Fluorescence life-time imaging microscopy (FLIM) monitors tumor cell death triggered by photothermal therapy with MoS2 nanosheets

Recently, photothermal therapy (PTT) has been proved to have great potential in tumor therapy. In the last several years, MoS2, as one novel member of nanomaterials, has been applied into PTT due to its excellent photothermal conversion e±cacy. In this work, we applied °uorescence lifetime imaging microscopy (FLIM) techniques into monitoring the PPTtriggered cell death under MoS2 nanosheet treatment. Two types of MoS2 nanosheets (single layer nanosheets and few layer nanosheets) were obtained, both of which exhibited presentable photothermal conversion e±cacy, leading to high cell death rates of 4T1 cells (mouse breast cancer cells) under PTT. Next, live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123, which were then continuously observed with FLIM technique. FLIM data showed that the °uorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS2 nanosheets signi ̄cantly increased during PTT treatment. By contrast, the °uorescence lifetime of the same dye in control cells (without nanomaterials) remained constant after laser irradiation. These ̄ndings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells, which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.


Introduction
At present, cancer is one of most lethal diseases in the world. There are still great limitations of traditional treatment strategies including surgery, chemotherapy, and radiotherapy, leading to development of a lot of alternative novel therapeutic approaches. [1][2][3] Among these various treatment approaches, photothermal therapy (PTT), which employs heat generated from visible or near infrared (NIR) absorbed materials to ablate tumors, has attracted more and more attention due to its advantages including minimal invasiveness and high e±ciency against drug-resistant tumors. [4][5][6][7][8] In the last several years, two-dimensional (2D) molybdenum disul¯de (MoS 2 ) nanomaterials have shown high light-absorption ability and satisfactory biocompatibility, making them potential powerful PTT agents for tumor treatment via their NIR absorption properties. [9][10][11][12][13] At present, the standard methods for measuring the e±ciency of the PTT agents against tumor cells includes biochemical methods such as cytotoxicity assays and microscopy methods such as live and dead cell imaging assays. However, these methods could not provide dynamic information of cell death at single cell level or even at subcelluar level. Therefore, the subcellular processes of PTT triggered cell death still needs further investigation. Compared with the above-mentioned methods,°u orescence lifetime imaging microscopy (FLIM) technique has great advantages in monitoring multiple cell physiological processes, including cell growth and cell death, since FLIM can provide real-time images and quantitative subcellular information. 14,15 In this study, we applied FLIM method to observe MoS 2 nanosheet treated 4T1 cells in order to study the mitochondria damage during cell death. We used two types of MoS 2 nanosheets, which are single layer nanosheets and few layer nanosheets (abbreviated as SL and FL, respectively), and obtained their photothermal conversion e±cacy. Then, we con¯rmed their PTT e±cacy on 4T1 cells under 808 nm laser irradiation. After the cell mitochondria were labeled with Rodamine123 (abbreviated as R123) dye, cells incubated with MoS 2 nanosheets or control cells under 808 nm laser irradiation were respectively monitored by using FLIM technique. Finally, FLIM data were analyzed to evaluate the antitumor e±cacy at single cell level.

Chemicals
Both of the MoS 2 single layer nanosheets and few layer nanosheets were purchased from Nanjing XFNANO Materials Tech. Rodamine123 were purchased from Beyotime Biotechnology. Cell cytotoxicity kits (CCK-8) were obtained from Nanjing KeyGen Biotech. All the cell culture reagents (medium, antibiotics and fetal bovine serum) were obtained from Invitrogen.

Material characterizations
The morphology of the MoS 2 nanosheets was obtained by a transmission electron microscopy (TEM) instrument (JEM-1230 CX, Jeol Ltd, Japan). The UV-Vis-NIR spectra of these nanosheets were recorded in a 10 mm path length cuvette on a spectrophotometer (UV1780, Shimadzu, Japan). The OD values in CCK-8 assays were determined by a microplate reader (In¯nite M200, Tecan, Switzerland).

Photothermal properties of the MoS 2 nanosheets
To evaluate the photothermal properties of the MoS 2 nanosheets, a series of MoS 2 aqueous solutions at di®erent concentrations (0, 25, 50, 100, 200 or 400 g/mL) were prepared and deposited into cuvettes. Then the cuvettes were illuminated by a continuous-wave laser (808 nm) at a power density of 1 W/cm 2 . The temperatures were continuously recorded by using a probe thermometer at every 30 s (for heating period) or every 1 min (for cooling period). The photothermal conversion e±ciency PTCE () was calculated as mentioned before 13,16,17 as follows: In Eq. (1), h represents the heat transfer coe±cient. A is the container surface area (1 cm 2 ). ÁT max is the maximum temperature change of the nanosheet solution at the corresponding time point. Qs is dened as the heat loss due to the light absorbed by the container. I refers to the incident power density of the 808 nm laser (1 W/cm 2 ) and A 808 is the absorbance value of the solution at 808 nm.
In order to get the hA in Eq. (1), a parameter is introduced as shown in Eq. (2), which is de¯ned as the ratio of ÁT to ÁT max.
ÁT stands for the temperature change during a cooling period. In addition, hA is calculated by applying the linear time data from the cooling period versus ln .

2.4.
Cell culture and cytotoxicity assay 4T1 cells (mouse breast cancer cells) were purchased from American Type Culture Collection (ATCC, USA). Cells were cultured in DMEM culture medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 C under a 5% CO 2 and humidity condition. In order to examine the biocompatibility of the MoS 2 nanosheets, a standard CCK-8 cell assay was carried on 4T1 cells. Around 10,000 of cells were seeded in each well of 96 well plates and grown overnight. The next day, medium with di®erent concentrations of the MoS 2 nanosheets (50, 100 or 200 g/mL) were added to the cells. Here, six replicates were applied for each condition. After 24 h incubation, the solutions were moved from cells and a mixture of 10 L of CCK-8 solution plus 100 L of fresh culture medium was added to each well, followed by another incubation for around 1-2 h. At the end of the chromogenic reaction, the OD value at 450 nm wavelength of each well of the plates was recorded by using the microplate reader.
Next, the PTT e±ciency of the MoS 2 nanosheets was measured by the CCK-8 assay in a similar way. Brie°y, 4T1 cells were seeded in 96-well plates and cultured overnight. Then, PBS or MoS 2 nanosheets (0, 100 or 200 g/mL) were added to the medium, and the cells were incubated in 5% CO 2 at 37 C. After incubation, the PBS or MoS 2 nanosheet treated samples (PTT groups) were illuminated under 808 nm laser irradiation (1 W/cm 2 ) for 5 mins, followed by the CCK-8 assay at the same procedure mentioned above.

FLIM experiments
In order to obtain FLIM images, around 100,000 of cells were seeded in each 35 mm confocal dish. After an overnight incubation, the cells were labeled with R123 and then incubated infresh medium with either PBS or MoS 2 nanosheets (200 g/mL). These cell dishes were then moved onto the microscope stage and observed for 18 mins. The images were obtained at every 3 mins during this period. Next, the image acquisition was continuously performed at the same frequency while introducing the 808 nm laser irradiation (1 W/cm 2 ) onto the cells for another 15 mins.
All microscopic imaging and FLIM analyses were performed with a Nikon Eclipse confocal system mounted on an inverted microscope. This microscope was equipped with a Fianium whitelase Supercontinuum laser (output laser from 470 to 670 nm, 6 ps pulse width, 80 MHz repetition rate) for single photon excitation. A 63 Â oil immersion objective lens (NA 1.40) was used for all images. To obtain FLIM data, the time-correlated singlephoton counting (TCSPC) technique was carried out with a Becker & Hickl SPC120 unit. In this study, the 514 nm excitation wavelength was selected to generate the°uorescence images of cells labeled with R123. A band-pass¯lter BP 535/30 was put in front of the PMT detector to acquire the speci¯c signals. All FLIM images were acquired at 256 Â 256 pixels and the collection time for one image was around 30 s. After image collection, the°u orescence lifetime image data was analyzed by using SPCImage (Becker & Hickl) software as described in our former studies, [18][19][20] providing the°u orescence decay signal of each image and lifetimes of the selected regions.
Next, to evaluate the photostability of R123 dye under NIR light irradiation, we carried out the following experiments. Brie°y, the R123 solution (50 M) or the cells labeled with R123 were prepared in confocal dishes. Then the dishes were moved onto the microscope stage and observed for 18 mins. The images were obtained at every 3 mins during this period and the photon numbers collected in 1 min were counted at each timepoint. After the 808 nm laser irradiation (1 W/cm 2 ) was turned on, the photon-counting image acquisition was continuously carried out for extra 15 mins.
In addition, we used FLIM technique to investigate the parameters that might in°uence the°uorescence lifetime of R123 dye. The R123 dye samples were prepared in a series of solutions (100 M) with di®erent pH values or with di®erent viscosities. Then these two series of R123 dye solutions were measured with FLIM system, providing images for data analysis.

Characterization of MoS 2 nanosheets
In this study, both MoS 2 nanosheets are stored in aqueous solutions. As shown in the TEM image ( Figs. 1(a) and 1(b)), a general view of the MoS 2 nanosheets showed that the lateral sizes of MoS 2 SL nanosheets and FL nanosheets are both in the range from around 50 to 200 nm, which is a suitable size range for e±cient endocytosis in the applications of nanoparticles into biological¯elds. 21,22 Moreover, the absorption range of both of the two types of MoS 2 nanosheets with the gradient concentrations (25,50,100,200 or 400 g/mL) expand to the NIR region from 700 to 900 nm (Figs. 1(c) and 1(d)), which is very similar to the results reported before. [9][10][11][12][13] This range is compliant with an 808 nm laser in the further investigation on their photothermal properties.

Photothermal properties of MoS 2 nanosheets
In order to examine the photothermal properties of MoS 2 SL nanosheets and FL nanosheets, we recorded the temperature trends of the aqueous solutions with a series of concentrations (0, 50, 100, and 200 g/mL) that were exposed to an 808-nm laser irradiation (1 W/cm 2 ) for 5 mins. slopes are positively associated with the increased concentrations of nanomaterials. For example, the temperature of the MoS 2 SL nanosheet solution (200 g/mL) dramatically increased from around 23.0 C to 61.8 C in 5 mins, achieving an excellent temperature high enough to an irreversible ablation to the tumor cells. 23 Under the same condition, the temperature change of the MoS 2 FL nanosheet solution (200 g/mL) exhibited even a greater increase to around 80 C after laser irradiation for 5 mins, which indicates the higher photothermal transduction e±cacy of MoS 2 FL than that of MoS 2 SL nanosheets.
To further investigate the photothermal transduction ability of these two types of nanosheet aqueous solution, we measured the temperature pro¯les of each solution with a concentration of 200 g/mL under a continuous laser irradiation (1 W/cm 2 ) for 5 mins, consequently followed by a natural cooling period till room temperature. The temperature pro¯le results were shown as in Figs. 2(c) and 2(d). These data were analyzed according to the reported method, resulting in the photothermal conversion e±ciency () of the MoS 2 SL and the MoS 2 FL nanosheets, which could achieve 17.4% and 24.8%, respectively, similar to that of previously reported MoS 2 nanosheets (24.37% or 26.7%). 9,13 Such suitable photothermal conversion capabilities indicate that MoS 2 nanosheets could o®er great advantages for PTT.

Photothermal therapy in vitro
In order to evaluate the potential PTT application abilities of MoS 2 SL or FL nanosheets, we carried out cell cytotoxicity assay as wells as PTT e®ect assay on 4T1 cells. Firstly, the potential toxicity of MoS 2 SL or MoS 2 FL was measured with a CCK-8 assay as the manufactory's instructions. Experimental data showed that there was little cytotoxicity for either MoS 2 SL or FL nanosheets after 24 h incubation, even at the highest concentration 200 g/mL (Fig. 3(a)), indicating their good biological compatibility.
In addition, we examined the e®ect of the PTT of MoS 2 SL or FL nanosheets in 4T1 cells by an exposure to the 808 nm laser irradiation. As presented in Figs. 3(b) and 3(c), in the PBS, only nanosheets, or only NIR (808 nm, 1 W/cm 2 , 5 mins) treated group, all the cell viabilities reached more than 90%, suggesting negligible damage e®ects. By contrast, in the MoS 2 SL or FL nanosheets plus NIR irradiation group, more than 80% of the cells were killed due to the nanosheet induced PTT e®ects, which were consistent with former studies. 9,17

FLIM data acquisition and analysis
Finally, we performed FLIM experiments for measuring the PTT e®ects of MoS 2 SL or FL nanosheets on 4T1 cells to acquire dynamic subcellular information. In order to monitor the local molecular environment changes in live 4T1 cells, the°uorescent dye R123 was used as a mitochondria localization marker in this process. 24,25 During each experiment, around 10-20 of live 4T1 cells were selected for continuously monitoring the°uorescence lifetime of R123 in cell mitochondria. The°u orescence lifetime image of these cells was obtained for every 3 mins in the¯rst period (no irradiation). Then an 808 nm laser was turned on to irritate the cells at 1 W/cm 2 . We recorded the°u orescence lifetime of R123 for 18 mins and then kept recording for another 15 mins after laser on. Figure 4(a) were a series of°uorescence lifetime images of typical 4T1 cells during laser irradiation, representing the R123°uorescence lifetime distributions of the dye in the cell mitochondria. Figure 4(b) was one representative raw result that collected the cells treated with MoS 2 FL nanosheets under NIR laser irradiation at 15 mins. This¯gure includes the°uorescence intensity image which indicates suitable signal intensity, the°uorescence lifetime image and the histogram of the lifetime distribution of the°uorescent R123 dye in the whole image. The inset in the lower part provides a decay curve for calculating the°uorescence lifetime. As shown in Fig. 4(c), the R123°uorescence lifetime from cells treated with both types of MoS 2 nanosheet showed slight°uctuations during thē rst 18 mins before laser on, indicating the nanosheet incubation had no e®ect on the°uorescence lifetime values. From the initial of laser irradiation, the°uorescence lifetimes of R123 in cells treated with both MoS 2 nanosheets signi¯cantly increased in an irreversible way. By contrast, the°u orescence lifetime of the same dye in the control cells remained constant. These above data showed that under physiological conditions, the°uorescence lifetimes of R123 in healthy 4T1 cells could keep constant with random°uctuation. The values of such°uctuation are usually in the range of 50-200 ps. The induction of NIR laser exposure onto nanosheet treated 4T1 cells lead to around 500 ps increases the obtained°uorescence lifetimes suggesting the signi¯cant correlation between the cell death and the PTT e®ects.
Moreover, the photostability of R123 were tested by exposing the R123 solution or the cells labeled with R123 under NIR laser irradiation. Both in vitro and in vivo results are shown in Figs. 5(a) and 5(b), respectively. The collected photon number of the in vitro sample keeps almost constant while the photon number from in vivo sample maintains around 90% after laser explosion, suggesting the satisfactory photostability of R123 through the whole experiment processes.
To further understand the potential microenvironment parameters that might in°uence the°uorescence lifetimes of R123, the R123 dye samples in di®erent solutions were then measured with FLIM system. The images were then obtained and analyzed, providing the data in Figs. 5(c) and 5(d). The°u orescence lifetimes of R123°uctuated slightly at di®erent pH conditions (Fig. 5(c)). By contrast, as shown in the Fig. 5(d), the R123 decreased as the percentage of glycerol in glycerol-water solutions increased from 0% to 80%, indicating that the°u orescence lifetime of R123 might be sensitive to the change of viscosity.
R123 has been considered as a satisfactory livecell probe for decades, which could target mitochondria due to its property of probing transmembrane potential. 24,25 In this work, when the 4T1 cells were killed during PTT procedures, the transmembrane potential of mitochondria might decline upon cells death, leading to the release of R123 dye from mitochondria into cytoplasm. As a result, the altered microenviroment might cause the changes of the R123°uorescence lifetime. Therefore, these results suggest that FLIM could provide realtime quantitative data in monitoring PTT triggered cancer cell death.

Summary
In this study, MoS 2 SL and FL nanosheets have been used for PTT in 4T1 cells. The NIR photothermal conversion e±ciency of MoS 2 SL and FL nanosheets reach the satisfactory values, ensuring them strong capabilities as powerful PTT agents. The CCK-8 data show that both the nanosheets have little toxicity and great cell killing e±cacy to 4T1 cells. In addition, FLIM techniques are used to monitor the local environment changes of the mitochondria compartments in live 4T1 cells. The signi¯cant°uorescence lifetime changes in mitochondria of the nanosheet treated cancer cells have been proved to be quantitative measurement methods for representing PTT induced cell death in a non invasive way. Currently, the organelletargeting and image-guided therapy has attracted more and more research interests. 26 Therefore, this work is expected to expand the utility of FLIM for speci¯c subcellular microenvironments into evaluating and examining the development of novel tumor thermo-chemo therapies.

Con°ict of Interest
The authors have declared that no competing interest exists.