Reagents and materials
DSPE-PEG2000 were obtained from Avanti Polar Lipids Inc. (Alabama, USA). ICG were obtained from Aladdin Chemical Co (Shanghai, China). Fetal bovine serum (FBS), penicillin/streptomycin (PS), and trypsin were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Other materials from Sigma-Aldrich (Saint Louis, MO, USA) were used without additional treatment.
Characterization
High-resolution transmission electron microscopy image of TPSe nanoparticles (NPs) was acquired by a Talos F200X transmission electron microscope. Particle size was obtained by a Malvern Zetasizer Nano ZS 90. UV-VIS absorption spectra was obtained by employing a HACH DR6000 UV-vis spectrophotometer. Infrared thermal image was collected using a Fluke Ti480 Infrared Camera.
Preparation of TPSe
Compound 4-(octyloxy)-N-(4-(octyloxy)phenyl)-N-(4-(5-(trimethylstannyl)thiophen − 2-yl)phenyl)aniline was synthesized according the reference [18], compounds 4,8-dibromo-6-(2-ethylhexyl)-[1, 2, 5]selenadiazolo[3,4-f]benzotriazole were obtained from WUXI Senior material (Wuxi, China).
Compound 4-(octyloxy)-N-(4-(octyloxy)phenyl)-N-(4-(5-(trimethylstannyl)thiophen-2-yl)phe nyl)aniline (149 mg, 0.2 mmol) and compound 4,8-dibromo-6-(2-ethylhexyl)-[1, 2, 5]selenadiazolo[3,4-f]benzotriazole (99 mg, 0.2 mmol) were added into the two-neck reactor, and then 10 mL of degassed toluene was added with a 10 min of N2 flushing. After that, 20 mg Pd(PPh3)4 catalyst was added into the noted mixture with the protection of N2. Afterward, the solution was directly heated and held the temperature of 110 ℃ for 24 h under N2 environment. The resulted crude product was firstly extracted using CH2Cl2, and further purified by column chromatography to offer a dark green solid (126.6 mg, 51%). 1H nuclear magnetic resonance (NMR) (400 MHz, Toluene-d8) δ 9.17 (d, J = 4.1 Hz, 2H), 7.67 (d, J = 8.7 Hz, 4H), 7.40 (d, J = 4.1 Hz, 2H), 7.12 (d, J = 5.9 Hz, 4H), 7.09 (t, J = 4.3 Hz, 8H), 6.80 (d, J = 8.9 Hz, 8H), 4.47 (d, J = 6.5 Hz, 2H), 3.67 (t, J = 6.4 Hz, 8H), 1.69–1.61 (m, 8H), 1.39–1.21 (m, 49H), 0.94–0.89 (m, 18H); 13C NMR (101 MHz, Toluene-d8) δ 155.93, 148.69, 143.33, 140.73, 137.51, 136.80, 132.67, 128.73, 128.50, 128.26, 127.83, 127.59, 127.35, 126.75, 126.53, 125.00, 124.76, 124.52, 120.81, 115.36, 67.84, 31.97, 29.55, 29.51, 29.45, 26.24, 23.06, 22.80, 14.01, 10.41ppm; high-resolution mass spectrometry (HRMS): m/z: [M] + calcd for C90H113N7O4S2Se, 1499.75; found, 1499.74556.
Preparation of TPSe NPs
TPSe molecule (0.5 mg) and DSPE-PEG2000 (0.5 mg) were weighted and dissolved in THF (1 mL). The solution was mixed thoroughly under sonication, and then injected into deionized water (9 mL) to obtain TPSe NPs under vigorous stirring. The obtained solution was successively purified with 0.22 µm filtration and concentrated by using centrifugal filter unit with a size of 100 kDa. The final NPs dispersion was kept at 4 ℃ until further experiments.
Examination of photothermal performance
The examination of PCE was performed as described in our previous report [41]. Typically, a 0.8 mL amount of TPSe NPs solutions with increased concentration from 0 to 40 µg mL− 1 was illuminated at 1 W cm− 2 for 6 min using an 808 nm light. After steady state temperature, excitation light was turned off until the temperature returns to the initial state. Thermal images were acquired and analyzed by a Fluke Ti480 Infrared Camera. The calculation of η was briefly described as following Eq. (1):
(1)
$$\eta =\frac{hS\left({T}_{max}-{T}_{surr}\right)-{Q}_{DI}}{I(1-{10}^{-{A}_{\lambda }})}$$
where η stands for photothermal conversion efficiency, h is the heat transfer coefficient, S is the surface area of cuvette exposed to laser irradiation. Tmax and Tsurr stand for the temperature reached at thermal equilibrium state and the surrounding temperature, respectively. QDI stands for the energy input from deionized water in the detected solution. I is the power density of 808 nm laser, Aλ is the absorbance intensity of TPSe NPs solution upon irradiation. hS could be calculated according to Eq. (2):
(2)
$${\tau }_{S}=\frac{{m}_{D}{C}_{D}}{hS}$$
where, mD and CD stand for the mass (0.8 g) and heat capacity (4.2 J g− 1) of DI water, respectively. τs stands for the time constant for heat transfer, which could be calculated according to Eq. (3):
(3)
$${t=-\tau }_{S}\text{ln}\left(\theta \right)={-\tau }_{S}\text{ln}\left(\frac{{T}_{t}-{T}_{Surr}}{{T}_{Max}-{T}_{Surr}}\right)$$
where, t stands for the cooling time points after laser irradiation for 10 mins, Tt stands for the recorded temperature of TPSe NPs solution during the cooling stage.
In vitro photostability test
To evaluate the photostability of TPSe NPs, NPs of 40 µg mL− 1 (0.8 mL) were illuminated at 1 W cm− 2 under 808 nm light for 1 h, while ICG of 30 µg mL− 1 was selected as the control sample. Furthermore, photothermal conversion capability of TPSe NPs solution was investigated under ten laser on/off irradiation cycles, and ICG was selected as the control group.
In vitro cytotoxicity of TPSe NPs
The cytotoxic effects of NPs on various cancer cell lines (HCT116, A549, 4T1, MCG-803, HepG2) were evaluated by CCK-8 assay (Beyotime Biotech Inc, China). Typically, the HCT116 human colorectal cancer cells were suspended in 1640 medium supplemented with 10% FBS and 1% PS in an incubator at 37°C, 5% CO2 atmosphere. Cell (5 × 103 cells/well) was cultured in a 96-well plate for 24 h and then challenged with NPs of various concentrations (6.25-30 µg mL− 1) for 4 h. After 5 min irradiation with or without an 808 nm laser (0.5 W cm− 2), cells were cultured for a further 24 h. Afterward, cell viability was determined using CCK-8 kits to evaluate the cytotoxicity of NPs.
Live-dead cell staining
In total, cells (2 × 105) were plated in confocal dishes. After attachment, the cell was treated with PBS or NPs (30 µg mL− 1) and then cultured for 4 h. Afterward, cells were illuminated with or without an 808 nm light for 5 min (0.5 W cm− 2). Cells were washed with PBS three times, followed by incubation with Calcein-AM/Propidium Iodide (Beyotime Biotechnology) in the dark for 30 min. After washing twice using PBS, fluorescence was monitored through a DMi8 Platform Live cell microscope (Leica Microsystems, Germany).
Tumor mouse model
All mice procedures were conducted according to the Animal Management and Ethics Committee of Soochow University. BALB/C nude mice of 4∼6 weeks were provided by Beijing Vital River Laboratory and housed for 1 week. Afterward, the HCT116 cell was injected in the right upper limb of nude mice (2 × 107 cells mL− 1, 100 µL), when the cell growth density is about 80%. Once tumors reached ~ 100 mm3, mice were subsequently used for NIR FLI and antitumor tests.
In vivo NIR-II FLI
In vivo NIR-II fluorescence image was acquired using an NIR-II imaging equipment with indium-gallium-arsenide (InGaAs) array. The equipment parameters were fixed and used for the following in vivo imaging (excitation wavelength 808 nm; power density 60 mW cm− 2; long pass filter 1050 nm; exposure time 300 ms). The BALB/c nude mice were injected with TPSe NPs (100 µL, 0.5 mg mL− 1) intravenously (n = 3) when the tumor volume is about 100 mm3. And they were kept anaesthetized via a nose cone during injection and imaging acquisition, which delivered 2 L min− 1 oxygen flow mixed with 3% isoflurane. NIR-II images of different tissues and tumors of the animals were acquired after 36 hours postinjection. The calculation of fluorescence signal intensities of images obtained was quantitatively performed using Image J. The biodistribution of TPSe NPs was analyzed based on the fluorescent intensity of excised organs.
In vivo antitumor activity
To investigate anti-tumor activity of NPs in vivo, the established tumor models were randomized into different groups (n = 4). The mice in experimental group were irradiated upon 808 nm light for 5 min with 0.5 W cm− 2 at 24 h after intravenous administration of either 100 µL of 0.5 mg mL− 1 NPs [TPSe NPs (0.5 mg mL− 1) + L] or with 100 µL of 1 mg mL− 1 NPs [TPSe NPs (1.0 mg mL− 1) + L]. Mice administrated with PBS, mice administrated with PBS and illuminated with laser (PBS + L), and mice injected with 0.5 mg mL− 1 of NPs [TPSe NPs (0.5 mg mL− 1)] were used as the control. The tumor temperatures were observed and recorded using a thermal camera. Tumor growth and mouse weight were monitored every 2 days during the whole experiment.
Biosafety assessment
On day 12, the tumor tissues were taken for weight measure, and several other tissues, including heart, liver, spleen, lung, kidney were obtained for H&E assay. The hematologic and biochemistry analysis of blood samples were performed using an automated hematology analyzer.