Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism

Craig van Bruggen, David Punihaole, Allison R. Keith, Andrew J. Schmitz, Jakub Tolar, Renee R. Frontiera, Theresa M. Reineke

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine-co-HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer–DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer–pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.

Original languageEnglish (US)
Pages (from-to)32919-32928
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number52
DOIs
StatePublished - Dec 29 2020

Bibliographical note

Funding Information:
We thank Dr. Jeffrey Ting and Dr. Zhe Tan for their contributions in polymer synthesis and Dr. William Boyle and Dr. Yogesh Dhande for their cell culture expertise. We also thank Dr. Guillermo Marqu?s, Dr. Thomas Pengo, and the University Imaging Center for acquisition and processing expertise of deconvolved widefield images and Samantha Linn for help with polymer characterization. Funding for this work was provided by the NIH (R35-GM9119441, D.P. and R.R.F.) and the NSF (DMR-1904853, C.V.B., D.P., A.J.S., T.M.R., and R.R.F.). D.P. gratefully acknowledges postdoctoral funding from the Ford Foundation. C.V.B. acknowledges graduate funding under the Frieda Martha Kunze and College of Science and Engineering Graduate Fellowships (University of Minnesota). The Raman microscopy experiments were carried out in the University of Minnesota?s Characterization Facility, which receives partial support from the NSF through the University of Minnesota Materials Research Science and Engineering Centers program under Award DMR-2011401.

Funding Information:
ACKNOWLEDGMENTS. We thank Dr. Jeffrey Ting and Dr. Zhe Tan for their contributions in polymer synthesis and Dr. William Boyle and Dr. Yogesh Dhande for their cell culture expertise. We also thank Dr. Guillermo Marqués, Dr. Thomas Pengo, and the University Imaging Center for acquisition and processing expertise of deconvolved widefield images and Saman-tha Linn for help with polymer characterization. Funding for this work was provided by the NIH (R35-GM9119441, D.P. and R.R.F.) and the NSF (DMR-1904853, C.V.B., D.P., A.J.S., T.M.R., and R.R.F.). D.P. gratefully acknowledges postdoctoral funding from the Ford Foundation. C.V.B. acknowledges graduate funding under the Frieda Martha Kunze and College of Science and Engineering Graduate Fellowships (University of Minnesota). The Raman microscopy experiments were carried out in the University of Minnesota’s Characterization Facility, which receives partial support from the NSF through the University of Minnesota Materials Research Science and Engineering Centers program under Award DMR-2011401.

Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.

Copyright © 2020 the Author(s). Published by PNAS.

Keywords

  • Biomaterial
  • Drug delivery
  • Gene editing
  • Gene therapy
  • Polymer

How much support was provided by MRSEC?

  • Shared

Reporting period for MRSEC

  • Period 1

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

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