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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.

SUBMITTER: Rui Y

PROVIDER: S-EPMC8730632 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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

Rui Yuan Y Wilson David R DR Tzeng Stephany Y SY Yamagata Hannah M HM Sudhakar Deepti D Conge Marranne M Berlinicke Cynthia A CA Zack Donald J DJ Tuesca Anthony A Green Jordan J JJ

Science advances 20220105 1

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 quan ...[more]

PMID: 34985952

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Project description:To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(D,L-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In conclusion, biomechanical interactions with membrane lipids are involved in cellular uptake and endosomal escape of NPs. Biophysical interaction studies could help us better understand the role of membrane lipids in cellular uptake and intracellular trafficking of NPs.

Dataset Information

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

Publications

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

Similar Datasets

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