figure 4. The results show that the effects of exosome electroporation loading of mRNA are similar when using buffer-2 and buffer-7.

[0207] Experiment 5:

[0208] 1. Cell preparation: Culture HFF-1 cells. HFF-1 cells are seeded and cultured in a six-well plate for later use.

[0209] 2. On the second day, the electroporation protocol of Experiment 1 was used to load mRNA into exosomes by electroporation using buffer-2. The experimental groups are shown in Table 5 below.

[0210] Table 5. Cell delivery experiments using Buffer-2 exosome electroporation to load mRNA.

[0211] 3. Add 100 μL of exosomes loaded with mRNA via electroporation to a six-well plate culture medium for HFF-1 cells, mix the medium well, and co-incubate the exosomes and cells for 24 hours.

[0212] 4. After co-incubating exosomes and cells for 24 hours, cells were collected, total RNA was extracted, and then RT-qPCR experiments were performed. The target genes for qPCR were the transferred mRNA fragments and the cell's internal reference gene β-actin, in order to detect whether there was mRNA delivered to the cells by exosomes.

[0213] 5. The experimental results of qPCR are shown in Figure 5. The figure shows the relative expression of the mRNA delivered into the cell by the exosomes relative to the cell's internal reference gene β-actin. The results show that the mRNA carried by the exosomes was delivered into the cell during co-incubation with the cell.

[0214] In the above embodiments, electroporation is used for drug loading onto extracellular vesicles. Electroporation is a physical drug loading method that, compared to chemical drug loading methods, does not introduce harmful chemicals during extracellular vesicle drug loading, thus meeting the regulatory requirements for clinical trial applications. A dedicated buffer solution with an osmotic pressure of 30-350 mOsm / kg was prepared for extracellular vesicle electroporation. Within this osmotic pressure range, the pH of the buffer solution was adjusted to 2.6-8.2 to ensure that the conductivity of the buffer solution was 3-17 mS / cm, and that this buffer solution environment would not cause substantial damage to extracellular vesicles or nucleic acid drugs. Based on this buffer solution and using appropriate electroporation parameters of the Yida Bio H1 electroporator, a complete extracellular vesicle drug loading process was formed.

[0215] The above embodiments of the scheme for loading nucleic acid drugs onto extracellular vesicles based on electroporation technology significantly improve the encapsulation and loading rates of drugs onto extracellular vesicles compared to chemical methods, and the experimental process cost is lower.

[0216] The buffer solution described in the above embodiments has significant advantages for loading large nucleic acid drugs (mRNA) into extracellular vesicles, maintaining stable and efficient loading of mRNA while ensuring good integrity of extracellular vesicle particles.

[0217] 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. A buffer solution for drug delivery in extracellular vesicles, characterized in that, The buffer solution is a liquid medium used in a method of loading RNA into extracellular vesicles. The buffer solution includes anions, the anions include acid radicals, the acid radicals include oxygen and at least one multivalent element other than oxygen, and the concentration of the acid radicals is 2-80 mM.

2. The buffer solution for drug delivery in extracellular vesicles according to claim 1, characterized in that, The concentration of the anion is 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

3. The buffer solution for drug delivery in extracellular vesicles according to claim 1, characterized in that, The at least one multivalent element other than oxygen includes at least one of carbon, silicon, phosphorus, sulfur, and arsenic.

4. The buffer solution for drug delivery in extracellular vesicles according to claim 1, characterized in that, The acid radical ions include one or more combinations selected from inorganic acid radical ions and / or organic acid radical ions.

5. The buffer solution for drug delivery in extracellular vesicles according to claim 4, characterized in that, The acid radical ions include one or more combinations selected from arsenate ions, arsenite ions, borate ions, carbonate ions, phosphate ions, phosphite ions, hydrogen phosphate ions, dihydrogen phosphate ions, thiosulfate ions, sulfate ions, sulfite ions, persulfate ions, silicate ions, metasilicate ions, oxalate ions, and citrate ions.

6. The buffer solution for drug delivery in extracellular vesicles according to claim 1, characterized in that, The anions include phosphate ions containing oxygen and phosphorus, and the concentration of the phosphate ions is 2-80 mM, or 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM; And / or, the anions include sulfate ions containing oxygen and sulfur, wherein the concentration of the sulfate ions is 2-80 mM, or 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM; And / or, the anions include carbonate ions containing oxygen and carbon, wherein the concentration of the carbonate ions is 2-80 mM, or 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM; And / or, the anion includes an oxygen- and carbon-containing citrate ion, the concentration of which is 2-80 mM, or 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM; And / or, the anion includes oxygen- and carbon-containing oxalate ions, the concentration of which is 2-80 mM, or 5-80 mM, or 5-75 mM, or 5-70 mM, or 5-65 mM, or 5-60 mM, or 10-80 mM, or 15-80 mM, or 20-80 mM, or 25-80 mM, or 10-75 mM, or 10-70 mM, or 10-65 mM, or 10-60 mM, or 15-75 mM, or 15-70 mM, or 15-65 mM, or 15-60 mM, or 20-75 mM, or 20-70 mM, or 20-65 mM, or 20-60 mM, or 25-60 mM, or 25-50 mM.

7. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The anion also includes a monovalent anion, which may be a chloride ion. The concentration of the monovalent anion is 2-150 mM, preferably 10-120 mM, more preferably 20-100 mM, and even more preferably 30-80 mM.

8. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The buffer solution includes cations, which include one or more combinations selected from sodium ions, potassium ions, magnesium ions and calcium ions; wherein the concentration of sodium ions is 10-150 mM, the concentration of potassium ions is 2-20 mM, the concentration of magnesium ions is 1-20 mM, and the concentration of calcium ions is 1-20 mM.

9. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The buffer solution includes a sugar, which includes one or more combinations selected from trehalose, sucrose, mannose, glucose, and fructose, at a concentration of 20-100 mM; and / or, the buffer solution includes iodixanol at a concentration of 1-25 (w / v)%.

10. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The solvent for the buffer solution includes water.

11. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The pH value of the buffer solution is 2.6-8.2, preferably 2.6-7.0, more preferably 2.6-6.5, and even more preferably 2.6-5.0 or 3.0-6.

5.

12. The buffer solution for drug delivery in extracellular vesicles according to claim 11, characterized in that, The osmotic pressure of the buffer solution is 30-350 mOsm / kg, preferably 30-300 mOsm / kg, and more preferably 100-250 mOsm / kg.

13. The buffer solution for drug delivery in extracellular vesicles according to claim 12, characterized in that, The conductivity of the buffer solution is 3-17 mS / cm.

14. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The buffer solution includes a pH adjuster, which may be one or more selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, sodium hydroxide, and potassium hydroxide.

15. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The raw materials for the buffer solution include: NaCl 10-150mM; KCl 2-20mM; MgCl2 1-20mM; Selected from one or more combinations of sulfates, carbonates, citrates, phosphates, and oxalates, 2-80 mM; Sugar 20-100mM; Iodixanol 1-25 (W / V)% pH adjuster 0.001-0.0000001M; The solvent is water.

16. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The buffer solution is a liquid medium used in a method for loading mRNA into extracellular vesicles based on electroporation.

17. The buffer solution for drug delivery in extracellular vesicles according to any one of claims 1 to 6, characterized in that, The RNA includes mRNA, siRNA, miRNA, shRNA, or circular RNA.

18. A method for drug delivery via extracellular vesicles, characterized in that, The method uses the buffer solution described in any one of claims 1 to 17 to load RNA into extracellular vesicles.

19. The extracellular vesicle drug delivery method according to claim 18, characterized in that, mRNA is loaded into extracellular vesicles using an electroporation method.

20. The extracellular vesicle drug delivery method according to claim 19, characterized in that, The method uses the X-Porator H1 electroporation instrument from Suzhou Yida Biotechnology Co., Ltd., and the matching electrode cups for electroporation.

21. The extracellular vesicle drug delivery method according to claim 19, characterized in that, The voltage of the electroporation is controlled to be 250-1000V / cm, and the total pulse width is 1000-10000us.

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