Cytotoxicity analysis of oxazine 4-perchlorate fluorescence nerve potential clinical biomarker for guided surgery

Abstract

Biological tissue discrimination is relevant in guided surgery. Nerve identification is critical to avoid potentially severe collateral damage. Fluorescence imaging by oxazine 4-perchlorate (O4P) has been recently proposed. In this work, the cytotoxicity of O4P on U87 human-derived glioma cells has been investigated as a function of concentration and operating room irradiation modes. A custom-built optical irradiation device was employed for controlled optical dosimetry. DNA damage and O4P intracellular localization was also investigated by immunofluorescence and confocal microscopy. The results show that concentration below 100 µM can be considered safe. These results contribute to the assessment of the feasibility of O4P as a nerve biomarker.

Fig. 1.

A) Schematic design of the custom-made cell irradiation system, consisting of a driver-controlled broadband LED, a diffuser, a collimation system and a filter wheel for bandwidth selection. The system can irradiate Petri dishes and multiwell plates. B) White light irradiation spectrum. C) 630 nm irradiation spectrum.

Fig. 2.

Cytotoxicity assay results, expressed as percentage of viability of the U87 cells in dark and after 15 minutes of 10 mW/cm² irradiation with white light (A) and λ=630 nm light (B). Cells were treated with different amounts of O4P (0, 50, 100, 200 and 400 μM) for 4 h. These data represent the relative (in percentage) cell viability with respect to the negative control after 4 independent experiments. The results were analyzed using a two-factor ANOVA test with 6 samples per group. Statistical significance is indicated by *p<0.05, **p<0.01, ***p<0.001.

Fig. 3.

DNA damage. A) Measurements of fluorescence intensity signals of ϒH2AX. B) High doses of O4P (100 uM) induce significant DNA damage (B.a), compared to control (B.b). In some of these cells, the amount of damage is dramatic (B.c, B.d). ϒH2AX (green), PI (red).

Fig. 4.

Representative examples of confocal microscopy images from dark control (A–H), white light (I–P) or at 630 nm irradiated cells (Q-X). Comparison between cells in the absence of fluorophore (controls A-D, I-L and Q-T) and cells treated with 100 µM of O4P. DAPI (blue), ϒH2AX (green), and PI (red). Scale bar = 50 µm.

Fig. 5.

O4P distribution (DAPI in blue, O4P in red). A) U87 confocal microscopy images treated with 50 and 100 µM of O4P either in dark or after irradiation at 630 nm for 15 and 30 min. Representative images of the localization of the fluorophore were taken after 4 hours of incubation. Nuclei labelled with DAPI are shown in blue. Scale bar = 25 μm. B) Higher magnification of merged images with no fluorophore and 100 μM. Scale bar = 20 μm.