Synthesis of a versatile mitochondria-targeting small molecule for cancer near-infrared fluorescent imaging and radio/photodynamic/photothermal synergistic therapies

Abstract

Although as a mainstay modal for cancer treatment, the clinical effect of radiotherapy (RT) does not yet meet the need of cancer patients. Developing tumour-preferential radiosensitizers or combining RT with other treatments has been acknowledged highly necessary to enhance the efficacy of RT. The present study reported a multifunctional bioactive small-molecule (designated as IR-83) simultaneously exhibiting tumour-preferential accumulation, near-infrared imaging and radio/photodynamic/photothermal therapeutic effects. IR-83 was designed and synthesized by introducing 2-nitroimidazole as a radiosensitizer into the framework of heptamethine cyanine dyes inherently with tumour-targeting and photosensitizing effects. As results, IR-83 preferentially accumulated in tumours, suppressed tumour growth and metastasis by integrating radio/photodynamic/photothermal multimodal therapies. Mechanism studies showed that IR-83 accumulated in cancer cell mitochondria, induced excessive reactive oxygen species (ROS), and generated high heat after laser irradiation. On one hand, these phenomena led to mitochondrial dysfunction and a sharp decline in oxidative phosphorylation to lessen tissue oxygen consumption. On the other hand, excessive ROS in mitochondria destroyed the balance of antioxidants and oxidative stress balance by down-regulating the intracellular antioxidant system, and subsequently sensitized ionizing radiation-generated irreversible DNA double-strand breaks. Therefore, this study presented a promising radiosensitizer and a new alternative strategy to enhance RT efficacy via mitochondria-targeting multimodal synergistic treatment.

Keywords: ALT, alanine aminotransferase; AST, aspartate amino transferase; CLSM, confocal laser scanning microscope; CREA, creatinine; DSBs, DNA double-strand breaks; GSH, glutathione; H&E, hematoxylin and eosin; HO-1, heme oxygenase 1; Heptamethine cyanine dyes; LLC, Lewis lung carcinoma; MMP, mitochondrial membrane potential; NADPH, nicotinamide adenine dinucleotide phosphate; NIR, near-infrared; NMR, nuclear magnetic resonance; NSCLC, non-small cell lung cancer; Near-infrared imaging; Nrf2, nuclear factor erythroid-derived 2-like 2; OXPHOS, oxidative phosphorylation; PBS, phosphate-buffered saline; PDT, photodynamic therapy; PI, propidium iodide; PLT, Platelet; PSs, photosensitizers; PTAs, photothermal agents; PTT, photothermal therapy; Phototherapy; RBC, red blood cell; ROS, reactive oxygen species; RT, radiotherapy; Radiosensitizer; Radiotherapy; SER, sensitizer enhancement ratio; SOSG, Singlet oxygen sensor green; WBC, white blood cell.

Graphical abstract

Scheme 1

Structure and synthetic route of IR-83.

Fig. 1

In vivo and ex vivo fluorescence imaging properties of IR-83. Real-time confocal fluorescence images of LLC cells after treatment with (A) IR-83 (2.5 ​μM) for different durations or B) incubated at normoxic (21% O₂) or hypoxic (5% O₂) condition. (C) In vivo fluorescence images of tumour-bearing mice taken at 24 ​h after -intravenous injection of IR-83 (0.25 ​mg/kg). (D). Quantitation of fluorescence intensities of the tumour areas after IR-83 intravenous injection (n ​= ​3). (E) Ex vivo fluorescence images of major organs and tumours dissected from LLC tumour-bearing mice at 24 ​h after intravenous injection of IR-83. (F) Semi-quantitative analysis of the average fluorescence intensities from excised tumour and vital organs (heart, liver, spleen, lung, and kidney) at 24 ​h post-IR-83 injection. The fluorescence intensity of intestine was served as reference to calculate relative fluorescence intensity of other organs.

Fig. 2

PDT and PTT effects of IR-83 in vitro and in vivo. (A) Relative cell viability of LLC cells after incubation with gradient concentrations of IR-83 for 24 ​h in the absence or presence of 808 ​nm NIR laser irradiation (2 ​W/cm², 5 ​min). (B) Live/dead staining images of LLC cells after incubation with PBS or IR-83 in the absence or presence of 808 ​nm laser. Live and dead cells were labelled with Calcein-AM (green) and propidium iodide (red), respectively. (C). Quantification of the generation of singlet oxygen in PBS or IR-83 solution (2.5 ​μM) post-exposure to NIR laser (2 ​W/cm²) for 5 ​min. (D) In vitro IR thermal images were recorded at different intervals in PBS (upper row) and IR-83 (2.5 ​μM) (lower row) groups under the presence of 808 ​nm laser (2 ​W/cm2). (E) Real-time IR thermal images of tumour-bearing mice post-intravenous injection of PBS or IR-83 (2.5 ​mg/kg) for 24 ​h under 808 ​nm laser (0.8 ​W/cm²) irradiation. (F) Temperature cures of tumour surface in PBS or IR-83-injected mice upon continuous exposure to 808 ​nm laser.

Fig. 3

In vitro radio-sensitization effect of IR-83 combined with phototherapy. (A) Fluorescence images of proliferative cells (red) after various treatments. Scale bar: 100 ​μm. (B) Representative colony formation images of LLC cells after receiving the indicated treatment. (C) Survival fractions of LLC cells in X-ray alone group or IR-83 plus NIR irradiation combined with the X-ray group after exposure to a range of radiation doses (0–8 ​Gy). (D) Flow cytometric analysis of the cell apoptosis of LLC cells after various treatments. (E) Representative images of comet phenomenon in LLC cells following various treatments. Scale bar: 75 ​μm. (F) The representative fluorescence images of γ-H2AX staining after various treatments. Blue and pink colors represented DAPI-stained nuclei and γ-H2AX foci, respectively. Scale bar: 75 ​μm.

Fig. 4

Effect of combined treatment on mitochondria morphology and function. (A) Confocal fluorescence images of LLC cells incubated with IR-83 (2.5 ​μM) for 4 ​h. Blue, green, and red colors represent DAPI-stained cell nuclei, Mitotracker, and IR-83 fluorescence, respectively. (B) CLSM images of mitochondria morphology in each group after labelling with mitochondria-specific probe (red). (C) TEM images of mitochondria in LLC cells after various treatments.

Fig. 5

Effect of combined treatment on Nrf2-mediated antioxidant system. (A) Generation of ROS in LLC cells after different treatments as indicated determined by DCFH-DA staining under the confocal microscope. (B) Relative GSH level and NADP⁺/NADPH ratio C) in LLC cells after receiving various treatments as indicated. (D) Western blotting analysis of Nrf2 and HO-1 protein expression in LLC cells after receiving various treatments for 24 ​h. (E) Semi-quantification expression levels of Nrf2 and HO-1 in cells. GAPDH and β-tubulin were selected as internal controls for Nrf2 and HO-1, respectively. “∗” Significantly different from control group; “#” Significantly different from X-ray alone group; ∗P ​< ​0.05, ∗∗P ​< ​0.01, #P ​< ​0.05, ##P ​< ​0.01.

Fig. 6

Anti-tumour activity of combined treatment in vivo. (A) Schematic illustration of the schedule for antitumor studies in LLC tumour-bearing mice. (B) Photographs of tumour tissues dissected from each group on day 21. (C) Tumour volume curves of different groups of mice after receiving the indicated treatments in 12 days (n ​= ​5 per group). (D) Tumour weight of mice over 12 days after different treatments (n ​= ​5 per group). (E) Probability of free survival of tumour-bearing mice in each group after receiving various treatments as indicated. (F) Representative images of lung tissues collected from different groups of mice. Black arrow indicated lung metastasis nodes. (G) Representative micrographs of H&E-stained lung sections were collected from different groups of mice. Scale bar: 20 ​μm. Error bars represent mean ​± ​S.D. “∗” Significantly different from vehicle group; “#” Significantly different from X-ray alone group; ∗P ​< ​0.05, ∗∗P ​< ​0.01, ##P ​< ​0.01.