Supporting information for Nano Letters Two-dimensional Ultrathin MXene Ceramic Nanosheets for Photothermal Conversion

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1 Supporting information for Nano Letters Two-dimensional Ultrathin MXene Ceramic Nanosheets for Photothermal Conversion Han Lin, 1,2 Xingang Wang, 1 Luodan Yu, 1,2 Yu Chen, 1 * and Jianlin Shi 1 * 1 State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, , P. R. China. 2 University of Chinese Academy of Sciences, Beijing, , P.R. China. chenyu@mail.sic.ac.cn; jlshi@mail.sic.ac.cn 1

2 Table of Contents Part A. Supplementary experimental details Part B. Supplementary figures and discussions Part C. References 2

3 Part A. Supplementary experimental details 1. Synthesis of Ti 3 C 2 nanosheets (MXenes) a. MAX phases Synthesis: The Ti 3 AlC 2 ceramics bulk was made by mixing elemental titanium, Ti (Alfa Aesar, Ward Hill, USA, 99.5 wt % purity; -325 mesh), aluminum, Al (Alfa Aesar, Ward Hill, USA, 99.5 wt % purity; -325 mesh), and graphite, C (Alfa Aesar, Ward Hill, USA, 99 wt % purity; particle size <48 µm, -300 mesh) powders in a 2:1:1 molar ratio. The powders were ball-milled for 10 h and then pressed into cylindrical discs, under pressure of 10 MPa. The discs were heated to 1500 C in a furnace for 2 h, under flowing argon, Ar. b. MXenes Synthesis: The resulting Ti 3 AlC 2 ceramics bulk was crushed using a mortar and pestle. Roughly 10 g powders were then immersed in 80 ml of a 40% HF aqueous solution (Sinopharm Chemical Reagents Co., Ltd., Shanghai, China) for 3 d at room temperature. After collecting by centrifugation and washing with water and ethanol, the precipitation were dispersed in 50 ml TPAOH (Tetrapropylammonium hydroxide 25 wt. % aqueous solution, J&K Scientific Co., Ltd., Beijing, China) under stirring for 3 d at room temperature. Then, the raw Ti 3 C 2 were collected by centrifugation and washed for three times with ethanol and water to remove the residual TPAOH. 2. Surface Modification of Ti 3 C 2 Nanosheets (Ti 3 C 2 -SP) The as-prepared Ti 3 C 2 nanosheets were hydrophilic and could not be used in biological application. For good biocompatibility, a thin-film approach to producing soybean phospholipid (Sigma-Aldrich, Shanghai, China) encapsulated Ti 3 C 2 nanosheets (Ti 3 C 2 -SP). Typically, 2 ml of Ti 3 C 2 nanosheets in ethanol solution (1 mg/ml) was added into 10 ml of 3

4 soybean phospholipid in chloroform solution (1 mg/ml) and then sonicated for 5 min. The mixture was incubated at 60 C under vacuum in a rotary evaporator for 30 min to evaporate the solvent. Subsequently, 10 ml phosphate buffer saline solution was added into the lipidic film and then sonicated for 10 min. 3. Synthesis of PLGA/Ti 3 C 2 Implant PLGA can be dissolved into organic solvent such as N-methylpyrrolidone (NMP) to form an oleosol and Ti 3 C 2 nanosheets also can be uniformly dispersed or dissolved into NMP. Therefore, a PLGA/Ti 3 C 2 oleosol can be obtained by homogenizing PLGA and Ti 3 C 2 into NMP. As a super hydrophobic polymer, PLGA chains in PLGA/Ti 3 C 2 oleosol will undergo an immediate liquid-solid phase transformation upon contacting water in vitro and in vivo (Video. S1). PLGA (Mw = g/mol, lactic acid/glycolic acid ratio of 50:50, Jinan Daigang Bio-technology Co., Ltd., China) was dissolved into NMP (99.5%, Sinopharm Chemical Reagents Co., Ltd., Shanghai, China) and stirred for 24 h to form a homogeneous PLGA NMP solution with a PLGA concentration of 0.5 g/ml. Then, 0.5 ml Ti 3 C 2 nanosheets in NMP solution (3 mg/ml) was then dispersed into the above 0.5 ml of PLGA NMP solution by ultraphonic and magnetic stirring to produce a PLGA/Ti 3 C 2 oleosol (1 ml, 1.5 mg/ml with Ti 3 C 2 concentration). Upon contacting with water, PLGA/Ti 3 C 2 oleosol solidified immediately with the quick diffusion of NMP forming a visible solid (called PLGA/Ti 3 C 2 implant). 4. Characterization X-ray diffraction (XRD) was performed on a Rigaku D/MAX-2200 PC XRD system with Cu Kα radiation (λ = 1.54 Å) at 40 kv and 40 ma. X-ray photoelectron spectroscopy (XPS) 4

5 spectrum was recorded by ESCAlab250 (Thermal Scientific). Size and Zeta potential measurements were measured on Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). Atomic force microscope (AFM) measurement was performed by means of Veeco DI Nanoscope Multi Mode V system. The Ti 3 C 2 concentration was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Agilent Technologies, US). UV-vis-NIR absorption spectra were recorded by UV-3600 Shimadzu UV-vis-NIR spectrometer with QS-grade quartz cuvettes at room temperature. Transmission electron microscopy (TEM) images were acquired on a JEM-2100F electron microscope operated at 200 kv. Scanning electron microscopy (SEM) images/scanning transmission electron microscopy (STEM) images and corresponding element mapping/eds spectrum were obtained on a field-emission Magellan 400 microscope (FEI Company). The confocal laser scanning microscopy (CLSM) images were recorded in FV1000 (Olympus Company, Japan). 5. Photothermal Performance of Ti 3 C 2 Nanosheets Photothermal performance of Ti 3 C 2 nanosheets was measured and analyzed by irradiating an individual hole of a 96-well culture plate containing 300 µl Ti 3 C 2 nanosheets dispersion with different concentrations. NIR laser was produced using an 808 nm high power multimode pump laser (Shanghai Connect Fiber Optics Company). The temperature and thermal images of the irradiated aqueous dispersion were recorded on an infrared thermal imaging instrument (FLIR TM A325SC camera, USA). a. Calculation of the Extinction Coefficient To evaluate the NIR photo-absorption capability of Ti 3 C 2 nanosheets, the extinction 5

6 coefficient α(λ) of the Ti 3 C 2 nanosheets is given, according to the Lambert-Beer Law: A(λ) = αlc (1) where A is the absorbance at a wavelength λ, α is the extinction coefficient, L is path-length (1 cm), and C is the concentration of the Ti 3 C 2 nanosheets (in g/l). The extinction coefficient α is determined by plotting the slope (in L g -1 cm -1 ) of each linear fit against wavelength. b. Calculation of the Photothermal Conversion Efficiency Following Roper [1] report, the total energy balance for the system is, = + (2) where m and C p are the mass and heat capacity of solvent (water), T is the solution temperature, is the energy inputted by Ti 3 C 2 nanosheets, is the baseline energy inputted by the sample cell, and is heat conduction away from the system surface by air. The NIR laser induced source term,, represents heat dissipated by electron-phonon relaxation of the plasmon on the Ti 3 C 2 nanosheets surface under the irradiation of 808 nm laser: = 1 10 (3) Where I is incident laser power (in unit of mw), is the absorbance of the Ti 3 C 2 nanosheets at wavelength of 808 nm, and η is the photothermal conversion efficiency from incident laser energy to thermal energy. In addition, expresses heat dissipated from light absorbed by the quartz cuvette sample cell itself, and it was measured independently using a sample cell containing pure water without Ti 3 C 2 nanosheets. is linear with temperature for the outgoing thermal energy, as the following equation: =h (4) 6

7 where h is heat transfer coefficient, S is the surface area of the container, and is ambient temperature of the surroundings. Once the laser power is defined, the heat input + will be finite. Since the heat output is increased along with the increase of the temperature according to the equation (4), the system temperature will rise to a maximum when the heat input is equal to heat output: + = =h (5) where is heat conduction away from the system surface by air when the sample cell reaches the equilibrium temperature, and is the equilibrium temperature. The 808 nm laser photothermal conversion efficiency (η) can be determined by substituting equation (3) for into equation (5) and rearranging to get = (6) where was measured independently to be mw, the was 18 C according to Figure S6a, I is 280 mw, is the absorbance ( ) of Ti 3 C 2 nanosheets at 808 nm (Figure 2d). Thus, only the hs remains unknown for calculating η. In order to get the hs, a dimensionless driving force temperature, θ is expressed using the maximum system temperature, = (7) and a sample system time constant =, (8) which is substituted into equation (2) and rearranged to yield = (9) At the cooling period of Ti 3 C 2 nanosheets aqueous dispersion, the light source was shut off, 7

8 the + =0, reducing the following equation = (10) and integrating, giving the expression = (11) Therefore, time constant for heat transfer from the system of determined to be = 302 s by applying the linear time data from the cooling stage (400 s) versus negative natural logarithm of driving force temperature (Figure S6b). In addition, the m is 0.3 g and the C is 4.2 J/g. Thus, according to equation (8), the hs is deduced to be 4.17 mw/ C. Substituting this value of hs into equation (6), the 808 nm laser photothermal conversion efficiency (η) of Ti 3 C 2 nanosheets can be calculated to be 30.6%. 6. Photothermal Performance of PLGA/Ti 3 C 2 Implant Photothermal performance of PLGA/Ti 3 C 2 implant was measured and analyzed by irradiating an individual hole of a 96-well culture plate containing PLGA/Ti 3 C 2 implant with different Ti concentrations. NIR laser was produced using an 808 nm high power multimode pump laser (Shanghai Connect Fiber Optics Company). The temperature and thermal images of the irradiated aqueous dispersion were recorded on an infrared thermal imaging instrument (FLIR TM A325SC camera, USA). 7. In Vitro Cytotoxicity Assay Murine breast cancer line 4T1 cells (noted as 4T1 cells, Shanghai Institute of Cells, Chinese Academy of Sciences) were maintained at 37 C under 5% CO 2 in Dulbecco s Modified Eagle s Medium (DMEM, high glucose, GIBCO, Invitrogen) and supplemented with 10% fetal bovine 8

9 serum (FBS), and 1% penicillin/streptomycin in a humidified incubator. 4T1 cells were preferred for the following in vitro and in vivo study. Cells were generally plated in cell culture flask (Corning, USA) and allowed to adhere for 24 h then harvested by treatment with 0.25% trypsin-edta solution (Gibco, USA). In vitro cytotoxicity of Ti 3 C 2 -SP was evaluated by a standard CCK-8 viability assay (Cell Counting Kit, Beyotime Institute of Biotechnology, Shanghai, China) of 4T1 cells. The cells were seeded in 96-well culture plates at a density of cells/well in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 C and 5% CO 2 for 24 h to allow the cells to attach. Then culture medium above was changed with fresh culture medium containing Ti 3 C 2 -SP with different concentration (0, 6, 12, 25, 50, 100, 200, and 400 µg/ml). After 24 or 48 h of incubation, the standard CCK-8 assay was used to evaluate the cell viabilities to the control group. 8. In Vitro Photothermal Ablation of Cancer cells 4T1 cells were first seeded in a 96-well plate at a density of cells/well in DMEM at a 37 C in the presence of 5% CO 2 for 24 h prior to treatment. Thereafter, the culture medium was removed, and then Ti 3 C 2 -SP of different concentrations (0, 6, 12, 25, 50, and 100 µg/ml) dispersed in DMEM were added into the wells. After 4 h of incubation, the culture medium with unbound nanosheets was removed and cells were rinsed three times with PBS (Shanghai Runcheng Bio-tech Co., Ltd.), and fresh complete medium was added into the wells. The cells were then irradiated for 3 min using an 808 nm laser at different output power densities (0, 0.25, 0.5, 1.0, 1.5, and 2.0 W cm -2 ). Finally, CCK-8 assay was used to evaluate the cell viabilities. Five replicates were done for each treatment group. 9

10 9. Confocal Fluorescence Imaging 4T1 cells were seeded in CLSM-exclusive culture dishes with a density of cells/well and allowed to adhere overnight. For examining the photothermal effect of Ti 3 C 2 -SP to 4T1 cells in vitro, DMEM solution of Ti 3 C 2 -SP (100 µl) was added into the dishes and subsequently incubated for 4 h, when cells of each disk reached 80% 90% confluence. The adherent cell solution was irradiated by an 808 nm laser for 5 min under a power density of 1.0 W cm -2. After removal of the medium, the adherent cells were rinsed with PBS for three times. 4T1 cells were incubated with calcein-am (100 µl) and PI solution (100 µl) for 15 min. Living cells and dead cells were stained with calcein-am (green fluorescence) and PI (red fluorescence) solution, respectively. 10. In Vivo Toxicity Assay Animal experiment procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments and the care regulations approved by the administrative committee of laboratory animals of Fudan University. Healthy female Kunming mice (~ 20 g) were obtained and raised at Laboratory animal center, shanghai medical college of Fudan University. These 4-week old mice were randomly divided into four groups (n = 7 for each group) and then intravenously administered with different doses: control, Ti 3 C 2 -SP in PBS (5 mg/kg), Ti 3 C 2 -SP in PBS (10 mg/kg) and Ti 3 C 2 -SP in PBS (20 mg/kg). In a month period, the body weight of mice was measured every two days and no significant behavioral changes were observed in three experimental groups compared to control group. The mice were anesthetized and dissected in 30 days of post-injection. The blood samples were collected for serum 10

11 biochemistry assay and complete blood panel test. Then the mice major organs (heart, liver, spleen, lung and kidney) were dissected, fixed in a 10% formalin and stained with hematoxylin and eosin (H & E) for histological analysis. 11. In Vivo Blood circulation and Biodistribution For blood circulation experiments, female Kunming mice were injected intravenously with Ti 3 C 2 -SP in PBS (n = 6). 15 µl blood was collected at varied time (2 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h) after injection. The blood was dispersed into 1 ml physiological saline contained heparin sodium injection (50 unit/ml). The concentration of Ti 3+ was measured by ICP-AES. The in vivo blood terminal half-life of Ti 3 C 2 -SP was calculated by a double-component pharmacokinetic model. Biodistribution of Ti 3 C 2 -SP in organs and tumor was performed in 4T1-bearing nude mice (n = 6). Mice were dissected after intravenous injection for 24 h of Ti 3 C 2 -SP in PBS (0.1 ml). Dissected organs and tumor were weighed and homogenized. The Ti 3 C 2 -SP distribution in different organs and tumor was calculated as the percentage of injected dose per gram of tissue. 12. In Vivo Photothermal Therapy by Intravenous Injection of Ti 3 C 2 -SP 4-week old female Balb/c nude mice (~ 14 g) were obtained and raised at Laboratory animal center, shanghai medical college of Fudan University. To develop the tumor model, Murine breast cancer line 4T1 cells ( cell/site) suspended in PBS (100 µl of dose for each mouse) were implanted subcutaneously into the back of mice. In vivo PTT by intravenous injection was performed when the tumors volume reached ~100 mm 3. The mice were randomly divided into four groups (n = 5 per group): The first group, as control group, was intravenously injected the saline solution (dose of 20 mg/kg); the second group, as Ti 3 C 2 -SP group, was 11

12 intravenously injected with Ti 3 C 2 -SP in PBS (dose of 20 mg/kg) without NIR laser irradiation; the third group, as NIR laser group, was only irradiated by NIR laser (808 nm) with a power density of 1.5 W cm -2 for 10 min; the fourth group, as Ti 3 C 2 -SP + NIR laser group, was intravenously injected with Ti 3 C 2 -SP in PBS (dose of 20 mg/kg) and then exposed to NIR laser with a power density of 1.5 W cm -2 for 10 min. Four hours after intravenous injection, the NIR laser irradiation could be carried out, and the mice were anesthetized before NIR laser irradiation. The volume of the tumors were monitored by a digital caliper every day during half a mouth after the corresponding experiments. The tumor volume was calculated according to the following formula: length width width / 2. The temperature and thermal images of the irradiated mice were recorded on an infrared thermal imaging instrument (FLIR TM A325SC camera, USA). The tumors were dissected after the corresponding treatments and fixed in a 10% formalin. Then the tumor issues were sectioned into slices and stained with H & E, TUNEL and Ki-67 for histological analysis. 13. In Vivo Photothermal Therapy by Intratumoral Injection of PLGA/Ti 3 C 2 Implant 4-week old female Balb/c nude mice (~ 14 g) were obtained and raised at Laboratory animal center, shanghai medical college of Fudan University. To develop the tumor model, Murine breast cancer line 4T1 cells ( cell/site) suspended in PBS (100 µl of dose for each mouse) were implanted subcutaneously into the back of mice. In vivo PTT by intratumoral injection was performed when the tumors volume reached ~100 mm 3. The mice were randomly divided into two groups (n = 5 per group): The first group, as control group, was intratumorally injected the saline solution (100 µl); the second group, as PLGA/Ti 3 C 2 implant + NIR laser group, was intratumorally injected the PLGA/Ti 3 C 2 oleosol (dose of 2 mg/kg) and then exposed 12

13 to NIR laser with a power density of 1.0 W cm -2 for 6 min. The mice were anesthetized before NIR laser irradiation. The volume of the tumors were monitored by a digital caliper every day during half a mouth after the corresponding experiments. The tumor volume was calculated according to the following formula: length width width / 2. The temperature and thermal images of the irradiated mice were recorded on an infrared thermal imaging instrument (FLIR TM A325SC camera, USA). The tumors were dissected after the corresponding treatments and fixed in a 10% formalin. Then the tumor issues were sectioned into slices and stained with H & E, TUNEL and Ki-67 for histological analysis. 13

14 Part B. Supplementary figures and discussions Figure S1. Transmission electron microscopy (TEM) images of HF-etched Ti3C2 powder at different magnifications. Figure S2. (a-c) High-resolution transmission electron microscopy (HRTEM) analysis of intercalated Ti3C2 nanosheets with one layer to three layers. (d) High-resolution TEM images (HRTEM) of intercalated Ti3C2 nanosheets. Inset in d is the SAED of Ti3C2 nanosheets. 14

15 Figure S3. XPS spectra of Ti 3 AlC 2 before and after HF etching. Figure S4. Elemental analysis of intercalated Ti 3 C 2 nanosheets was measured by TEM-EDS, and the presence of Cu content result from Cu grid. Table S1. XPS survey of Ti 3 C 2 nanosheets (Ti 3 AlC 2 sample after HF etching) 15

16 Figure S5. Schematic illustration of the theoretical atom model of Ti 3 AlC 2 phase. Figure S6. (a) Photothermal effect of aqueous dispersion of Ti 3 C 2 nanosheets under irradiation with the NIR laser (808 nm, 1.0 W cm -2 ), in which the irradiation lasted for 400 s, and then the laser was shut off. (b) Time constant for heat transfer from the system is determined to be = 302 s by applying the linear time data from the cooling period (after 400 s) versus negative natural logarithm of driving force temperature, which is obtained from the cooling stage of panel a. 16

17 Figure S7. Photothermal property and stability of Ti 3 C 2 nanosheets. Ti 3 C 2 nanosheets of varied concentrations (72, 36, 18, 9, 4.5 ppm and pure water) were exposed to an 808 nm laser at different densities of 0.25 W cm -2 (a), 0.5 W cm -2 (b) and 1.0 W cm -2 (c). (d) Recycling heating profile of a Ti 3 C 2 nanosheet suspension dispersed in water (36 µg/ml, 100 µl) with an 808 nm laser irradiation (1.0 W cm -2 ) for five laser on/off cycles. Figure S8. (a) Dynamic light scattering (DLS) and (b) Zeta potential profile of Ti 3 C 2 nanosheets and Ti 3 C 2 -SP nanosheets dispersed in water. 17

18 Figure S9. Scanning transmission electron microscopy (STEM) analysis of ultrathin Ti 3 C 2 -SP nanosheets with (a) bright-field image, (b) dark-field image, (c) HAADF image and (d) secondary electron image. (e-h) The corresponding element-linear scanning of ultrathin Ti 3 C 2 -SP nanosheets. The obvious signal of P element from SP indicates the coating of SP on the surface of Ti 3 C 2 nanosheets. Figure S10. Digital images of Ti 3 C 2 nanosheets and Ti 3 C 2 -SP nanosheets dispersed in various solvents. 18

19 Table S2. Polydispersity index (PDI) of Ti 3 C 2 -SP nanosheets dispersed in different solvents (H 2 O, PBS, SBF, Saline and DMEM) at time point of 0, 1, 2, 4, 8, 12, 24, 48, and 72 hour. Figure S11. Photothermal-heating curves and stability of Ti 3 C 2 -SP nanosheets. (a) Ti 3 C 2 -SP nanosheets of varied concentrations were exposed to an 808 nm laser at different densities of 1.5 W cm -2. (b) Recycling heating profile of a Ti 3 C 2 -SP nanosheet suspension dispersed in water (72 µg/ml, 100 µl) with an 808 nm laser irradiation (1.5 W cm -2 ) for five laser on/off cycles. 19

20 Figure S12. Confocal laser scanning microscopic (CLSM) images of 4T1 cells incubated with Ti 3 C 2 -SP at the concentration of 200 µg/ml for 0, 1, 2, 4, and 8 h. For each panel, the images from top to bottom show cell nuclei stained by DAPI (blue: DAPI = 4, 6-diamidino-2-phenylindole), FITC fluorescence from Ti 3 C 2 -SP in cells (green), the overlays of the former two images, three-dimensional (3D) confocal fluorescence reconstructions of Ti 3 C 2 -SP endocytosed 4T1 cells, and 2D mappings of FITC fluorescence intensity of selected regions. All images share the same scale bar (50 µm). 20

21 Figure S13. Hematological assay of mice from the control group and three treatment groups (at different Ti 3 C 2 -SP dosages of 5 mg/kg, 10 mg/kg and 20 mg/kg) for 30 days. 21

22 Figure S14. Temperature elevation at the tumor site of nude mice in groups of control and Ti 3 C 2 -SP (i.v.) + NIR laser during laser irradiation. Figure S15. Digital photos of 4T1 tumor-bearing mice in the groups of the control, Ti 3 C 2 -SP only, NIR laser only and Ti 3 C 2 -SP + NIR laser taken during 16 days period after different treatments. 22

23 Figure S16. Photographs of 4T1 tumor-bearing mice and tumor region (the control group and Ti 3 C 2 -SP + NIR laser group) at the 16 th day. Figure S17. In vitro phase transformation of PLGA/Ti 3 C 2 oleosol. (a) PLGA/Ti 3 C 2 oleosol in a syringe. (b, c) The oleosol dropped into water and underwent a rapid liquid-solid phase transformation to from a solid implant. (d) Solid PLGA/Ti 3 C 2 implant with elasticity. (e) Solid PLGA/Ti 3 C 2 implant after high temperature treatment shows that the Ti 3 C 2 nanosheets are uniformly distributed in the PLGA oleosol. 23

24 Figure S18. (a, b) Field emission scanning electron microscopy (FESEM) image of PLGA/Ti 3 C 2 implant. (c, e, f) Corresponding element mappings of Ti, O and C. (d) Merged image of FESEM image of PLGA/Ti 3 C 2 implant and element mapping of Ti. Figure S19. Photothermal performance of PLGA/Ti 3 C 2 implant under exposure to an 808 nm laser of different power densities based on varied Ti 3 C 2 concentrations of (a) 0.5 mg/ml, (b) 1 mg/ml and (c) 2 mg/ml. 24

25 Figure S20. (a) Scheme of intratumoral administration of PLGA/Ti 3 C 2. (b) Tumor tissue extracted from mouse after the intratumor injection of PLGA/Ti 3 C 2 implant. (c) Cross section of tumor tissue showing the distribution of PLGA/Ti 3 C 2 implant in the solid tumor. Figure S21. The temperature elevation at the tumor site of nude mice in groups of control and PLGA/Ti 3 C 2 implant + NIR laser during laser irradiation. Figure S22. Digital photos of 4T1 tumor-bearing mice in groups of control and PLGA/Ti 3 C 2 implant taken within 16 days period after different treatments. 25

26 Part C. References (1) D. K. Roper, W. Ahn, M. Hoepfner, J. Phys. Chem. C 2007, 111,