ABSTRACT
Lipid nanoparticles (LNPs) have emerged as promising platforms for efficient in vivo mRNA delivery owing to advancements in ionizable lipids. However, maintaining the thermostability of mRNA/LNP systems remains challenging. While the importance of only a small amount of lipid impurities on mRNA inactivation is clear, a fundamental solution has not yet been proposed. In this study, we investigate an approach to limit the generation of aldehyde impurities that react with mRNA nucleosides through the chemical engineering of lipids. We demonstrated that piperidine-based lipids improve the long-term storage stability of mRNA/LNPs at refrigeration temperature as a liquid formulation. High-performance liquid chromatography analysis and additional lipid synthesis revealed that amine moieties of ionizable lipids play a vital role in limiting reactive aldehyde generation, mRNA-lipid adduct formation, and loss of mRNA function during mRNA/LNP storage. These findings highlight the importance of lipid design and help enhance the shelf-life of mRNA/LNP systems.
Subject(s)
Lipids , Nanoparticles , Piperidines , RNA Stability , RNA, Messenger , Nanoparticles/chemistry , RNA, Messenger/metabolism , RNA, Messenger/genetics , Lipids/chemistry , Piperidines/chemistry , Humans , Temperature , LiposomesABSTRACT
Cationic liposomes composed of dialkyl cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can efficiently deliver siRNA to the lungs following the intravenous injection of cationic liposome/siRNA complexes (lipoplexes). In this study, we examined the effect of cationic lipid of cationic liposomes on siRNA delivery to the lungs after intravenous injection. We used six kinds of cationic cholesterol derivatives and 11 kinds of dialkyl or trialkyl cationic lipids as cationic lipids, and prepared 17 kinds of cationic liposomes composed of a cationic lipid and 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) for evaluation of siRNA biodistribution and in vivo gene silencing effects. Among cationic liposomes, those composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18), 2-((1,5-bis(octadecyloxy)-1,5-dioxopentan-2-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-3-18D), 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), or cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) with DOPE exhibited high accumulation of siRNA in the lung and significant suppression of Tie2 mRNA expression after the intravenous injection of cationic lipoplexes with Tie2 siRNA. Furthermore, DC-1-16/DOPE and DC-1-18/DOPE lipoplexes with protein kinase N3 (PKN3) siRNA could suppress the tumour growth when intravenously injected into mice with lung LLC metastasis. These findings indicate that the siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of cationic lipoplexes were strongly affected by the type of cationic lipid of cationic liposomes.
Subject(s)
Lipids/chemistry , Lung/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , Animals , Cations , Drug Delivery Systems , Female , Gene Expression Regulation , Gene Silencing , Humans , Liposomes , Luciferases/metabolism , Lung Neoplasms/drug therapy , MCF-7 Cells , Mice , Mice, Inbred BALB C , Molecular Structure , RNA, Messenger , Receptor, TIE-2/genetics , Receptor, TIE-2/metabolismABSTRACT
Various N-propargylanilines bearing a conjugated diyne moiety at the 2-position were converted to tetracyclic fused carbazoles by treatment with a homogeneous gold(I) catalyst. This cascade reaction proceeds through indole formation with concomitant rearrangement of the N-propargyl group, intramolecular nucleophilic addition toward the resulting allene moiety, and subsequent hydroalkenylation. This transformation enables a one-pot synthesis of fused carbazoles from readily accessible substrates with 100% atom economy.
