Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles.

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.

Chemistry of Lipid Nanoparticles for RNA Delivery.

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.

Self-assembled mRNA vaccines.

mRNA vaccines have evolved from being a mere curiosity to emerging as COVID-19 vaccine front-runners. Recent advancements in the field of RNA technology, vaccinology, and nanotechnology have generated interest in delivering safe and effective mRNA therapeutics. In this review, we discuss design and self-assembly of mRNA vaccines. Self-assembly, a spontaneous organization of individual molecules, allows for design of nanoparticles with customizable properties. We highlight the materials commonly utilized to deliver mRNA, their physicochemical characteristics, and other relevant considerations, such as mRNA optimization, routes of administration, cellular fate, and immune activation, that are important for successful mRNA vaccination. We also examine the COVID-19 mRNA vaccines currently in clinical trials. mRNA vaccines are ready for the clinic, showing tremendous promise in the COVID-19 vaccine race, and have pushed the boundaries of gene therapy.

Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery.

Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.