Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2

Rotem, Rany; Bertolini, Jessica; Salvioni, Lucia; Barbieri, Linda; Rizzuto, Maria Antonietta; Tinelli, Veronica; Gori, Alessandro; Adams, Stephen; Colombo, Miriam; Prosperi, Davide

doi:10.1016/j.nano.2022.102626

Cited by 3 publications

(2 citation statements)

References 32 publications

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“…However, measuring cell penetration of biomolecules is not straightforward. In recent years, chemical biologists have developed a variety of strategies for quantifying passive penetration, total cellular uptake, and cytosolic/nuclear penetration with different detection methods including fluorescence microscopy, [4][5][6][7] mass-spectrometry, [8][9][10][11][12][13] NMR, 14 flow cytometry, [15][16][17] FACS, 18,19 qPCR, 20 luminescence, 21,22 or ELISA. 23 For cell-based assays, the choice of cell line is critical and measurement with different methods and/or in different cell lines can lead to different results.…”

Section: Introductionmentioning

confidence: 99%

Comparing cell penetration of biotherapeutics across human cell lines

Batistatou,

Kritzer

2024

Preprint

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A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types, independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure data could be compared across cell lines. We compared the penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides, but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

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Section: Introductionmentioning

confidence: 99%

Comparing cell penetration of biotherapeutics across human cell lines

Batistatou,

Kritzer

2024

Preprint

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“…However, measuring cell penetration of biomolecules is not straightforward. In recent years, chemical biologists have developed a variety of strategies for quantifying passive penetration, total cellular uptake, and cytosolic/nuclear penetration with different detection methods including, but not limited to, fluorescence microscopy, − mass spectrometry, − liquid chromatography–mass spectrometry, fluorescence spectroscopy, − NMR, flow cytometry, − FACS, , qPCR, luminescence, , and ELISA . For cell-based assays, the choice of cell line is critical, and measurements with different methods and/or in different cell lines often produce different results. − In addition, model cell lines such as HeLa are often convenient, but they do not always recapitulate normal cellular physiology.…”

Section: Introductionmentioning

confidence: 99%

Comparing Cell Penetration of Biotherapeutics across Human Cell Lines

Batistatou,

Kritzer

2024

ACS Chem. Biol.

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A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

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Recent advances in methods for quantifying the cell penetration of macromolecules

Batistatou,

Kritzer

2024

Current Opinion in Chemical Biology

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Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2

Cited by 3 publications

References 32 publications

Comparing cell penetration of biotherapeutics across human cell lines

Comparing cell penetration of biotherapeutics across human cell lines

Comparing Cell Penetration of Biotherapeutics across Human Cell Lines

Recent advances in methods for quantifying the cell penetration of macromolecules

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