Autophagy-Activated Self-reporting Photosensitizer Promoting Cell Mortality in Cancer Starvation Therapy

Abstract

Cancer starvation therapy have received continuous attention as an efficient method to fight against wide-spectrum cancer. However, during cancer starvation therapy, the protective autophagy promotes cancer cells survival, compromising the therapeutic effect. Herein, a novel strategy by combination of autophagy-activated fluorescent photosensitizers (PSs) and cancer starvation therapy to realize the controllable and efficient ablation of tumor is conceived. Two dual-emissive self-reporting aggregation-induced emission luminogens (AIEgens), TPAQ and TPAP, with autophagy-activated reactive oxygen species (ROS) generation are prepared to fight against the protective autophagy in cancer starvation therapy. When protective autophagy occurs, a portion of TPAQ and TPAP will translocate from lipid droplets to acidic lysosomes with significant redshift in fluorescence emission and enhanced ROS generation ability. The accumulation of ROS induced by TPAQ-H and TPAP-H causes lysosomal membrane permeabilization (LMP), which further results in cell apoptosis and promotes cell death. In addition, TPAQ and TPAP can enable the real-time self-reporting to cell autophagy and cell death process by observing the change of red-emissive fluorescence signals. Particularly, the efficient ablation of tumor via the combination of cancer starvation therapy and photodynamic therapy (PDT) induced by TPAQ has been successfully confirmed in 3D tumor spheroid chip, suggesting the validation of this strategy.

Keywords: 3D tumor spheroid chip; autophagy-activated photosensitizer; cancer starvation therapy; dual-emissive self-reporting AIEgen; photodynamic therapy.

Scheme 1

A) Chemical structures of TPAQ and TPAP in normal and acidic conditions. B) The schematic illustration of the effect mechanism of TPAQ and TPAP on A549 cells under normal and starving state.

Figure 1

Normalized absorption a) and FL b) spectra of TPAQ (A) and TPAP (B) in different solvents; FL spectra c) of TPAQ (A) and TPAP (B) in EtOH and EtOH/H₂O mixtures with different H₂O fractions (f _H2O); d) Changes in the FL peak intensities (I) of the solutions of TPAQ (A) and TPAP (B) with the H₂O contents in the EtOH/H₂O mixtures. I ₀ is the intensity in pure EtOH. Concentration: 10 µM.

Figure 2

a) Absorption spectra of TPAQ (A) and TPAP (B) in EtOH/PBS (v:v = 1:1) solvents at different pH values; b) Plots of the absorbance of TPAQ (A) and TPAP (B) at different pH values and the fitted curve; c) FL spectra of TPAQ (A) and TPAP (B) excited at 400 and 380 nm, respectively; d) FL spectra of TPAQ (A) and TPAP (B) excited at 500 and 450 nm, respectively. Concentration: 10 µM.

Figure 3

Confocal laser scanning microscopy (CLSM) images of live A549 cells stained with 2 µM TPAQ and TPAP under normal and starving conditions, respectively A), and under starving and CQ‐treated starving conditions, respectively B). Blue channel: λ _ex = 405 nm, λ _em = 410–480 nm; Red channel: λ _ex = 488 nm, λ _em = 600–700 nm. Scale bar = 20 µm.

Figure 4

A) Optimized geometries and the frontier orbitals of TPAQ, TPAQ‐H, TPAP, and TPAP‐H; B) Diagram of energy level of singlet and triplet states of TPAQ, TPAQ‐H, TPAP, and TPAP‐H based on optimized singlet geometries; C) Changes of FL intensity at 535 nm of DCF‐DA in the presence or absence of TPAQ/TPAP in PBS with pH = 4.8 or pH = 7.4 under light irradiation for different time points; D) CLSM images of live A549 cells under starvation treated with “DCF‐DA + TPAQ/TPAP”, “DCF‐DA”, “DCF‐DA + chloroquine + TPAQ/TPAP”, “DCF‐DA + NAC + TPAQ/TPAP” with light irradiation for 5 min. Scale bar = 20 µm.

Figure 5

A) CLSM images of live A549 cells pre‐stained with 20 µM TPAQ and TPAP, respectively, and then treated with Alexa Fluor 647‐Dextran in “normal”, “starvation”, “starvation + light” conditions (a) and the relevant mean FL intensity of Alexa Fluor 647‐Dextran in a (b); for Alexa Fluor 647‐Dextran, λ _ex = 640 nm, λ _em = 650–720 nm; B) CLSM images of live A549 cells stained with TPAQ and TPAP, respectively, in “normal”, “starvation”, “starvation + light”, and “fixed” conditions; Blue channel: λ _ex = 405 nm, λ _em = 410–480 nm; Red channel: λ _ex = 488 nm, λ _em = 600–700 nm. Scale bar = 20 µm.

Figure 6

A) TUNEL staining of fixed A549 cells pre‐stained with 20 µM TPAQ and TPAP, respectively, in “normal”, “starvation”, and “starvation + light” conditions (a) and statistics of TUNEL positive cells (b) in relevant conditions in a; TUNEL: λ _ex = 488 nm, λ _em = 500–600 nm, DAPI: λ _ex = 405 nm, λ _em = 420–480 nm, Scale bar = 50 µm; B) Cell viability of A549 cells stained with 20 µM TPAQ and TPAP in “normal”, “starvation”, and “starvation + light” conditions. Power density: 50 mW cm⁻². Bar graphs are presented as mean ± S.E. ^*P<0.05, ^**P<0.01 versus control group, ^###P<0.001 versus no‐light group in unpaired two‐tailed t test.

Figure 7

A) The physical object and schematic diagram of the tumor spheroid chip; B) CLSM images of A549 tumor spheres stained with 20 µM TPAQ in starving condition (a); bright field pictures of tumor spheres pre‐stained with 20 µM TPAQ in chip without or with white light irradiation in starving condition (b) and the statistics of the size of tumor spheres in b (c). Scale bar = 50 µm. Bar graphs are presented as mean ± S.E. ^***P< 0.001 versus no‐light group in unpaired two‐tailed t test.