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, 11 (1), 446

H 2 S-activatable Near-Infrared Afterglow Luminescent Probes for Sensitive Molecular Imaging in Vivo

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H 2 S-activatable Near-Infrared Afterglow Luminescent Probes for Sensitive Molecular Imaging in Vivo

Luyan Wu et al. Nat Commun.

Abstract

Afterglow luminescent probes with high signal-to-background ratio show promise for in vivo imaging; however, such probes that can be selectively delivered into target sites and switch on afterglow luminescence remain limited. We optimize an organic electrochromic material and integrate it into near-infrared (NIR) photosensitizer (silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) and (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) containing nanoparticles, developing an H2S-activatable NIR afterglow probe (F12+-ANP). F12+-ANP displays a fast reaction rate (1563 ± 141 M-1 s-1) and large afterglow turn-on ratio (~122-fold) toward H2S, enabling high-sensitivity and -specificity measurement of H2S concentration in bloods from healthy persons, hepatic or colorectal cancer patients. We further construct a hepatic-tumor-targeting and H2S-activatable afterglow probe (F12+-ANP-Gal) for noninvasive, real-time imaging of tiny subcutaneous HepG2 tumors (<3 mm in diameter) and orthotopic liver tumors in mice. Strikingly, F12+-ANP-Gal accurately delineates tumor margins in excised hepatic cancer specimens, which may facilitate intraoperative guidance of hepatic cancer surgery.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Schematic illustration of the design of F12+-ANP.
a Optimization of dication EM 12+ into F12+ by introducing two electron-withdrawing pentafluorophenyl groups, and proposed chemical conversion of F12+ into diene F2 upon reduction by H2S. b Preparation of F12+-ANP and proposed mechanism of H2S-mediated fast activation of NIR afterglow luminescence at 780 nm following pre-irradiation with an 808-nm laser. c The detailed photoreaction processes to produce NIR afterglow luminescence within activated F12+-ANP (i.e., F2-ANP). F12+-ANP was designed to contain hydrophobic EM F12+, MEH-PPV, and NIR775 via DSPE-PEG2000-assisted nanoprecipitation. F12+-ANP is initially afterglow “off” owing to the presence of F12+ that can quench both the fluorescence and 1O2 production of MEH-PPV and NIR775 via two efficient FRET processes. Upon reaction with H2S, F12+ was reduced into F2, and the FRET-based quenching processes were eliminated within F2-ANP, resulting in the recovery of fluorescence and 1O2 production capacity. Upon irradiation with the 808-nm laser, NIR775 can sensitize 3O2 to generate 1O2, which then oxidizes the vinylene bond of MEH-PPV to form unstable dioxetane. After cessation of irradiation, the subsequent slow degradation into MEH-PPV-aldehyde and releases photons at 580 nm, followed by efficient intraparticle ET to NIR775, ultimately switching “on” NIR afterglow luminescence at 780 nm.
Fig. 2
Fig. 2. Characterization of F12+-ANP in vitro.
a Comparison of the UV–visible-NIR absorption spectra of F12+-ANP, F12+, NIR775, and MEH-PPV. b DLS and transmission electron microscopy (TEM) image (inset) of F12+-ANP. c Fluorescence and d absorption spectra of F12+-ANP (58/28/2.2 μg mL−1 F12+(BF4)2/MEH-PPV/NIR775) in the absence or presence of NaHS (200 μM, 1 min). Inset: photographs of F12+-ANP before (−) and after (+) incubation with NaHS in PBS buffer. Fluorescence spectra was acquired by synchronous fluorescence scanning (λex = 400‒800 nm, offset = 100 nm). e Afterglow luminescence spectra and images (inset) of F12+-ANP (58/28/2.2 μg mL−1 F12+(BF4)2/MEH-PPV/NIR775) with and without incubation with NaHS (200 μM) in PBS buffer (pH 7.4) at 37 °C for 1 min, followed by irradiation with 808-nm laser (1 W cm−2, 1 min). After cessation of laser, the afterglow images were acquired under an open filter, with an acquisition time of 60 s. f Decay of afterglow luminescence of H2S-activated F12+-ANP in PBS buffer at 37 °C. g Afterglow luminescence images of F12+-ANP (58/28/2.2 μg mL−1 F12+(BF4)2/MEH-PPV/NIR775) upon incubation with varying concentrations of NaHS (0, 1.5, 3, 5, 10, 15, 20, 25, 30, 40, 50, 80, 125, 200, and 250 μM) at 37 °C for 1 min. h Plot of the afterglow luminescence intensity of F12+-ANP and the concentration of NaHS from 0 to 50 μM. i Afterglow luminescence intensities and images (inset) of F12+-ANP upon incubation with different reductants or ROS (200 µM NaHS, 1.25 mM l-cysteine (l-Cys), 10 mM glutathione (GSH), 1 mM homocysteine (Hcy), 1.25 mM ascorbic acid (VC), 1.25 mM dithiothreitol (DTT), 100 μM β-mercaptoethanol (BME), 1 mM H2O2, 1 mM ClO, ONOO (1 mM NaNO2 + 1 mM H2O2), O2. (100 μM xanthine + 22 mU xanthine oxidase)) for 10 min. The solutions were then irradiated with the 808-nm laser (1 W cm−2, 1 min), and the NIR afterglow images were collected for 60 s with a 790 nm filter after the end of irradiation. Data denote mean ± standard deviation (s.d.) (n = 3). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Preparation of F12+-ANP-Gal for imaging of H2S in cells.
a Schematic for the preparation of F12+-ANP-Gal. b Fluorescence imaging of HepG2 cells after incubation with F12+-ANP, F12+-ANP-Gal, F12+-ANP-Gal plus 20 mM free β-Gal, or A549 cells incubated with F12+-ANP-Gal for 3 h. Scale bar: 20 μm. c Afterglow luminescence images and intensities (d) of cells (~1 × 105 cells) treated with indicated conditions in b. e Fluorescence imaging of HepG2 cells incubated with F12+-ANP-Gal, F12+-ANP-Gal together with 300 µM ZnCl2, 1 mM NaHS, 200 μM l-Cys, or 200 μM l-Cys plus 50 mg L−1 PAG and 20 μM AOAA. Scale bar: 20 μm. f Afterglow luminescence images and intensities g of HepG2 cells (~3 × 104 cells) treated with indicated conditions in e. All the cells were incubated with F12+-ANP or F12+-ANP-Gal at a concentration of 58/28/2.2 μg mL−1 F12+(BF4)2/MEH-PPV/NIR775 for 3 h. For afterglow luminescence imaging, the cell pellets were irradiated with the 808-nm laser (1 W cm−2) for 1 min. After cessation of laser, the afterglow images were acquired for 60 s with an open filter. Data denote mean ± s.d. (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3). Statistical differences were analyzed by Student’s t test. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Afterglow imaging of H2S in s.c. HepG2 tumors.
a Schematic for noninvasive fluorescence and afterglow imaging of H2S in HepG2 tumor-bearing mice. b Afterglow (left) and fluorescence (right) imaging of HepG2 tumors in mice at 0, 4, 8, 12, and 24 h following i.v. injection of F12+-ANP-Gal or F12+-ANP in saline (saline), F12+-ANP-Gal with i.t. injection of l-Cys or ZnCl2. l-Cys (1 mM, 25 µL) or ZnCl2 (1 mM, 25 µL) was injected into tumors at 3.5 h post i.v. injection of F12+-ANP-Gal (211/100/8 μg F12+(BF4)2/MEH-PPV/NIR775, 200 μL). c Quantification of SBRs for afterglow (solid) and fluorescence (dash) imaging of HepG2 tumors in mice treated with F12+-ANP-Gal or F12+-ANP alone, F12+-ANP-Gal plus l-Cys, or ZnCl2 at indicated time point. d Afterglow (up) and fluorescence (down) imaging of HepG2 tumors at size of ~12, ~45, and ~100 mm3 in mice at 12 h post i.v. injection of F12+-ANP-Gal. e Quantification of afterglow and fluorescence intensities of HepG2 tumors at different size. f Plots of the SBRs for afterglow and fluorescence imaging versus the tumor size revealed a strong correlation for F12+-ANP-Gal (Person’s r = 0.99). For afterglow imaging, the mouse body was irradiated with the 808-nm laser (1 W cm−2) for 1 min. After cessation of laser, the afterglow images were acquired for 60 s with an open filter. The fluorescence images were acquired with λex/em = 740/790 nm. Red arrows indicate the locations of HepG2 tumors in mice, and black circles indicate the background locations. Data denote mean ± s.d. (*P < 0.05, **P < 0.01, n = 3). Statistical differences were analyzed by Student’s t test. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Noninvasive imaging of orthotopic liver tumors in mice.
a Schematic for afterglow imaging of orthotopic HepG2 tumors in living mice. b Bioluminescence (BL), fluorescence (FL), and afterglow imaging of control mice (Control) and orthotopic HepG2 tumor (liver tumor) bearing mice at 12 h post i.v. injection of F12+-ANP-Gal (211/100/8 μg F12+(BF4)2/MEH-PPV/NIR775, 200 μL). c Afterglow (red) and FL (blue) intensities in the livers of control mice and orthotopic liver tumor-bearing mice. d Comparison of the SBRs for afterglow and fluorescence imaging of livers in control mice and orthotopic liver tumor-bearing mice. Red arrows indicate the locations of livers, and black circles indicate the background locations. e Representative ex vivo afterglow images of main organs (e.g., liver (Li), lung (Lu), heart (He), kidneys (Ki), intestines (In), stomach (St), spleen (Sp), and tumor (Tu)) resected from control mice (left) and orthotopic HepG2 tumor-bearing mice (right) at 12 h post i.v. injection of F12+-ANP-Gal. Red arrow and yellow dash box indicate the locations of HepG2 tumor in the liver and normal liver tissues chosen for region of interest (ROI), respectively. f Comparison of the average afterglow intensities of tumors and main organs resected from control (blue) and orthotopic liver tumor (red) mice. g WB analysis shows the relative CBS and CSE protein levels in the liver tissues resected from control mice and HepG2 tumor tissues resected from orthotopic liver tumor mice. All afterglow luminescence images were acquired for 60 s with an open filter, after pre-irradiation of mouse body or organs with the 808-nm laser (1 W cm−2, 1 min). All fluorescence images were acquired with λex/em = 740/790 nm. Data denote mean ± s.d. (*P < 0.05, **P < 0.01, n = 3). Statistical differences were analyzed by Student’s t test. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Detection of H2S in blood samples.
a Afterglow images of F12+-ANP in blood samples freshly collected from 10 healthy persons, 10 HCC patients, and 10 CRC patients. Freshly collected bloods were 2-fold diluted with PBS buffer (1×, pH 7.4), and then incubated with F12+-ANP (58/28/2.2 μg mL−1 F12+(BF4)2/MEH-PPV/NIR775) at 37 °C for 1 min, followed by irradiation with the 808-nm laser (1 W cm−2) for 1 min. After removal of the laser, the afterglow luminescence images were immediately acquired for 60 s with an 790 nm filter. b Afterglow luminescence intensities of F12+-ANP in 2-fold diluted blood samples. c Quantification of H2S concentrations in whole blood of healthy persons and HCC or CRC patients. Data denote mean ± s.d. (***P < 0.001, n = 10). Statistical differences were analyzed by Student’s t test. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Afterglow imaging of liver tumor tissues in HCC specimens.
a Schematic for afterglow imaging of tumor tissues in clinically excised liver specimens using F12+-ANP-Gal. b Representative photograph (bright field), afterglow, and FL images of the liver specimen resected from an HCC patient. The specimen was incubated with F12+-ANP-Gal (58/28/2.2 μg mL−1 F12+(BF4)2/MEH-PPV/NIR775) in PBS buffer (1×, pH 7.4) at 37 °C for 3 h, and then rinsed with PBS buffer for three times. The whole specimen was irradiated with the 808-nm laser (1 W cm−2, 1 min). After cessation of the laser, the afterglow image was acquired under an open filter, with an acquisition time of 60 s. The fluorescence image was collected with λex/em = 740/790 nm. c Fluorescence images of liver tissue slices were dissected from the HCC specimen after incubation with F12+-ANP-Gal (green) for 3 h and stained with DAPI (blue). d H&E staining of the liver tissue slice dissected from the HCC specimen. Black dash boxes indicate the enlarged areas, in which box ROI 1 shows the tumor tissue and box ROI 2 indicates the normal liver tissue, respectively. e Quantitative analysis of the average SBRs for afterglow and fluorescence imaging of liver specimens resected from HCC patients. Data denote mean ± s.d. (n = 4). Black dash boxes in b indicate the location of tumors, and green dash boxes indicate the location of normal liver tissues selected as the background. Source data are provided as a Source Data file.

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