Selective imaging and cancer cell death via pH switchable near-infrared fluorescence and photothermal effects

A theranostic probe is designed that specifically illuminates and photoablates cancer cells by sensing pH changes in the lysosomes and mitochondria.


Supporting Information
IR2 showed minimized cytotoxicity in both cancer and normal cells. Concentration dependent cytotoxicities of IR2 in HepG2, HeLa, HL-7702, HUVEC, and HEK-293 cells after incubation for 24 h. The relative cell viabilities were determined by CCK-8 assay. The data were presented as mean ± SD, n = 5 wells of 96-well plates for each group. The bars were shown as mean ± SD, n = 12 microscopic fields from three independent tests for each value. (C) Mander's coefficients of co-localization for IR2 with respect to lysotracker quantified from confocal microscopic images. The bars were shown as mean ± SD, n = 5 microscopic fields for each value. (D) Fluorescence microscopic images of live HepG2, HeLa and HL-7702 cells stained with IR2 (20 μM, 12 h) and mitochondrial marker. NIR fluorescence were displayed in red and mitochondrial fluorescence were displayed in green.
(E) Confocal fluorescence microscopic images of HeLa, HUVEC, and HEK-293 cells at 12 h post incubation of IR2 (20 μM). NIR fluorescence were displayed in red and fluorescence of mitochondrial marker were displayed in green. Cells were suspended in acidic buffer (pH 4.0) for 10 min before imaging. Bar, 20 μm. (F) The colocalization coefficients between IR2 and mitochondrial marker under intra-plasmatic acidification (pH 4.0). The bars were shown as mean ± SD, n = 5 microscopic fields for each value. (G) Mean fluorescence intensities of IR2 (20 μM, 12 h) in HepG2, HeLa, HL-7702, HUVEC, and HEK-293 cells quantified from microscopic images after cytoplasmic acidification. The bars were shown as mean ± SD, n = 12 microscopic fields from three independent tests for each value. (H) Cells were treated with IR2 (20 μΜ, 12 h)/laser (750 nm, 6.0 W/cm 2 , 10 min), IR2 alone, laser alone or blank. Relative cell viabilities were measured by CCK8 assay at 24 h post treatment. The bars were shown as mean ± standard deviation (SD), n = 5 wells for each group.
All pH measurements were performed with a Mettler Toledo (Switzerland) MP220 pH meter. Absorption spectra were performed on a SHIMADZU (Japan) UV-2550 spectrophotometer. Fluorescence spectra were collected on a SHIMAZDU (Japan) RF-5301PC fluorophotometer.
In vitro fluorescence images were acquired in IVIS Spectrum In Vivo Imaging System (Caliper Life Sciences, USA).
The MDL-III-750nm-2W laser was obtained from Changchun New Industries Optoelectronics Tech. Co. Ltd. (China). The thermal profiles were determined with an A150-15-M infrared thermal camera (IRtech Ltd. China). Fluorescence microscopic images were performed from a Leica (Germany) DMI4000D inverted microscope or a Zeiss

Synthesis and Characterization
Compound 1. A mixture of 40 mL dimethylformamide (0.5 mol) and 40 mL methylene chloride was chilled in an ice bath for 30 min. 37 mL phosphorus oxychloride (0.4 mol) and cyclohexanone (10 g, 0.1 mol) were added dropwise to the mixture with stirring. The solution was refluxed for 3 h, cooled to room temperature, poured onto 200 g of ice, and allowed to stand overnight. The yellow solid was collected and weighted 8.0 g (46.5%).
Compound 2. 5-nitro-2,3,3-trimethyl-3H-indolium (205 mg, 1.0 mmol) was added to a solution of stannous chloride (1.6 g, 7.0 mmol) in hydrochloric acid (32%, 10 mL) with stirring. The mixture was stirred for 16 hours and allowed to cool to room temperature. The reaction mixture was poured onto crushed ice and the solution was rendered alkaline with sodium hydroxide (5.0 M). The mixture was filtered and dissolved in chloromethane and washed with water (3 x 10 mL). The organic phase was dried over anhydrous sodium sulfate and decolorized with a small amount of active carbon. The solvent was removed by rotary evaporation and the targeted compound was obtained as light yellow solid (128.2 mg 73.2%). 1

Compound 3a.
Compound 2 (175.1 mg, 1.0 mmol), di-tert-butyl dicarbonate (261.9 mg, 1.2 mmol) and sodium bicarbonate (168.0 mg, 2.0 mmol) were added into a mixture of distilled water and dioxane (V:V = 1:1) in an ice-bath and stirred for 45 min. The reaction bottle was then heated and refluxed under argon for another 12 h and the solvent was removed. The crude product was purified by silica gel based column chromatography with an eluting solvent (petroleum ether : ethyl acetate = 1:1) to offer compound 3a as a light yellow powder (226.1 mg, 82.2%). 1  the red product was filtered and re-dissolved in 20 mL saturated sodium chloride and was extracted with chloroform (3 x 20 mL). After that, the water extract was evaporated and the product was dried under vacuum without further purification. The yields of 4a-4c were determined in a range of 46-75%.
Compound 5a-5c. General synthetic procedure of compound 5a-5c. Compound 6a-6c. General synthetic procedure of compound 6a-6c. Compound 5a-5c (0.4 mmol), desiccant K 2 CO 3 (118.8 mg, 0.9 mmol), tetrakis (triphenylphosphine) palladium (26.5 mg, 0.02 mmol) and 4-carboxyphenylboronic acid (119.4 mg, 0.7 mmol) were mixed in 2.0 mL distilled water and stirred at 95 °C for 3 h under argon. The crude product was purified by silica gel based column chromatography with an eluting solvent system in which the gradient of CH 2 Cl 2 /CH 3 OH changed from 90% to 70%. Removal of solvent and dehydration under vacuum afforded the purified product as green powder.   Compound IR1-3. General synthetic procedure of compound IR1-3. Deprotection of compound 6a-6c (0.1 mmol) in a mixture of TFA and CH 2 Cl 2 (V/V = 1:1) offered compound IR1-3. The crude product was purified by silica gel based column chromatography with an eluting solvent system in which the gradient of CH 2 Cl 2 /CH 3 OH (containing 0.1% trimethylamine) changed from 90% to 60%. Removal of solvent and dehydration under vacuum afforded the purified product as green powder. The mixture was dropped into the ice-cold diethyl ether and a brown precipitate was filtrated.
The crude product was purified by column chromatography with an eluting solvent system in which the gradient of CH 2 Cl 2 /CH 3 OH changed from 90% to 75%. Removal of solvent afforded pure IR4 as a brown-yellow powder (20.4 mg，41.6 %). 1

Photospectroscopic Studies
Preparation of the stock solution. Stock solutions of IR1-4 were prepared in dimethyl sulfoxide (DMSO) with a concentration of 10 mM.
And then, the solutions were subpackaged and stored at -40 °C.

Absorption spectra.
Working solutions (1.0 μM) were prepared by diluting the stock solution in disodium hydrogen phosphate-citric acid buffer solution with pH ranging from 7.4 to 2.4 (pH changed with an interval of 0.2 units). All absorption spectra were recorded in a quartz cuvette (10 × 10 mm) at room temperature (r.t.). The scanning wavelength range is 400-900 nm and scanning speed is 1.0 nm/s (slit width: 5 nm).
Emission spectra. Working solutions (1.0 μM) were prepared under the same procedures as absorption test. All emission spectra were performed with a photomultiplier tube at r.t. Fluorescence emission spectra of all samples were collected at a 90-degree angle relative to the excitation light path. All fluorophores were excited at 750 nm and recorded from 760 nm to 900 nm. pH dependent temperature profile.
Laser power dependent temperature profile. Disodium hydrogen phosphate-citric acid buffer solution with pH 7.4 and 4.0 were prepared. A final concentration of 10 μM IR1-4 were added into centrifuge tubes in the above buffered solutions (100 μL) that mimic neutral and acidic environment. The tubes were irradiated under 750 nm laser at selected laser powers (1.2, 2.4, 3.6, 4.8 and 6.0 W/cm 2 ) for 180 s respectively. The temperature curves were obtained with an infrared thermal imaging camera. : In Equation 1, subscripts s and r refer to sample and reference respectively; Φ represents the fluorescence quantum yield; I stands for the measured integrated emission intensity; η is the refractive index; A is the optical density. Thermalgraphic images. A final concentration of 10 μM IR1-4 in buffered solutions (100 μL) with same pH range as above were added to centrifuge tubes. The solutions were irradiated under 750 nm laser at a power density of 6.0 W/cm 2 for 180s. The thermographic maps were captured when the solutions reached their maximal temperature values by an infrared thermal imaging camera.
Measurements of pKa ∆T . pH dependent temperature increment (∆T = T -T 0 ) were determined as the maximal values (T) minus the starting temperature (T 0 ) at each pH point according to the pH dependent temperature profiles. All the temperature increment values were normalized to the values under pH 7.4 respectively. The data analysis were similar as the procedures of pKa ∆T measurement.

ROS levels detection in solutions.
A final concentration of 10 μM IR2 and 5 μM DCFH-DA in buffered solutions (100 μL) with same pH range as above were added to a 96-well plate. The solutions were irradiated under a 750 nm laser at a power density of 6.0 W/cm 2 for 10 min or not. The fluorescence images were collected by an IVIS Spectrum equipped with a 500/540 filter set.
Photothermal stability evaluation. Working solutions (10 μM) of IR1-4 were prepared in disodium hydrogen phosphate-citric acid buffer solutions (pH 4.0 and pH 7.4) respectively. All fluorophores were excited at 750 nm (6.0 W/cm 2 , 10 min) and the temperature curves were obtained with an infrared thermal imaging camera.
All fluorophores were excited at 750 nm and recorded at maximal emission wavelengths (755 nm, 761 nm, 756 nm, and 760 nm for IR1-4 respectively). Fluorescence intensities were measured every 5 min for 1 h at r.t.
pH values of working solutions were reversibly adjusted between 7.4 and 4.0 by adding hydrochloric acid (5.0 M) and sodium hydroxide (5.0 M) solutions. All fluorophores were excited at 750 nm and recorded at maximal emission wavelengths (same as mentioned above). Fluorescence signals were measured for five rounds.

Selectivity evaluation.
A final concentration of 200 mM K + , Ca 2+ , Mg 2+ , Zn 2+ , Co 2+ , Fe 3+ , Fe 2+ , NH 4 + , cysteine, proline, glycine, histidine and H 2 O 2 were prepared in disodium hydrogen phosphate-citric acid buffer solution with pH value determined as 4.0 and 7.4 respectively. Stock solutions of IR1-4 were diluted to 1.0 μM in the above solutions. Fluorescent intensities were measured at their maximal emission wavelengths (same as mentioned above) upon excitation at 750 nm.

In Vitro Microscopic and Flow Cytometric Studies
Cell culture. Human adenocarcinoma cell line HeLa, human hepatocellular liver carcinoma cell line HepG2, human normal liver cell line HL-7702, human embryonic kidney cell line HEK-293, and human umbilical vein endothelial cell line HUVEC were used for in vitro studies. Cells were grown in DMEM (for HeLa, HepG2, HUVEC, and HEK-293) or RMPI 1640 (for HL-7702) culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (PS) at 37 °C in a humidified incubator of 5% CO 2 . Cells were carefully harvested and split when they reached 80% confluence to maintain exponential growth.
Cytotoxicity studies. The CCK-8 cell proliferation assay was applied to determine the viabilities of the cells treated with IR2.
A cell monolayer in exponential growth was harvested using 0.25% trypsin, and a single-cell suspension was obtained.
Cell suspensions with a density of 1.0 × 10 4 cells/well in 100 μL cell culture medium were added to 96-well plates.
After cell attachment, the cells were treated by IR2 with different concentrations (0.01-200 μM) that was sterile filtered through MILLEX®-HV 0.22 μm syringe filter in DMEM/RMPI 1640. After another 24 h incubation, cells were washed twice with phosphate buffer saline (PBS) and incubated with fresh medium containing 10 μL CCK-8. The cell viabilities were measured in an iMark Microplate Absorbance Reader according to the protocol. Cell viability (%) = mean of absorbance value of treatment group / mean of absorbance value of control × 100%.
Time dependent cellular uptake. HepG2 cells (2 × 10 5 cells/well) were seeded into 35 mm glass bottom cell culture dishes (φ20 mm, NEST Biotechnology) in 2.0 mL culture medium. After cell attachment, the culture medium was replaced by the DMEM containing IR2 (20 μM) and incubated for another 0.5, 4, 12, and 24 h respectively. The cells were washed twice with PBS ad incubated with PBS (pH 4.0) for another 10 min before cellular imaging. The fluorescent images were obtained by Zeiss LSM 880 confocal laser scanning microscope using a 20X objective lenses. The red fluorescence of IR2 were obtained by using a 633 nm laser. Filter set: Long pass (LP) 645 nm.
In vitro cellular uptake. HepG2 cells (2 × 10 5 cells/well) were seeded into 35 mm glass bottom cell culture dishes (φ20 mm, NEST Biotechnology) in 2.0 mL culture medium. The culture medium was replaced by the DMEM containing IR2 (20 μM) after cell attachment and incubated for another 12 h.
LysoTracker Green DND-26 (500 nM for 30 min) and MitoTracker Green FM (100 nM for 30 min) were added respectively before cellular imaging. The cells were washed twice with PBS and observed by Zeiss LSM 880 confocal laser scanning microscope using a 63X oil objective lenses. A portion of the cells pre-treated with IR2 and MitoTracker were incubated with PBS (pH 4.0) for another 10 min before cellular imaging. The green fluorescence of LysoTracker Green DND-26 and MitoTracker Green FM were collected by using a 488 nm laser. Filter set: Band pass (BP) 495-550 nm. The NIR fluorescence of IR2 were obtained by using a 633 nm laser. Filter set: Long pass (LP) 645 nm. Similar procedures were taken for HeLa, HL-7702, HUVEC and HEK-293 cell imaging.
Lysosomal activation of IR2. HepG2 cells (2 × 10 5 cells/well) were seeded on 35 mm glass bottom cell culture dishes and cultured for 24 h in 2.0 mL medium. The cells were incubated in DMEM containing IR2 (20 μM) for 12 h and meanwhile spiked with or without BFA (100 nM) for 2 h, and then Lysotracker green DND-26 (500 nM) for 30 min. A portion of the cells pre-treated with BFA, IR2 and LysoTracker were incubated with PBS (pH 4.0) for another 10 min before cell imaging. The cells were imaged by a Zeiss LSM 880 confocal fluorescence microscope with filter sets the same as the cellular uptake imaging above.
Morphology change studies. Morphology changes of different cells lines after IR2/laser treatment were studied under a Leica DMI4000D inverted microscope. HepG2 cells (2 × 10 5 cells/well) were seeded into 35 mm cell culture dishes in 2.0 mL medium. 24 h after the cell attachment, the medium was replaced by the DMEM containing 20 μM IR2. The cells were incubated for another 12 h and washed twice with PBS prior to the application of laser. The dishes were placed on the Labnet AccublockTM digital dry bath incubator so that the cells were remained at 37 °C. The cells were irradiated with a 750 nm laser (6.0 W/cm 2 ) for 10 min. Bright filed pictures of the cells prior to laser, 30 min and 24 h after laser treatment were collected respectively. Similar procedures were taken for HeLa and HL-7702 cell imaging.

Calcein-AM/PI staining.
The live/dead cells after laser treatment were visualized by calcein-AM/PI double staining kit. HepG2 cells (2 × 10 5 cells/well) were seeded into 35 mm glass bottom cell culture dishes in 2.0 mL culture medium. 24 h after the cell attachment, the culture medium was replaced by the DMEM containing 20 μM IR2 or DMEM only as control. As methods mentioned previously, both the groups were irradiated with a 750 nm laser (6.0 W/cm 2 ) for 10 min. After another 24 h incubation, cells were washed twice by PBS and stained with calcein-AM and propidium iodide (PI) for visualization of live/dead cells according to the protocol. The fluorescence images were obtained by Zeiss LSM 710 confocal microscope with proper filter sets (Calcein-AM: Ex: 488 nm; Em: 493-552 nm. PI: Ex: 561 nm Em: 566-697 nm). Similar procedures were taken for HeLa and HL-7702 cell imaging.

Quantification of hyperthermia induced cell death.
Quantitative evaluation of photothermal efficacy was determined by CCK-8 assay. HepG2, HeLa, HL-7702, HUVEC, and HEK-293 cells (1 × 10 4 cells/well) were seeded into 96-well plate and incubated with 100 μL media alone or containing 20 μM IR2 for 12 h after cell adherence. The two groups of cells were then exposed to 6.0 W/cm 2 750 nm laser irradiation for 10 min or not respectively.
After incubation for another 24 h and the cell viabilities of the four groups were quantified by the CCK-8 in an iMark Microplate Absorbance Reader.
Time lapse imaging. HepG2 cells were plated in 35 mm glass-bottomed dishes with 2 mL culture medium.
The cells were then treated with 20 μM IR2 for 12 h and 100 nM MitoTracker for 30 min prior to imaging.
The bright filed images of cells and green fluorescent images of mitochondrial marker were obtained every 2 min for 12 min. And the cells were undergone photo-bleaching by a 633 laser (5.0 mW) for the rest of time during the whole acquisition process. The fluorescence images were taken with filter sets (Ex: 488 nm, Em: 495-550). Similar procedures were taken for HeLa and HL-7702 cell imaging.
Detecting ROS levels within cells. HepG2 cells (1 × 10 4 cells/well) were seeded into a 96-well plate and incubated with 20 μM IR2 for 12 h and 10 μM DCFH-DA for 30 min. The cells were then exposed to laser irradiation (6.0 W/cm 2 , 750 nm) for 10 min prior imaging. As a positive control, the cells were treated with a Rosup (1 μL/mL) and DCFH-DA (10 μM) for 30 min prior imaging. Bright filed and fluorescent pictures of the cells were collected under a Leica DMI4000D inverted microscope. Filter sets (Ex: 480±20 nm, Em: 527±15 nm).

JC-1 flow cytometric analysis.
Mitochondrial membrane potential was determined using JC-1 mitochondrial membrane potential assay kit. Briefly, after treatment with 20 μM IR2 for 12 h followed by laser irradiation (6 W/cm 2 , 10 min), the cells were collected and stained with 10 μg/mL JC-1 solution at 37 °C, 5% CO 2 for 15 min. The cells were washed with PBS twice and resuspended in 500 μL PBS. Cells were analyzed on BD FACSAria II flow cytometer using a 488-nm excitation light source and a band-pass filters 515-545 nm (green fluorescence) and a band-pass filter 564-606 nm (red fluorescence).
The data were analyzed by FlowJo software. The report generated by plotting and gating for cell samples excluding debris. The cell population percentages of each quadrant were displayed.
Annexin V-FITC/PI flow cytometric analysis. The cell death pathway upon the IR2/laser irradiation was studied by flow cytometry after staining with annexin V-FITC/PI kit. After the same treatment of IR2/laser as described above, the cells were collected and washed with PBS twice. The cells were resuspended in 500 μL PBS and stained with Annexin V-FITC and PI solution at r.t. for 15 min. Cells were analyzed on BD FACSAria II flow cytometer using 488 nm excitation, a 515-545 nm band pass filter for FITC detection and a filter 665-685 nm for PI detection. The data report were obtained under similar process as JC-1 analysis above.