Romanian Society of Pharmaceutical Sciences

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A QUALITY BY DESIGN APPROACH TO OPTIMISE THE TRANSFECTION EFFICIENCY OF POLY(AMIDOAMINE)-BASED NANOPARTICLES WITH mRNA

SAKETH REDDY RANAMALLA 1, 2, ALINA SILVIA PORFIRE 2*, MANUELA BANCIU 3#, IOAN TOMUȚA 2#

1Doctoral School in Integrative Biology, Faculty of Biology and Geology, “Babeș-Bolyai” University, 400015 Cluj-Napoca, Romania
2Department of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, University of Medicine and Pharmacy “Iuliu Hațieganu”, 400010, Cluj-Napoca, Romania
3Department of Molecular Biology and Biotechnology, Centre of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babeș-Bolyai” University, 400015 Cluj-Napoca, Romania

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Disulfide bond poly (amido amine) based (PAA) nanoparticles (NPs), characterized by cationic, bio-reducible properties, are promising vehicles for mRNA delivery. This study aimed to boost the transfection efficiency of PAA-based NPs at reduced mRNA concentrations, focusing on higher levels of cell transfection with low cytotoxicity. NPs loaded with β-galactosidase (β-gal) mRNA were tested in three cell lines, HEK293, C28/I2 and Jurkat cells, with varying transfection efficiencies. To identify the most effective PAA-based polymer, genetic material to polymer ratios and optimal RNA dosages, a Quality by Design (QbD) approach was employed, including a Failure Mode and Effects Analysis (FMEA) and Design of Experiments (DoE). Results indicated that a polymer-to-genetic material ratio (P:G) of 10:1 to 18:1 for polymer B and at an RNA dosage of 408 to 480 ng were optimal. Validation experiments confirmed the model's accuracy in predicting peak cell transfection and viability. This research effectively determined the design space for the P:G ratio and RNA dosage, enhancing transfection across different cell types, including hard-to-transfect Jurkat cells, with minimized toxicity. Our studies demonstrate the benefit of a QbD approach to optimize gene delivery, accelerating in vitro or ex-vivo gene therapy development stages.