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The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA


Pratik Gurnani

Anna K. Blakney

Roberto Terracciano

Joshua E. Petch

Andrew J. Blok


Paul F. McKay

Robin J. Shattock


RNA technology has the potential to revolutionize vaccination. However, the lack of clear structure-property relationships in relevant biological models mean there is no clear consensus on the chemical motifs necessary to improve RNA delivery. In this work, we describe the synthesis of a series of copolymers based on the self-hydrolyzing charge-reversible polycation poly(dimethylaminoethyl acrylate) (pDMAEA), varying the lipophilicity of the additional co-monomers. All copolymers formed stable polyplexes, showing efficient complexation with model nucleic acids from nitrogen/phosphate (N/P) ratios of N/P = 5, with more hydrophobic complexes exhibiting slower charge reversal and disassembly compared to hydrophilic analogues. The more hydrophobic copolymers outperformed hydrophilic versions, homopolymer controls and the reference standard polymer (polyethylenimine), in transfection assays on 2D cell monolayers, albeit with significantly higher toxicities. Similarly, hydrophobic derivatives displayed up to a 4-fold higher efficacy in terms of the numbers of cells expressing green fluorescent protein (GFP+) cells in ex vivo human skin (10%) compared to free RNA (2%), attributed to transfection enrichment in epithelial cells. In contrast, in a mouse model, we observed the reverse trend in terms of RNA transfection, with no observable protein production in more hydrophobic analogues, whereas hydrophilic copolymers induced the highest transfection in vivo. Overall, our results suggest an important relationship between the vector lipophilicity and RNA transfection in vaccine settings, with polymer biocompatibility potentially a key parameter in effective in vivo protein production.


Gurnani, P., Blakney, A. K., Terracciano, R., Petch, J. E., Blok, A. J., Bouton, C. R., …Alexander, C. (2020). The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA. Biomacromolecules, 21(8), 3242-3253.

Journal Article Type Article
Acceptance Date Jul 9, 2020
Online Publication Date Jul 9, 2020
Publication Date Aug 10, 2020
Deposit Date Jul 14, 2020
Publicly Available Date Jul 10, 2021
Journal Biomacromolecules
Print ISSN 1525-7797
Electronic ISSN 1526-4602
Publisher American Chemical Society
Peer Reviewed Peer Reviewed
Volume 21
Issue 8
Pages 3242-3253
Keywords Materials Chemistry; Bioengineering; Polymers and Plastics; Biomaterials
Public URL
Publisher URL


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