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CN-122012367-A - Engineering exosome and preparation method and application thereof

CN122012367ACN 122012367 ACN122012367 ACN 122012367ACN-122012367-A

Abstract

The invention relates to an engineering exosome, a preparation method and application thereof. The invention develops an integrated platform technology, designs composite nano particles with specific structures and compositions, and develops an exosome preparation process based on the composite nano particles so as to simultaneously improve four steps of an engineering exosome production flow, thereby realizing efficient preparation of the engineering exosome, and the obtained exosome has good stability, can efficiently load medicines, and can be applied to treatment of cerebral nerve diseases, respiratory diseases and the like.

Inventors

  • RUAN GANG
  • WEN XIAOWEI
  • XU ZIXING
  • Hao Zerun
  • CHEN YANMING
  • Yin Haofan
  • XIE KAI
  • WANG XUEYING
  • MIN JIE

Assignees

  • 西交利物浦大学

Dates

Publication Date
20260512
Application Date
20260204

Claims (10)

  1. 1. A composite nanoparticle is characterized in that, the composite nanoparticle comprises a nanoparticle and an exosome biosynthesis stimulating ligand coupled with the nanoparticle; The nanoparticles include self-assemblable proteins and superparamagnetic iron oxide nanoparticles.
  2. 2. The composite nanoparticle of claim 1, wherein the exosome biosynthesis stimulating ligand comprises a transmembrane peptide; Optionally, the transmembrane peptide comprises Tat transmembrane peptide; Optionally, the amino acid sequence of the Tat transmembrane peptide comprises a sequence shown in SEQ ID NO. 1.
  3. 3. The composite nanoparticle of claim 1 or 2, wherein the protein comprises at least one of bovine serum albumin, human serum albumin, egg white lysozyme, bovine lactalbumin, zein, β -casein, egg white albumin, β -lactoglobulin, or lactoferrin.
  4. 4. A composite nanoparticle according to any one of claims 1 to 3, further comprising a drug and/or detection agent, the drug and/or detection agent being supported on the nanoparticle; optionally, the drug comprises at least one of a small molecule, a nucleic acid, or a polypeptide; optionally, the detection agent comprises a molecular imaging probe.
  5. 5. The method of preparing composite nanoparticles of claim 1, comprising: dissolving a protein solution in an aqueous solution to prepare a protein solution; dissolving superparamagnetic iron oxide nanoparticles in an organic solvent to prepare a nanocrystal solution; Mixing the nanocrystal solution with the protein solution, incubating the mixed solution at room temperature to obtain an incubated mixed solution, centrifuging the incubated mixed solution, removing supernatant, and collecting precipitate to obtain nanoparticles; Coupling the nanoparticle with an exosome biosynthesis stimulating ligand to obtain the composite nanoparticle.
  6. 6. Use of the composite nanoparticle of any one of claims 1-4 for the preparation of exosomes.
  7. 7. A method of preparing an exosome, the method comprising: mixing and incubating exosome synthesized cells with the composite nano-particles according to any one of claims 1-4, and magnetically separating exosome to obtain exosome.
  8. 8. The method of preparing an exosome of claim 7, wherein the method of magnetic separation comprises: The modularized magnet array is assembled into a cuboid by using a permanent magnet, the modularized magnet array is placed in the separation container, a driving magnet is arranged outside the separation container, and the driving motor and a transmission device are linked with the driving magnet, so that the modularized magnet array in the separation container slowly moves according to a set rotating speed, and an exosome containing composite nano particles is captured on the surface of the modularized magnet array; The exosomes are then dissociated from the modular magnet array surface for harvesting using physical ultrasound or chemical dissociation liquid assisted flushing.
  9. 9. An engineering exosome is characterized in that, the engineered exosome is produced by the method of producing exosome of claims 7 or 8.
  10. 10. Use of an engineered exosome of claim 9 in the manufacture of a medicament for treating a disease comprising at least one of a neurological disease, respiratory disease, skin disease, cardiovascular disease, reproductive disease, cancer, metabolic disease, immune system disease, or ophthalmic disease.

Description

Engineering exosome and preparation method and application thereof Technical Field The invention belongs to the technical field of biology, and relates to an engineering exosome, a preparation method and application thereof. Background Extracellular Vesicles (EVs), such as exosomes, have shown great potential as emerging cell therapies and nanomedicines. Exosomes (common diameter ranges of 50-150 nm) are considered more suitable for therapy than other types of EVs (e.g., microvesicles and apoptotic bodies) because the biogenesis of exosomes involves unique intracellular regulatory processes that may determine their composition and function, resulting in a more controlled therapeutic effect. However, the therapeutic effect of exosomes themselves is often insufficient to produce clinical value, and therefore "engineered exosomes" have been developed that confer enhanced and/or additional functions by binding to drugs (including small molecules, nucleic acids, protein drugs that have therapeutic effects). For example, CN116271097A discloses an engineering exosome based on a metal organic framework, which takes a tumor homing peptide modified exosome as a shell, takes a metal organic framework ZIF-8 as an inner core, and loads superparamagnetic iron oxide nano particles and doxorubicin as a multifunctional tumor diagnosis and treatment reagent. Currently, clinical transformations of engineered exosomes present challenges in production, including insufficient quantity and purity, difficulty in efficient high-volume loading of cargo, low efficiency, poor scalability, the need for expensive separation equipment, poor storage stability, and high overall costs. To improve the production of engineered exosomes, techniques have been developed such as starving the cells of origin to stimulate their production, separating the exosomes using tangential flow filtration to increase the scalability of separation, mechanically treating the exosomes to temporarily open the exosome membrane to increase the cargo load, using cryoprotectants to reduce the stability loss of the exosomes during freeze-drying storage during freeze-thawing cycles, etc. However, in general, each of these techniques can only improve one of the four steps of the engineered exosome manufacturing process (or in rare cases two steps), namely exosome biosynthesis (secretion), cargo loading, isolation and storage, and it is difficult to meet the increasing demands of clinical transformations for the efficiency and quality of exosome manufacturing. In conclusion, the development of a method for efficiently preparing high-quality exosomes has important significance for the application field of exosomes. Disclosure of Invention Aiming at the defects and actual demands of the prior art, the invention provides an engineering exosome, and a preparation method and application thereof, so as to prepare high-quality exosome with high efficiency. In order to achieve the above purpose, the invention adopts the following technical scheme: in a first aspect, the present invention provides a composite nanoparticle comprising a nanoparticle and an exosome biosynthesis stimulating ligand coupled thereto, the nanoparticle comprising a self-assemblable protein and a superparamagnetic iron oxide nanoparticle. The composite nano particles with specific structures can be used as a ligament, so that the effect of all main steps (including secretion, drug loading, separation and storage) of the production flow of the engineering exosome is greatly improved, and the efficient preparation of the high-quality exosome is realized. Optionally, the exosome biosynthesis-stimulating ligand comprises a transmembrane peptide. Optionally, the transmembrane peptide comprises Tat transmembrane peptide. Optionally, the amino acid sequence of the Tat transmembrane peptide comprises a sequence shown in SEQ ID NO. 1. Alternatively, the protein comprises any one or a combination of at least two of bovine serum albumin, human serum albumin, egg white lysozyme, bovine lactalbumin, zein, beta-casein, ovalbumin, beta-lactoglobulin, or lactoferrin. . Optionally, the composite nanoparticle further comprises a drug and/or detection agent, which is supported on the nanoparticle. Optionally, the drug comprises at least one of a small molecule, a nucleic acid, or a polypeptide. Optionally, the detection agent comprises a molecular imaging probe. In a second aspect, the present invention provides a method for preparing the composite nanoparticle according to the first aspect, comprising: dissolving a protein solution in an aqueous solution to prepare a protein solution; dissolving superparamagnetic iron oxide nanoparticles in an organic solvent to prepare a nanocrystal solution; Mixing the nanocrystal solution with the protein solution, incubating the mixed solution at room temperature to obtain an incubated mixed solution, centrifuging the incubated mixed solution, removing supernatant, and collecting preci