Nitric oxide activatable photosensitizer accompanying extremely elevated two-photon absorption for efficient fluorescence imaging and photodynamic therapy† †Electronic supplementary information (ESI) available: Experimental details of synthesis, characterization and supplementary figures. See DOI: 10.1039/c7sc04044j

A nitric oxide (NO) activatable photosensitizer was constructed for efficient fluorescence imaging and photodynamic therapy.

Instruments: All the calculations were done with Gaussian09 program (Revision B.01). NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz spectrometer ( 1 H: 400 MHz, 13 C: 100 MHz) and referenced to tetramethylsilane (TMS) as the internal standard, the following abbreviations are used to explain the multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet. Mass spectra were obtained on a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker AutoFlex III system). The steady-state absorption data and photoluminescence spectra were measured by a Shimadzu UV-3600 Plus ultraviolet-visible-near-infrared spectrophotometer and an Edinburgh FLSP920 spectrofluorometer, respectively. The absolute fluorescence quantum yield measured using an Edinburgh FLSP920 fluorescence spectrophotometer equipped with an integrating sphere and a xenon lamp. Two-photon absorption cross-sections were measured by the Z-scan technique using home-built experimental setup. 2 The methyl thiazolyl tetrazolium (MTT) assay was performed by a PowerWave XS/XS2 microplate spectrophotometer (BioTek, Winooski, VT). The laser source is a Ti:sapphire system that produced 100 fs pulses at a repetition of 80 MHz. All images were acquired on Leica TCS SP5X Confocal Microscope System equipped with Leica HCX PL APO 63x/1.20 W CORR CS, 405 nm Diode laser, white laser (470 nm to laser lines, each excitation wavelength provides 1.5 mV), and Ti-Sapphire laser (~4 W at 800 nm) which corresponded to approximately 1% (~40 mW at 800 nm) average power in the focal plane.
Statistical analysis: The statistical analysis of the samples was undertaken using a Student's t-test, and p-values < 0.05 were considered statistically significant. All data reported are means ± standard deviations, unless otherwise noted.

Theoretical calculation
The corresponding excitation energy and molecular orbitals of DBB and OPD were calculated using Density Functional Theory (DFT). All the calculations are completed in Gaussian 09.

DBB
Scheme S1 Synthetic route of DBB-NO.

Optimal experiment of DBB-NO
Absolute fluorescence quantum yield. We employed an Edinburgh FLSP920 fluorescence spectrophotometer equipped with an integrating sphere to measure the absolute fluorescence quantum yield according to the following formula: The calculated Ф F of DBB-NO and DBB in PBS buffer solution is 0.17% and 9.3 %, respectively.
Determination of the detection limit. The detection limit was calculated according to the method reported in the previous literature 3 . The fluorescence emission spectrum of DBB-NO was measured by five times and the standard deviation of blank measurement was achieved. The fluorescence intensity at 415 nm was plotted as a concentration of NO. The detection limit was calculated with the following equation: Where σ is the standard deviation of blank measurement, k is the slope between the fluorescence intensity versus NO concentration.
Two-photon absorption cross-sections (δ) spectra. The δ spectrum of target molecule was acquired via a point-to-point method. Briefly, the δ of different sample in PBS at different TP-excitation wavelength (710,720,730,740,750,760,770,780,790,800,810,820,830) were tested, respectively. Then the δ against different TPE wavelength were ploted as the two-photon absorption cross-sections spectra (Fig. 2b).   and 10 μL DEA·NONOate (200 equiv) was added to probe (5 μM) in PB buffer solution, and light (365 nm) with a power density of 6.5 mW cm -2 was employed as the irradiation source. The absorption of ADMA at 261 nm was recorded at various irradiation times to obtain the decay rate of the photosensitizing process. TMPyP 4 in PB buffer solution was used as the reference (r), and Φ r = 74% in water. 4  After that, macrophages were exposed to 1 kHz femtosecond laser TP-irradiation at 750 nm with power density of ∼1.0 W cm −2 for 5 min (without specifically states, all the TP-irradiation in following at 750 is same as this) and then these macrophages were incubated for 4 hours before testing cellular viability. In the third group, RAW 264.7 macrophages were incubated with 2 μM DBB-NO for 6 h, washed by PBS buffer and subsequently incubated with LPS (1 μg/mL), IFN-γ (1 μg/mL) and N-acetylcysteine (NAC, 2 mM) respectively for 1 h. After that, macrophages were exposed to TP-irradiation at 750 nm for 5 min and then these macrophages were incubated for 4 hours before testing cellular viability. In the fourth group, normal RAW 264.7 macrophages were incubated with 2 μM DBB-NO for 6 h.

Dark cytotoxicity and biocompatibility of DBB-NO
After that, macrophages were exposed to TP-irradiation at 750 nm for 5 min and then these macrophages were incubated for 4 hours before testing cellular viability. For cellular viability assessment, the macrophages were further incubated 1 h in a medium containing 1 μM of calcein AM and 500 nM of propidium iodide (PI). Calcein AM with green emission represents living cells and PI with red emission represent dead cells. For Calcein AM, the excitation wavelength is 488 nm and the collection wavelength range is from 510-530 nm. For PI, the collection wavelength range is from 610-630 nm upon excitation at 559 nm.
in vitro TP-PDT using MTT assay. For evaluating the NO-activatable TP-PDT of DBB-NO in activated macrophage, RAW 264.7 macrophages were incubated with DBB-NO at different concentration for 6 h, washed by PBS buffer and subsequently incubated with LPS (1 μg/mL) and IFN-γ (1 μg/mL). After 1 h, these prepared macrophages were exposed to the TP-irradiation at 750 nm for 5 min. After incubating another 12 h, the cell viability of macrophages was measured by typical MTT assay. HeLa cells and normal RAW 264.7 macrophage (without treatment of LPS and IFN-γ) under the same experimental conditions were also performed for direct comparison.
in vitro TP-PDT using flow cytometric assay. To get a quantitative evaluation of TP-PDT effect of DBB-NO, the same four groups as above mentioned for were prepared in 4 well plates. The first group is that RAW 264.7 macrophages were incubated with 2 μM DBB-NO for 6 h. Then macrophages were washed by PBS buffer and subsequently incubated with LPS (1 μg/mL) and IFN-γ (1 μg/mL) for 1 h to activate macrophage before testing cellular viability. In the second group, RAW 264.7 macrophage cells were incubated with Lipopolysaccharide (LPS, 1 μg/mL) and interferon-γ (IFN-γ, 1 μg/mL) for 1 h to activate macrophage. After that, macrophages were exposed to TP-irradiation at 750 nm for 5 min and then these macrophages were incubated for 4 hours before testing cellular viability. In the third group, RAW 264.7 macrophages were incubated with 2 μM DBB-NO for 6 h, washed by PBS buffer and subsequently incubated with LPS (1 μg/mL), IFN-γ (1 μg/mL) and N-acetylcysteine (NAC, 2 mM) respectively for 1 h. After that, macrophages were exposed to TPirradiation at 750 nm for 5 min and then these macrophages were incubated for 4 hours before testing cellular viability. In the fourth group, RAW 264.7 macrophages were incubated with 2 μM DBB-NO for 6 h. After that, macrophages were exposed to TP-irradiation at 750 nm for 5 min and then these macrophages were incubated for 4 hours before testing cellular viability. For cellular viability assessment, the collected         Flow cytometric assay about TP-PDT effect of DBB-NO in which using Annexin FITC/propidium iodide (PI) cell apoptosis to distinguish viable cells from dead cells. The activated macrophages treated with either DBB-NO or TP-irradiation alone exhibited a high cell viability (Annexin V-FITC − /PI − ) with value exceeding 91.1%. After treated with DBB-NO under TP-irradiation, the viable macrophages (Annexin V-FITC − /PI − ) decreased to 25.9% and the sum population of early apoptotic (Annexin V-FITC + /PI − ) and latest-age apoptotic (Annexin V-FITC + /PI + ) macrophages significantly increased (88.7%), indicating a highly efficient TP-PDT. For the control where normal macrophages treated with DBB-NO under TP-irradiation, no obvious cell apoptosis was observed. From these results, we concluded that DBB-NO is endogenous NO-activatable and can serve as a smart photosensitizer to selectively destroy activated macrophage from normal ones for a precision therapy.