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 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.