Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery

Malfanti, Alessio; Sami, Haider; Balasso, Anna; Marostica, Giulia; Arpac, Busra; Mastrotto, Francesca; Mantovani, Giuseppe; Cola, Elisa; Anton, Martina; Caliceti, Paolo; Ogris, Manfred; Salmaso, Stefano

Abstract

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

Citation

Malfanti, A., Sami, H., Balasso, A., Marostica, G., Arpac, B., Mastrotto, F., Mantovani, G., Cola, E., Anton, M., Caliceti, P., Ogris, M., & Salmaso, S. (2023). Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery. Journal of Controlled Release, 363, 101-113. https://doi.org/10.1016/j.jconrel.2023.09.022

• Journal Article Type: Article
• Acceptance Date: Sep 14, 2023
• Online Publication Date: Sep 26, 2023
• Publication Date: Nov 1, 2023
• Deposit Date: Apr 25, 2025
• Publicly Available Date: Apr 28, 2025
• Journal: Journal of Controlled Release
• Print ISSN: 0168-3659
• Electronic ISSN: 1873-4995
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 363
• Pages: 101-113
• DOI: https://doi.org/10.1016/j.jconrel.2023.09.022
• Keywords: Lipoplex engineering, Oligo-cationic condensing lipids, Cell penetration enhancers, Endosomal escape
• Public URL: https://nottingham-repository.worktribe.com/output/48097028
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0168365923006107?via%3Dihub
• Additional Information: This article is maintained by: Elsevier; Article Title: Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery; Journal Title: Journal of Controlled Release; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.jconrel.2023.09.022; Content Type: article; Copyright: © 2023 The Authors. Published by Elsevier B.V.