Ionizable Lipids with Triazole Moiety from Click Reaction for LNP-Based mRNA Delivery

Abstract

Ionizable lipid-containing lipid nanoparticles (LNPs) as a non-viral vector with good safety and potency have been considered as an ideal delivery system for gene therapy. The screening of ionizable lipid libraries with common features but diverse structures holds the promise of finding new candidates for LNPs to deliver different nucleic acid drugs such as messenger RNAs (mRNAs). Chemical strategies for the facile construction of ionizable lipid libraries with diverse structure are in high demand. Here, we report on the ionizable lipids containing the triazole moiety prepared by the copper-catalyzed alkyne–azide click reaction (CuAAC). We demonstrated that these lipids served well as the major component of LNPs, in order to encapsulate mRNA using luciferase mRNA as the model system. Thus, this study shows the potential of click chemistry in the preparation of lipid libraries for LNP assembly and mRNA delivery.

Ionizable Lipids from Click Reactions for Lipid Nanoparticle Assembling and mRNA Delivery

“…For initial analysis on the influence of triazole substitution in the ionizable lipid molecules on different physical and chemical parameters of the LNPs containing these lipids as well as mRNAs, we first chose six molecules, A (SM-102), AzA , TzA-1 , B , AzB , and TzB-1 , for parallel comparison. We prepared LNPs based on the protocol of FDA-approved LNPs for the mRNA vaccine against COVID-19 by Moderna and replaced SM-102 with other lipids . We measured the apparent p K a of LNPs with or without luciferase mRNA encapsulation using the 2-( p -toluidinyl) naphthalene-6-sulfonic acid (TNS) fluorescence method that has been ubiquitously utilized in p K a characterization .…”

“…We used two types of mRNA (luciferase mRNA and eGFP mRNA) in the tests, and the mass ratio of ionizable lipids to mRNA was controlled at 20:1. The molar ratios of the ionizable lipid:DSPC:cholesterol:PEGylated lipids in the mixture was 50:10:38.5:1.5 as used in the Moderna formulation . LNPs were prepared by the mixture using the extrusion mechanism of the liposome (see Materials and Methods for details).…”

“…In addition, enzyme-assisted ligation reactions also increased the efficiency of library construction . We demonstrated the construction of lipid libraries by using the copper-catalyzed azide–alkyne cycloaddition (CuAAC), which is also known as click chemistry to assemble the hydrophobic tail similar to that found in SM-102 with different head groups …”

“…22 We demonstrated the construction of lipid libraries by using the copper-catalyzed azide−alkyne cycloaddition (CuAAC), which is also known as click chemistry to assemble the hydrophobic tail similar to that found in SM-102 with different head groups. 23 Lipid molecules generated from the click reactions share the common structural feature, that is, the presence of the triazole moiety. It is necessary to rationalize what is the influence of the triazole moiety on the pK a value change of the ionizable lipids and the following effect on the LNP assembly as well as mRNA delivery.…”

Cationic lipids via multi-component Passerini reaction for non-viral gene delivery

“…21 The transfection performance of LNPs is significantly affected by the structural features of cationic lipids, such as the structure of amine head group, the degradability of the connection bond, and the length and symmetry of the hydrophobic tails. 22,23 At present, some chemical strategies such as Michael addition, 24 epoxide ring-opening reaction, 25 and alkyne–azide click reaction 26 have been used in the synthesis of cationic lipids. However, most synthesis strategies involve complicated multi-step reactions to obtain target lipids with special functions.…”