Figure 4. Contribution of oxygen free radicals to necrosis caused by pan IR700 based PIT.

A. Pan IR700 and IR700 mediated PIT produces reactive oxygen species. UMUC5 cells pre-treated with DCFDA (20µM) were incubated with pan IR700 (10µg/ml) or an equivalent amount of IR700 with and without Trolox (1mM) followed by irradiation with NIR 4 J/cm² or no NIR (Supplemental Fig. 9). Immediately following NIR irradiation, production of ROS was measured as a function of DCA generation, a fluorescent substance with excitation maxima 488 nm and emission maxima 535 nm. There is an approximately three- to four-fold increase in the amount of ROS production in both pan IR700 and IR700 treated cells immediately after NIR treatment which is inhibited in the presence of Trolox.

B. Pan IR700 and IR700 mediated PIT produces singlet oxygen species. UMUC5 cells pre-treated with singlet oxygen sensor green were incubated with pan IR700 (10µg/ml) or an equivalent amount of IR700 with and without NaN₃ (10mM) followed by irradiation with NIR 4 J/cm². Immediately after NIR irradiation, the SOS production was measured at 535nm. There is an approximately four-fold increase in the amount of ROS production in both pan IR700 and IR700 treated cells immediately after NIR treatment which is quenched in the presence of NaN₃. In untreated cells or panitumumab treated cells, NIR exposure did not change SOS production compared to non NIR treated cells.

C. SOS quencher, NaN₃, protects the cells from pan IR700 PIT-induced cell death. UMUC5 cells were incubated with pan IR700 in the presence or absence of a ROS quencher (Trolox 1mM) or a SOS quencher (NaN₃, 10mM) or both followed by irradiation with no NIR or NIR 4J/cm². One hour later the resultant cell survival/death was measured using MTS reagent. The SOS quencher NaN₃ is able to completely rescue pan IR700 PIT induced cell death.