RRC ID |
71948
|
Author |
Munson MJ, O'Driscoll G, Silva AM, Lázaro-Ibáñez E, Gallud A, Wilson JT, Collén A, Esbjörner EK, Sabirsh A.
|
Title |
A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery.
|
Journal |
Commun Biol
|
Abstract |
RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.
|
Volume |
4(1)
|
Pages |
211
|
Published |
2021-2-16
|
DOI |
10.1038/s42003-021-01728-8
|
PII |
10.1038/s42003-021-01728-8
|
PMID |
33594247
|
PMC |
PMC7887203
|
MeSH |
Endosomes / metabolism*
Galectins / genetics
Galectins / metabolism*
Gene Transfer Techniques*
Genes, Reporter
HeLa Cells
Hep G2 Cells
Humans
Lipids / chemistry*
Luminescent Proteins / genetics
Luminescent Proteins / metabolism
Microscopy, Fluorescence
Nanoparticles*
RNA Transport*
RNA, Messenger / genetics
RNA, Messenger / metabolism*
Recombinant Fusion Proteins / metabolism
Time Factors
Time-Lapse Imaging
|
IF |
4.165
|
Resource |
Human and Animal Cells |
HuH-7(RCB1366) |