Loading of Extracellular Vesicles through Imparting of Mechanical Shear

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 homogenization,

isolating the modified extracellular vesicle containing the payload molecule, and

optionally formulating the isolated modified extracellular vesicle into a pharmaceutical composition.

2 . The method of claim 1 , wherein the vesicle is an exosome, a nanovesicle, an apoptotic body, a microvesicle, a lysosome, an endosome, an enveloped virus, a viral vector, a liposome, a lipid nanoparticle, a micelle, a multilamellar structure, a revesiculated vesicle, or an extruded cell.

3 . The method of claim 1 , wherein the payload molecule is a therapeutic molecule.

4 . The method of claim 1 , wherein the homogenization is microfluidization.

5 . The method of claim 4 , wherein the microfluidization is a single pass.

6 . The method of claim 4 , wherein the microfluidization is performed between 10,000 to 30,000 psi.

7 . (canceled)

8 . (canceled)

9 . (canceled)

10 . The method of claim 1 , wherein the microfluidization is multiple passes, wherein the pressure is different in the multiple passes.

11 . (canceled)

12 . The method of claim 10 , wherein the pressure is different in the multiple passes.

13 . The method of claim 1 , wherein the vesicle is in a buffered solution.

14 . (canceled)

15 . (canceled)

16 . The method of claim 13 , wherein the buffer is between pH 7 and 8.

17 . (canceled)

18 . (canceled)

19 . (canceled)

20 . The method of claim 1 , wherein homogenization occurs in a volume of at least 1 ml.

21 . The method of claim 1 , wherein the temperature is 15° C. to 80° C.

22 . (canceled)

23 . (canceled)

24 . The method of claim 1 , wherein the payload 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 is siRNA.

26 . The method of claim 1 , wherein the vesicle and the payload are first mixed in a solution and the solution is homogenized.

27 . The method of claim 1 , wherein a solution comprising the vesicle is homogenized first and the payload is added to the homogenized vesicle solution.

28 . The method of claim 1 , wherein the vesicle is modified with a plurality of payload molecules.

29 . (canceled)

30 . (canceled)

31 . The method of claim 1 , wherein the vesicle in solution is both homogenized and treated with another technology for loading the payload molecules.

32 . The method of claim 31 , wherein the vesicle is pre-treated before homogenization or post-treated after homogenization.

33 . (canceled)

34 . The method of claim 31 , wherein the additional treatment is a chemical treatment.

35 . (canceled)

36 . The method of claim 25 , wherein the siRNA is conjugated to a cholesterol moiety.