Molecular Size of Linear and Branched Polyethylemine Influences the Transfection Efficacy of mRNA Polyplexes Aerosolised using a Vibrating Mesh Nebulizer
Abhimata Paramanandana, Hao Ying Li & Ben Forbes
King’s College London, Institute of Pharmaceutical Science, London SE1 9NH, UK
Summary
Messenger ribonucleic acid (mRNA) therapeutics have gained considerable attention in recent years due to their wide range of potential medical applications. However, they face challenges in terms of drug delivery; notably their cellular uptake and stability. In this study, the production of a reporter mRNA sequence was optimised and the stability of nanoparticle polyplexes to aerolisation was evaluated by measuring transfection in cells. The goal was to explore the influence of delivery vectors on the stability and the efficiency of respirable mRNA therapeutics. Delivery by inhalation offers advantages for nucleic acid delivery including direct lung targeting and bypassing metabolic and other clearance mechanisms. The study used green fluorescent protein (GFP) mRNA as a model nucleic acid therapeutic and utilized linear and branched polyethyleneimine (PEI) polymers of different molecular weight for nanoparticle formulation. Plasmid DNA linearization was achieved using AfeI and PmeI restriction enzymes and in vitro transcription was effected using a MAXIScript™ Kit. Gel retardation assays confirmed effective mRNA complexation within the PEI polyplexes and evaluated physical polyplex stability after nebulization. Transfection studies on HEK 289T cells showed successful uptake of the polyplexes and expression of GFP. This study demonstrated that it is possible to formulate mRNA polyplexes that are physically stable to aerosolization using a vibrating mesh nebuliser, and used cell transfection to show that their biological effects are retained but diminished. These findings highlight the potential of inhalation-based delivery as a promising strategy for lung-targeted mRNA therapeutics