LOADING OF EXTRACELLULAR VESICLES THROUGH IMPARTING OF MECHANICAL SHEAR
Methods of loading extracellular vesicles with payload molecules via homogenization are disclosed herein.
1 . A method for producing an isolated extracellular vesicle for delivery of a payload molecule, the method comprising:
modifying an extracellular vesicle with a payload molecule via microfluidization, wherein the payload molecule is a therapeutic molecule, and
isolating the modified extracellular vesicle containing the payload molecule.
2 . The method of claim 1 , comprising formulating the modified isolated extracellular vesicle into a pharmaceutical composition.
3 - 4 . (canceled)
5 . The method of claim 1 , wherein the microfluidization is a single pass.
6 . The method of claim 1 , wherein the microfluidization is performed between 10,000 to 30,000 psi.
7 - 8 . (canceled)
9 . The method of claim 5 , wherein the single pass microfluidization is performed at 30,000 psi.
10 . The method of claim 1 , wherein the microfluidization is multiple passes.
11 - 12 . (canceled)
13 . The method of claim 1 , wherein the extracellular vesicle is in a buffered solution between pH 7 and 8, comprising phosphate buffered saline and 0.5-5% sucrose.
14 - 19 . (canceled)
20 . The method of claim 1 , wherein the microfluidization occurs in a volume of at least 1 ml.
21 . The method of claim 1 , wherein the method is performed at a temperature of 15° C. to 80° C.
22 - 23 . (canceled)
24 . The method of claim 1 , wherein the payload molecule is an siRNA, an miRNA, an antisense RNA, a DNA, a plasmid, an mRNA, a tRNA, a protein, a carbohydrate, a lipid, a small molecule drug, a STING agonist, a toxin, an antibody, a recombinant protein, a viral vector, or a vaccine.
25 . The method of claim 1 , wherein the payload molecule is a STING agonist.
26 . The method of claim 1 , wherein the extracellular vesicle and the payload molecule are first mixed in a solution and the solution is homogenized.
27 . The method of claim 26 , wherein a the payload molecule is added to the homogenized extracellular vesicle solution.
28 . The method of claim 1 , wherein the extracellular vesicle is modified with a plurality of payload molecules.
29 - 30 . (canceled)
31 . The method of claim 1 , wherein the extracellular vesicle in solution is both microfluidized and treated with a technology for loading the payload molecules.
32 . The method of claim 31 , wherein the extracellular vesicle is pre-treated before microfluidization or post-treated after microfluidization.
33 . (canceled)
34 . The method of claim 31 , wherein the technology is a chemical treatment.
35 . The method of claim 1 , wherein the microfluidization is done comprises a batch, a semi-batch, or a continuous process.
36 . An extracellular vesicle comprising a payload molecule, wherein the extracellular vesicle is prepared by the method of claim 1 .
37 . An extracellular vesicle comprising a payload molecule, wherein the payload molecule is loaded into the extracellular vesicle via homogenization.