Bottom-up assembly of synthetic extracellular vesicles
Inventors
Staufer, Oskar • Plazman, Yilia • Spatz, Joachim P.
Assignees
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Publication Number
US-12569439-B2
Publication Date
2026-03-10
Expiration Date
Abstract
The present invention relates to a method for producing synthetic extracellular vesicles comprising a lipid bilayer including at least two lipids, one or more extracellular vesicle associated proteins, and optionally one or more nucleic acid molecules. The inventive synthetic extracellular vesicles are formed by emulsification using a mechanic emulsifier in the form of polymer shell stabilized synthetic extracellular vesicles. The inventive method allows producing synthetic extracellular vesicles miming the composition and function of natural extracellular vesicles. Therefore, synthetic extracellular vesicles with specific protein and nucleic acids compositions are also disclosed herein, as well as their therapeutic uses.
Core Innovation
The invention relates to synthetic extracellular vesicles, including synthetic exosomes, made as polymer shell-stabilized synthetic extracellular vesicles with defined lipid bilayers and extracellular vesicle associated proteins or fragments thereof. The vesicles are produced with a bottom-up emulsification-based assembly in which a water phase is combined with an oil phase to generate vesicles that are homogenous in size, having a coefficient of variation in size lower than 13% and a hydrodynamic radius between 70 nm and 5000 nm.
The method combines a water phase comprising at least two lipids, including a negative charged lipid and a lipid coupled to a functional ligand for conjugation to one or more extracellular vesicle associated proteins or fragments thereof, together with the extracellular vesicle associated proteins or fragments. An amphiphilic copolymer dissolved in an oil phase is used, where the amphiphilic copolymer is a diblock copolymer with a hydrophobic polymer block and a hydrophilic polymer block or a triblock copolymer with two hydrophobic polymer blocks and a hydrophilic polymer block, and the oil phase comprises a fluorosurfactant triblock.
The combined phases are emulsified using a mechanic or electronic emulsifier to produce vesicles in which the amphiphilic copolymer forms a polymer shell stabilizing the synthetic extracellular vesicle. The vesicles have an arrangement in which the one or two hydrophobic polymer blocks are arranged on the outer side and the hydrophilic polymer block is arranged on the inner side of the polymer shell.
Claims Coverage
The provided claim set includes two independent claims. The first independent claim covers a bottom-up method of producing polymer shell-stabilized synthetic extracellular vesicles by combining a lipid/protein water phase with a fluorosurfactant/oil-phase amphiphilic diblock or triblock copolymer and emulsifying the combined phases to obtain vesicles with defined size metrics and hydrodynamic radius. The second independent claim covers a synthetic extracellular vesicle composition having a defined hydrodynamic radius, a specified lipid bilayer composition, and one or more extracellular vesicle associated proteins selected from a specified group.
Bottom-up production of polymer shell-stabilized synthetic extracellular vesicles by fluorosurfactant/oil-phase emulsification
A method for producing polymer shell-stabilized synthetic extracellular vesicles comprising: providing a water phase comprising at least two lipids including a negative charged lipid and a lipid coupled to a functional ligand for conjugation to one or more extracellular vesicle associated proteins or fragments thereof; providing an amphiphilic copolymer dissolved in an oil phase as a diblock copolymer with a hydrophobic polymer block and a hydrophilic polymer block or as a triblock copolymer with two hydrophobic polymer blocks and a hydrophilic polymer block, wherein the oil phase comprises a fluorosurfactant triblock; combining the water phase and the oil phase; producing polymer shell-stabilized synthetic extracellular vesicles by emulsifying the combined phases using a mechanic or electronic emulsifier; forming a polymer shell stabilizing the synthetic extracellular vesicle with the hydrophobic block(s) arranged on the outer side and the hydrophilic block arranged on the inner side; wherein the vesicles are homogenous in size showing a coefficient of variation in size lower than 13% and have a hydrodynamic radius between 70 nm and 5000 nm.
Synthetic extracellular vesicle composition with defined hydrodynamic radius, lipid bilayer classes, ligand-coupled lipids, and extracellular vesicle associated proteins
A synthetic extracellular vesicle having a hydrodynamic radius between 70 nm and 5000 nm comprising a lipid bilayer comprising at least two lipids selected from the group comprising neutral lipids, anionic lipids, cationic lipids, pH-sensitive lipids, photoswitchable lipids, and related listed lipid classes; one or more extracellular vesicle associated proteins selected from a group comprising specified membrane proteins and other specified protein classes, including fragments thereof; and one or more lipids coupled to a functional ligand selected from the group comprising biotin, N-hydroxysuccinimide ester, nitrilotriacetic acid-nickel, maleimides, aromatic maleimid, dithiopyridinyl, pyridyl disulfide, pyridyldithiopropionate, N-benzylguanine, cyanuric chloride, a magnetic resonance imaging reagent, and a chelator, and/or one or more lipids coupled to polyethyleneglycol with a molecular weight comprised between 350 and 50,000 g/mole.
Overall, the claim coverage centers on a production method defined by the specific combination of a lipid/protein water phase, a fluorosurfactant-containing oil phase with a specified amphiphilic diblock/triblock copolymer, and emulsification to obtain size-homogeneous polymer shell-stabilized synthetic extracellular vesicles with specified hydrodynamic radius, and on a synthetic extracellular vesicle composition defined by lipid bilayer classes, ligand-coupled lipids, optional PEG coupling, and one or more extracellular vesicle associated proteins selected from specified groups.
Stated Advantages
Higher stability compared to prior art.
Controlled composition.
High-efficiency nucleic-acid encapsulation.
Size homogeneity with a coefficient of variation (CV) lower than 13%.
Higher purity and batch reproducibility.
Documented Applications
Treating or ameliorating a disorder in a patient by administering a therapeutically effective amount of a synthetic extracellular vesicle, where the disorder is selected from a listed group of disease categories including inflammation, cancer, rheumatic disorders, GVHD, osteoarthritis, cardiovascular/epithelial/neurodegenerative/autoimmune/bone/cartilage disorders, osteoporosis, renal osteodystrophy, Paget’s disease of bone, rickets, intoxication, neuroendocrinology/endocrinology disorders, genetic/infectious/dental disorders, cosmetic procedures, coagulation disorders, dermatoses, diabetes, and age-associated disorders.
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