Vacuole-inducing compounds, methods of making and using the same

Abstract

Vacuole- or exosome-inducing indole-based chalcone and substituted triazole-hydrazone compounds that induce endosomal vacuolization and increase exosome yield, but do not trigger growth arrest or cell death, and methods of making and using are described.

What is claimed is: 
     
         1 . A method for increasing exosome production in a cell, comprising: exposing the cell to an indole-pyridinyl-propenone compound that induces endosomal vacuolization but does not trigger growth arrest or cell death. 
     
     
         2 . The method of  claim 1  wherein, the compound has the following chemical structure: 
       
         
           
           
               
               
           
         
         R═OH, OCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , NHCOCH 3  or NHCO 2 C(CH 3 ) 3 ; and 
         R′═H, CH 3 , CH 2 CH 2 CH 3 , CH(CH 3 ) 2 , or CH 2 CH(CH 3 ) 2 ; 
         excluding where R═OCH 3  and R′═CH 3 . 
       
     
     
         3 . The method of  claim 2 , wherein R═OCH 3 ; and, R′═H, CH 2 CH 2 CH 3 , CH(CH 3 ) 2 , or CH 2 CH(CH 3 ) 2 . 
     
     
         4 . The method of  claim 3 , wherein R═OCH 3 , and R′═CH 2 CH 2 CH 3 . 
     
     
         5 . The method of  claim 2 , wherein R═OH, OCH 2 CH 3 , OCH(CH 3 ) 2 , NHCOCH 3  or NHCO 2 C(CH 3 ) 3 ; and, R′═CH 3 . 
     
     
         6 . The method of  claim 5 , wherein R═OCH 2 CH 3 ; and, R′═CH 3 . 
     
     
         7 . The method of  claim 2 , wherein R═OCH 2 CH 3 ; and R′═CH 2 CH 2 CH 3 . 
     
     
         8 . The method of  claim 1 , wherein exosomes are collected by one or more of the following: ultracentrifugation; column chromatography; size exclusion; and filtration through a device containing an affinity matrix that is selective toward exosomes. 
     
     
         9 . A method using exosomes produced by  claim 1 , comprising harvesting the exosomes for use as nanocarriers for packaging and delivery of a therapeutic material to a subject in need thereof. 
     
     
         10 . The method of  claim 9 , wherein the therapeutic material comprises one or more of: small molecules, miRNAs, and proteins. 
     
     
         11 . The method of  claim 1 , wherein the cell comprises one or more of: multipotent mesenchymal stromal cells; stem cells; and, cancer cells. 
     
     
         12 . The method of  claim 1 , wherein the cell is a mammalian cell. 
     
     
         13 . A method of producing transiently expressed miRNA containing cells, comprising:
 transfecting a cell population of cells capable of producing exosomes produced according to the method of  claim 1  with one or more plasmids encoding miRNA;   harvesting cells from the cell population after transfection; and,   confirming the presence of the miRNA in one or more of the harvested cells.   
     
     
         14 . A method of treating a subject in need thereof with modified exosomes, comprising:
 transfecting exosome-producing cells produced by the method of  claim 1 , with one or more carriers encoding a therapeutic material;   harvesting the exosomes from the cell population or media containing same after transfection;   confirming the presence of the therapeutic material in one or more of the harvested exosomes; and,   administering to the subject one or more of the harvested exosomes in a pharmaceutically effective amount to treat the subject.   
     
     
         15 . A method for increasing exosome production in a cell culture, comprising: exposing the culture to a substituted triazole-hydrazone compound that induces endosomal vacuolization but does not trigger cell death or attenuate cell growth. 
     
     
         16 . The method of  claim 15 , wherein the compound has the following chemical structure: 
       
         
           
           
               
               
           
         
         wherein R═CH 2 , NH, O; and R′=an electron withdrawing group. 
       
     
     
         17 . The method of  claim 16 , wherein R═O; and R′=Iodo. 
     
     
         18 . The method of  claim 16 , wherein the electron withdrawing groups are selected from: halides (F, Cl, Br and I), trifluoromethyl (CF 3 ), nitro (NO 2 ), acetyl (—COCH 3 ) and trimethylammonium (N(CH 3 ) 3 .