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An ultrasensitive near-infrared ratiometric fluorescent probe for imaging mitochondrial polarity in live cells and in vivo.

ABSTRACT: Mitochondrial polarity is a crucial characteristic of these indispensable organelles, and tremendously impacts cellular events. Herein, we describe a new mitochondria-targeting fluorescent probe MCY-BF2 that is singularly sensitive and specifically responsive to mitochondrial polarity. The pull-push system in the conjugated structure of MCY-BF2 is responsible for the polarity-ultrasensitivity due to the excited state intramolecular charge transfer (ICT). By combining with cardiolipin, MCY-BF2 preferentially accumulates in mitochondria. Because the fluorescence emission wavelengths exhibit an obvious red-shift with increasing media polarity, the fluorescence intensity ratio at two different wavelengths versus the solvent dielectric constant can quantify the mitochondrial polarity. Experimental results demonstrate that the fluorescent intensity of MCY-BF2 in a non-polar solvent, dioxane, is 120 times higher than that in a polar solvent, dimethyl sulfoxide. As the first near-infrared (NIR) and most sensitive fluorescent imaging probe for polarity, MCY-BF2 can locate exclusively in mitochondria in various cells and discriminate polarity differences between normal and cancer cells. Also, the intrinsic polarity variance at different developmental stages in Caenorhabditis elegans (C. elegans) was reported here for the first time. Interestingly, the embryonic development stage has a more non-polar environment with a dielectric constant of 7.20, and in contrast the polarity at the young adult stage changes to 10.07. In addition, in vivo imaging results suggest that the tumor tissues of mice have an obviously lower polarity than that in normal tissues. Altogether, the merits of the NIR property, high sensitivity and moderate Stokes shift all greatly promote the accuracy of imaging. This probe will be a promising tool for studying biological processes and the pathological mechanism of polarity-related diseases.

SUBMITTER: Xiao H

PROVIDER: S-EPMC5964966 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeCurrent checkpoint inhibitor immunotherapy strategies in glioblastoma are challenged by mechanisms of resistance including an immunosuppressive tumor microenvironment. T cell immunoglobulin domain and mucin domain 3 (TIM3) is a late-phase checkpoint receptor traditionally associated with T cell exhaustion. We apply fluorescent imaging techniques to explore feasibility of in vivo visualization of the immune state in a glioblastoma mouse model.ProceduresTIM3 monoclonal antibody was conjugated to a near-infrared fluorescent dye, IRDye-800CW (800CW). The TIM3 experimental conjugate and isotype control were assessed for specificity with immunofluorescent staining and flow cytometry in murine cell lines (GL261 glioma and RAW264.7 macrophages). C57BL/6 mice with orthotopically implanted GL261 cells were imaged in vivo over 4 days after intravenous TIM3-800CW injection to assess tumor-specific uptake. Cell-specific uptake was then assessed on histologic sections.ResultsThe experimental TIM3-800CW, but not its isotype control, bound to RAW264.7 macrophages in vitro. Specificity to RAW264.7 macrophages and not GL261 tumor cells was quantitatively confirmed with the corresponding clone of TIM3 on flow cytometry. In vivo fluorescence imaging of the 800CW signal was localized to the intracranial tumor and significantly higher for the TIM3-800CW cohort, relative to non-targeting isotype control, immediately after tail vein injection and for up to 48 h after injection. Resected organs of tumor bearing mice showed significantly higher uptake in the liver and spleen. TIM3-800CW was seen to co-stain with CD3 (13%), CD11b (29%), and CD206 (26%).ConclusionsWe propose fluorescent imaging of immune cell imaging as a potential strategy for monitoring and localizing immunologically relevant foci in the setting of brain tumors. Alternative markers and target validation will further clarify the temporal relationship of immunosuppressive effector cells throughout glioma resistance.

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An ultrasensitive near-infrared ratiometric fluorescent probe for imaging mitochondrial polarity in live cells and in vivo.

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