Poster W13 - Rational Design of Thermostable Ionic Liquid Lipid Nanocarriers for Self-Amplifying RNA Delivery

Introduction: Self-amplifying RNA (saRNA) encapsulated in lipid nanocarriers (LNCs) are attractive vaccine formulations for global pandemics, demonstrating successful therapeutic outcomes at a minimal dose compared to messenger RNA. However, the greatest issues of existing LNCs for RNA therapeutics include limited intracellular delivery achieved and poor formulation thermostability. This restricts therapeutic effects and global formulation distribution. Ionic liquids (ILs) are highly tuneable compounds, comprised of cations and anions. ILs can be designed to possess unique physicochemical properties to enhance the thermal, structural and thermodynamic stability of diverse therapeutics. The focus of this research is to demonstrate a rational formulation engineering approach, utilising the biocompatible ILs choline chloride ([Cho][Cl]), choline dihydrogen phosphate ([Cho][DHP]), and choline acetate ([Cho][OAc]) to engineer IL-LNCs for improved intracellular delivery and thermostability of saRNA. To our knowledge, this proof-of-concept study is the first to explore the systematic engineering of thermostable IL-LNCs for the delivery of saRNA.

Materials and

Methods: To engineer the formulations, cholesterol and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine were dissolved and mixed in chloroform/methanol solutions and dried into thin films via rotary evaporation. Then, the thin films were hydrated with solutions including saRNA and each of the select ILs. For comparison, the thin films were also hydrated utilising conventional aqueous solutions. Finally, following extrusion through polycarbonate membranes, 0.2μm, the IL-LNCs and conventional LNCs were obtained.
For each formulation the hydrodynamic diameter and surface charge was determined via dynamic light scattering and zeta potential measurements. The saRNA loading in each formulation was quantified using a Quant-iT RiboGreen assay. Finally, in vitro transfections were performed in HEK293T cells and the transfection efficiency for each formulation was determined using firefly luciferase as a reporter protein.

Results, Conclusions, and Discussions: Overall, the LNCs and IL-LNCs exhibited hydrodynamic diameter values of approximately 120 nm and 150-170 nm, respectively. Additionally, the IL-LNCs showed surplus surface charges of approximately -40 mV compared to approximately -10 mV for the conventional LNCs. Moreover, the IL-LNCs presented saRNA encapsulation efficiencies between 80-90%, highest for the [Cho][DHP]-LNCs and lowest for the [Cho][OAc]-LNCs. In contrast, the conventional LNCs presented an saRNA encapsulation efficiency of approximately 60 %. Furthermore, confocal microscopy images showed higher cellular uptake of the IL-LNCs compared to the conventional LNCs. Qualitative analysis demonstrated that cellular uptake was similar for the [Cho][DHP]-LNCs and [Cho][Cl]-LNCs and relatively lower for [Cho][OAc]-LNCs. We found that the transfection efficiency of each fresh IL-LNC formulation was approximately 10^7 relative light units (RLU) compared to approximately 10^4 RLU for the fresh conventional LNC formulation. The formulations were stored under 25 °C and the transfection efficiency of each was evaluated periodically over the course of 50 days. Based on the luciferase expression, on day 50 the [Cho][DHP]-LNCs showed transfection efficiency of approximately 10^5 RLU, while the [Cho][Cl]-LNCs and [Cho][OAc]-LNCs exhibited transfection efficiencies of approximately 10^3 RLU. Moreover, the conventional LNCs showed transfection efficiency of approximately 10^1 RLU.

Based on the experimental data and quantum mechanics/molecular mechanics calculations performed, we suggest that the highest encapsulation efficiency, cellular uptake, transfection efficiency and thermostability of the [Cho][DHP]-LNCs is attributed to strong steric and confinement effects induced by the dihydrogen phosphate anions, serving to increase system structural stability. Notably, while both showed reduced transfection efficiencies following 50 days under 25 °C, the [Cho][Cl]-LNCs exhibited higher cellular uptake and encapsulation efficiency compared to the [Cho][OAc]-LNCs. We rationalise that the [Cho][Cl]-LNCs possess higher charge density compared to [Cho][OAc]-LNCs suggesting that charge-mediated delivery into cells is a major mechanism at play. Holistically, this research demonstrates a novel rational design approach to engineer IL-LNCs equipped to efficiently deliver saRNA, and in particular [Cho][DHP]-LNCs show immense promise for improving formulation storage stability. Optimisation of these proof-of-concept platforms could influence the future of rational design of LNCs for a broad array of therapeutics and for eliminating the costly cold chain.

Acknowledgements (Optional): This research is funded by the Department of Health and Social Care using UK Aid funding and is managed by the Engineering and Physical Sciences Research Council (EPSRC, grant EP/R013764/1). Grateful acknowledgement to Dr Apanpreet Kaur for her contributions to this research.