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.

Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy.

Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions.

Hybrid core-shell particles for mRNA systemic delivery.

mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines.

Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA.

BACKGROUND: RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. Here, we have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was in vitro transcribed in cell lines and evaluated for expression. RNA-lipoplex nanoparticles were then assembled by complexing RNA with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice. RESULTS: NIS expression was detected on the membrane of two cell lines as early as 6 h after transfection and gradually decreased over 48 h. In vivo and ex vivo PET/MRI of anionic spleen-targeting or cationic lung-targeting NIS-RNA lipoplexes revealed a visually detectable rapid increase of Iodine-124 uptake in the spleen or lung compared to control-RNA-lipoplexes, respectively, with minimal background in other organs except from thyroid, stomach and salivary gland. CONCLUSIONS: The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.