Loading of extracellular vesicles through imparting of mechanical shear

Abstract

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.