Abstract
Lipid nanoparticles (LNPs) exhibit high potential as carriers of messenger RNA (mRNA). However, the arduous preparation process of mRNA-loaded LNPs remains a huge obstacle for their widespread clinical application. Herein, we tackled this issue by mRNA PEGylation through hybridization with polyethylene glycol (PEG)-conjugated RNA oligonucleotides (PEG-OligoRNAs). Importantly, mRNA translational activity was preserved even after hybridization of 20 PEG-OligoRNAs per mRNA. The straightforward mixing of the PEGylated mRNA with lipofectamine LTX, a commercial lipid-based carrier, just by pipetting in aqueous solution, allowed the successful preparation of mRNA-loaded LNPs with a diameter below 100 nm, whereas the use of non-PEGylated mRNA provided large aggregates above 100- and 1000-nm. In vivo, LNPs prepared from PEG-OligoRNA-hybridized mRNA exhibited high structural stability in biological milieu, without forming detectable aggregates in mouse blood after intravenous injection. In contrast, LNPs from non-PEGylated mRNA formed several micrometer-sized aggregates in blood, leading to rapid clearance from blood circulation and deposition of the aggregates in lung capillaries. Our strategy of mRNA PEGylation was also versatile to prevent aggregation of another type of mRNA-loaded LNP, DOTAP/Chol liposomes. Together, our approach provides a simple and robust preparation method to LNPs for in vivo application.
Highlights
Messenger RNA encoding therapeutic proteins has recently demonstrated its potential to treat a variety of diseases in preclinical and clinical studies [1,2]
Using a commercially-available reagent, lipofectamine LTX, and a widely-used liposomal formulation, 1,2-bis(dioleoyloxy)-3-(trimethylamonio)propane (DOTAP)/cholesterol (Chol), as model Lipid nanoparticles (LNPs) to load messenger RNA (mRNA) hybridized with polyethylene glycol (PEG)-OligoRNAs (PEG-OligoRNAs/mRNA), we studied the feasibility of our strategy through detailed physicochemical characterization and functional analyses using cultured cells and mice
Region was avoided (Supplementary Table S1), by using a software that predicts RNA secondary structure [22], unless no unstructured regions were found nearby, because endogenous base-paring in mRNA might hamper the hybridization of PEG-OligoRNAs to the mRNA
Summary
Messenger RNA (mRNA) encoding therapeutic proteins has recently demonstrated its potential to treat a variety of diseases in preclinical and clinical studies [1,2]. The development of a simple and scalable technique capable of preventing such aggregate formation in buffer would be highly valuable for the straightforward formulation of clinically relevant mRNA-loaded LNPs. LTX-Based LNPs to Cultured Cells. Efficiency of mRNA cellular uptake was evaluated by measuring GLuc mRNA amount in the cells through quantitative real-time PCR (qRT-PCR). LNP loaded with non-PEGylated mRNA exhibited 2–4 orders higher efficiency of GLuc protein expression compared to that loaded with 20 PEG-OligoRNAs/mRNA (Figure 7a). (a) GLuc. PEGylated and that loaded with 20 PEG-OligoRNAs/mRNA were transfected protein concentration in the culture medium h after the transfection.
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