High-efficiency transfection of biological cells using sonoporation

Abstract

A method is provided for achieving transfection of host cells using sonoporation. An acoustic radiation generator is positioned in acoustic coupling relationship with respect to a reservoir containing host cells to be transfected, exogenous material to be incorporated into the host cells, and a cell-compatible fluid medium. The acoustic radiation generator is activated to generate acoustic radiation and direct the acoustic radiation into the reservoir in a manner effective to enable transfection of the host cells with the exogenous material.

We claim: 
     
         1 . An acoustic method for transfecting an exogenous material into cells, the method comprising:
 (a) providing a system that comprises (i) a reservoir containing host cells, gas-filled cationic microbubbles, and the exogenous material to be introduced into the host cells, the host cells and the exogenous material contained within a fluid medium, and (ii) an acoustic radiation generator to generate and direct acoustic radiation;   (b) acoustically coupling the acoustic radiation generator to the reservoir;   (c) activating the acoustic radiation generator to generate and direct acoustic radiation into the reservoir in a manner that acoustically activates the microbubbles, wherein acoustic radiation is transferred from the microbubbles to the host cells to provide sonoporated host cells, thereby facilitating introduction of the exogenous material into the sonoporated host cells; and   (d) acoustically decoupling the acoustic radiation generator from the reservoir.   
     
     
         2 . The method of  claim 1 , wherein the reservoir is one of a plurality of reservoirs each containing host cells, gas-filled cationic microbubbles, and the exogenous material to be introduced into the host cells, and step (b) comprises acoustically coupling the acoustic radiation generator to the reservoir without simultaneously acoustically coupling the acoustic radiation generator to any other of the plurality of reservoirs. 
     
     
         3 . The method of  claim 2 , further comprising, after step (d):
 (e) acoustically coupling the acoustic radiation generator to a second reservoir in the plurality of reservoirs without simultaneously coupling the acoustic radiation generator to any other reservoir.   
     
     
         4 . The method of  claim 1 , wherein the host cells in the reservoir are conjugated to the microbubbles. 
     
     
         5 . The method of  claim 4 , wherein the host cells in the reservoir are conjugated to the microbubbles by at least: functionalizing each of the microbubbles with a first binding moiety, functionalizing each of a plurality of host-cell specific antibodies with a second binding moiety configured to link to the first binding moiety, and combining the microbubbles with the antibodies. 
     
     
         6 . The method of  claim 1 , wherein the fluid medium comprises an isotonic buffer solution. 
     
     
         7 . The method of  claim 1 , wherein the cells are plated on a surface of the reservoir. 
     
     
         8 . The method of  claim 1 , wherein the cells are non-adherent. 
     
     
         9 . The method of  claim 1 , wherein the exogenous material comprises a nucleic acid, a plasmid, peptide, a protein, a lipid, a polysaccharide, a small molecule, or a combination thereof. 
     
     
         10 . The method of  claim 9 , wherein the exogenous material comprises a DNA plasmid. 
     
     
         11 . The method of  claim 9 , wherein the exogenous material comprises a ribonucleoprotein. 
     
     
         12 . The method of  claim 11 , wherein the ribonucleoprotein is capable of altering host cell nucleic acids. 
     
     
         13 . The method of  claim 12 , wherein the ribonucleoprotein comprises a guide RNA and a CRISPR-associated RNA-programmable DNA or RNA nuclease protein or protein complex. 
     
     
         14 . The method of  claim 13 , wherein the ribonucleoprotein comprises a guide RNA and Cas 9 protein. 
     
     
         15 . The method of  claim 14 , wherein the ribonucleoprotein comprises a guide RNA and a catalytically inactive CRISPR-associated RNA-programmable DNA or RNA nuclease protein or protein complex. 
     
     
         16 . The method of  claim 1 , wherein step (c) comprises irradiating the reservoir for 15 seconds to 40 seconds with acoustic tonebursts at a rate of 10 to 25 tonebursts per second. 
     
     
         17 . The method of  claim 16 , wherein each cyclic acoustic toneburst is an approximately 5-cycle to 10-cycle toneburst. 
     
     
         18 . The method of  claim 1 , wherein the acoustic radiation generator comprises a first transducer and a second transducer, wherein the second transducer comprises an annular transducer disposed around a first transducer, wherein the first transducer is configured to operate at a different frequency than the second transducer. 
     
     
         19 . The method of  claim 18 , wherein the annular transducer operates at a frequency in the range of about 1 MHz to about 2.5 MHz, and the standard transducer operates at a frequency in the range of about 6 MHz to about 20 MHz. 
     
     
         20 . The method of  claim 18 , wherein one of the two transducers primarily functions to supply the acoustic energy for sonoporation and the other transducer delivers acoustic energy to (a) enable acoustic ejection of sonoporated cells from the fluid medium or (b) change the relative position of the microbubbles with respect to the host cells.