Dataset Information

Activatable Photosensitizer for Targeted Ablation of lacZ-Positive Cells with Single-Cell Resolution.

ABSTRACT: To achieve highly selective ablation of lacZ-positive cells in a biological milieu in vivo, we developed an activatable photosensitizer, SPiDER-killer-?Gal, targeted to ?-galactosidase encoded by the lacZ reporter gene. Hydrolysis of SPiDER-killer-?Gal by ?-galactosidase simultaneously activates both its photosensitizing ability and its reactivity to nucleophiles, so that the phototoxic products generated by light irradiation are trapped inside the lacZ-positive cells. The combination of SPiDER-killer-?Gal and light irradiation specifically killed lacZ-positive cells in coculture with cells without lacZ expression. Furthermore, ?-galactosidase-expressing cells in the posterior region of cultured Drosophila wing discs and in pupal notum of live Drosophila pupae were selectively killed with single-cell resolution. This photosensitizer should be useful for specific ablation of targeted cells in living organisms, for example, to investigate cellular functions in complex networks.

SUBMITTER: Chiba M

PROVIDER: S-EPMC6813548 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Activatable Photosensitizer for Targeted Ablation of lacZ-Positive Cells with Single-Cell Resolution.

Chiba Mayumi M Kamiya Mako M Tsuda-Sakurai Kayoko K Fujisawa Yuya Y Kosakamoto Hina H Kojima Ryosuke R Miura Masayuki M Urano Yasuteru Y

ACS central science 20190826 10

To achieve highly selective ablation of lacZ-positive cells in a biological milieu in vivo, we developed an activatable photosensitizer, SPiDER-killer-βGal, targeted to β-galactosidase encoded by the lacZ reporter gene. Hydrolysis of SPiDER-killer-βGal by β-galactosidase simultaneously activates both its photosensitizing ability and its reactivity to nucleophiles, so that the phototoxic products generated by light irradiation are trapped inside the lacZ-positive cells ...[more]

PMID: 31660435

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Project description:Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging standard for functional evaluation of future probe analogues and provides a basis for extending this strategy into glaucoma-specific animal models.

Dataset Information

Activatable Photosensitizer for Targeted Ablation of lacZ-Positive Cells with Single-Cell Resolution.

Publications

Activatable Photosensitizer for Targeted Ablation of <i>lacZ</i>-Positive Cells with Single-Cell Resolution.

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