Lipid nanoparticle topology regulates endosomal escape and delivery of RNA to the cytoplasm

Abstract

RNA therapeutics have the potential to resolve a myriad of genetic diseases. Lipid nanoparticles (LNPs) are among the most successful RNA delivery systems. Expanding their use for the treatment of more genetic diseases hinges on our ability to continuously evolve the design of LNPs with high potency, cellular-specific targeting, and low side effects. Overcoming the difficulty of releasing cargo from endocytosed LNPs remains a significant hurdle. Here, we investigate the fundamental properties of nonviral RNA nanoparticles pertaining to the activation of topological transformations of endosomal membranes and RNA translocation into the cytosol. We show that, beyond composition, LNP fusogenicity can be prescribed by designing LNP nanostructures that lower the energetic cost of fusion and fusion–pore formation with a target membrane. The inclusion of structurally active lipids leads to enhanced LNP endosomal fusion, fast evasion of endosomal entrapment, and efficacious RNA delivery. For example, conserving the lipid make-up, RNA–LNPs having cuboplex nanostructures are significantly more efficacious at endosomal escape than traditional lipoplex constructs.

Which Lipid Nanoparticle (LNP) Designs Work? A Simple Kinetic Model Linking LNP Chemical Structure to In Vivo Delivery Performance

“…Specifically, current observations suggest that our lipophilicity parameter is not able to fully capture the effects of configurational isomers of lipids. Recent literature indicates that lipid shape and packing can be key determinants of the pH response of LNPs. − This shift in the ionization state directly contributes to the endosomal escape of nucleic acid-containing LNPs. Thus, a refined model could include lipid packing parameters to account for the different lipid head geometries and intrinsic curvature of the lipid components that affect how lipids interact with each other.…”

Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin

“…Once internalized, LNPs enter endosomes, from which they need to escape to yield functional effects. In general, less than ≤2% eventually reach the cytosol ultimately limiting LNP efficacy and requiring higher doses to induce a therapeutic effect. , The endosomal route of a particle once more depends on the LNP composition, size, zeta potential, and the cell type. , Hence, we investigated the endosomal localization of mRNA and RNP-loaded LNPs in KCs (Figure , Figure S5) noting a different compartmentalization for these genetic cargos. RNP-LNPs predominantly colocalized in recycling (RAB11A+) and late (LAMP1+) endosomes, whereas only a few mRNA-loaded LNPs were detected in late endosomes.…”

“…48,49 The endosomal route of a particle once more depends on the LNP composition, size, zeta potential, and the cell type. 50,51 Hence, we investigated the endosomal localization of mRNA and RNP-loaded LNPs in KCs (Figure 3, Figure S5) noting a different compartmentalization for these genetic cargos. RNP-LNPs predominantly colocalized in recycling (RAB11A+) and late (LAMP1+) endosomes, whereas only a few mRNA-loaded LNPs were detected in late endosomes.…”

Self-Assembly of Unusually Stable Thermotropic Network Phases by Cellobiose-Based Guerbet Glycolipids

“…Supramolecular network morphologies stemming from the selfassembly of molecular amphiphiles offer opportunities for the bottom-up construction of materials with applications including organic semiconductors and energy storage media, 1 size-selective molecular separations membranes, 2 membranebound protein crystallization platforms, 3,4 and internally structured lipid nanoparticles for therapeutic delivery. 5,6 Exemplified by functional bicontinuous double gyroid (DG), double diamond, and other periodic networks, applications of these materials crucially depend on their interpenetrating, labyrinthine nanochannels with tailored chemical constitutions and physical properties. These intricate self-assembled structures are known to arise from thermotropic self-assembly of various liquid crystal (LC) mesogens 7 comprising chemically dissimilar segments of widely varied structures, including polycatenars, 8 rod-like polyphiles, 9,10 wedge-shaped molecules, 11 disk-like molecules, 12 bolamphiphiles, 13,14 and glyco-lipids.…”

“…Supramolecular network morphologies stemming from the self-assembly of molecular amphiphiles offer opportunities for the bottom-up construction of materials with applications including organic semiconductors and energy storage media, size-selective molecular separations membranes, membrane-bound protein crystallization platforms, , and internally structured lipid nanoparticles for therapeutic delivery. , Exemplified by functional bicontinuous double gyroid (DG), double diamond, and other periodic networks, applications of these materials crucially depend on their interpenetrating, labyrinthine nanochannels with tailored chemical constitutions and physical properties. These intricate self-assembled structures are known to arise from thermotropic self-assembly of various liquid crystal (LC) mesogens comprising chemically dissimilar segments of widely varied structures, including polycatenars, rod-like polyphiles, , wedge-shaped molecules, disk-like molecules, bolamphiphiles, , and glycolipids. − However, these thermotropic phases often form only with restricted molecular compositions and in narrow thermal stability windows, since the negative Gaussian (“saddle splay”) curvature microdomain interfaces of these assemblies exhibit substantial mean curvature deviations that result in molecular packing frustration. − The lack of robust molecular designs that drive mesoscale network self-assembly substantially curtails exciting current and future applications of these nanostructures.…”