High-throughput and high-content bioassay enables tuning of polyester nanoparticles for cellular uptake, endosomal escape, and systemic in vivo delivery of mRNA

Abstract

Nanoparticle-based mRNA therapeutics hold great promise, but cellular internalization and endosomal escape remain key barriers for cytosolic delivery. We developed a dual nanoparticle uptake and endosomal disruption assay using high-throughput and high-content image-based screening. Using a genetically encoded Galectin 8 fluorescent fusion protein sensor, endosomal disruption could be detected via sensor clustering on damaged endosomal membranes. Simultaneously, nucleic acid endocytosis was quantified using fluorescently tagged mRNA. We used an array of biodegradable poly(beta-amino ester)s as well as Lipofectamine and PEI to demonstrate that this assay has higher predictive capacity for mRNA delivery compared to conventional polymer and nanoparticle physiochemical characteristics. Top nanoparticle formulations enabled safe and efficacious mRNA expression in multiple tissues following intravenous injection, demonstrating that the in vitro screening method is also predictive of in vivo performance. Efficacious nonviral systemic delivery of mRNA with biodegradable particles opens up new avenues for genetic medicine and human health.

Helper-Polymer Based Five-Element Nanoparticles (FNPs) for Lung-Specific mRNA Delivery with Long-Term Stability after Lyophilization

“…Due to the large molecular weight and negative charge of mRNA, it cannot passively cross the cell membrane. − Additionally, it can be easily degraded by nuclease existing in the extracellular space. − Lipid nanoparticles (LNP) are so far the most successful delivery platform that can facilitate cellular uptake of mRNA while preventing it from degradation. , Lung-specific delivery of mRNA has been achieved by high-throughput screening or rational design. − For example, in 2016, Anderson et al achieved systemic delivery of mRNA to the lung first using hybrid polymer–lipid nanoparticles, which were composed of poly­(β-amino esters) (PBAEs) and lipid-polyethylene glycol (PEG) . Motivated by this potential outcome, they optimized polymer structures as well as nanoparticle formulations in 2018 and developed new PBAEs that were universal for the systemic delivery of mRNA and pDNA to the lung in 2021. , Furthermore, Xu et al identified lipidoids whose tails containing amide bonds were able to deliver mRNA to the lung .…”

mRNA-Loaded Lipid Nanoparticles Targeting Immune Cells in the Spleen for Use as Cancer Vaccines

“…It is also possible that types of cells, other than immune cells, may be transfected. In the case of nanoparticles capable of delivering mRNA to the spleen, gene expression has been confirmed in cells other than immune cells, including endothelial cells [26]. However, transfection of these cells would not be expected to affect the immune response.…”

Poly(Beta-Amino Ester)s as High-Yield Transfection Reagents for Recombinant Protein Production

“…PBAE nanoparticle binding, uptake and escape have been previously described in comparison to PEI, 7 , 15 , 29 and the development of a galectin 8-based biosensor enabled detection and comparison of endosomal escape of PBAEs versus various PEI constructs. 30 Bishop and Kozielski et al review various structural elements, such as charge density, hydrophobicity, and molecular weight, of PBAEs, PEI, and other biomaterials that help facilitate intracellular delivery of nucleic acids. 15 Karlsson et al review additional PBAE gene delivery mechanisms, giving a comprehensive description of studies investigating topics such as the binding strength of PBAEs, the effect of surface charge on toxicity, and the buffering capacity of PBAEs based on secondary and tertiary amines leading to endosomal escape.…”