Diaminorhodamine and Light-Activatable NO Donors: Photorelease Quantification and Potential Pitfalls.

Light-activatable nitric oxide (NO) donors have become of interest in the recent years. They produce NO when illuminated by light, which enables the control of its local concentration and is promising for biomedical applications. Several successful prototypes of photodonors have been published, but further research is needed to improve their properties such as water-solubility, activation wavelength, biocompatibility etc. One of major challenges on this way is to evaluate the efficiency of NO generation. Several methods may be used to track NO, including spin traps, specific electrodes and fluorescence-based probes. We have studied the applicability of well-known fluorescent reporter, diaminorhodamine (DAR-2), for the evaluation of NO production by photodonors. Our results indicate that DAR-2 can be used for the quantification of NO photorelease if this process is not accompanied by the singlet oxygen formation. Otherwise the oxidation of probe results in huge fluorescence increase, which interferes with signal due to reaction with NO. This issue should be taken into account when studying hybrids releasing both NO and 1O2, which are promising for photodynamic therapy.

Photolysis of dimethoxynitrobenzyl-"caged" acids yields fluorescent products.

Carboxylic acids conjugated with 4,5-dimethoxy-2-nitrobenzyl photoremovable protecting group are well known and widely used for biological studies. In this paper, we study the photolysis of likewise "caged" acetic, caprylic and arachidonic acids. Unexpectedly, we observed huge growth of fluorescence emission at ~430 nm during photolysis. Following further UV irradiation, a product with fluorescence at longer wavelength was formed (470 nm excitation / ~500-600 nm emission). While it may be used to monitor the "uncaging", these fluorescent products may interfere with widespread dyes such as fluorescein in biomedical experiments. This effect might be negligible if the photolysis products dissolve in the medium. On the other hand, we observed that arachidonic and caprylic acids derivatives self-organize in emulsion droplets in water environment due to long lipophilic chains. Illumination of droplets by UV rapidly induces orange fluorescence excited by 488 nm light. This fluorescence turn-on was fast (~0.1 s) and apparently caused by the accumulation of water-insoluble fluorescent residuals inside droplets. These self-organized lipophilic structures with fluorescence turn-on capability may be of interest for biomedical and other application. We have identified and hypothesized some compounds which may be responsible for the observed fluorescense.