Abstract
BackgroundMessenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA’s transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases.ResultsA lipid–polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA.ConclusionsCharge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid–polymer NPs.
Highlights
Messenger RNA has gained remarkable attention as an alternative to DNA-based therapies in biomedical research
Design and characterization of blank lipid‐coated and chitosan‐coated PLGA nanoparticles Two different core–shell structured delivery systems have been manufactured using either PLGA, a biodegradable and biocompatible polymer as the core, which was coated with a cationic lipid (DOTMA), or a cationic polymer, to achieve a positive surface charge
Cell viability and cytotoxicity assay It is common knowledge that cationic charged particles are associated with higher cytotoxic effects [37], partly explicable due to potentially enhanced interaction of Kinetics of cellular internalization for blank and Messenger RNA (mRNA)‐loaded nanoparticles Prior to transfection studies, we evaluated the kinetics of cellular internalization for blank and mRNA-loaded NPs, as the knowledge about the efficiency of uptake towards dendritic cells might help to understand the subsequent transfection
Summary
Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. Su et al [11] produced a phospholipid-coated poly-(βamino ester) (PBAE) hybrid system mediating a transfection rate of around 30% in a dendritic cell line (DC2.4 cell line), while Perche et al [23] improved the transfection efficiency, even further, up to 60% with mannosylated histidylated lipoplexes Besides these criteria of manufacturing safe and efficient systems, an auxiliary knowledge about the mRNA translation kinetics is important for a better understanding of mRNA’s transient course [9] for tailoring therapeutic strategies. To our knowledge, there is still no study available, which quantifies protein translation kinetics using tailor-made mRNA nanocarriers
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