Highly efficient singlet oxygen generation, two-photon photodynamic therapy and melanoma ablation by rationally designed mitochondria-specific near-infrared AIEgens

Photosensitizers (PSs) with multiple characteristics, including efficient singlet oxygen (1O2) generation, cancer cell-selective accumulation and subsequent mitochondrial localization as well as near-infrared (NIR) excitation and bright NIR emission, are promising candidates for imaging-guided photodynamic therapy (PDT) but rarely concerned. Herein, a simple rational strategy, namely modulation of donor–acceptor (D–A) strength, for molecular engineering of mitochondria-targeting aggregation-induced emission (AIE) PSs with desirable characteristics including highly improved 1O2 generation efficiency, NIR emission (736 nm), high specificity to mitochondria, good biocompatibility, high brightness and superior photostability is demonstrated. Impressively, upon light irradiation, the optimal NIR AIE PS (DCQu) can generate 1O2 with efficiency much higher than those of commercially available PSs. The excellent two-photon absorption properties of DCQu allow two-photon fluorescence imaging of mitochondria and subsequent two-photon excited PDT. DCQu can selectively differentiate cancer cells from normal cells without the aid of extra targeting ligands. Upon ultralow-power light irradiation at 4.2 mW cm−2, in situ mitochondrial photodynamic activation to specifically damage cancer cells and efficient in vivo melanoma ablation are demonstrated, suggesting superior potency of the AIE PS in imaging-guided PDT with minimal side effects, which is promising for future precision medicine.

For cell culture, minimum essential medium (MEM), fetal bovine serum (FBS), penicillinstreptomycin solution, MitoTracker Green were purchased from Invitrogen. 1 H and 13 C NMR spectra were measured on a Bruker ARX 400 NMR spectrometer using CDCl 3 and DMSO-d 6 as solvents and tetramethylsilane (TMS; δ = 0 ppm) was chosen as internal reference. High-resolution mass spectra (HR-MS) were obtained on a Finnigan MAT TSQ 7000 Mass Spectrometer System operated in a MALDI-TOF mode. Absorption spectra were measured on a Milton Roy Spectronic 3000 Array spectrophotometer. Steady-state photoluminescence (PL) spectra were measured on a Perkin-Elmer spectrofluorometer LS 55. Absolute fluorescence quantum yield was measured by a calibrated integrating sphere (Labsphere). Single crystal data was collected on a SuperNova, Dual, Cu at zero, Atlas diffractometer. The crystal was kept at 100.01(10) K during data collection. Using Olex2, the structure was solved with the Superflip structure solution program using Charge Flipping and refined with the ShelXL refinement package using Least Squares minimisation. Two-photon excitation fluorescence cross-section was measured by two-photon excitation fluorescence method using rhodamine B as reference. The excitation source for two-photon excitation was a femtosecond optical parametric amplifier (Coherent OPerA Solo) pumped by an amplified Ti:Sapphire system (Coherent Legend Elite system) and then detected with a spectrometer (Acton SpectraPro-500i) coupled to an CCD. Simulation was carried out with the Gaussian 09 package.
Laser confocal scanning microscope images were collected on Zeiss laser scanning confocal microscope (LSM 710) and analyzed using ZEN 2009 software (Carl Zeiss).

Cytotoxicity of DCQu to cells under light irradiation.
Cytotoxicity was evaluated by the 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in accordance to the manufacturer manual. Cells were seeded in 96-well plates (Costar, IL, USA) at a density of 6000-8000 cells per well. After overnight culturing, medium in each well was replaced by 100 μL fresh medium containing different concentrations of DCQu or Ce6. The volume fraction of DMSO is below 0.2%. After incubation for 30 min, plates containing cells with fresh medium were exposed to white light (4.2 mW/cm -2 ) for 90 min and another array of plates with cells was kept in dark as control. Then, the plates were conducted the same treatment as the biocompatibility test. After 24 h, 10 μL of MTT solution (5 mg/mL in PBS) was added into each well. After 4 h incubation, DMSO was added into each well and the plate was gently shaken to dissolve all the precipitated formed.
Finally, the absorption of each well at 570 nm was recorded via a plate reader (Perkin-Elmer Victor3TM). Each trial was performed with five wells parallel.

Cell apoptosis study. HeLa cells were stained with DCQu for 30 mins, washed with PBS for 3
times and irradiated with white light for 30 min. Then, cells were further stained with Annexin V-FITC for 15 min and imaged, excitation filter = 488 nm and emission filer = 500600 nm. Cells with DCQu and Annexin V-FITC staining but without light irradiation were taken as control group.

Two-photon fluorescence imaging in cells.
HeLa cells used for two-photon microscopy were stained with DCQu (5 μM) refer to the procedure described for confocal fluorescence imaging.

Two-photon fluorescence images of HeLa cells were collected using a Stimulated emission depletion (STED) microscopy (Leica Stimulated Emission Depletion Microscope) equipped with a multiphoton laser (Coherent Chameleon Ultra II Multiphoton laser). Excitation wavelength = 900
nm; emission filter = 600740 nm.
Two-photon PDT. HeLa cells were seeded 210 5 / dish in confocal image dish with 2 mL MEM medium supplied with 10% FBS and 1% PLS. After 24 hours, cells were stained with 5 μM DCQu for 30 min at 37 ℃, and then kept with fresh medium. Cells were then imaged under STED microscope equipped with two-photon laser with 900 nm excitation, 2500 W (67% gain). And images were taken after 1, 2, 4, 8, 16 and 32 scans.

O 2 generation detection. ABDA was used as the O 2 -monitoring agent. In the experiments, 13
μL of ABDA stock solution (7.5 mM) was added to 2 mL of each AIEgen (CPy, CQu, DCPy and DCQu) suspension (5 μM) and white light (4.2 mW/cm -2 ) was employed as the irradiation source.
The absorption of ABDA at 378 nm was recorded at various irradiation time to obtain the decay rate of the photosensitizing process.

ROS generation detection by H2DCF-DA in cells.
The HeLa cells were seeded in 35 mm petri dish with cover slip at a density of 100 000 cells and incubated for 24 h. Cells were pre-incubated with 10 μM H2DCF-DA for 4 hours followed by staining with or without 5 μM DCQu for 30 min and then washed with 1PBS for 3 times. The cells were carefully made into imaging slides and the slide was subjected to white light irradiation of maximum white light of CLSM 810 for 0, 0.5, 1.0 and 5 min followed by CLSM imaging (excitation: 488 nm, emission: 500-530 nm).
In vivo photodynamic therapy of melanoma. Mouse B16 melanoma cells were cultured in 75T culture flasks in culture medium supplemented with 10% FBS at 37 o C in a 5% CO 2 incubator.
Female C57BL/6 mice (6-7 weeks) were inoculated with B16 cells (2 x 10 6 cells/mL) on the rear dorsal area of each mouse. After ~7 days, the tumor reached a volume of ~50 mm 3 and used for followed therapy experiments. The tumor-bearing mice were randomly divided into 6 groups, including (1) PBS, (2) Light irradiation for 10 min, (3) Ce6 (10 μM) at dark, (4) DCQu (10 μM) at dark, (5) Ce6 with light irradiation, and (6) DCQu with light irradiation, respectively. The light irradiation was performed using a LED light with ultralow power density at 4.2 mW cm −2 . Typical intratumoral treatments were performed every three days and the mice photographs were recorded.
Tumor growth was measured by measuring the tumor diameter with a caliper. The tumor volume was calculated using the following equation: V = a * b 2 /2, where a and b are the largest and smallest diameters of the tumor. After diverse treatments, tumor tissues and major organs were harvested for histological analysis.