O'Dea, Shirley and Annibaldi, Valeria and Gallagher, Louise and Mulholland, Joanne and Molloy, Emer L. and Breen, Conor and Gilbert, Jennifer L. and Martin, Darren S. and Maguire, Michael and Curry, Fitz-Roy (2017) Vector-free intracellular delivery by reversible permeabilization. PLoS ONE, 12 (3). e0174779. ISSN 1932-6203

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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.

Item Type:	Article
Additional Information:	Citation: O’Dea S, Annibaldi V, Gallagher L, Mulholland J, Molloy EL, Breen CJ, et al. (2017) Vector-free intracellular delivery by reversible permeabilization. PLoS ONE 12(3): e0174779. https://doi.org/10.1371/journal.pone.0174779 Copyright: © 2017 O’Dea et al. This is an open access article distributed under the terms of the Creative Commons Attribution License https://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords:	Vector-free intracellular delivery; reversible permeabilization;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	10984
Identification Number:	https://doi.org/10.1371/journal.pone.0174779
Depositing User:	Conor Breen
Date Deposited:	21 Aug 2019 14:19
Journal or Publication Title:	PLoS ONE
Publisher:	Public Library of Science
Refereed:	Yes
URI:
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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