Intracellular delivery and transfection methods and devices

Abstract

Embodiments of the present disclosure provide a multistage procedure for treatment of biological samples (e.g., living cells with membranes, and the like) with a substance (e.g., a drug, DNA, RNA, plasmids, and other biomolecules or materials) to achieve more efficacious intracellular delivery and transfection.

We claim: 
     
         1 . A system for delivering a substance into a living cell having a cell membrane, comprising:
 a) a permeabilization system configured for permeabilization of the cell membrane to form pores in the membrane of a cell in suspension, and   b) an insertion system configured to actively transport and incorporate a substance into one or more of the cell interior, a vesicle, a nanostructure defined by a membrane, and a microstructure defined by a membrane.   
     
     
         2 . The system of  claim 1 , wherein the permeabilization system is further configured for permeabilization of the cell membrane using a technique selected from the group consisting of: mechanical poration, electrical poration, thermal poration, and a combination of two or more of these techniques. 
     
     
         3 . The system of clam  2 , wherein the mechanical poration includes poration using an acoustic field, an action of a shear field, an action of a tension field, or a combination thereof. 
     
     
         4 . The system of  claim 1 , wherein the transporting and incorporating a substance is conducted using an active insertion technique selected from the group consisting of: an electrophoretic technique, a magnetophoretic technique, an acoustophoretic technique, and a combination of two or more of these techniques. 
     
     
         5 . The system of  claim 1 , wherein the transporting and incorporating includes delivering the substance to one or more of the cell interior, the vesicle, the nanostructure defined by a membrane, and the microstructure defined by a membrane. 
     
     
         6 . The system of  claim 1 , wherein the transporting and incorporating includes delivering the substance to a sub-cellular target. 
     
     
         7 . The system of  claim 6 , wherein delivery includes delivering the substance to the nucleus, mitochondria, or vesicle. 
     
     
         8 . The system of  claim 1 , wherein permeabilization is conducted on a cell suspension that does not include the substance. 
     
     
         9 . The system of  claim 8 , wherein the substance is added to the cell suspension during step b). 
     
     
         10 . The system of  claim 1 , wherein steps a) and b) are performed sequentially in a single chamber. 
     
     
         11 . The system of  claim 1 , wherein steps a) and b) are performed separately. 
     
     
         12 . The system of  claim 1 , wherein the substance is selected from the group consisting of: a charged substance, a magnetic substance, a substance that moves under the action of an acoustic field, and a combination of two or more of these. 
     
     
         13 . The system of  claim 12 , wherein the charged substance is selected from the group consisting of: a drug, a polynucleotide, an antigen, a polypeptide, an antibody, an antigen, a hapten, and an enzyme. 
     
     
         14 . The system of  claim 13 , wherein the magnetic substance is a magnetic nanoparticle. 
     
     
         15 . The system of  claim 14 , wherein the magnetic nanoparticle is attached to a substance selected from the group consisting of: a drug, a polynucleotide, an antigen, a polypeptide, an antibody, an antigen, a hapten, and an enzyme. 
     
     
         16 . The system of  claim 1 , wherein transporting and incorporating step includes applying an electric field of about 0.001 volts to 1 kilovolt per centimeter to the suspension. 
     
     
         17 . The system of  claim 1 , wherein transporting and incorporating step includes applying an electric field of about 50 volts to 100 volt per centimeter to the suspension. 
     
     
         18 . A system for delivering a substance into a living cell having a cell membrane, comprising:
 a) a permeabilization system configured for permeabilization of the cell membrane to form pores in the membrane of a cell in suspension,
 wherein the permeabilization system comprises a lumen configured for microliter volumes and an orifice, and the permeabilization system is further configured to provide a mechanical shear force that induces a tension in the cell membrane upon ejection of the cell from the orifice; and 
   b) an insertion system configured to actively transport and incorporate a substance into the cell interior.   
     
     
         19 . The system of  claim 18 , wherein the system is configured for continuous flow of a suspension of one or more cells or drop-wise flow of a suspension of one or more cells. 
     
     
         20 . The system of  claim 19 , wherein the system is self-pumping. 
     
     
         21 . The system of  claim 18 , wherein the system is configured to perform (a) and (b) sequentially. 
     
     
         22 . The system of  claim 18 , wherein the system is configured to perform (a) and (b) simultaneously. 
     
     
         23 . A system for delivering a substance into a living cell having a cell membrane, comprising:
 a) a permeabilization system configured for permeabilization of the cell membrane by providing a mechanical shear force that induces a tension in the cell membrane to form pores in the membrane of a cell in suspension; and   b) an insertion system configured to actively transport and incorporate a substance into one or more of the cell interior, a vesicle, a nanostructure defined by a membrane, and a microstructure defined by a membrane.   
     
     
         24 . The system of  claim 23 , wherein the system is configured for continuous flow of a suspension of one or more cells or drop-wise flow of a suspension of one or more cells. 
     
     
         25 . The system of  claim 23 , wherein the system is self-pumping. 
     
     
         26 . The system of  claim 23 , wherein the system is configured to perform (a) and (b) sequentially. 
     
     
         27 . The system of  claim 23 , wherein the system is configured to perform (a) and (b) simultaneously.