Detection and Elimination of Senescent Cells with a Self-Assembled Senescence-Associated β-Galactosidase-Activatable Nanophotosensitizer

Abstract

Senescent cells have become an important therapeutic target for many age-related dysfunctions and diseases. We report herein a novel nanophotosensitizing system that is responsive to the senescence-associated β-galactosidase (β-gal) for selective detection and elimination of these cells. It involves a dimeric zinc(II) phthalocyanine linked to a β-galactose unit via a self-immolative linker. This compound can self-assemble in aqueous media, forming stable nanoscale particles in which the phthalocyanine units are stacked and self-quenched for fluorescence emission and singlet oxygen production. Upon internalization into senescent HeLa cells, these nanoparticles interact with the overproduced senescence-associated β-gal inside the cells to trigger the disassembly process through enzymatic cleavage of the glycosidic bonds, followed by self-immolation to release the photoactive monomeric phthalocyanine units. These senescent cells can then be lit up with fluorescence and eliminated through the photodynamic action upon light irradiation with a half-maximal inhibitory concentration of 0.06 μM.

Figure 1

Schematic illustration of the mechanistic action of the senescence-associated β-gal-activatable nanophotosensitizing system for the detection and elimination of senescent cells.

Scheme 1. Synthetic Scheme of Gal-(ZnPc*)₂

Figure 2

(a) TEM image of Gal-(ZnPc*)₂-NP. (b) Hydrodynamic diameter distribution of Gal-(ZnPc*)₂-NP in water. Change in hydrodynamic diameter and PDI (inset) of Gal-(ZnPc*)₂-NP (c) in water over a period of 5 days and (d) in RPMI 1640 medium over a period of 24 h at room temperature.

Figure 3

(a) Electronic absorption and (b) fluorescence (λ_ex = 610 nm) spectra of Gal-(ZnPc*)₂-NP (1 μM) in water, PBS, and DMF, respectively, and ZnPc* (2 μM) in DMF. (c) Rates of consumption of DPBF (initial concentration = 30 μM) sensitized by Gal-(ZnPc*)₂-NP (1 μM) in water, PBS, and DMF, respectively, and ZnPc* (2 μM) in DMF upon light irradiation (λ > 610 nm). Change in fluorescence spectrum of Gal-(ZnPc*)₂-NP (1 μM) in the (d) absence and (e) presence of β-gal (10 unit mL^–1) in PBS with Tween 80 (0.01% v/v) at 37 °C over a period of 30 h. The inset of each figure shows the change in fluorescence intensity at 703 nm with time. (f) Percentage of fluorescence recovery of Gal-(ZnPc*)₂-NP (1 μM) in the absence and presence of β-gal (10 unit mL^–1) in PBS with Tween 80 (0.01% v/v) at 37 °C over a period of 30 h. (g) Change in fluorescence spectrum of Gal-(ZnPc*)₂-NP (1 μM) in PBS with Tween 80 (0.01% v/v) after mixing with different concentrations of β-gal at 37 °C for 30 h. The inset shows the change in fluorescence intensity at 703 nm with a concentration of β-gal from 0 to 0.05 unit mL^–1. (h) Rates of consumption of DPBF (initial concentration = 30 μM) sensitized by Gal-(ZnPc*)₂-NP (1 μM) with and without pretreatment with β-gal (10 unit mL^–1) at 37 °C for 30 h and ZnPc* (2 μM) in PBS with Tween 80 (0.01% v/v) upon light irradiation (λ > 610 nm).

Figure 4

(a) Fluorescence intensities in proliferating and senescent HeLa cells after incubation with Gal-(ZnPc*)₂-NP (2 μM) for 2 h, followed by incubation in the culture medium for different periods of time measured by flow cytometry. Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments. (b) Confocal images of proliferating and senescent HeLa cells after incubation with Gal-(ZnPc*)₂-NP (2 μM) for 2 h and then in the culture medium for a further 2 h. (c) Visualization of the intracellular fluorescence of the activated form of Gal-(ZnPc*)₂-NP and various subcellular trackers in senescent HeLa cells as well as the corresponding fluorescence intensity profiles. (d) Intracellular ROS as shown by the fluorescence of DCF in proliferating and senescent HeLa cells after sequential incubation with Gal-(ZnPc*)₂-NP (0.5 μM) for 2 h, in the culture medium for a further 2 h, and then with H₂DCFDA (10 μM) for 30 min, followed by dark or light (λ > 610 nm, fluence rate = 23 mW cm^–2) treatment for 5 min. (e) Cytotoxicity of Gal-(ZnPc*)₂-NP against proliferating and senescent HeLa cells for both dark and light (λ > 610 nm, fluence rate = 23 mW cm^–2) treatment for 20 min. Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate.