A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery.
A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery.
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用于定量纳米颗粒摄取、内体逃逸和功能性RNA递送的高通量半乳糖凝集素-9成像测定。
DOI：
10.1038/s42003-021-01728-8
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发表时间：
2021-02-16
影响因子：
5.9
通讯作者：
Sabirsh A
中科院分区：
文献类型：
--
作者：
Munson MJ;O'Driscoll G;Silva AM;Lázaro-Ibáñez E;Gallud A;Wilson JT;Collén A;Esbjörner EK;Sabirsh A
基于rna的疗法在治疗许多无法治愈的人类疾病方面具有巨大的潜力。然而，它们的功效，特别是mRNA，仍然受到细胞递送不良和有限的内体逃逸的阻碍。开发和优化递送载体，如脂质纳米颗粒（LNPs），受到有限的筛选方法的阻碍，无法充分详细地探测LNPs的细胞内加工。我们利用半乳糖凝集素-9报告基因和荧光标记的mRNA，开发了一种基于高通量成像的内体逃逸试验，以探测纳米颗粒介导的摄取、内体逃逸频率和mRNA翻译之间的相关性。此外，该分析已集成在筛选平台中，用于优化脂质纳米颗粒配方。我们发现半乳糖凝集素-9募集是由不同mRNA递送纳米颗粒和小分子诱导的内体逃逸事件的一个强大的定量报告。我们确定了具有优异逃逸特性的纳米颗粒，并证明了内体逃逸反应的细胞系差异，强调了针对特定应用微调递送配方的必要性。Munson等人开发了一种基于半乳糖凝集素-9报告因子的高通量内体逃逸成像检测，以探测纳米颗粒介导的摄取、内体逃逸频率和mRNA翻译之间的相关性。通过筛选各种递送系统、调制剂和纳米颗粒组分的组合，作者展示了纳米药物如何在特定应用中优化其优越的逃逸特性和功效。
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. Munson et al. develop a high-throughput Galectin-9 reporter based endosomal escape imaging assay to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. By screening various delivery systems, modulators and combinations of nanoparticle components, the authors demonstrate how nanomedicines can be optimized for superior escape properties and efficacy in specific applications.
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DOI：
10.1126/science.aag1417
发表时间：
2017-03-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Castellano BM;Thelen AM;Moldavski O;Feltes M;van der Welle RE;Mydock-McGrane L;Jiang X;van Eijkeren RJ;Davis OB;Louie SM;Perera RM;Covey DF;Nomura DK;Ory DS;Zoncu R
通讯作者：
Zoncu R
影响因子：
46.9
作者：
Gilleron, Jerome;Querbes, William;Zerial, Marino
通讯作者：
Zerial, Marino
影响因子：
10.8
作者：
Kauffman, Kevin J.;Dorkin, J. Robert;Anderson, Daniel G.
通讯作者：
Anderson, Daniel G.
影响因子：
17.1
作者：
Joshi, Bhagyashree S.;de Beer, Marit A.;Zuhorn, Inge S.
通讯作者：
Zuhorn, Inge S.
影响因子：
4.3
作者：
Nishi, Nozomu;Itoh, Aiko;Nakamura, Takanori
通讯作者：
Nakamura, Takanori