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Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity

Gene Therapy volume 30, pages 783–791 (2023)Cite this article

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Abstract

Nonviral transfection of mammalian cells can be performed with electrostatic complexes (polyplexes) between a plasmid DNA (pDNA) encoding a foreign gene and a cationic polymer. However, an excess of the cationic polymer is required for pDNA condensation and polyplexes formation, which generate in vivo toxicity. Here, we present a new concept of polyplexes preparation aiming to reduce the polymer quantity. pDNA was functionalized with 3,6,9-trioxaundecan-1- {4 - [(2-chloroethyl) ethylamino)] - benzylamino}, 11-azide, and polyethyleneimine (lPEI) with reducible dibenzocyclooctyl (SS-DBCO) groups allowing azide-alkyne cycloaddition between pDNA and lPEI after condensation. The size of polyplexes with DBCO-SS-lPEI was smaller than with lPEI due to a stronger DNA condensation thanks to linkages between polymer and pDNA preventing dissociation until disulfide bridges reduction. In vitro transfection showed that the amount of DBCO-SS-lPEI leading to the most efficient polyplexes was three times lower than lPEI. As expected, toxicity in mice was significantly reduced upon intravenous injection of DBCO-SS-lPEI polyplexes at doses where the lPEI polyplexes killed mice. This is probably due to the high stability of the DBCO-SS-lPEI polyplexes which prevented their aggregation in the pulmonary capillaries. Overall, this new concept of polyplexes with DBCO-SS-lPEI offering the possibility of administering higher doses of polyplexes than lPEI and their ability to pass the pulmonary barrier could be favorably exploited for transfection of distant organs or tissues, such as tumors.

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Fig. 1: Plasmid DNA functionalization.
Fig. 2: lPEI functionalization.
Fig. 3: Agarose gel electrophoresis shift assay.
Fig. 4: Size and ζ potential of polyplexes.
Fig. 5: Transfection of HeLa and HepG2 cells.
Fig. 6: Cytotoxicity.
Fig. 7: Transfection of HeLa cells with pDNA encoding EGFP.
Fig. 8: In vivo transfection.
Fig. 9: Transfection of nondividing cells.

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Data availability

The data generated and analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Stéphanie RÉTIF, Marilyne LE MEE and Sharuja NATKUNARAJAH (CIPA, CNRS-TAAM UPS44 Orléans, France) for their technical assistance in in vivo experiments.

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  1. Centre de Biophysique Moléculaire, CNRS UPR4301, Inserm and University of Orléans, F-45071, Orléans cedex 02, France

    Delphine Maze, Chantal Pichon & Patrick Midoux

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PM conceived and designed the work that led to the submission, DM and PM acquired data, and/or played an important role in interpreting the results. PM and CP drafted and revised the manuscript. PM, DM, and CP approved the final version. DM, CP, and PM agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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Correspondence to Patrick Midoux.

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Maze, D., Pichon, C. & Midoux, P. Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity. Gene Ther 30, 783–791 (2023). https://doi.org/10.1038/s41434-023-00386-1

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