Fig 2: DHA@MIL-101 NRs facilitated DNA and mitochondria damage of Lewis cells. A Schematic illustration of DNA and mitochondria damage by DHA@MIL-101 NRs sourced ROS and LPO. B, C Lewis cells were incubated with DHA, MIL-101 NRs and DHA@MIL-101 NRs respectively. Comet experiments were applied to detect the DNA double-strand breaks (DDSB). The tail moment length was analyzed quantitatively via randomly selected cells. D The expression of p53 and ?-H2A.X, which are biomarkers of DNA damage, was assayed by WB. E The expression of cGAS, STING, which indicates DNA damage response (DDR), was assayed by WB. F The nuclear-translocation of NF-?B, which is direct evidence of NF-?B activation, was assayed by immunofluorescent staining. G Mitochondrial membrane potential was detected using a JC-1 probe with confocal microscopy. H The morphology of mitochondria was observed with TEM. All values are means ± SD (n = 3, * p < 0.05)

Fig 3: PDT induced by Ce6 promoted ROS production and DNA damage in vitro. (A,B) Intracellular ROS generation was detected by the DCFH-DA probe and flow cytometry. Geometric means were used to quantify the MFI. (C) The DNA breakage in macrophages was assayed using the comet assay. (D,E) The activity of DDR characterized by the enhanced expression of p53 and ?-H2A.X was detected by WB. (F,G) The expression of apoptotic proteins Bax and caspase-3 was measured by WB. (H–K) The blots were quantitatively analyzed using the ratio of mean gray. (L) The cellular distribution and expression of ?-H2A.X were analyzed by immunofluorescence staining. Values were means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 4: D-gal induced changes of senescence makers in C6 cells. (A) Western blot detected the expression of p53 and Lamin B1 in C6 cells treated with 0 mM, 28 mM, 111 mM, and 222 mM D-gal for 8 d. (B, C) Quantification of p53 (n=7) and Lamin B1 (n=5) expression as shown in (A). (D) Immunostaining analysis of Lamin B1 (green) in control and senescent C6 cells (treated with 222 mM D-gal for 8 d). (E) qPCR analysis of p16 mRNA level in control cells and senescent C6 cells (treated with 222 mM D-gal for 8 d) (n=3). (F) qPCR analysis of NF-?B mRNA level in control cells and senescent C6 cells (treated with 222 mM D-gal for 8 d) (n=3). (G) Western blot detected the expression of NF-?B and p53 in control cells and senescent C6 cells (treated with 222 mM D-gal for 8 d). (H) Quantification of NF-?B expression as shown in (G) (n=12). Scale bars, 20 µm. Data shown are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: DHA@MIL-101 NRs exhibited significant efficacy in vivo and induced powerful apoptosis, ferroptosis, and DNA damage in tumor grafts. Caudal vein injection of DHA, MIL-101 NRs and DHA@MIL-101 NRs in 200 µL of PBS per mouse. A The mice were sacrificed and tumor grafts were harvested, then photographed. B The weight of tumor grafts was measured. C The tumor growth of Lewis cells-bearing mice was monitored during the whole experiment. D The tumor tissues were prepared for paraffin sections, then labeled with DAPI, Tunel and PI. The apoptosis and necrosis were assayed using confocal microscopy. E, F Expression of GPX4 (biomarkers of ferroptosis) and p53 (biomarkers of DNA damage) was detected with IHC staining. Values are means ± SD (n = 5, * p < 0.05)