Development and in vitro evaluation of lipid nanoparticle formulations to mitigate the ABC phenomenon
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Date
2025-07-08
Authors
Soncin, Matteo
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Abstract
Methylmalonic acidemia (MMA) is a rare genetic disorder caused by a deficiency in the mitochondrial enzyme methylmalonyl-CoA mutase (MMUT), leading to the toxic accumulation of methylmalonic acid. Lipid nanoparticles (LNPs) represent a promising strategy for delivering mRNA encoding MMUT, building on their clinical success in mRNA vaccines and siRNA therapies. However, PEGylated LNPs can induce immunological side effects. While polyethylene glycol (PEG) improves colloidal stability and circulation time, repeated administration may trigger anti-PEG antibody responses. These can lead to accelerated blood clearance (ABC) and complement activation-related pseudoallergy (CARPA). Structural studies indicate that the Fab region of anti-PEG antibodies recognizes PEG epitopes of ~16 ethylene glycol units (~700 Da), highlighting the importance of PEG structure in immunogenicity. This thesis aimed to optimize mRNA-LNP performance by evaluating the effects of PEGlipid molecular weight (350–5,000 Da) and molar ratio (1.5–10%) on nanoparticle properties and biological activity. Thirteen GFP mRNA-loaded LNP formulations were prepared by microfluidic mixing and characterized for size, polydispersity, zeta potential, and encapsulation efficiency. In vitro studies using HepG2 cells assessed GFP expression, Cy5 uptake, and endosomal escape. Results showed that PEG-lipid structure and density strongly affect particle size, stability, uptake, and intracellular trafficking. LNPs with 14:0 PEG550 PE (3.5–5%) achieved optimal delivery performance comparable to the gold-standard DMG-PEG2000, while 14:0 PEG350 PE and 14:0 PEG5000 PE formulations were less effective. These findings highlight the critical role of PEG-lipid design in mRNA-LNP optimization and support rational strategies for developing safer, more efficient gene therapies.
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Keywords
Lipid nanoparticles, Microfluidic, PEG-lipids, mRNA delivery