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 delivery system for delivering a substance into a living cell having a cell membrane, comprising:
 a) a permeabilization system comprising one or more ejection devices, wherein the one or more ejection devices comprises an ejector structure and one or more orifices, and wherein the permeabilization system is configured for permeabilization of the cell membrane to form pores in the membrane of a cell in suspension; and   b) an active 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 delivery system of  claim 1 , wherein the ejector structure is a two-dimensional groove. 
     
     
         3 . The delivery system of  claim 1 , wherein the ejector structure has a three-dimensional tapered geometry. 
     
     
         4 . The delivery system of  claim 1 , wherein the one or more orifices is a single slot orifice. 
     
     
         5 . The delivery system of  claim 1 , wherein the ejector structure is not tapered and is terminated by an orifice of smaller dimension than the ejector structure. 
     
     
         6 . The delivery system of  claim 1 , wherein the one or more ejection devices is a plurality of ejection devices configured as a one-dimensional array. 
     
     
         7 . The delivery system of  claim 1 , wherein the one or more ejection devices is a plurality of ejection devices configured as a two-dimensional matrix. 
     
     
         8 . The delivery system of  claim 1 , wherein the delivery 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. 
     
     
         9 . The delivery 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. 
     
     
         10 . The delivery system of  claim 9 , 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. 
     
     
         11 . The delivery system of  claim 1 , wherein the permeabilization system further comprises the forced flow of gas configured to move the suspension through the one or more ejector devices. 
     
     
         12 . The delivery 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. 
     
     
         13 . The delivery system of  claim 1 , wherein the delivery 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. 
     
     
         14 . The delivery system of  claim 1 , wherein the delivery system is configured for high-throughput and multiplexed operation in a microarray format. 
     
     
         15 . The delivery system of  claim 1 , wherein the transporting and incorporating includes delivering the substance with the active transport system to one or more of the cell interior, the vesicle, the nanostructure defined by a membrane, and the microstructure defined by a membrane. 
     
     
         16 . The delivery system of  claim 1 , wherein the transporting and incorporating includes delivering the substance with the active transport system to a sub-cellular target. 
     
     
         17 . The delivery system of  claim 16 , wherein delivery includes delivering the substance with the active transport system to the nucleus, mitochondria, or vesicle. 
     
     
         18 . The delivery system of  claim 1 , wherein permeabilization is conducted with the permeabilization system on a cell suspension that does not include the substance. 
     
     
         19 . The delivery system of  claim 17 , wherein the substance is added to the cell suspension during active insertion with the active transport system. 
     
     
         20 . The delivery system of  claim 1 , wherein permeabilization and active insertion are performed sequentially in a single chamber. 
     
     
         21 . The delivery system of  claim 1 , wherein permeabilization and active insertion are performed separately. 
     
     
         22 . The delivery system of  claim 1 , wherein the substance inserted with the active insertion system 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. 
     
     
         23 . The delivery system of  claim 22 , 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. 
     
     
         24 . The delivery system of  claim 23 , wherein the magnetic substance is a magnetic nanoparticle. 
     
     
         25 . The delivery system of  claim 24 , 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. 
     
     
         26 . The delivery 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. 
     
     
         27 . The delivery 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. 
     
     
         28 . A delivery 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 active insertion system configured to actively transport and incorporate a substance into the cell interior.   
     
     
         29 . The delivery system of  claim 28 , wherein the delivery 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. 
     
     
         30 . The delivery system of  claim 29 , wherein the delivery system is self-pumping. 
     
     
         31 . The delivery system of  claim 28 , wherein the delivery system is configured to perform permeabilization and active insertion sequentially. 
     
     
         32 . The delivery system of  claim 28 , wherein the delivery system is configured to perform permeabilization and active insertion simultaneously. 
     
     
         33 . A delivery 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 active 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.   
     
     
         34 . The delivery system of  claim 33 , wherein the delivery 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. 
     
     
         35 . The delivery system of  claim 33 , wherein the delivery system is self-pumping. 
     
     
         36 . The delivery system of  claim 33 , wherein the delivery system is configured to perform permeabilization and active insertion sequentially. 
     
     
         37 . The delivery system of  claim 33 , wherein the delivery system is configured to perform permeabilization and active insertion simultaneously. 
     
     
         38 . The delivery 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; and
 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.   
     
     
         39 . The delivery system of  claim 1 , wherein the permeabilization system configured for permeabilization of the cell membrane is selected from the group consisting of: a mechanical poration device, an electrical poration device, thermal poration device, and a combination of two or more of these devices. 
     
     
         40 . The delivery system of  claim 1 , wherein the 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 is selected from the group consisting of: an electrophoretic device, a magnetophoretic device, an acoustophoretic device, and a combination of two or more of these devices. 
     
     
         41 . The delivery system of  claim 1 , wherein the permeabilization system configured for permeabilization of the cell membrane is selected from the group consisting of: a mechanical poration device, an electrical poration device, thermal poration device, and a combination of two or more of these devices; and
 wherein the 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 is selected from the group consisting of: an electrophoretic device, a magnetophoretic device, an acoustophoretic device, and a combination of two or more of these devices.   
     
     
         42 . The delivery system of  claim 28 , 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; and
 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.   
     
     
         43 . The delivery system of  claim 28 , wherein the permeabilization system configured for permeabilization of the cell membrane is selected from the group consisting of: a mechanical poration device, an electrical poration device, thermal poration device, and a combination of two or more of these devices. 
     
     
         44 . The delivery system of  claim 28 , wherein the 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 is selected from the group consisting of: an electrophoretic device, a magnetophoretic device, an acoustophoretic device, and a combination of two or more of these devices. 
     
     
         43 . The delivery system of  claim 28 , wherein the permeabilization system configured for permeabilization of the cell membrane is selected from the group consisting of: a mechanical poration device, an electrical poration device, thermal poration device, and a combination of two or more of these devices; and
 wherein the 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 is selected from the group consisting of: an electrophoretic device, a magnetophoretic device, an acoustophoretic device, and a combination of two or more of these devices.