Abstract
Cationic liposomes are attractive carriers for mRNA delivery. Here, mRNA lipoplexes (LX) were prepared with the cationic lipids α-aminolipophosphonate (3b) or imidazolium lipophosphoramidate (2) associated with various α-aminolipophosphonates co-lipids comprising protonable groups (imidazole or pyridine) and DOPE. Physicochemical parameters of liposomes and their membrane fusion activity were measured. LXs comprising either 3b- or 2- allowed transfection of ~25% and 40% of dendritic cells with low cytotoxicity, respectively; the efficiency increased up to 80% when 2 was combined with the imidazole-based co-lipid 1. The transfections were high with 3b/1, 3b/DOPE, 2/1 and 2/DOPE LXs. We observed that the transfection level was not well correlated with the acid-mediated membrane fusion activity of liposomes supposed to destabilize endosomes. The mRNA release from LXs and its translation capacity after release were studied for the most efficient LXs. The results showed that the more mRNA was condensed, the poorer the translation efficiency after release was. In contrast to DNA, circular dichroism performed on mRNA complexed with 2/DOPE revealed the presence of denatured mRNA in LXs explaining this lack of translation efficiency. This is an important parameter that should be stressed for the preparation of mRNA LXs with a conserved mRNA translation activity.
Highlights
Today, the effectiveness of mRNA vaccines is demonstrated through the development and use of approved vaccines for the COVID-19 virus [1–6]
The membrane fusion activity of liposomes were measured at pH 7.4 and pH 5.5 and transfection efficiencies of lipoplexes with mRNA encoding enhanced green fluorescent protein (EGFP) were and pH 5.5 and transfection efficiencies of lipoplexes with mRNA encoding EGFP were evaluated in the murine dendritic DC2.4 cell line
The culture medium was discarded; cells were washed with serum-free medium and incubated for 4 h at 37 ◦ C with LX (2.5 μg of mRNA or Plasmid DNA (pDNA) diluted in 500 mL serum-free culture medium)
Summary
The effectiveness of mRNA vaccines is demonstrated through the development and use of approved vaccines for the COVID-19 virus [1–6] This efficiency makes it possible to consider using synthetic mRNAs for multiple therapeutic applications such as viral infections, cancer, gene therapy, cell reprogramming, and genome editing (CRISPR/Cas9) [7–18]. We evaluated mRNA transfection with cationic liposomes comprising either the lipid 2 or the α-amino-lipophosphonate featuring tertiary amine (3b) as cationic lipids the lipid 2 or the α-amino-lipophosphonate featuring tertiary amine (3b) as cationic lipids associated with either DOPE or pH responsive α-amino-lipophosphonate lipids as coassociated with either DOPE or pH responsive α-amino-lipophosphonate lipids as co-lilipids (Figure 1) [33]. The membrane fusion activity of liposomes were measured at pH 7.4 and pH 5.5 and transfection efficiencies of lipoplexes with mRNA encoding EGFP were and pH 5.5 and transfection efficiencies of lipoplexes with mRNA encoding EGFP were evaluated in the murine dendritic DC2.4 cell line.
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