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Genome-wide forward genetic screening to identify receptors and proteins mediating nanoparticle uptake and intracellular processing

Nature Nanotechnology (2024)Cite this article

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Abstract

Understanding how cells process nanoparticles is crucial to optimize nanomedicine efficacy. However, characterizing cellular pathways is challenging, especially if non-canonical mechanisms are involved. In this Article a genome-wide forward genetic screening based on insertional mutagenesis is applied to discover receptors and proteins involved in the intracellular accumulation (uptake and intracellular processing) of silica nanoparticles. The nanoparticles are covered by a human serum corona known to target the low-density lipoprotein receptor (LDLR). By sorting cells with reduced nanoparticle accumulation and deep sequencing after each sorting, 80 enriched genes are identified. We find that, as well as LDLR, the scavenger receptor SCARB1 also mediates nanoparticle accumulation. Additionally, heparan sulfate acts as a specific nanoparticle receptor, and its role varies depending on cell and nanoparticle type. Furthermore, some of the identified targets affect nanoparticle trafficking to the lysosomes. These results show the potential of genetic screening to characterize nanoparticle pathways. Additionally, they indicate that corona-coated nanoparticles are internalized via multiple receptors.

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Fig. 1: Mutagenized cells with reduced nanoparticle accumulation are enriched after multiple selections.
Fig. 2: Time-resolved analysis of enriched genes identified in multiple sorts.
Fig. 3: Effect of the identified targets on nanoparticle intracellular accumulation and adhesion.
Fig. 4: Role of receptors LDLR and SCARB1, and heparan sulfate, in nanoparticle intracellular accumulation.
Fig. 5: Role of heparan sulfate and apolipoprotein receptors in the intracellular accumulation of different nanoparticles.
Fig. 6: Effect of the identified targets in nanoparticle trafficking to the lysosomes.

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Data availability

The data that support the findings of this study are available within the article and its Supplementary Information and Supplementary Data. The raw sequencing data are deposited at the European Nucleotide Archive under accession code PRJEB71925. The complete analysis of the sequencing results is included as Supplementary Data 1. The raw imaging and nanoparticle physicochemical characterization data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The code to analyse the sequencing data generated in this study is available at https://github.com/Vityay/NanoScreen.

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Acknowledgements

This study was funded by the European Research Council (ERC) as part of the European Union’s Horizon 2020 research and innovation programme under grant agreement 637614 (A.S.) (NanoPaths). We acknowledge T. Brummelkamp (Netherlands Cancer Institute, Amsterdam, The Netherlands) for providing the plasmids for retroviral mutagenesis, and J. Carette (Stanford University, Stanford, CA, USA) for sharing detailed information on the mutagenesis and LAM-PCR methods. We also thank H. Haisma (GRIP) and J. van den Born for suggestions on retroviral production and heparan sulfate detection respectively, P. Ettema (GRIP) and K. Hoekstra-Wakker (ERIBA) for technical assistance with bacterial culture and preparation of samples for next generation sequencing, respectively, and K. Yang (GRIP) for liposome preparation. E. Frijlink and W. Hinrichs (GRIP) are acknowledged for access to nanoparticle tracking analysis (NTA), H. van der Mei (University Medical Center Groningen) for access to dynamic light scattering (DLS) and Herman Sillje (University Medical Center Groningen) for access to epifluorescence microscopy. FACS was performed at the flow cytometry facility of the University Medical Center Groningen. Confocal fluorescence imaging was performed at the imaging facility of the University Medical Center Groningen.

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Authors and Affiliations

  1. Department of Nanomedicine & Drug Targeting, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, The Netherlands

    Daphne Montizaan, Roberta Bartucci, Catharina Reker-Smit, Sander de Weerd & Anna Salvati

  2. Department of Pharmaceutical Analysis, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, The Netherlands

    Christoffer Åberg

  3. European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

    Victor Guryev & Diana C. J. Spierings

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Contributions

D.M. designed and performed all experiments for the forward genetic screening (from the production of the viral particles, the preparation of the mutagenized library and the selection of the cells with reduced nanoparticle intracellular accumulation to the LAM-PCR preparation for sequencing, pathway analysis on the results and all their validation with the exceptions specified below), analysed and interpreted the data and wrote the manuscript. R.B. performed nanoparticle intracellular accumulation and adhesion kinetics in silenced cells, with competitors and after inhibition of SCARB1, as well as the imaging and quantification of nanoparticle colocalization with lysosomes, and tested the panel of targets in A549 cells and with the negatively charged liposomes together with contributions by C.R.-S. and analysed and interpreted these data. In addition, C.R.S. also assisted in cell sorting for forward genetic screening and performed some of the digestion and competition experiments with the panel of nanoparticles. S.d.W. performed nanoparticle tracking analysis experiments and analysis. C.Å. contributed to the analysis of the results. V.G. performed the mapping of the sequenced inserts to the genome and supervised the analysis of the sequencing results. D.C.J.S. supervised the LAM-PCR-based next-generation sequencing library preparation and sequencing. A.S. designed and supervised the whole project, analysed and interpreted the data and wrote the manuscript. All authors have read and revised the manuscript.

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Correspondence to Anna Salvati.

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Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–20, Tables 1–6 and References.

Reporting Summary

Supplementary Data 1

Number of insertions per gene before and after each selection of mutagenized HAP1 cells with reduced nanoparticle intracellular accumulation (up to six sorts).

Supplementary Data 2

Description of the role of the 80 enriched genes identified by time-resolved forward genetic screening.

Supplementary Data 3

Source data for Supplementary Figures.

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Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

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Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

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Source Data Fig. 6

Statistical source data.

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Montizaan, D., Bartucci, R., Reker-Smit, C. et al. Genome-wide forward genetic screening to identify receptors and proteins mediating nanoparticle uptake and intracellular processing. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-024-01629-x

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