Acidic pH-induced changes in lipid nanoparticle membrane packing

Koitabashi, Kyoka; Nagumo, Hiroki; Nakao, Mizuka; Machida, Tomoko; Yoshida, Kohki; Sakai‐Kato, Kumiko

doi:10.1016/j.bbamem.2021.183627

Cited by 16 publications

(13 citation statements)

References 36 publications

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“…Higher water content inside the lipid membrane thus may affect the kinetics of acidification and assist rapid membrane destabilization. Koitabashi et al examined lipid membrane destabilization in response to pH in siRNA-LNPs using a Laurdan assay and found a positive correlation between membrane hydration and gene silencing efficiency; however, this report did not focus on the kinetics of acidification. An interesting point regarding membrane hydration is that siRNA-LNPs have less water content than mRNA-LNPs of identical formulation as suggested by 31 P NMR experiments, which likely stems from the much longer length of hydrophilic RNA chains.…”

Section: Propertiesmentioning

confidence: 96%

Chemistry of Lipid Nanoparticles for RNA Delivery

et al. 2021

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Metrics & MoreArticle Recommendations CONSPECTUS: Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a homogeneous lipid core. These vesicles are widely used in small-molecule drug and nucleic acid delivery and recently gained much attention because of their remarkable success as a delivery platform for COVID-19 mRNA vaccines. Nonetheless, the utility of transient protein expression induced by mRNA extends far beyond vaccines against infectious diseasesthey also hold promise as cancer vaccines, protein replacement therapies, and gene editing components for rare genetic diseases. However, naked mRNA is inherently unstable and prone to rapid degradation by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects mRNA from extracellular ribonucleases and assists with intracellular mRNA delivery.In this Account, we discuss the core features of LNPs for RNA delivery. We focus our attention on LNPs designed to deliver mRNA; however, we also include examples of siRNA-LNP delivery where appropriate to highlight the commonalities and the dissimilarities due to the nucleic acid structure. First, we introduce the concept of LNPs, the advantages and disadvantages of utilizing nucleic acids as therapeutic agents, and the general reasoning behind the molecular makeup of LNPs. We also briefly highlight the most recent clinical successes of LNP-based nucleic acid therapies. Second, we describe the theory and methods of LNP self-assembly. The common idea behind all of the preparation methods is inducing electrostatic interactions between the nucleic acid and charged lipids and promoting nanoparticle growth via hydrophobic interactions. Third, we break down the LNP composition with special attention to the fundamental properties and purposes of each component. This includes the identified molecular design criteria, commercial sourcing, impact on intracellular trafficking, and contribution to the properties of LNPs. One of the key components of LNPs is ionizable lipids, which initiate electrostatic binding with endosomal membranes and facilitate cytosolic release; however, the roles of other lipid components should not be disregarded, as they are associated with stability, clearance, and distribution of LNPs. Fourth, we review the attributes of LNP constructs as a whole that can heavily influence RNA delivery. These attributes are LNP size, charge, internal structure, lipid packing, lipid membrane hydration, stability, and affinity toward biomacromolecules. We also discuss the specific techniques used to examine these attributes and how they can be adjusted. Finally, we offer our perspective on the future of RNA therapies and some questions that remain in the realm of LNP formulation and optimization.

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Section: Propertiesmentioning

confidence: 96%

Chemistry of Lipid Nanoparticles for RNA Delivery

et al. 2021

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“…Regarding the latter, an ionizable library based on a dilinoleyl hydrophobic tail and varying headgroups exhibited a wide range of apparent pK a values (4.17-8.12) [10] . Hence, the pK a of the ionizable lipid measured in LNPs likely depends on the molecular structure of its headgroup [10] , its hydrophobic tail groups [32] , and neighboring lipid packing, including LNP lipid composition [33] . Another study [29] showed that the measured pK a of ionizable lipids when dissolved in micelles (consisting of neutral surfactants) decreased when the number of ionizable lipids per micelle increased.…”

Section: Role Of Different Lipids In Lnpsmentioning

confidence: 99%

The role of lipid components in lipid nanoparticles for vaccines and gene therapy

Albertsen

Kulkarni

Witzigmann

et al. 2022

Advanced Drug Delivery Reviews

417

224

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“…Koitabashi et al previously used the fluorescent Laurdan dye to identify the hydration of LNPs formulated with ionizable lipid, KC2, by measuring GP. 25 setting, LNPs are taken up into the cells by endocytosis, gradually acidify during endosome maturation, and initiate the release of encapsulated nucleic acid cargo by the binding of positively charged ionizable lipid (such as MC3) to a negatively charged endosomal membrane. 26 As the ionizable lipid transitions to a protonated state, it attracts more water molecules and destabilizes the lipid membrane.…”

Section: Development Of Gal9-gfp Reporter Cell Line and Visualization...mentioning

confidence: 99%

“…Therefore, a high GP value is indicative of low membrane hydration or denser packing of the phospholipid bilayer. 25,27 Results of the Laurdan assay comparing fresh LNPs to thawed are shown in Figure 2a,b. Overall, LNPs were agnostic toward the buffer in this assay.…”

Section: Development Of Gal9-gfp Reporter Cell Line and Visualization...mentioning

confidence: 99%

Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles

et al. 2022

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Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers�HEPES, Tris, and phosphate-buffered saline�with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze−thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.

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Acidic pH-induced changes in lipid nanoparticle membrane packing

Cited by 16 publications

References 36 publications

Chemistry of Lipid Nanoparticles for RNA Delivery

Chemistry of Lipid Nanoparticles for RNA Delivery

The role of lipid components in lipid nanoparticles for vaccines and gene therapy

Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles

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