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Development of miRNA delivery AuNP vectors with targeting properties

par Dutour, Raphael
Président du jury Maenhaut, Carine
Promoteur Laurent, Pascal
Co-Promoteur Bruylants, Gilles
Publication Non publié, 2026-06-04

Thèse de doctorat

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Résumé :

The development of new therapeutics benefiting from both high specificity and biocompatibility is necessary to improve the treatment of severe diseases such as cancer. Gene therapy represents a promising avenue notably via the RNA delivery affording to modulate genetic expression without affecting the genome. Currently, the main carriers used for RNA delivery are the lipid nanoparticles. However, commercially available delivery vectors such as lipofectamine still suffer from transfection control, biological stability and biocompatibility, and RNA therapeutics carriers need to be improved. The use of AuNPs as miRNA or siRNA delivery vehicles is a promising alternative to reach these objectives. Indeed, AuNPs present several advantages as a presumed low cytotoxicity or high control over their surface composition, or the possibility to use them in various theranostic applications thanks to their optical properties. The main reported AuNP vectors for miRNA and siRNA delivery are developed by covalent attachment of NA modified with thiol anchoring groups, or by adsorption of NA to AuNPs coated with positively charged species (Chapter I). The existing methods for the coating of AuNPs with RNA are however limited.The general objective of this thesis was to develop stable, tunable and biocompatible miRNA delivery platforms based on AuNPs and presenting targeting activities via the grafting of targeting ligands at their surface in addition to the nucleic acids.In Chapter II, we first assessed some reported methods to coat AuNPs with SH-RNA and highlighted difficulties to obtain stable and well-covered AuNPs-S-RNA conjugates via a reproducible procedure. As expected, the salt-aging method was efficient with both SH-DNA and SH-RNA, but we highlighted that numerous environmental factors could affect the efficiency of this conjugation approach. Also, we observed that the reported low pH-assisted method was not efficient with SH-RNA while promising results were obtained to develop AuNPs-S-RNA via the freezing strategy.With the aim to improve the stability of the gold vectors, we chose to work with AuNPs coated with calixarenes as they demonstrated great robustness and improved stability in comparison with the commonly used AuNPs-citrate (Chapter III). Also, some studies reported the development of AuNPs-calixarenes coated with biomolecules with a tunable bioconjugation density. We notably observed some differences in the stability of AuNPs-calixarenes in PBS depending on the synthetic pathway and identified interesting approaches to limit the reproducibility issues of the calixarene grafting onto the gold surface.With the objective to improve the stability of the AuNPs-NA conjugates and the NA grafted onto their surface, we tried to form a covalent bond between NA and calixarenes (Chapter IV). The main approach that has been used is the coupling using EDC/sNHS to form an amide bond as many studies reported the efficiency of this method for the coupling of biomolecules, such as proteins, on particles. Despite we demonstrated the efficiency of this coupling strategy with both DNA and RNA, we faced important difficulties to obtain stable AuNPs-calixarenes coatedviwith NA once incubated in PBS. However, we found a simple approach to strongly adsorb the NA on AuNPs-calixarenes via salt addition. Also, we also found some promising methods to improve the colloidal stability of these AuNP vectors.In Chapter V, we found a promising way to passivate these AuNP conjugates by adding SH- NA between the calixarenes. We thus used this approach to develop bifunctional nanodevices by coupling a targeting ligand to the calixarenes via the formation of an amide bond while attaching the miRNA strands with a thiol anchor. This bioconjugation strategy is particularly promising for the development of multifunctional nanodevices. Moreover, an assessment of the RNA stability grafted on the gold surface was performed in biological conditions by RT-qPCR and highlighted the considerable influence of some factors including the AuNP surface composition and the conjugation strategy used to anchor the NA. Very encouraging results were obtained with some AuNP designs with an improvement in RNA stability compared to the reference AuNPs-S-RNA system. We notably showed that the bioconjugation of the targeting ligands to the calixarenes via an amide bond allowed to significantly improve the RNA stability.Finally, we assessed the therapeutic potential of the AuNPs. In the context of this thesis, we tried to reduce the side effects of the anticancer drug cyclophosphamide through the delivery of the Let-7a miRNA to ovarian cells. In Chapter VI, we confirmed the low toxicity and cellular uptake of these vectors but could only identify poor transfection. Considering that the main vectors used for these biological studies presented some stability issues in PBS and low ability to protect RNA from degradation, we believe that a large screening of different AuNP designs could allow to identify the key factors to improve the transfection efficiency of these gold carriers. A promising result was obtained by highlighting a targeting activity of AuNPs coated with a targeting ligand and with miRNA duplexes, but the delivery activity of these AuNPs still requires investigation.In summary, we found different conjugation avenues allowing the obtention of stable AuNPs- NA conjugates and an improved control over the surface composition. These AuNPs could be of high interest for numerous biomedical applications, but some efforts are still required to improve their delivery efficiency including a broader understanding of the link between the AuNP surface composition and the RNA release mechanism inside the cells.