Figure 2.

l-Cysteine accelerates ligand release after NIR light exposure, but NaN₃ inhibits this reaction. (a) Mean 700 nm fluorescence intensity in 25 μM Pc 3 with various concentrations of l-cysteine in PBS was decreased in both an NIR light- and l-cysteine dose-dependent manner (n = 4, mean ± SEM). (b) Aggregation after NIR light exposure in Pc 3 with l-cysteine mixture. The cyan color of IR700 decreased and aggregation appeared, while 700 nm fluorescence was lost in an l-cysteine dose-dependent manner. (c) 3 μM pan–IR700 with various concentrations of l-cysteine in PBS was irradiated with NIR light and imaged using 700 nm fluorescence imaging. Under more than 1 mM l-cysteine condition in PBS, mean 700 nm fluorescence intensity was decreased in an NIR light dose-dependent manner (n = 4, mean ± SEM). (d) Under more than 1 mM NaN₃ with 3 μM pan–IR700 in PBS, mean 700 nm fluorescence intensity after NIR light exposure was significantly decreased (n = 4, mean ± SEM, vs 0 mM NaN₃; two-way ANOVA repeated measures, followed by Dunnett’s test; *p < 0.05; **p < 0.01; ****p < 0.0001). (e) In 3 μM pan–IR700 in PBS with both 1 mM l-NaAA and l-cysteine, NaN₃ did not affect mean 700 nm fluorescence intensity after NIR light exposure (n = 4, mean ± SEM). (f) ESR spectra recorded from samples containing IR700 (0.5 mM) and l-cysteine (10 mM) in PBS when irradiated with NIR light under argon saturation conditions. (i) Control spectrum of a sample containing IR700 (0.5 mM) and l-cysteine (10 mM) in PBS without NIR light exposure. (ii) ESR spectra recorded when the samples were irradiated with NIR light. (iii) ESR spectra shown in red were obtained by the addition of 100 mM NaN₃ to the samples containing IR700 (0.5 mM) and l-cysteine (10 mM) in PBS and irradiating them with NIR under argon-saturated conditions. The ESR spectrum overlaid in gray is identical to the ESR spectrum recorded in the absence of NaN₃ shown in (ii). ▲, an undesirable but negligible signal originated from the quartz ESR tube. Cys, l-cysteine. (g) Remaining IR700% (post/pre) under conditions (ii) and (iii). The averaged values are represented (n = 2). (h) HPLC chart of l-cystine and l-cysteine in the presence of IR700 under several conditions. When the solution of IR700 containing l-cysteine was irradiated under hypoxic conditions, the peaks of l-cysteine and l-cystine were observed, although there was no peak of l-cystine without irradiation. On the other hand, in the case of aerobic conditions, the peak of l-cysteine almost disappeared after irradiation.