All starting materials were obtained from commercial supplies and used without further purification. The chemicals of Sodium tetrachloropalladate(II) (Na2PdCl4), Sodium bromide (NaBr), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), Ascorbic acid (AA) and Hyaluronic acid (HA) were purchased from Macklin Co., Ltd. The chemicals of acetone, polyvinylpyrrolidone (PVP, 58000w), N,N-Dimethylformamide (DMF), Ethanol and Acetic acid from Aladdin Co., Ltd. (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Beyotime Biotech Co., Ltd. (China). Calcein AM/PI Kit and Annexin V-FITC/PI Apoptosis Detection Kit was obtained from Shanghai Bestbio (China). Ultrapure water was used throughout.
Synthesis Of Pd Nanocubes
PVP (53 mg), AA (30 mg), NaBr (130 mg) were dissolved into ultrapure water (4 mL), the mixture was then stirred at 80 ℃ over 5 minutes. 30 mg of Na2PdCl4 (dissolved in 1.5 mL of ultrapure water) was poured in the above mixture and stirred for 3 hours. After been cooled to room temperature, Pd nanocubes were collected by centrifugation and then stored it in 1mL of DMF solution.
3 mL of the synthesized Pd nanocubes were added to 2 mL DMF solution containing 100 mg Zr6 clusters), then the solution was stirred at room temperature for 4 hours (solution A). TCPP (50 mg) was then dissolved into DMF solution (5 mL) and dispersed by ultrasound (solution B). Then acetic acid (6 mL) was added to mixed mixture of solution A and solution B, and stirred for 12 hours. Finally, the product was collected after centrifugation and washing.
HA (10 mg) was dispersed in the ultrapure water (100 mL), 5 mg of [email protected] was added after ultrasonic. After stirring for 24 hours, washed with ultrapure water, and the final product was stored in ultrapure water.
UV-Vis absorption spectra were recorded on a UV-265 spectrophotometer. SEM was detected by REGULUS8230*. TEM was carried on a JEM-2100. PXRD patterns were recorded on SmartLab 9KW. Fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer. One-photon and two-photon imaging data acquisition and processing were performed using Lecia TCS SP8 DIVE FALCON which equipped with single-wavelength laser and femtosecond laser (adjustable output wavelength: 680 - 1080 nm, 80 MHz, 140 fs).
Singlet Oxygen (O) Detection
The 1O2 was detected by 9,10-anthracenedipropanoic acid (ABDA, a singlet oxygen sensor) due to the generated 1O2 would react with ABDA and reduce the absorbance around 378 nm. [email protected]@HA (50 µg mL−1), ABDA (100 µM) and H2O2 (100 µM) were incubated together under white light and ultrasound (1.2 W cm−2) irradiation within 0-5 minutes. The absorbance of the mixture was measured at the different time.
Electron Spin Resonance (Esr) Assay
The spin traps 2,2,6,6-tetramethylpiperidine (TEMP, trapping 1O2, 20 µL) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, trapping •OH, 20 µL) were employed to detect the species of ROS generated by [email protected]@HA (50 µg mL−1). The ESR signals of the [email protected]@HA before and after LED light (range from 400 to 700 nm, 40 mW cm−2) and ultrasound (1.2 W cm−2) irradiation were recorded.
Cellular Uptake Analysis
QSG-7701 cells (CD44-negative) and HepG2 cells (CD44-positive) were seeded onto the cell culture dishes and grown to about 70% confluency for next using. QSG-7701 cells and HepG2 cells were treated with [email protected]@HA (100 µg mL−1, and another dish HepG2 cells were precultured with 10 times of HA before incubation with [email protected]@HA. And after 8 hours of incubation, the cellular uptake ability of [email protected]@HA were analyzed using CLSM.
Cytotoxicity Assays In Cells
The PDT/CDT/SDT effect of [email protected]@HA was studied by the methylthiazolyldiphenyltetrazolium bromide (MTT) assay. The [email protected]@HA stock solution is diluted with fresh medium to the required concentration (0, 30, 60, 90, 120, 150 µg mL−1). Before the experiment, HepG2 cells were cultured for 24 hours in 96-well plates. Then exchange the cell culture medium with different concentrations of [email protected]@HA medium solution. They were incubated at 37°C for 8 hours in 5% CO2 atmosphere, and then irradiated by laser (800 nm, 1 W cm−2) and ultrasound (1.2 W cm−2) for 15 min. 100 µL of fresh medium were used to exchange the cell medium solutions 20 µL (5 mg mL−1) MTT solution were added to each well following. The cell plates were then incubated for another 4 hours. After removing the MTT medium, the formazan crystals were dissolved in DMSO (100 µL well−1) and the absorbance was detected at 490 nm using a microplate reader. And duplicated experiments have been tested.
Singlet Oxygen Detection In Cells
HepG2 cells were treated with [email protected]@HA (100 µg mL−1) for 8 hours, and then incubated with 1 µM singlet oxygen sensor green (SOSG) for 10 minutes. Next, HepG2 cells were washed with PBS and irradiated for 15 minutes under laser (800 nm, 1 W cm−2) and ultrasound (1.2 W cm−2). The green fluorescence was observed by CLSM with the excitation wavelength of 504 nm (λem: 500-550nm).
Live/dead Assay With Calcein Am/pi
After the HepG2 cells were washed with PBS solution twice, [email protected]@HA (100 µg mL−1) was added to the above medium and incubated for 8 hours, and then the cells were treated under different conditions. Calcein AM and PI were added to detect the cells vitality of HepG2 cells. Fluorescence images are collected by CLSM.
Determination Of Annexin V-fitc And Pi
HepG2 cells were incubated with [email protected]@HA (100 µg mL−1) at 35°C and 5% CO2 for 8 hours. After adding H2O2, they were irradiated with laser (800 nm, 1 W cm−2) and ultrasound (1.2 W cm−2) for 15 minutes. Then, the Annexin V-FITC (1 µM) and PI (1 µM) were added and incubated for 20 minutes. Fluorescence images of the cells were collected by a confocal laser scanning microscope.
Flow Cytometry Study
Cells seeded into the 6-well plates were incubated for 24 hours, the medium containing [email protected]@HA (100 µg mL−1) was used.. After irradiated with laser (800 nm, 1W cm−2) and ultrasound (1.2 W cm−2) 15 minutes, the cells were collected after centrifugation and then resuspended in binding buffer containing Propidium Iodide (PI, 10 µL) and Annexin-V FITC (5 µL) for 15 minutes in darkness. The signal was collected by a BD FACS Calibur flow cytometer (Beckaman/Gallios).
The One/two-photon Fluorescence Imaging Study Of [email protected]@ha
A Lecia TCS SP8 DIVE FALCON which equipped with single-wavelength laser and femtosecond laser (adjustable output wavelength: 680 - 1080 nm, 80 MHz, 140 fs) was employed to achieve one/two-photon fluorescence imaging. HepG2 cells were treated with [email protected]@HA for 8 hours. And then, slices were prepared from cardiac muscle tissue in Balb/c mice. The tissue sections were cut to 200 mm thickness. The tissue sections was incubated with [email protected]@HA for 30 minutes. The one-photon fluorescence emission was observed excitation at 458 nm (0.2 W/cm2). The two-photon fluorescence emission was observed excitation at 800 nm (0.2 W/cm2).