In situ label-free X-ray imaging for visualizing the localization of nanomedicines and subcellular architecture in intact single cells

doi:10.1038/s41596-023-00902-y

Review

. 2024 Jan;19(1):30-59.

doi: 10.1038/s41596-023-00902-y. Epub 2023 Nov 13.

In situ label-free X-ray imaging for visualizing the localization of nanomedicines and subcellular architecture in intact single cells

Mingjing Cao^#¹, Yaling Wang^#¹, Liming Wang^{1

2}, Kai Zhang², Yong Guan³, Yuecong Guo¹, Chunying Chen^{4

5

6}

Affiliations

¹ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing, China.
² CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China.
³ National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, China.
⁴ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing, China. chenchy@nanoctr.cn.
⁵ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China. chenchy@nanoctr.cn.
⁶ GBA National Institute for Nanotechnology Innovation, Guangzhou, China. chenchy@nanoctr.cn.

^# Contributed equally.

PMID: 37957402
DOI: 10.1038/s41596-023-00902-y

Review

In situ label-free X-ray imaging for visualizing the localization of nanomedicines and subcellular architecture in intact single cells

Mingjing Cao et al. Nat Protoc. 2024 Jan.

. 2024 Jan;19(1):30-59.

doi: 10.1038/s41596-023-00902-y. Epub 2023 Nov 13.

Authors

Mingjing Cao^#¹, Yaling Wang^#¹, Liming Wang^{1

2}, Kai Zhang², Yong Guan³, Yuecong Guo¹, Chunying Chen^{4

5

6}

Affiliations

¹ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing, China.
² CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China.
³ National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, China.
⁴ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing, China. chenchy@nanoctr.cn.
⁵ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China. chenchy@nanoctr.cn.
⁶ GBA National Institute for Nanotechnology Innovation, Guangzhou, China. chenchy@nanoctr.cn.

^# Contributed equally.

PMID: 37957402
DOI: 10.1038/s41596-023-00902-y

Abstract

Understanding the intracellular behaviors of nanomedicines and morphology variation of subcellular architecture impacted by nanomaterial-biology (nano-bio) interactions could help guide the safe-by-design, manufacturing and evaluation of nanomedicines for clinical translation. The in situ and label-free analysis of nano-bio interactions in intact single cells at nanoscale remains challenging. We developed an approach based on X-ray microscopy to directly visualize the 2D or 3D intracellular distribution without labeling at nanometer resolution and analyze the chemical transformation of nanomedicines in situ. Here, we describe an optimized workflow for cell sample preparation, beamline selection, data acquisition and analysis. With several model bionanomaterials as examples, we analyze the localization of nanomedicines in various primary blood cells, macrophages, dendritic cells, monocytes and cancer cells, as well as the morphology of some organelles with soft and hard X-rays. Our protocol has been successfully implemented at three beamline facilities: 4W1A of Beijing Synchrotron Radiation Facility, BL08U1A of Shanghai Synchrotron Radiation Facility and BL07W of the National Synchrotron Radiation Laboratory. This protocol can be completed in ~2-5 d, depending on the cell types, their incubation times with nanomaterials and the selected X-ray beamline. The protocol enables the in situ analysis of the varieties of metal-containing nanomaterials, visualization of intracellular endocytosis, distribution and excretion and corresponding subcellular morphological variation influenced by nanomedicines in cell lines or primary cells by using this universal and robust platform. The results facilitate the understanding of the true principle and mechanism underlying the nano-bio interaction.

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[1] Zhang, C. et al. Progress, challenges, and future of nanomedicine. Nano Today 35, 101008 (2020). - DOI

[2] Zhang, C. et al. Progress, challenges, and future of nanomedicine. Nano Today 35, 101008 (2020). - DOI

[3] Poon, W., Kingston, B., Ouyang, B., Ngo, W. & Chan, W. A framework for designing delivery systems. Nat. Nanotechnol. 15, 819–829 (2020). - DOI - PubMed

[4] Poon, W., Kingston, B., Ouyang, B., Ngo, W. & Chan, W. A framework for designing delivery systems. Nat. Nanotechnol. 15, 819–829 (2020). - DOI - PubMed

[5] Bourzac, K. Nanotechnology: carrying drugs. Nature 491, S58–S60 (2012). - DOI - PubMed

[6] Bourzac, K. Nanotechnology: carrying drugs. Nature 491, S58–S60 (2012). - DOI - PubMed

[7] Gewin, V. Big opportunities in a small world. Nature 460, 540–541 (2009). - DOI - PubMed

[8] Gewin, V. Big opportunities in a small world. Nature 460, 540–541 (2009). - DOI - PubMed

[9] Kim, B. Y., Rutka, J. T. & Chan, W. C. Nanomedicine. N. Engl. J. Med. 363, 2434–2443 (2010). - DOI - PubMed

[10] Kim, B. Y., Rutka, J. T. & Chan, W. C. Nanomedicine. N. Engl. J. Med. 363, 2434–2443 (2010). - DOI - PubMed

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In situ label-free X-ray imaging for visualizing the localization of nanomedicines and subcellular architecture in intact single cells

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In situ label-free X-ray imaging for visualizing the localization of nanomedicines and subcellular architecture in intact single cells

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