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Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery

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Abstract

Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure–performance relationship of chitosan-based gene delivery systems.

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Acknowledgments

This work was supported by the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council (NSERC), le Fond de la Recherche en Santé du Québec (FRSQ), and the Canada Foundation for Innovation (CFI). The authors thank Nicolas Tran-Khanh for comments that greatly improved the manuscript.

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    Authors and Affiliations

    1. Department of Chemical Engineering, École Polytecyhnique de Montréal, Université de Montréal Campus, 2900, boul. Édouard-Montpetit, 2500, Chemin de Polytechnique, Montreal, QC, H3T 1J4, Canada

      Marc Thibault, Marc Lavertu, Mélina Astolfi & Michael D. Buschmann

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    1. Marc Thibault
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    2. Marc Lavertu
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    3. Mélina Astolfi
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    4. Michael D. Buschmann
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    Correspondence to Michael D. Buschmann.

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    Marc Thibault and Marc Lavertu have contributed equally to this work.

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    Thibault, M., Lavertu, M., Astolfi, M. et al. Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery. Mol Biotechnol 58, 648–656 (2016). https://doi.org/10.1007/s12033-016-9964-8

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