Cell culture
Human colon cancer cell line HCT116 and RKO, and normal colon cell line NCM460 purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) were used in this study. HCT116 and RKO cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with high glucose (HyClone, Logan, UT, USA), while NCM460 cells were cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium (GIBCO, Invitrogen, Carlsbad, CA, USA). Both media were supplemented with 10% fetal bovine serum (FBS; BI, Biological Industries, Israel) and 1% penicillin and streptomycin (GIBCO, Invitrogen, Carlsbad, CA, USA). Cells were maintained in an incubator at 37 °C under a humidified atmosphere with 5% CO2. Cells at the exponential growth phase were used for all experiments.
DVDMS
DVDMS (molecular formula: C68H66N8O9Na4, molecular weight: 1230.265) with purity as high as 98.5% was generously provided by Jiangxi Qinglong Hi-tech Co., Ltd (Jiangxi, China). DVDMS was dissolved in phosphate-buffered saline (PBS, pH 7.2, BioScience, Shanghai, China) at a storage concentration of 2 mg/mL and sterilized using a 22-µm flitter (Jet Bio-Filtration Co., Ltd., Guangzhou, China), followed by storage in the dark at − 20 °C prior to use. The chemical structure of DVDMS is shown in Fig. 7.
Spectral analysis of DVDMS
The absorption spectra of DVDMS at 10 µg/mL concentration in PBS were recorded between 300 and 700 nm wavelength using an ultraviolet spectrophotometer system (G6860A, Agilent Technologies, Malaysia), while its emission spectra at various concentrations (5, 10, 20, 30, and 40 µg/mL) in PBS were recorded using a microplate reader (BioTek, Winooski, VT, USA) under an appropriate excitation wavelength at 37 °C.
Cytotoxicity assessment of DVDMS
HCT116, RKO and NCM460 cells were harvested and seeded in a 96-well plate at a density of about 1 × 104 cells/well. After incubation for 24 h, the cells were treated with DVDMS at different concentrations (0, 1, 2, 5, 10, 15, and 20 µg/mL). Cell viability was analyzed 6 h post-incubation with DVDMS using CCK8 assay, which is based on that WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt) is reduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells and is measured by absorbance at 450 nm. In brief, each well was treated with a mixture of 10 µL CCK8 solution (Solarbio, Beijing, China) and 90 µL serum-free medium after washing the cells twice with PBS, and the plate was incubated for 1 h at 37 °C. Optical density (OD) was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). Cell survival rate was calculated using the following equation:
$${\text{Cell survival }}\left( \% \right) \, = \, \left( {{\text{OD}}_{\text{treatment}} - {\text{ OD}}_{\text{blank}} } \right)/\left( {{\text{OD}}_{\text{control}} - {\text{ OD}}_{\text{blank}} } \right) \, \times { 1}00\% .$$
Intracellular uptake of DVDMS
To analyze the intracellular localization of DVDMS in HCT116 cells and RKO cells, about 5 × 104 cells/well were cultured in a glass-bottom culture dish. After 24 h, the cells were incubated with DVDMS (5 µg/mL) for 6 h, followed by gent washing with cold PBS twice and fixation with 4% paraformaldehyde for 20 min. The cells were stained with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and the intracellular fluorescence of DVDMS was observed under a laser scanning confocal microscope (LSM880, Carl Zeiss, Germany). To quantitatively investigate the effects of different incubation time points on the intracellular accumulation of DVDMS, the NCM460, RKO and HCT116 cells (2.5 × 105 cell/well) were seeded into six-well plates and incubated with 5 µg/mL DVDMS for 0, 1, 3, 6, 9, and 12 h. The mean fluorescence intensity of intracellular DVDMS was analyzed using a flow cytometer (BD Accuri C6 Plus, BD Biosciences, Franklin Lakes, NJ, USA) and a microplate reader (BioTek, Winooski, VT, USA).
Ultrasonic system and SDT protocol
The experimental apparatus used in this study is shown in Fig. 8. A homemade single-element spherical transducer (center frequency: 0.970 MHz, lateral and axial full-width at half-maximum intensity of the beam: 3.5 and 15 mm, respectively) was used, and the acoustic peak rarefactional pressure map at the focal area in the lateral plane was measured using a needle hydrophone (HNR-0500, Onda Corp, Sunnyvale, CA, USA). The transducer was driven by a 50-dB power amplifier (2100L, Electronics & Innovation, Rochester, N.Y., USA), which was connected to a functional generator (AFG3102C, Tektronix, Inc., Beaverton, O.R., USA) to produce ultrasound waves. The transducer was seamlessly immersed in a cone filled with degassed water. The cone tip was sealed with a thin polyurethane membrane, and the focal zone of the ultrasound beam was left 2 mm beneath the cone tip. Cells (8 × 104 cells/slip) were seeded into an 8-mm-diameter circular coverslip. During the treatment, the coverslip was placed in the center of a cell culture dish filled with degassed water. The cone tip was immersed and placed at a distance of 2 mm above the coverslip to ensure that the cells were in the focal zone of the acoustic field. The cell culture dish was placed above a water tank filled with degassed water and a piece of ultrasound-absorbing material at the bottom.
The study had four groups as follows: a control group without any treatment (Control), group incubated with DVDMS alone for 6 h (DVDMS), group treated with ultrasound alone (US), and a group treated with SDT using DVDMS (SDT). The ultrasonic parameters used in US and SDT groups were the same (center frequency: 0.970 MHz; acoustic power: 3.45 W; duration: 3 min; duty cycle: 30%). For each group, cells on five coverslips were used to undergone the treatment and collected for further flow cytometry analysis. The treatments were repeated five times. The cell coverslip was placed slightly at the bottom of the cell culture dish and exposed to ultrasound. Cellular apoptosis and necrosis were evaluated after different treatments.
Analyses of cellular apoptosis and necrosis
Cells were seeded (8 × 104 cells/well) on circular coverslips placed in a 24-well plate. Cells were randomly divided into four groups (Control, DVDMS, US, and SDT) as mentioned above. The cells in DVDMS and SDT groups were incubated with DVDMS (5 µg/mL) in the dark for 6 h. Then, the cells in US and SDT groups were exposed to ultrasound for 3 min. After treatments, cells were cultured for 2 h before flow cytometry analyses. Cellular apoptosis and necrosis were analyzed with an Annexin V-FITC and PI detection kit (BD Biosciences, San Diego, CA, USA). In brief, the cells were harvested and suspended in a flow tube using 200 µL 1× binding buffer. To adjust the fluorescence compensation, a blank control group (without staining) and two groups stained with PI or FITC solution separately were prepared. The cells in the four groups (Control, DVDMS, US and SDT) were stained with both FITC and PI solution. After staining in the dark, the apoptosis and necrosis of cells were immediately evaluated with flow cytometry (BD Accuri C6 Plus, BD Biosciences, Franklin Lakes, NJ, USA) using the FL-1 filter (excitation 488 nm, emission 525 nm) and FL-2 filter (excitation 488 nm, emission 590 nm).
Detection of intracellular ROS after treatment
The probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) is a non-fluorescent and cell-permeable agent that is easily hydrolyzed to its de-esterified product DCFH, which could be oxidized into its highly fluorescent form 2,7-dichlorofluorescein (DCF) by intracellular ROS. Therefore, the fluorescence intensity of DCF is used to probing the intracellular ROS level. For qualitative analysis, HCT116 or RKO cells (8 × 104 cells/well) were seeded on circular coverslips placed in 24-well plates. After incubation for 24 h, cells were treated with 10 μM DCFH-DA (Solarbio, Beijing, China) for 20 min at 37 °C with gentle shaking in the dark and then were divided into the four groups. At 2 h post-treatment, cells were subjected to fluorescence imaging or flow cytometry analysis. For fluorescence imaging, cells were carefully washed twice with PBS and then the fluorescence of DCF was observed using an inverted fluorescence microscope (ECLIPSE Ti, Nikon, Tokyo, Japan) with excitation and emission wavelengths of 498 and 522 nm, respectively. To quantify the fluorescence intensity of DCF, cells were harvested, washed with PBS, filtered, and analyzed immediately with flow cytometry (BD Accuri C6 Plus, BD Biosciences, Franklin Lakes, NJ, USA).
Statistical analysis
All data are expressed as mean ± standard deviation, and statistical significance was determined using one-way analysis of variance (ANOVA). A value of p < 0.01 was considered statistically significant.
