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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., Graphical abstract Unlabelled Image, Highlights • mRNA vaccines showed spectacular success in the race for COVID-19 vaccines. • Design of both mRNA and the delivery vectors help in fine-tuning efficacy. • Self-assembled mRNA delivery vectors include lipid and polymer nanoparticles. • Lipid nanoparticle mRNA vaccines have ca. 95% efficacy and earned EUA FDA approval. • These unprecedented results may pave the road for future mRNA vaccine development.

Lipid-based nanoparticles (LNPs) for the delivery of mRNA have jumped to the forefront of non-viral gene delivery. Despite this exciting development, poor endosomal escape after LNP cell entry remains an unsolved, rate-limiting bottleneck. Here we report the use of galectin 8-GFP (Gal8-GFP) cell reporter system to visualize the endosomal escaping capabilities of LNPs encapsulating mRNA. LNPs substituted with phytosterols in place of cholesterol exhibited various levels of Gal8 recruitment in the Gal8-GFP reporter system. In live-cell imaging, the results of the LNPs containing β-sitosterol (LNP-Sito) showed a 10-fold increase in detectable endosomal perturbation events when compared to the standard cholesterol LNPs (LNP-Chol), suggesting the superior capability of LNP-Sito to escape from endosomal entrapment. Trafficking studies of these LNPs showed strong localization with late endosomes. This highly sensitive and robust Gal8-GFP reporter system can be a valuable tool to elucidate intricacies of LNP trafficking and ephemeral endosomal escape events, enabling the advancement in gene 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.

We studied the diffusion of three model proteins, lysozyme (Lz), bovine hemoglobin (BHb), and bovine serum albumin (BSA), normal to the (111) plane of sintered silica colloidal crystals with three different pore "radii" (7.5, 19, and 27 nm). We demonstrated that these colloidal crystals exhibit size selectivity when the nanopores are sufficiently small (7.5 and 19 nm). Because these nanopores are still larger than the diffusing proteins, the observed size selectivity can be attributed to the tortuosity of the colloidal nanopores. Larger (27 nm) nanopores led to higher transport rates but at the cost of selectivity. In addition to the size selectivity, we also demonstrated that 19 nm nanopores possess shape selectivity for the proteins of comparable molecular weights. We showed that the high temperature sintering required for the preparation of sintered colloidal crystals reduces the extent of interactions between the proteins and the nanopore surface, which appear to play a minor role in the diffusion, and that transport selectivity is decided solely by protein size and shape. Taken together, our observations suggest that sintered silica colloidal crystals constitute promising nanoporous membranes for protein separations, with easily controllable pore size, size and shape selectivity, and minimal surface fouling.

We report the preparation and characterization of charged nanoporous membranes by self-assembly of "hairy" silica nanoparticles (HNPs) functionalized with polyelectrolyte copolymer brushes. We show that HNP membranes possess high water flux, have well-defined pore sizes, and rejection up to 80% of charged species in solution. The properties of these membranes can be tuned by controlling the length and composition of polymer brushes and the electrolyte concentration in solution. We demonstrate that membrane pore sizes undergo changes of up to 40% in response to changes in the ionic strength of the salt solution. Using MD computer simulations of a coarse-grained model, we link these tunable properties to the conformations of polymer chains in the spaces between randomly packed HNPs. As polymer length increases, the polymers fill the interparticle gaps, and the pore size decreases markedly. On the basis of their straightforward fabrication and tunable properties, HNP membranes may find applications in size- and charge-selective separations, water desalination, and responsive devices.

Water dispersible boron nanoparticles have great potential as materials for boron neutron capture therapy of cancer and magnetic resonance imaging, if they are prepared on a large scale with uniform size and shape and hydrophilic modifiable surface. We report the first method to prepare spherical, monodisperse, water dispersible boron core silica shell nanoparticles (B@SiO2 NPs) suitable for aforementioned biomedical applications. In this method, 40 nm elemental boron nanoparticles, easily prepared by mechanical milling and carrying 10-undecenoic acid surface ligands, are hydrosilylated using triethoxysilane, followed by base-catalyzed hydrolysis of tetraethoxysilane, which forms a 10-nm silica shell around the boron core. This simple two-step process converts irregularly shaped hydrophobic boron particles into the spherically shaped uniform nanoparticles. The B@SiO2 NPs are dispersible in water and the silica shell surface can be modified with primary amines that allow for the attachment of a fluorophore and, potentially, of targeting moieties.