Nucleophile responsive charge-reversing polycations for pDNA transfection

23 January 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Polycationic carriers promise low cost and scalable gene therapy treatments, however inefficient intracellular unpacking of the genetic cargo has limited transfection efficiency. Charge-reversing polycations, which transition from cationic to neutral or negative charge, can offer targeted intracellular DNA release. We describe a new class of charge-reversing polycation which undergoes a cationic-to-neutral conversion by a reaction with cellular nucleophiles. The deionization reaction is relatively slow with primary amines, and much faster with thiols. In mammalian cells, the intracellular environment has elevated concentrations of amino acids (~10x) and the thiol glutathione (~1000x). We propose this allows for decationization of the polymeric carrier slowly in the extracellular space and then rapidly in the intracellular milleu for DNA release. We demonstrate that in a lipopolyplex formulation this leads to both improved transfection and reduced cytotoxicity when compared to a non-responsive polycationic control.



Supplementary materials

SI for Nucleophile responsive charge-reversing polycations for pDNA transfection
Additional experimental details and methods, including cell culture protocols, fluorescently labelled polymer synthesis, 1H NMR spectra, GPC chromatograms, TEM micrographs, CLSM images and DLS data.


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