title: Vector-free intracellular delivery by reversible permeabilization.
abstract: Despite advances in intracellular delivery technologies, efficient methods are still required that are vector-free, can address a wide range of cargo types and can be applied to cells that are difficult to transfect whilst maintaining cell viability. We have developed a novel vector-free method that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying composition, properties and size. A permeabilizing delivery solution was developed that contains a low level of ethanol as the permeabilizing agent. Reversal of cell permeabilization is achieved by temporally and volumetrically controlling the contact of the target cells with this solution. Cells are seeded in conventional multi-well plates. Following removal of the supernatant, the cargo is mixed with the delivery solution and applied directly to the cells using an atomizer. After a short incubation period, permeabilization is halted by incubating the cells in a phosphate buffer saline solution that dilutes the ethanol and is non-toxic to the permeabilized cells. Normal culture medium is then added. The procedure lasts less than 5 min. With this method, proteins, mRNA, plasmid DNA and other molecules have been delivered to a variety of cell types, including primary cells, with low toxicity and cargo functionality has been confirmed in proof-of-principle studies. Co-delivery of different cargo types has also been demonstrated. Importantly, delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner. Unlike some other vector-free methods, adherent cells are addressed in situ without the need for detachment from their substratum. The method has also been adapted to address suspension cells. This delivery method is gentle yet highly reproducible, compatible with high throughput and automated cell-based assays and has the potential to enable a broad range of research, drug discovery and clinical applications.
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Shirley
authors.identifier: http://orcid.org/0000-0002-7197-4676
authors.initials: S
authors.lastname: O'Dea
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Valeria
authors.initials: V
authors.lastname: Annibaldi
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Louise
authors.initials: L
authors.lastname: Gallagher
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Joanne
authors.initials: J
authors.lastname: Mulholland
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Emer L
authors.initials: EL
authors.lastname: Molloy
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Conor J
authors.initials: CJ
authors.lastname: Breen
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Gilbert
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Darren S
authors.initials: DS
authors.lastname: Martin
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.
authors.forename: Michael
authors.initials: M
authors.lastname: Maguire
authors.affiliation: Avectas Ltd., Maynooth, Co. Kildare, Ireland.|Department of Physiology & Membrane Biology, University of California, Davis, California, United States of America.
authors.forename: Fitz-Roy
authors.initials: FR
authors.lastname: Curry
mesh_terms: D000072283:A549 Cells; D002463:Cell Membrane Permeability / Q000235:genetics / Q000502:physiology*; D016503:Drug Delivery Systems; D018274:Electroporation; D005434:Flow Cytometry; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D007770:L-Lactate Dehydrogenase