Poly(ADP-ribose) polymerase (PARP) is a validated therapeutic target and an attractive biomarker for fluorescence-guided tumor imaging. Because PARP resides in the nucleus, fluorescent probes must permit nuclear entry of the targeting ligand without compromising binding. The authors aimed to expand available PARP-directed imaging agents for preclinical tumor detection, with an emphasis on probes suitable for intraoperative tracking of oral squamous cell carcinoma.
The reported series comprises 5-azetidinyl BODIPY cores functionalized at the 5-position with olaparib-derived ligands linked through flexible spacers. This design intended to (1) retain conformational flexibility of the targeting moiety, (2) incorporate a donor–acceptor fluorophore architecture to enlarge the Stokes shift, and (3) provide nuclear penetration compatible with PARP targeting. Structures of the designed compounds are shown in the source figures and include multiple derivatives; the probe referred to as 1a emerged as the lead based on imaging performance.
In cell-based assays, 1a produced efficient nuclear localization in PARP-overexpressing cell lines, specifically U87 (a glioma line) and Cal27 (an oral squamous cell carcinoma line). Confocal microscopy used excitation at 488 nm for the probe (detection 500–700 nm) and Hoechst 33342 nuclear counterstain excited at 405 nm (detection 420–500 nm). Colocalization analysis yielded high Pearson correlation between the probe and the nuclear stain, supporting nuclear localization. Grayscale intensity profiles demonstrated overlapping nuclear signals, with expected partial nonoverlap in nucleolar regions where Hoechst signal is reduced.
Specificity for PARP was supported by two experimental approaches reported in the source: competitive blocking with excess olaparib and comparison with HEK293 cells (a line with lower PARP expression). Pretreatment of Cal27 cells with olaparib reduced 1a nuclear signal, consistent with receptor- or target-mediated engagement. Imaging intensity quantification accompanied the microscopy images; statistical comparisons in the figures indicate significant reductions in signal with blocking conditions.
The lead probe 1a was evaluated in vivo in both subcutaneous Cal27 xenografts and orthotopic oral tumor models. Intravenous administration at the reported dose (5 μmol/kg) led to rapid tumor accumulation and enabled clear fluorescence-based tumor visualization. Time-course whole-animal imaging in orthotopic models showed signal at multiple early time points. A comparator probe, PARPi-FL, was imaged in parallel in several experiments to benchmark performance.
Ex vivo fluorescence images of tumors and major organs were collected at early postinjection time points (1.5 and 3 h) from subcutaneous Cal27 xenograft-bearing mice. Quantitative measures presented in the source include average fluorescence radiant efficiency values and tumor-to-normal tongue signal ratios. These metrics showed tumor accumulation of 1a and were used to assess contrast relative to PARPi-FL. In the orthotopic model, blocking with olaparib reduced in vivo signal measured as average radiant efficiency, supporting in vivo specificity.
By connecting an olaparib-derived targeting ligand to a 5-azetidinyl BODIPY fluorophore through flexible linkers, the authors generated a probe that balances nuclear access and favorable photophysical properties (including an enlarged Stokes shift). Probe 1a demonstrated PARP-dependent nuclear labeling in cultured cells and rapid, specific accumulation in Cal27 tumor models in vivo, suggesting utility for fluorescence-guided visualization of oral tumors. The work expands the repertoire of PARP-targeted fluorescent probes and highlights design considerations when targeting nuclear enzymes.
The authors declared no conflicts of interest. Detailed synthetic methods, full quantitative datasets, and all experimental procedures are reported in the full article available via the source; specific procedural details and extended numerical results are provided there.