A Comprehensive Review of mRNA Vaccines.

mRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clinical development, and potential for rapid, low-cost manufacturing. These vaccines have progressed from being a mere curiosity to emerging as COVID-19 pandemic vaccine front-runners. The advancements in the field of nanotechnology for developing delivery vehicles for mRNA vaccines are highly significant. In this review we have summarized each and every aspect of the mRNA vaccine. The article describes the mRNA structure, its pharmacological function of immunity induction, lipid nanoparticles (LNPs), and the upstream, downstream, and formulation process of mRNA vaccine manufacturing. Additionally, mRNA vaccines in clinical trials are also described. A deep dive into the future perspectives of mRNA vaccines, such as its freeze-drying, delivery systems, and LNPs targeting antigen-presenting cells and dendritic cells, are also summarized.

Hydrophobic Domain Structure of Linear-Dendritic Poly(ethylene glycol) Lipids Affects RNA Delivery of Lipid Nanoparticles.

In this work, a series of linear-dendritic poly(ethylene glycol) (PEG) lipids (PEG-GnCm) were synthesized through a strategy using sequential aza- and sulfa-Michael addition reactions. The effect of modulating the hydrophobic domain of linear-dendritic PEG lipids was systematically investigated for in vitro and in vivo small RNA delivery as the surface-stabilizing component of 5A2-SC8 dendrimer lipid-based nanoparticles (DLNPs). The lipid alkyl lengths (C8, C12, and C16) and dendrimer generations (G1, G2, and G3) were altered to create PEG-GnCm with different physical properties and anchoring potential. The tail chemical structure of PEG-GnCm did not affect the formulation of 5A2-SC8 DLNPs, including the nanoparticle size, RNA encapsulation, and stability. However, the tail chemical structure did dramatically affect the RNA delivery efficacy of the formed 5A2-SC8 DLNPs with different PEG-GnCm. First-generation PEG lipids (PEG-G1C8, PEG-G1C12, and PEG-G1C16) and a second-generation PEG lipid (PEG-G2C8) formed 5A2-SC8 DLNPs that could deliver siRNAs effectively in vitro and in vivo. 5A2-SC8 DLNPs formulated with second-generation PEG lipids (PEG-G2C12 and PEG-G2C16) and all three third-generation PEG lipids (PEG-G3C8, PEG-G3C12, and PEG-G3C16) lost the ability to deliver siRNA effectively in vitro and in vivo. Overall, we determined that the hydrophobic domain chemical structure of linear-dendritic poly(ethylene glycol) lipids affected the RNA delivery of DLNPs by impacting the escape of 5A2-SC8 DLNPs from endosomes at early cell incubation times, thereby indicating how PEG lipid anchoring and chemical structure can modulate in vitro and in vivo siRNA delivery efficacies.

On the Influence of Fabrication Methods and Materials for mRNA-LNP Production: From Size and Morphology to Internal Structure and mRNA Delivery Performance In Vitro and In Vivo.

Lipid nanoparticle (LNP) remains the most advanced platform for messenger RNA (mRNA) delivery. To date, mRNA LNPs synthesis is mostly performed by mixing lipids and mRNA with microfluidics. In this study, a cost-effective microfluidic setup for synthesizing mRNA LNPs is developed. It allows to fine-tune the LNPs characteristics without compromising LNP properties. It is compared with a commercial device (NanoAssemblr) and ethanol injection and the influence of manufacturing conditions on the performance of mRNA LNPs is investigated. LNPs prepared by ethanol injection exhibit broader size distributions and more inhomogeneous internal structure (e.g., bleb-like substructures), while other LNPs show uniform structure with dense cores. Small angel X-ray scattering (SAXS) data indicate a tighter interaction between mRNA and lipids within LNPs synthesized by custom device, compared to LNPs produced by NanoAssemblr. Interestingly, the better transfection efficiency of polysarcosine (pSar)-modified LNPs correlates with a higher surface roughness than that of PEGylated ones. The manufacturing approach, however, shows modest influence on mRNA expression in vivo. In summary, the home-developed cost-effective microfluidic device can synthesize LNPs and represents a potent alternative to NanoAssemblr. The preparation methods show notable effect on LNPs' structure but a minor influence on mRNA delivery in vitro and in vivo.

A closer look at calcium-induced interactions between phosphatidylserine-(PS) doped liposomes and the structural effects caused by inclusion of gangliosides or polyethylene glycol- (PEG) modified lipids.

The effects of polyethylene glycol- (PEG) modified lipids and gangliosides on the Ca2+ induced interaction between liposomes composed of palmitoyl-oleoyl phosphatidylethanolamine (POPE) and palmitoyl-oleoyl phosphatidylserine (POPS) was investigated at physiological ionic strength. Förster resonance energy transfer (FRET) studies complemented with dynamic light scattering (DLS) and cryo-transmission electron microscopy (Cryo-EM) show that naked liposomes tend to adhere, rupture, and collapse on each other's surfaces upon addition of Ca2+, eventually resulting in the formation of large multilamellar aggregates and bilayer sheets. Noteworthy, the presence of gangliosides or PEGylated lipids does not prevent the adhesion-rupture process, but leads to the formation of small, long-lived bilayer fragments/disks. PEGylated lipids seem to be more effective than gangliosides at stabilizing these structures. Attractive interactions arising from ion correlation are proposed to be a driving force for the liposome-liposome adhesion and rupture processes. The results suggest that, in contrast with the conclusions drawn from previous solely FRET-based studies, direct liposome-liposome fusion is not the dominating process triggered by Ca2+ in the systems studied.

Psychotropic drugs interaction with the lipid nanoparticle of COVID-19 mRNA therapeutics.

The messenger RNA (mRNA) vaccines for COVID-19, Pfizer-BioNTech and Moderna, were authorized in the US on an emergency basis in December of 2020. The rapid distribution of these therapeutics around the country and the world led to millions of people being vaccinated in a short time span, an action that decreased hospitalization and death but also heightened the concerns about adverse effects and drug-vaccine interactions. The COVID-19 mRNA vaccines are of particular interest as they form the vanguard of a range of other mRNA therapeutics that are currently in the development pipeline, focusing both on infectious diseases as well as oncological applications. The Vaccine Adverse Event Reporting System (VAERS) has gained additional attention during the COVID-19 pandemic, specifically regarding the rollout of mRNA therapeutics. However, for VAERS, absence of a reporting platform for drug-vaccine interactions left these events poorly defined. For example, chemotherapy, anticonvulsants, and antimalarials were documented to interfere with the mRNA vaccines, but much less is known about the other drugs that could interact with these therapeutics, causing adverse events or decreased efficacy. In addition, SARS-CoV-2 exploitation of host cytochrome P450 enzymes, reported in COVID-19 critical illness, highlights viral interference with drug metabolism. For example, patients with severe psychiatric illness (SPI) in treatment with clozapine often displayed elevated drug levels, emphasizing drug-vaccine interaction.

Synthesis and characterization of PEGylated bolaamphiphiles with enhanced retention in liposomes.

Long-circulating liposomes are typically prepared with poly(ethylene glycol)- (PEG-) modified lipids, where the lipid portion is inserted in the lipid bilayers as an anchor and the hydrophilic PEG coats the surface to prevent liposome aggregation and rapid clearance in vivo. However, these steric protection effects are compromised upon systemic administration due to low retention of PEGylated lipids within liposome membranes upon dilution. Hence, a series of PEGylated bolaamphiphiles (PEG-bolas) were for the first time developed to increase retention in the lipid bilayer, presumably leading to enhanced integrity of the PEG protective layer upon dilution. We hypothesized that PEG-bolas with a sufficiently long hydrophobic domain and rigid central group could predominantly adopt a membrane-spanning configuration, taking full advantage of steric protection offered by PEG and enhanced retention in liposomes enabled by the bola geometry. In this paper, liposomes stabilized by PEG-bolas comprised of a biphenyl core and twelve-carbon alkyl chain not only exhibited similar storage and biological stability compared to conventional PEGylated lipid stabilized liposomes, but also significantly improved retention upon dilution. Our findings facilitate new designs of liposome-stabilizing agents and can be applied to improve the delivery efficiency of liposomal delivery vehicles in vivo.

Probing adsorption of DSPE-PEG2000 and DSPE-PEG5000 to the surface of felodipine and griseofulvin nanocrystals.

Nanosized formulations of poorly water-soluble drugs show great potential due to improved bioavailability. In order to retain colloidal stability, the nanocrystals need to be stabilized. Here we explore the use of the poly(ethylene glycol) (PEG) conjugated phospholipids DSPE-PEG2000 and DSPE-PEG5000 as stabilizers of felodipine and griseofulvin nanocrystals. Nanocrystal stability and physicochemical properties were examined and the interaction between the PEGylated lipids and the nanocrystal surface as well as a macroscopic model surface was investigated. Using quartz crystal microbalance with dissipation monitoring both mass adsorption and the thickness of the adsorbed layer were estimated. The results indicate that the PEGylated lipids are adsorbed as flat layers of around 1-3nm, and that DSPE-PEG5000 forms a thicker layer compared with DSPE-PEG2000. In addition, the mass adsorption to the drug crystals and the model surface are seemingly comparable. Furthermore, both DSPE-PEG2000 and DSPE-PEG5000 rendered stable drug nanocrystals, with a somewhat higher surface binding and stability seen for DSPE-PEG2000. These results suggest DSPE-PEG2000 and DSPE-PEG5000 as efficient nanocrystal stabilizers, with DSPE-PEG2000 giving a somewhat higher surface coverage and superior colloidal stability, whereas DSPE-PEG5000 shows a more extended structure that may have advantages for prolongation of circulation time in vivo and facilitation for targeting modifications.