細胞内のナノスケール薬物分子を明らかにする新手法を開発(New method reveals nano-scale drug molecules in cells)

2023-02-02 スウェーデン王国・チャルマース工科大学

<関連情報>

• https://news.cision.com/chalmers/r/new-method-reveals-nano-scale-drug-molecules-in-cells,c3707022
• https://pubs.acs.org/doi/10.1021/acs.analchem.2c02111
• https://pubs.acs.org/doi/10.1021/acsnano.9b09804

NanoSIMSによるオリゴヌクレオチド治療薬の細胞内絶対定量化 Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS

Cécile Becquart, Rouven Stulz, Aurélien Thomen, Maryam Dost, Neda Najafinobar, Anders Dahlén, Shalini Andersson, Andrew G. Ewing and Michael E. Kurczy
Analytical Chemistry  Published:July 13, 2022
DOI:https://doi.org/10.1021/acs.analchem.2c02111

Abstract

Antisense oligonucleotide (ASO)-based therapeutics hold great potential for the treatment of a variety of diseases. Therefore, a better understanding of cellular delivery, uptake, and trafficking mechanisms of ASOs is highly important for early-stage drug discovery. In particular, understanding the biodistribution and quantifying the abundance of ASOs at the subcellular level are needed to fully characterize their activity. Here, we used a combination of electron microscopy and NanoSIMS to assess the subcellular concentrations of a ³⁴S-labeled GalNAc-ASO and a naked ASO in the organelles of primary human hepatocytes. We first cross-validated the method by including a ¹²⁷I-labeled ASO, finding that the absolute concentration of the lysosomal ASO using two independent labeling strategies gave matching results, demonstrating the strength of our approach. This work also describes the preparation of external standards for absolute quantification by NanoSIMS. For both the ³⁴S and ¹²⁷I approaches used for our quantification methodology, we established the limit of detection (5 and 2 μM, respectively) and the lower limit of quantification (14 and 5 μM, respectively).

細胞内質量分析イメージングと小器官を横断する絶対定量分析 Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles

Aurélien Thomen, Neda Najafinobar, Florent Penen, Emma Kay, Pratik P. Upadhyay, Xianchan Li, Nhu T. N. Phan, Per Malmberg, Magnus Klarqvist, Shalini Andersson, Michael E. Kurczy and Andrew G. Ewing
ACS Nano  Published:April 2, 2020
DOI:https://doi.org/10.1021/acsnano.9b09804

Abstract

Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.