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Photoactivatable senolysis with single-cell resolution delays aging

Abstract

Strategies that can selectively eliminate senescent cells (SnCs), namely senolytics, have been shown to promote healthy lifespan. However, it is challenging to achieve precise, broad-spectrum and tractable senolysis. Here, we integrate multiple technologies that combine the enzyme substrate of senescence-associated β-galactosidase (SA-β-gal) with fluorescence tag for the precise tracking of SnCs, construction of a bioorthogonal receptor triggered by SA-β-gal to target and anchor SnCs with single-cell resolution and incorporation of a selenium atom to generate singlet oxygen and achieve precise senolysis through controllable photodynamic therapy (PDT). We generate KSL0608-Se, a photosensitive senolytic prodrug, which is selectively activated by SA-β-gal. In naturally-aged mice, KSL0608-Se-mediated PDT prevented upregulation of age-related SnCs markers and senescence-associated secretory phenotype factors. This treatment also countered age-induced losses in liver and renal function and inhibited the age-associated physical dysfunction in mice. We therefore provide a strategy to monitor and selectively eliminate SnCs to regulate aging.

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Fig. 1: Integrated design strategy.
Fig. 2: Photochemical properties and β-gal-triggered protein labeling.
Fig. 3: Cell imaging and intracellular localization of KSL0608-O and KSL0608-Se.
Fig. 4: Photo-induced ROS generation and PDT effect in vitro.
Fig. 5: Fluorescence imaging using KSL0608-O in vivo.
Fig. 6: Selective removal of SnCs in doxo-treated mice.
Fig. 7: Selective removal of SnCs in naturally aged mice.
Fig. 8: The expression of SASP factors, evaluation of physical function in mice and RNA sequencing analysis.

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Data availability

All data during the current study are available within the paper and its Supplementary Information or from the corresponding author upon reasonable request.

Code availability

Source code used for RNA-sequencing analysis can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE186522. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213846.

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Acknowledgements

We gratefully appreciate the financial support from the National Natural Science Foundation of China (grants 22037002 to J.L. and Y. Guo, 32121005 to J.L., 21977082 to Y. Guo and 22007032 to Xinming Li), the Natural Science Basic Research Program of Shaanxi (grant 2020JC-38 to Y. Guo), the Innovation Program of Shanghai Municipal Education Commission (grant 2021-01-07-00-02-E00104 to J.L.), the Shanghai Frontier Science Research Base of Optogenetic Techniques for Cell Metabolism (grant 2021 Sci & Tech 03-28 to J.L.), the Innovative Research Team of High-level Local Universities in Shanghai (grant SHSMU-ZDCX20212702 to J.L.) and the Chinese Special Fund for State Key Laboratory of Bioreactor Engineering (2060204 to J.L.). T.D.J. wishes to thank the Royal Society for a Wolfson Research Merit Award and the Open Research Fund of the School of Chemistry and Chemical Engineering, Henan Normal University for support (Grant 2020ZD01 to T.D.J.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Y. Guo and J.L. conceived and designed the project. Y. Guo, J.L., D.S., Y. Gao and T.D.J. wrote and revised the manuscript. D.S. and Xinming Li performed the synthetic work. D.S. and W.L. performed and analyzed the experiments. Y.H. performed modeling assay. Xiaokang Li assisted with data analysis.

Corresponding authors

Correspondence to Yuan Guo or Jian Li.

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Extended data

Extended Data Fig. 1 Optical properties of KSL0608-O and KSL0608-Se.

a,b, The normalized fluorescence intensity of KSL0608-O (10 µM, a) and KSL0608-Se (10 µM, b) after the addition of different analytes, including E. coli β-gal (10 U/mL), E. coli β-gal (10 U/mL) + BSA (1 mg/mL), E. coli β-gal (10 U/mL) + L-Cys (1 mg/mL), E. coli β-gal (10 U/mL) + GSH (1 mg/mL), E. coli β-gal (10 U/mL) + NAC (1 mg/mL), E. coli β-gal (10 U/mL) + H2S (25 µM). c,d, Time kinetic curves of KSL0608-O (10 µM, c) and KSL0608-Se (10 µM, d) after the addition of E. coli β-gal (10 U/mL) in PBS solution containing 1 mg/mL BSA. e,f, pH-dependent fluorescence intensity of KSL0608-O (10 µM, e) and KSL0608-Se (10 µM, f) before and after the addition of E. coli β-gal (10 U/mL) in PBS solution containing 1 mg/mL BSA. g,h, Time-dependent fluorescence intensity of KSL0608-O (10 µM, g) and KSL0608-Se (10 µM, h) before and after the addition of E. coli β-gal (10 U/mL) in PBS solution containing 1 mg/mL BSA. Error bars (a and b) represent mean values (± S.D.). n = 3 independent samples.

Source data

Extended Data Fig. 2 Fluorescence imaging in live cells.

a, Confocal imaging of SKOV3 cells and HepG2 cells incubated with KSL0608-O (10 μM) for 30 min, respectively. b, Confocal imaging of SKOV3 cells and HepG2 cells incubated with KSL0608-Se (10 μM) for 30 min, respectively. c, Confocal imaging of SKOV3 cells treated without or with D-galactose (1 mM) or PETG (1 µM) for 2 h and then incubated with KSL0608-O (10 μM) for 30 min. d, Confocal imaging of SKOV3 cells treated without or with D-galactose (1 mM) or PETG (1 µM) for 2 h and then incubated with KSL0608-Se (10 μM) for 30 min. Cells were incubated with Hoechst (1 μM) at 37 °C for 10 min. Blue channel: λexem = 405/420−470 nm. NIR channel (KSL0608-O): λexem = 561/600−700 nm. NIR channel (KSL0608-Se): λexem = 561/650−750 nm. Scale bar: 50 μm.

Extended Data Fig. 3 The cytotoxicity of light dose and KSL0608-Se-mediated PDT efficacy in vitro.

a-d, Young and MitoC-induced senescent A549 cells (a), young and ROS-induced senescent NRK-52E cells (b), young and doxo-induced senescent HL-7702 cells (c), MRC-5 cells (P28) and MRC-5 cells (P40) (d) separately treated with irradiation for different times (0-20 min). Then, these cells were further cultured for 24 h and the cytotoxicity of light dose to these cells was measured by a CCK-8 assay. e-h, Young and MitoC-induced senescent A549 cells (e), young and ROS-induced senescent NRK-52E cells (f), young and doxo-induced senescent HL-7702 cells (g), MRC-5 cells (P28) and MRC-5 cells (P40) (h) separately incubated with different concentrations of KSL0608-Se for 30 min, then treated with the irradiation for 20 min. These cells were further cultured for 48 h and the cytotoxicity of KSL0608-Se-mediated PDT to these cells was measured by a CCK-8 assay. i-l, Young and MitoC-induced senescent A549 cells (i), young and ROS-induced senescent NRK-52E cells (j), young and doxo-induced senescent HL-7702 cells (k), MRC-5 cells (P28) and MRC-5 cells (P40) (l) separately incubated with different concentrations of KSL0608-Se for 30 min, then treated with the irradiation for 20 min. These cells were further cultured for 72 h and the cytotoxicity of KSL0608-Se-mediated PDT to these cells was measured by a CCK-8 assay. Error bars represent mean values (± S.D.). n = 3 independent samples.

Source data

Extended Data Fig. 4 Dark toxicity of KSL0608-Se in vitro.

a-d, Young and MitoC-induced senescent A549 cells (a), young and ROS-induced senescent NRK-52E cells (b), young and doxo-induced senescent HL-7702 cells (c), MRC-5 cells (P28) and MRC-5 cells (P40) (d) separately incubated with different concentrations of KSL0608-Se. These cells were further cultured for 24 h and the cytotoxicity of KSL0608-Se to these cells was measured by a CCK-8 assay. e-h, Young and MitoC-induced senescent A549 cells (e), young and ROS-induced senescent NRK-52E cells (f), young and doxo-induced senescent HL-7702 cells (g), MRC-5 cells (P28) and MRC-5 cells (P40) (h) separately incubated with different concentrations of KSL0608-Se. These cells were further cultured for 48 h and the cytotoxicity of KSL0608-Se to these cells was measured by a CCK-8 assay. i-l, Young and MitoC-induced senescent A549 cells (i), young and ROS-induced senescent NRK-52E cells (j), young and doxo-induced senescent HL-7702 cells (k), MRC-5 cells (P28) and MRC-5 cells (P40) (l) separately incubated with different concentrations of KSL0608-Se. These cells were further cultured for 72 h and the cytotoxicity of KSL0608-Se to these cells was measured by a CCK-8 assay. Error bars represent mean values (± S.D.). n = 3 independent samples.

Source data

Extended Data Fig. 5 Fluorescence imaging of cells in co-culture system.

a, Schematic of workflow for co-culturing young and senescent cells. Young cells were pre-labeled by CellTracker Green (5 μM). b, The fluorescent image of co-cultured cells (young HL-7702 cells and doxo-induced senescent HL-7702 cells). Green channel: λexem = 488/510 − 530 nm. Scale bar: 100 μm. c, The fluorescent image of co-cultured cells incubated with KSL0608-O (10 μM). d, The relative fluorescence intensity in NIR channel of young HL-7702 cells, doxo-induced senescent HL-7702 cells, MRC-5 cells (P28) and senescent MRC-5 cells (P40). HL-7702, n = 21; MRC-5, n = 10. e, The fluorescent image of co-cultured cells incubated with KSL0608-Se (10 μM). f, The relative fluorescence intensity in NIR channel of young HL-7702 cells, doxo-induced senescent HL-7702 cells, MRC-5 cells (P28) and senescent MRC-5 cells (P40). HL-7702, n = 19; MRC-5, n = 16. Young HL7702 cells and MRC-5 cells (P28) were pre-labeled by CellTracker Green (5 μM) and all cells were pre-stained by Hoechst (1 μM). Blue channel: λexem = 405/420−470 nm. Green channel: λexem = 488/510−530 nm. NIR channel (KSL0608-O): λexem = 561/600−700 nm. NIR channel (KSL0608-Se): λexem = 561/650−750 nm. Scale bar: 50 μm. Error bars (d and f) represent mean values (± S.D.). ‘n’ stands for the number of images and the images in each group from three biological replicates. Significant differences were obtained by analysis with two-sided Student’s t-test.

Source data

Extended Data Fig. 6 Expression levels of SASP factors and p16.

a-g, The expression of CXCL1 (a), CXCL3 (b), IL-1β (c), IL-6 (d), MMP-1 (e), MMP-7 (f) and TNF-α (g) in kidneys from the mice in different groups (young control, aged control, KSL0608-Se, KSL0608-Se + irradiation; n = 5 for each group). h-n, The expression of CXCL1 (h), CXCL3 (i), IL-1β (j), IL-6 (k), MMP-1 (l), MMP-7 (m) and TNF-α (n) in livers from the mice in different groups (young control, aged control, KSL0608-Se, KSL0608-Se + irradiation; n = 5 for each group). o, The expression level of cell cycle regulators p16 in serum from the mice in different groups (young control, n = 12; aged control, n = 9; KSL0608-Se, n = 11; KSL0608-Se + irradiation, n = 11). Error bars represent mean values (± S.D.). ‘n’ stands for the number of mice. Significant differences (ns, not significant) were obtained by analysis with one-way ANOVA followed by Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 7 RNA sequencing of mice.

a, GO annotations analysis of 146 common differentially expressed genes between the two comparisons (aged control vs. young control and KSL0608-Se + irradiation vs. aged control). b, The heatmap of the expression of 146 common differently expressed genes in livers of mice in different groups. c-l, The normalized expression of 146 differently expressed genes in mice of different groups. n = 5 for each group, ‘n’ stands for the number of mice.

Source data

Supplementary information

Supplementary Information

Supplementary materials and instruments; Supplementary Table 1; Supplementary Figs. 1-11; synthesis and characterization; Supplementary references; Unprocessed western blots

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Supplementary Video 1

Fluorescence-guided photoactivatable senolysis with single-cell resolution.

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Numerical source data for Fig. 2.

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Unprocessed gels for Fig. 2c.

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Unprocessed western blots for Fig. 6b.

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Unprocessed western blots for Fig. 7b.

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Numerical source data for Fig. 8.

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Shi, D., Liu, W., Gao, Y. et al. Photoactivatable senolysis with single-cell resolution delays aging. Nat Aging 3, 297–312 (2023). https://doi.org/10.1038/s43587-023-00360-x

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