Design and synthesis of ionisable amino-polyester lipid nanoparticles for extrahepatic RNA delivery

Messenger RNA (mRNA)-based therapeutics represent a powerful platform with the potential to transform modern medicine by targeting diseases at their molecular origins. Advances in mRNA chemistry and delivery technologies have enabled applications ranging from vaccines and cancer immunotherapy to protein replacement and regenerative medicine. Polymeric nanoparticles offer a promising route for extrahepatic mRNA delivery, although challenges such as polydispersity and biocompatibility have hindered their clinical translation. Controlled polymerisation methods, such as ring-opening polymerisation (ROP), can help overcome these limitations. Ionisable amino polyesters (APEs) synthesised via ROP and co-formulated with lipids into nanoparticles (APE-LNPs) have demonstrated selective mRNA delivery to non-liver tissues. In the first part of this work, 36 ionisable APEs were synthesised to investigate how polymer composition affects APE-LNP physicochemical properties and mRNA delivery. The library of APE-LNPs encapsulating Firefly Luciferase (FLuc) mRNA was evaluated in vitro and in vivo, revealing that the interplay between tertiary amino-alcohols and lactone side chains influences nanoparticle formation, transfection efficiency, and organ specificity, particularly in lungs and spleen. The second part focused on enhancing mRNA delivery efficacy and tissue specificity through post-polymerisation functionalisation of APEs with different linkers and end-capping amine compositions. While these modifications had minimal impact on physicochemical properties, they significantly influenced delivery outcomes. Cationic and zwitterionic linkers enhanced spleen selectivity, whereas neutral linkers with non-polar amines favoured lung targeting, demonstrating the importance of polymer design for targeted mRNA delivery. To address the challenge of accurate RNA quantification in complex formulations, Scatter-Free Absorption Spectroscopy (SFAS) was investigated. This technique eliminates interference from light scattering and nanoparticle components, enabling precise quantification of total RNA in intact nanoparticles. Finally, the transfection performance of the selected APE-LNPs was assessed in M0 and M1 macrophages to explore their potential for targeting immune cells. APE-LNPs achieved efficient mRNA delivery in both phenotypes, with distinct transfection profiles between activation states, providing insights into how macrophage polarisation influences mRNA delivery. Overall, this work highlights APEs as a promising alternative to ionisable lipids for mRNA-based therapies and underscores the critical role of polymer structure and functionalisation in achieving efficient and tissue-selective mRNA delivery.

Grantor dc:publisher

University College Cork

Year dc:date.issued

2025

Author dc:creator

• Lopez Espinar, Aida

Advisors dc:contributor.advisor

• Kowalski, Piotr
• Vucen, Sonja

• Ionisable amino-polyesters
• Nanoparticles
• Extrahepatic delivery

Statement dc:rights

• © 2025, Aida Lopez Espinar.

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc/4.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10468/18925

OAI identifier oai:identifier

oai:cora.ucc.ie:10468/18925