CN-122005498-A - Engineering exosome drug delivery system with enhanced membrane fluidity and preparation method and application thereof

Abstract

The invention relates to an engineering exosome drug delivery system with enhanced membrane fluidity, and a preparation method and application thereof. According to the system, unsaturated phospholipid is inserted into an exosome membrane, so that the membrane fluidity of the exosome is enhanced on the premise of not damaging the natural structure and the protein composition of the exosome membrane, and the entrapment efficiency of polypeptide proteins, nucleic acids and small molecular chemical drugs is remarkably improved. The enhancement of the fluidity of the membrane synchronously improves the diffusion capacity, the cellular uptake efficiency and the transepithelial transport performance of exosomes in intestinal mucus, and can effectively overcome the physiological barrier of oral delivery. In animal models of type I and type II diabetes, the system significantly improves the oral bioavailability of insulin and somalunin, and exhibits excellent glycemic control effects. The preparation method is simple and convenient, has good biocompatibility, and provides a novel delivery strategy with good conversion prospect for oral biological macromolecules and micromolecular chemical medicines.

Inventors

• HUANG YUAN
• NI MINGJIE
• HUA ZICHUN

Assignees

• 四川大学

Dates

Publication Date

20260512

Application Date

20260313

Claims (9)

1. 1. The membrane fluidity enhanced engineering exosome drug delivery system is characterized by comprising an extracted natural exosome, unsaturated phospholipids inserted into a phospholipid bilayer membrane of the exosome and an entrapped drug, wherein the exosome is at least one of milk, human milk, goat milk and macrophages, the unsaturated phospholipids are dioleoyl phospholipids, and the entrapped drug is positioned in the exosome or combined with the exosome membrane.
2. 2. The engineered exosome delivery system of claim 1, wherein the dioleoyl phospholipid comprises dioleoyl lecithin (DOPC), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), dioleoyl phosphatidic acid (DOPA), and dioleoyl phosphatidylserine (DOPS), preferably dioleoyl lecithin (DOPC).
3. 3. The engineered exosome delivery system according to claim 1, wherein the drug is selected from at least one of the following classes (1) biomacromolecule drugs including at least one selected from the group consisting of insulin, insulin analogues, somalupeptide, glucagon-like peptide-1 receptor agonists, growth hormone, octreotide acetate, antibodies or antibody fragments, preferably insulin or somalupeptide or TNF- α nanobody, ② nucleic acid drugs selected from at least one selected from the group consisting of small interfering ribonucleic acid, microribonucleic acid, messenger ribonucleic acid, deoxyribonucleic acid or plasmid deoxyribonucleic acid, preferably microribonucleic acid, (2) small molecule chemical drugs selected from at least one of 5-fluorouracil, paclitaxel, colchicine, carbamazepine, celecoxib, glimepiride, doxorubicin or curcumin, preferably 5-fluorouracil or paclitaxel.
4. 4. The engineered exosome delivery system according to claim 1, wherein the mass ratio of extracted natural exosomes to unsaturated phospholipids inserted into their phospholipid bilayer membrane is 10:1, 8:1, 4:1 and 1:1 (w/w), preferably 1:1 (w/w).
5. 5. A process according to claim 1 The method of engineering an exosome drug delivery system according to any one of claims 4, comprising the steps of: (1) Extracting exosomes by an ultra-high speed centrifugation method; (2) Incubating the unsaturated phospholipid with the extracted exosomes so that the unsaturated phospholipid is inserted into the exosome membrane, wherein the mass ratio of the exosomes to the unsaturated phospholipid is 10:1, 8:1, 4:1 and 1:1 (w/w); (3) Purifying by ultracentrifugation or size exclusion chromatography to obtain an engineered exosome with enhanced membrane fluidity; (4) The preparation method comprises the steps of uniformly mixing a drug solution and an engineering exosome dispersion liquid with enhanced membrane fluidity according to a certain proportion, coating the drug in the exosome or combining the drug on the exosome membrane by an incubation method, and removing the non-coated free drug by an ultrafiltration method.
6. 6. The method according to claim 5, wherein the incubation temperature in step (2) is 35-40 ℃ and the incubation time is 1-5h.
7. 7. An oral drug delivery system comprising the engineered exosome delivery system of any one of claims 1-4, and a pharmaceutically acceptable carrier or adjuvant.
8. 8. Use of the engineered exosome delivery system of any one of claims 1-4 or the oral drug delivery system of claim 7 in the preparation of a formulation for oral administration.
9. 9. The use according to claim 8, wherein the formulation is a solution, a capsule, a tablet or a granule.

Description

Engineering exosome drug delivery system with enhanced membrane fluidity and preparation method and application thereof Technical Field The invention belongs to the field of pharmaceutical preparations, and particularly relates to an engineering exosome drug delivery system based on membrane fluidity regulation and control, and a preparation method and application thereof. Background Exosomes are nanoscale extracellular vesicles actively secreted by cells, have good biocompatibility, low immunogenicity and natural ability to cross biological barriers, and have become a very promising drug delivery vehicle in recent years. Among the many sources, milk exosomes are readily available in raw materials, low in cost, mass producible, and demonstrated to be absorbable by the oral route, exhibiting unique industrialization and clinical application potential. The polypeptide and protein drugs, the nucleic acid drugs and part of small molecule chemical drugs have remarkable effects in the treatment of diseases. However, the oral delivery of the polypeptide protein and the nucleic acid drugs all face great challenges that polypeptide protein and nucleic acid drugs are easy to degrade in the gastrointestinal tract and difficult to cross intestinal epithelial barriers, and small molecule chemical drugs can be partially absorbed, but have the problems of low solubility, poor targeting, high systemic toxicity and the like. Together, these factors result in non-ideal oral bioavailability, and many drugs still rely heavily on injection administration at present, greatly affecting patient compliance and therapeutic effect. The natural exosomes are used as oral carriers, so that a new idea is provided for the difficult delivery of the medicines. However, exosomes are generally low in entrapment efficiency for various drugs, and their ability to cross complex intestinal barriers is limited, limiting their practical use. Studies have shown that exosome function is closely related to its membrane physical properties, where membrane fluidity is a key physical parameter that determines membrane fusion, substance transport across membranes, and efficiency of interaction with cells. However, in the prior art, chemical modification or targeted ligand coupling is focused on an exosome membrane, how to accurately regulate and control the fluidity of the exosome membrane by an engineering means so as to synchronously realize efficient entrapment of polypeptides, nucleic acids and small molecule chemical drugs and enhance the oral delivery efficiency of the polypeptides, nucleic acids and small molecule chemical drugs, and no systematic research report and effective technical scheme are still available at present. Therefore, a new strategy capable of regulating and controlling the membrane fluidity by physical means on the premise of not damaging the natural structure and function of exosomes is developed, so that the drug loading efficiency, intestinal penetration and oral bioavailability of various drugs are remarkably improved, and the novel strategy has important innovation significance and wide application value. Disclosure of Invention Aiming at the problems that polypeptide proteins and nucleic acid medicines are easy to degrade in the gastrointestinal tract and difficult to pass through intestinal epithelial barriers in the prior art, small molecular chemical medicines can be partially absorbed, but have low solubility, poor targeting property, high systemic toxicity and the like, the invention provides an exosome oral drug delivery system based on membrane fluidity regulation and control, and a preparation method and application thereof. It is an object of the present invention to provide an engineered exosome delivery system with enhanced membrane fluidity comprising an extracted natural exosome from at least one of milk, human milk, goat milk or macrophages, an unsaturated phospholipid inserted into the exosome phospholipid bilayer membrane, and a drug entrapped inside the exosome or bound to its membrane. The unsaturated phospholipid has unsaturated double bond in its molecular chain, so that the fluidity of lipid molecules is effectively enhanced, and the dynamic disorder of exosome membrane is obviously improved, while saturated phospholipid with similar structure (such as distearoyl phosphatidylcholine (DSPC)) has no effect of enhancing the fluidity of the same membrane due to lack of unsaturated double bond. The unsaturated phospholipids are dioleoyl phospholipids including, but not limited to, dioleoyl lecithin (DOPC), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), dioleoyl phosphatidic acid (DOPA), and dioleoyl phosphatidylserine (DOPS). The engineered exosomes have a controllable membrane fluidity, useful for efficient entrapment and oral delivery of biological macromolecular and small molecule chemical classes of drugs, wherein the unsaturated phospholipid is preferably dioleoyl lecith