Intracellular delivery and transfection methods and devices
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.
1. A method of delivering a substance into a living cell having a cell membrane, comprising:
a) permeabilization of the cell membrane in a suspension to form pores in the cells using a permeabilization system, and
b) actively transporting and incorporating a substance into the cell interior through pores formed during permeabilization of the membrane using an insertion system.
2. The method of claim 1 , wherein permeabilization includes 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 method of claim 2 , wherein the mechanical poration includes poration using an acoustic field, an action of a shear field, or a combination of an acoustic field and a shear field.
4. The method of claim 1 , wherein the transporting and incorporating is conducted using an energetic 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 method of claim 1 , wherein the transporting and incorporating includes delivering the substance to the cell interior.
6. The method of claim 1 , wherein the transporting and incorporating includes delivering the substance to a sub-cellular target.
7. The method of claim 6 , wherein delivery includes delivering the substance to the nucleus or mitochondria.
8. The method of claim 1 , wherein permeabilization is conducted on a cell suspension that does not include the substance.
9. The method of claim 8 , wherein the substance is added to the cell suspension during step b).
10. The method of claim 1 , wherein steps a) and b) are performed sequentially in a single chamber.
11. The method of claim 1 , wherein steps a) and b) are performed separately.
12. The method 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 method 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 method of claim 13 , wherein the magnetic substance is a magnetic nanoparticle.
15. The method 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 method of claim 1 , wherein transporting and incorporating includes applying an electric field of about 0.001 volts to 1 kilovolt per centimeter to the suspension.
17. The method of claim 1 , wherein transporting and incorporating includes applying an electric field of about 50 volts to 100 volt per centimeter to the suspension.