Flower-like PEGylated MoS2 nanoflakes for near-infrared photothermal cancer therapy

Photothermal cancer therapy has attracted considerable interest for cancer treatment in recent years, but the effective photothermal agents remain to be explored before this strategy can be applied clinically. In this study, we therefore develop flower-like molybdenum disulfide (MoS2) nanoflakes and investigate their potential for photothermal ablation of cancer cells. MoS2 nanoflakes are synthesized via a facile hydrothermal method and then modified with lipoic acid-terminated polyethylene glycol (LA-PEG), endowing the obtained nanoflakes with high colloidal stability and very low cytotoxicity. Upon irradiation with near infrared (NIR) laser at 808 nm, the nanoflakes showed powerful ability of inducing higher temperature, good photothermal stability and high photothermal conversion efficiency. The in vitro photothermal effects of MoS2-PEG nanoflakes with different concentrations were also evaluated under various power densities of NIR 808-nm laser irradiation, and the results indicated that an effective photothermal killing of cancer cells could be achieved by a low concentration of nanoflakes under a low power NIR 808-nm laser irradiation. Furthermore, cancer cell in vivo could be efficiently destroyed via the photothermal effect of MoS2-PEG nanoflakes under the irradiation. These results thus suggest that the MoS2-PEG nanoflakes would be as promising photothermal agents for future photothermal cancer therapy.

A culture medium without nanoflakes was used as the blank control. After discarding the medium with or without MoS 2 -PEG samples, the cells were washed twice with phosphate buffer saline (PBS) to remove the residual MoS 2 -PEG, 90 μL fresh RPMI 1640 serum-free medium containing 10 μL CCK-8 solutions was added and the cells were incubated for another 2 h. The absorbance of each well was monitored at 450 nm on a microplate reader (MK3, Thermo, USA). The results of cytotoxicity were expressed as the percentage of cell viability. The relative cell viability was expressed as the Equation 2, and five parallel experiments were carried out for each treatment group. (1) Where

In vitro photothermal ablation of MoS 2 -PEG nanoflakes against HeLa cells.
HeLa cells were first plated in a 96-well plate at a density of 10 4 cells per well for 24 h to allow cell attachment. Thereafter, the culture medium was removed, and cells were divided into four groups: group I, blank control cells; group II, MoS 2 -PEG nanoflakes (60 μg/mL) alone; group III, NIR 808-nm treatment only; and group IV, MoS 2 -PEG nanoflakes (60 μg/mL) + NIR 808-nm. At the end of incubation for 2 h, the cells of group III and IV were exposed to an 808-nm laser at a power density of 2.0 W/cm 2 for 10 min. At the end of incubation for 2 h, the cells of group III and IV were exposed to an 808-nm laser at a power density of 2.0 W/cm 2 for 10 min. After irradiation treatment, all the cells were then incubated at 37 °C for another 24 h. CCK-8 assay was performed to evaluate the cell viabilities.
For acridine orange (AO)/propidium iodide (PI) staining, 4T1 cells were seeded and divided into four groups as described above. After different treatment, culture medium was discarded and 4T1 cells were rinsed twice with PBS. 300 μL of dye mixture containing 50 nM AO and 300 nM PI were then added to the wells. After incubation for 10 min, the sample was washed by PBS solution and the images of the labeled cells were observed immediately by using a fluorescence microscope (Olympus IX71). Each experiment was performed three times.

Photothermal conversion efficiency calculation
The photothermal conversion efficiency (η) of MoS 2 -PEG nanoflake was calculated according to the previously reported methods, detailed calculation as following: 3,4 During the photothermal heating process, the total energy balance for the system can be expressed as: For Q NF , Equation (4) can be given as: Where I = 2000 mW/cm 2 is the laser power which is incident on the system, A 808 is defined as the absorbance of the MoS 2 -PEG nanoflakes at the wavelength of 808 nm, and η is known as the photothermal conversion efficiency from the absorbed laser energy to thermal energy.

S8
Furthermore, the energy dissipation mainly occurs through the heat conduction and thermal radiation. Q Loss is linear with temperature for the outgoing thermal energy, then take the form as Equation (5): Where h (mW/(m 2 ·°C )) is heat transfer coefficient, S (m 2 ) is the surface area of the container, △T is the temperature change which is defined as T-T sur , T (°C ) is the water temperature and T Surr (°C ) is the solution temperature ambient temperature of surrounding environment.
When the temperature rises at a maximum steady-state temperature T Max (°C ), the system reaches the steady state. In this case, the heat input is equal to heat output, and the left side of Equation (3) becomes zero. So we then obtain Then η can be determined by combining Equation (3-6) and rearranging: (1−10 − 808 ) Where Q S is measured independently to be 9.0 mW, the (T Max -T Sur ) is 46.7°C , I is 2000 mW/cm 2 , A 808 is 0.96. Thus, in the Equation (7), only the hS remains unknown parameter for calculating η.
In order to solve hS, the following notation θ is used herein, which is defined as the ratio of (T-T surr ) to (T Max -T sur ): And a sample system time constant τ s (s) is introduced: Substituting Equations (8) and (9) into Equation (3) and rearranging to obtain: When at the cooling stage of MoS 2 -PEG nanoflakes aqueous dispersion, the laser source has been shut off, so the Q NF + Q S = 0. Under this condition, Equation (10) becomes: Note that after integration Equation (11), the Equation expresses as: Therefore, time constant for heat transfer from the system is determined to be τ s = 157s by applying the linear time data from the cooling period (after 300 s) vs -lnθ ( Fig. 3D). In addition, the m is 0.4 g and the C is 4.2 J/g°C. Thus, according to Equation (9), the hS is calculated to be 10.7 mW/°C . Substituting hS = 10.7 mW/°C into Equation (7), the result photothermal conversion efficiency (η) of MoS 2 -PEG nanoflakes can be calculated to be 27.6 %.